AFAB Volume 4 Issue 2 by cielo shabatura

ISSN: 2159-8967 www.AFABjournal.com

Volume 4, Issue 2 2014

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

EDITORIAL BOARD Sooyoun Ahn

Hae-Yeong Kim

University of Florida, USA

Kyung Hee University, South Korea

Walid Q. Alali

Woo-Kyun Kim

University of Georgia, USA

Kenneth M. Bischoff

M.B. Kirkham

NCAUR, USDA-ARS, USA

Kansas State University, USA

Debabrata Biswas

Todd Kostman

University of Maryland, USA

University of Wisconsin, Oshkosh, USA

Claudia S. Dunkley

Y. M. Kwon

University of Georgia, USA

University of Arkansas, USA

Michael Flythe

Maria Luz Sanz

USDA, Agricultural Research Service

MuriasInstituto de Quimica Organic General, Spain

Lawrence Goodridge

Melanie R. Mormile

McGill University, Canada

Missouri University of Science and Tech., USA

Leluo Guan

Rama Nannapaneni

University of Alberta, Canada

Mississippi State University, USA

Joshua Gurtler

Jack A. Neal, Jr.

ERRC, USDA-ARS, USA

University of Houston, USA

Yong D. Hang

Benedict Okeke

Cornell University, USA

Auburn University at Montgomery, USA

Armitra Jackson-Davis

John Patterson

Alabama A&M University, USA

Purdue University, USA

Divya Jaroni

Toni Poole

Oklahoma State University, USA

FFSRU, USDA-ARS, USA

Weihong Jiang

Marcos Rostagno

Shanghai Institute for Biol. Sciences, P.R. China

LBRU, USDA-ARS, USA

Michael Johnson

Roni Shapira

University of Arkansas, USA

Hebrew University of Jerusalem, Israel

Timothy Kelly

Kalidas Shetty

East Carolina University, USA

North Dakota State University, USA

William R. Kenealy Mascoma Corporation, USA Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

EDITORIAL STAFF EDITOR-IN-CHIEF Steven C. Ricke University of Arkansas, USA

EDITORS Todd R. Callaway FFSRU, USADA-ARS, USA Philip G. Crandall University of Arkansas, USA Janet Donaldson Mississippi State University, USA

MANAGING and LAYOUT EDITOR Ellen J. Van Loo Ghent, Belgium

TECHNICAL EDITOR Jessica C. Shabatura Fayetteville, USA

ONLINE EDITION EDITOR C.S. Shabatura Fayetteville, USA

Ok-Kyung Koo Korea Food Research Institute, South Korea

ABOUT THIS PUBLICATION Mailing Address: 2138 Revere Place . Fayetteville, AR . 72701 Agriculture, Food & Analytical Bacteriology (ISSN 2159-8967) is published quarterly. Instructions for Authors may be obtained at the back of this issue, or online via our website at www.afabjournal.com Manuscripts: All correspondence regarding pending manuscripts should be addressed Ellen Van Loo, Managing Editor, Agriculture, Food & Analytical Bacteriology: ellen@afabjournal.com Information for Potential Editors: If you are interested in becoming a part of our editorial board, please contact Editor-in-Chief, Steven Ricke, Agriculture, Food & Analytical Bacteriology: editor@afabjournal.com

Website: www.AFABjournal.com

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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

TABLE OF CONTENTS ARTICLES 96

Contribution of Chemical and Physical Factors to Zoonotic Pathogen Inactivation during Chicken Manure Composting M.C. Erickson, J. Liao, X. Jiang, and M.P. Doyle

REVIEW 76

Antibiotic Use in Livestock Production Broadway, P. R., J. A. Carroll, and T. R. Callaway

Effects of Co-nutrients in Foods and Bioremediation in the Environment on Methylmercury P. G. Crandall, C. A. O’Bryan

109 Alternative antimicrobial supplements that positively impact animal health and food safety Broadway, P. R., J. A. Carroll, and T. R. Callaway

122 Human Health Benefits of Isoflavones from Soybeans k. Kushwaha, C. A. O’Bryan, D. Babu, P. G. Crandall, P. Chen, and S.-O. Lee

Introduction to Authors 147 Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors. Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

REVIEW Antibiotic Use in Livestock Production P. R. Broadway1, J. A. Carroll2, and T. R. Callaway3 Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX Livestock Issues Research Unit, Agricultural Research Service, USDA, Lubbock, TX 3 Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, USDA, College Station, TX 1

“Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, or exclusion of others that may be suitable.” USDA is an equal opportunity provider and employer

ABSTRACT Antibiotic usage is a useful and commonly implemented practice in livestock and production agriculture that has progressively gained attention in recent years from consumers of animal products due to concerns about human and environmental health. Sub-therapeutic usage of antibiotics has led to a concern that prophylactic supplementation leads to antimicrobial resistance, and this particular practice has come under public scrutiny. The consumer and media misconceptions about antibiotic usage and production strategies utilized in livestock production have caused a shift in consumer demands. Antibiotics directly and indirectly affect the livestock industry by treating illness and promoting the overall health of the animal, which may enhance production parameters such as growth and profitability. However, pending legislation threatens to eliminate the current antibiotic usage strategies implemented by producers. This review will address the historical and current use of antibiotics as it pertains to production animal agriculture to summarize how antibiotics promote animal health and growth performance. Keywords: Antibiotic, livestock, animal health, review

INTRODUCTION Antibiotic usage in meat animal production is a hotly debated issue in the livestock industry that has acquired more attention as consumers seek to place more “natural” and “safer” products on their Correspondence: Todd Callaway, todd.callaway@ars.usda.gov Tel: +1-979-260-9374 Fax: +1-979-260-9332.

Agric. Food Anal. Bacteriol. 4: 76-85, 2014

table (Gilbert and McBain, 2003). Consumer perception can greatly influence food animal production as has been recently observed for some common food production practices; such as lean finely textured beef (“pink slime”) which was removed from meat formulations of producers due to negative media attention and consumer perception (Flock, 2012). The use of gestation crates in swine production has also drawn increasing attention, leading to the refusal of

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some retailers and restaurants to purchase pork from producers that utilize gestation crates (Food Safety News, 2013). Furthermore, antibiotic usage in animals for health benefits and growth promotion has continued to be a concern of the American consumer in recent years. In response to similar concerns, the European Union (EU) banned sub-therapeutic supplementation of animal feeds with antibiotics (Pradella, 2006). Recently, the U.S. Food and Drug Administration issued a guidance directive on the judicious use of antibiotics in food animals, and this measure has led some to believe that this is phase one of an agenda to remove sub-therapeutic antibiotic use from livestock production.

years (Moore et al., 1946; Jukes et al., 1950; Rogers et al., 1995; Salinas-Chavira et al., 2009). Performance parameters can be quantitatively measured in a variety of ways including, but not limited to: mortality, weight gain, meat/milk quality, and feed efficiency. While the mode of action by which antibiotics improve feed efficiency has not been fully elucidated, growth performance may be enhanced due to decreased inflammation in the small intestine (Feighner and Dashkevicz, 1987; Eyssen and DeSomer, 1963). To further explain how antibiotics may work in conjunction to promote animal health and food safety, McCracken and Gaskins (1999) indicated that the development of the intestinal immune system occurs

The gastrointestinal tract of animals is populated with a complex microbial ecosystem that is essential for the function, growth, and overall health of the animal (Chaucheyras-Durand and Durand, 2010). Many livestock producers currently utilize feeding and production strategies, including the use of antibiotics, that alter the microbial ecology of the gastrointestinal tract of the animal to benefit the overall health and production efficiency of their animals. As a bonus to the consumer, some of these strategies may also help eliminate or reduce foodborne pathogens that may contaminate the food supply (Perlman, 1973). If and when sub-therapeutic antibiotic use in food animals is banned in the U.S., alternative strategies must be implemented to replicate these positive effects in order for the livestock industry to remain viable.

in conjunction with the development of the normal microflora of the animal; however chronic stimulation of the immune system may decrease the amount of protein available for growth (Gordon et al., 1963). Studies comparing germ-free and conventionally raised animals have demonstrated this phenomenon and have reported alterations in immune function of these animals in conjunction with the development of the intestinal microflora (McCraken and Lorenz, 2001). Thinning of the intestinal epithelium in conjunction with the use of antibiotics may be the result of decreased microbial production of polyamines and volatile fatty acids (VFAs) that enhance intestinal cell growth and activity (Ferket et al., 2002). Ferket et al. (2002) states that intestinal mucosal thinning that may occur with the use of antibiotics may increase energy availability for growth because the animal does not have to maintain a larger intestinal mucosal layer.

CURRENT USE OF ANTIBIOTICS IN LIVESTOCK

Cattle

Antibiotics are used in the livestock industry for a variety of reasons including treatment of disease, prophylaxis, as well as improving feed efficiency and overall growth performance (Berge et al., 2005;

Antibiotics have been used for decades in cattle, and some of the most commonly used antibiotics in the feedlot setting are a class of compounds known as ionophores (Russell and Strobel, 1989). Ionophores were approved for use in ruminants in the

Brown et al., 1975). While antibiotics do not make label claims that suggest alteration of growth parameters in livestock, the association between their use and growth promotion has been reported in many species such as cattle, swine, and poultry for over 50

1970s (Russell and Strobel, 1989). The ionophore monensin was fed to chickens as a coccidiostat, and the manure from these poultry houses was spread on cattle pastures as a fertilizer. Cattle grazing these pastures grew more rapidly than cattle grazing pas-

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tures fertilized with manure from poultry houses where the chickens were not fed monensin (Callaway, 2013). As a result, the ionophore monensin was directly incorporated into cattle rations beginning in the 1970’s, and this compound has been reported to enhance growth performance through a variety of modifications of the ruminal microbiome (Raun et al., 1976; Callaway et al., 2003). Ionophores primarily inhibit bacteria with Gram positive physiology, including lactic acid bacteria, and this improves growth efficiency, average daily gain (ADG), reduces wasteful protein degradation (by hyperammonia producing bacteria), reduces methanogenesis, and reduces ruminal acidosis via lower lactate production (Russell

disease for the first 28 days at the feedlot (Guillermo and Berg, 1995; Smith et al., 1993). Another commonly used antibiotic in beef production is Tilmicosin which is a broad spectrum antibiotic used to treat and prevent BRD. Tilmicosin works to inhibit protein synthesis of bacteria such as Pasteurella hemolytica that may lead to the onset of BRD. Treatment of cattle upon arrival into feedlots with Micotil®, a solution of Tilmicosin, was shown to decrease BRD symptoms and increase dry matter intake (Galyean et al., 1995). Antibiotics are also used in livestock to prevent specific physiologic disorders such as ruminal lactic acidosis, a common problem in grain fed cattle that can be chronic or acute and range from moderate to

and Strobel, 1988). Ionophores have been reported to reduce liver abscesses by inhibiting epithelial keratinization caused by lactic acidosis and subsequent Fusobacterium necrophorum infections (Nagaraja and Chengappa, 1998; Lechtenberg et al., 1998). While compounds such as ionophores alter the microbial ecology of the gastrointestinal tract to promote overall health and performance, other antibiotics are used to treat specific bacterial disease and illness. Some of these antibiotics may also elicit a dual effect, promoting both health and performance in the animals. Bovine respiratory disease (BRD) is the most common and expensive disease present in American cattle, and the use of antibiotics to treat/ prevent this disease is a great example of this dual effect of antibiotics (Smith, 1998; Snowder et al., 2006). Bovine respiratory disease is a complex disease caused by exposure to various viral (e.g., Infectious Bovine Rhino-tracheitis, Bovine Viral Diarrhea, Bovine Respiratory Syncytial Virus, and Parainfluenza Virus) and/or bacterial (e.g.., Pasteurella hemolytica, Pasteurella multocida, Haemophilussomnus, Mycoplasmasp. and Actinomycespyogenes) pathogens. Bovine respiratory disease may be mitigated in a number of ways including vaccination, management practices, and antibiotic treatments to prevent and/

severe (Nagaraja and Titgemeyer, 2007; Slyter, 1976; Muir et al., 1981; Nagaraja et al., 1982). Ruminal acidosis is the accumulation of lactate in the rumen resulting in a lowered pH that decreases animal growth performance parameters, and leads to the development of other health problems such as laminitis, bloat, and liver abscesses (Nagaraja and Chengappa, 1998; Nocek, 1997; Enemark, 2008). In acute clinical lactic acidosis, D-lactate is the acid primarily responsible for this condition (Dunlop, 1965); however, the role of lactate in sub-acute acidosis is not fully understood (Enemark, 2009). The onset of acidosis is linked with feeding readily fermentable carbohydrates that are commonly associated with a high concentrate ration as would normally be fed in the cattle feedlot or swine finishing production systems (Owens et al., 1998; Russell and Hino, 1985). Antibiotics/antimicrobials and other feedstuffs have been reported to be effective strategies to prevent the onset of ruminal acidosis (Owens et al.,1998; Callaway et al., 2003). Antibiotics may decrease the incidence of liver abscesses in cattle which may be the result of ruminal acidosis and may predict carcass performance (Rogers et al., 1995; Brown and Lawrence, 2010). Virginamycin is an antibiotic used to prevent necrotic enteritis in cattle and has also been

or treat the disease. Addition of chloratetracycline and sulfamethazine to treat enteritis, coccidiosis, and bovine respiratory disease (BRD) in the ration of cattle arriving at the feed lot was also reported to increase ADG while decreasing the risk of bovine respiratory

reported to increase the gain to feed ratio in cattle (Salinas-Chavira et al., 2009). Rogers et al. (1995) reported an increase in ADG and feed conversion, and a decrease in liver abscesses in cattle fed virginamycin.

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Swine As in ruminants, such as cattle, antibiotics are used in swine production for many of the same reasons. These pharmaceuticals are used in swine for both prophylactic and treatment therapies, and in some cases, these antibiotics can also effect performance parameters. Jensen et al. (1955) reported increased gains and feed conversion in swine fed the antibiotic aureomycin. While aeuromycin was also initially reported to enhance reproductive performance in swine (Yestal et al., 1952), subsequent work by Davey et al. (1955) reported no difference in reproductive performance when swine were fed various concentrations of the antibiotic. Via-

Tylosin supplemented in the drinking water of swine for 17 days decreased clinical signs of gastrointestinal infection and promoted growth performance (Paradis et al., 2004). Tylosin-supplemented swine showed no clinical or pathological signs of proliferative enteropathy (ileitis) after experimental infection with Lawsonia intracellularis (McOrist et al., 1997). The mitigation of disease in concert with enhanced growth and reproductive performance as a result of antibiotic usage in swine help make the use of antibiotics a profitable production strategy (Zimmerman, 1986).

Poultry

bility and performance of newborn and suckling piglets was also unaffected when swine were supplemented with aureomycin (Davey et al., 1955). Aureomycin was further reported to increase profitability by increasing belly weight and decreasing backfat thickness (Perry et al., 1953). Zimmerman (1986) reported that antibiotics such as chloratetracyline, furazolidone, lincomycin, salinomycin, tylosin, and virginamycin may improve average weight gain by approximately 15%. Additionally, Zimmerman (1986) reported that combined use of chloratetracycline, penicillin, and sulfamethazine (2:1:2) increased ADG in starter pigs by 25%. Multiple studies in swine also indicate that treatment by any of the aforementioned antibiotics can increase farrowing rate (Zimmerman, 1986; Ruiz et al., 1968; Anderson, 1969; Hays 1978). Litter size may also be increased with the addition of a combination of antibiotics (Zimmerman, 1986; Ruiz et al., 1968; Hays 1978). The antibiotics penicillin and streptomycin increased the growth rate of swine fed to market weight (Bridges et al., 1952). Penicillin and streptomycin used in conjunction are still approved for use in the swine industry, as well as bovine, equine, and ovine species, to treat bacteria such as Arcanobacterium, Klebsiella pneumonia, Listeria spp., Mannheimia haemolytica, Pasteurella, Staphylococcus, and Salmonella (Norbrook Laboratories,

Antibiotic usage is an extremely important aspect of poultry production and has been used in production and researched extensively since the 1950s (Feighner and Dashkevicz, 1987). Antibiotics used in poultry production are believed to be effective growth promotants due to the alterations they induce in the microflora of the gastrointestinal tract (Feighner and Dashkevicz, 1987). This theory is supported by experiments that report germ-free chickens grow more efficiently than commercially raised poultry, and germ-free animals do not grow faster when given antibiotics with growth promoting capabilities (Coates et al., 1963; Forbes and Pank, 1959). In poultry, antibiotic feeding has been reported to increase weight gain and feed conversion efficiency (feed/gain; Feighner and Dashkevicz, 1987; Bunyan et al., 1977). Feed efficiency has been reported to be improved in poultry supplemented with antibiotics by reducing microbial populations in competition for nutrients and reduction of pathogenic bacteria (Feighner and Dashkevicz, 1987; Eyssen and deSomer, 1963; Barnes et al., 1978). Studies have reported that ammonia production by bacteria in the GI tract of monogastrics may suppress growth (Dang and Visek, 1960; Harbers et al., 1963; Visek, 1978). Deconjugation of bile salts may also play a role in

2013). Tylosin is another antibiotic approved for use in swine that can be provided via intramuscular injection, feed, or water, and is effective in preventing and controlling porcine proliferative enteropathy (ileitis; Paradis, 2004; Marseller et al., 2001; McOrist et al., 1997).

growth suppression due to Streptococcus faecium in the small intestine; however, the use of antibiotics has been reported to reduce attachment of this bacterium to intestinal epithelia (Cole and Fuller, 1984; Fuller et al., 1984; Fuller et al., 1983).

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Table 1. Some antibiotics used in animal agriculture that may be used to promote overall animal health and impact pathogen colonization and shedding

Some Antibiotics used in Animal Agriculture By Species Cattlea

Swineb

Poultryc

Aquacultured

Amoxicillin

Apramycin

Ardacin

Amoxicillin

Ampicillin

Bacitracin

Avilamycin

Ampicillin

Enroflaxin

Bambermycin

Avoparcin

Chloramphenicol

Erythromycin

Carbadox

Bacitracin manganese

Cortimoxazole

Florfenicol

Chloratetracycline

Erythromycin

Enroflaxin

Oxytetracycline

Furazolidone

Lincomycine

Erythromycin

Penicillin

Lincomycin

Mocimycin

Florfenicol

Sulfadimethoxine

Nosiheptide

Neomycin

Furazolidine

Tilmicosin

Salinomycin

Nosiheptide

Nitrofurans

Tylosin

Tiamulin

Penicillin

Oxolinic acid

Tylosin

Soframycin

Oxytetracycline

Virginamycin

Tetracycline

Sarafloxacin

Tylosin

Streptomycin

Virginamycin

Sulphadizine TrimethoprimSulfamethoxazole

Currin and Whittier, 2009

Zimmerman, 1986

Castanon, 2007

Defoirdt et al., 2011

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The intestinal epithelia in poultry and other species play a large role in the growth capabilities of animals, and antibiotics can alter the intestinal microflora as well as the intestinal epithelia of animals to promote growth. As mentioned previously, thinner intestinal epithelia may result in more efficient nutrient uptake and absorption (Eyessen and deSomer, 1963; Ford and Coates, 1971; Siddons and Coates, 1972; Sieburth et al., 1951). Also, antibiotics reduce populations of bacteria in the intestines, thereby making more nutrients available for animal growth (Eyssen, 1962; Monson et al., 1954). When antibiotics reduce the microbial population in the GI tract, they may inherently reduce pathogens responsible for dis-

of antibiotics in aquaculture (Alderman and Hastings, 1998). As in many food-producing species, antibioticresistant bacteria such as Aeromonas salmonicida, A. hydrophila, Vibrio anguillarum, Pseudomonas fluorescens, Pasteurella piscida, and Edwardsiella tarda have been documented in aquaculture species (Aoki, 1988).

ease or subclinical infections (Eyssen and deSomer, 1963a; Eyssen and deSomer, 1963b; Eyssen and deSomer, 1967; Sieburth et al., 1951). The combination of all these effects elicited by antibiotics provides a possible explanation as to why antibiotics enhance growth performance and feed efficiency.

ficacy and safety of these pharmaceuticals. These compounds are used not only to treat disease, but can also be used effectively as a prophylactic treatment. Such strategies to control pathogens in foodproducing animals may, in some cases, improve growth performance parameters while simultaneously promoting the overall health of the animal. Thus, antibiotics are a critical player in the profitability of agriculture in the U.S. and throughout the world and play a vital role in feeding the ever growing world population. However, an ever changing population and shifts in consumer demand have placed pressure on the agricultural industry and governments to reduce and/or eliminate the use of antibiotics in food production. While this potential change could possibly be detrimental to current management strategies, there are potential alternatives to antibiotics that have been extensively researched in livestock to promote health, performance, profitability, and food safety.

Aquaculture As in mammalian production, antibiotics also play a critical role in the aquaculture industry. Diseases in production aquaculture are estimated to cause losses of approximately 3 billion dollars per year globally (Subasinghe, et al., 2001). There are more than 100 known pathogens to fish; however, some of these are opportunistic pathogens (Alderman and Hastings, 1998). One of the main bacterial culprits are Vibrio bacteria (harveyi, cambellii, and parahaemolyticus; Defoirdt et al., 2007). While these pathogens are detrimental to the health of the aquaculture, some bacteria such as Vibrio cholera and vulnificus, may cause human disease as well (Thompson et al., 2004). Some of the antibiotics used in aquaculture are chloramphenicol, gentamycin, trimethorprim, tiamulin, tetracyclines, quinolones, and sulfonamides (Table 1; Defoirdt et al., 2007). Most of these antibiotics are incorporated into the feed of the aquaculture at specified dosages with required withdrawal times (Alderman and Hastings, 1998). However, countries around the world have vastly different regulations regarding the administration, dosage, withdrawal, and control

SUMMARY Antibiotics are an important part of agriculture and food production originating from the cattle, swine, poultry, and aquaculture industries, and much research has been conducted to determine the ef-

REFERENCES Alderman, D.J. and T.S. Hastings. 1998. Antibiotic use in aquaculture: development of antibiotic resistance- potential for consumer health risk. Int. J. Food Sci. & Technol. 33:139-155. Anderson, M.D. 1969. Effect of feeding furazilidone to gilts and sows at breeding. Hess & Clark, Research Digest. 7(5):1.

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Aoki, T. 1988. Drug-resistant plasmids from fish pathogens. Microbiol. Sci.. 5:219-223. Barnes, E.M., G.C. Mead, C.S. Impey, and B.W. Adams. 1978. The effect of dietary bacitracin on the incidence of Streptococcus faecalis subspecies liquefaciencs and related streptococci in the intestines of young chicks. Br. Poult. Sci. 19:713-723. Berge, A.C.B., P. Lindeque, D.A. Moore, and W.M. Sischo. 2005. A clinical trial evaluating prophylactic and therapeutic antibiotic use on health and performance of preweaned calves. J. Dairy Sci. 88:3036. Bridges, J.H., I.A. Dyer, and W.C. Burkhart. 1952. Effects on penicillin and streptomycin on the growth

Chaucheyras-Durand, F. and Durand, H. 2010. Probiotics in animal nutrition and health. Benef. Microbes. 1:3-9. Coates, M.E., R. Fuller, G.F. Harrison, M. Lev, and S.F. Suffolk. 1963. A comparison of the growth of chicks in the Gustafsson germ-free apparatus and in a conventional environment, with and without dietary supplements of penicillin. Br. J. Nutr. 17:141-150. Cole, C.B. and R. Fuller. 1984. Bile acid deconjugation and attachment of chicken gut bacteria; their possible role in growth depression. Br. Poult. Sci. 25:227-231. Currin, J.F. and D. Whittier. 2009. Recognition and

rate and bacterial count in the feces of pigs. J. Anim. Sci. 11:474-479. Brown, H., R.F. Bind, H.P. Grueter, J.W. McAskill, C.O. Cooley, and R.P. Rathmacher. 1975. Tylosin and chloratetracycline for the prevention of liver abcesses, improved weight gains and feed efficiency in feedlot cattle. J. Anim. Sci. 40:207-213. Brown, T.R. and T.E. Lawrence. 2010. Association of liver abnormalaties with carcass grading performance and value. J. Anim. Sci. 88:4037-4043. Bunyan, J., L. Jeffries, J.R. Sayers, A.L. Gulliver, and K. Coleman. 1977. Antimicrobial substances and chick growth promotion: the growth-promoting activities of antimicrobial substances, including fifty-two used either in therapy or as dietary additives. Br. Poult. Sci. 18:283-294. Callaway, 2013. Personal communication about monenesin on cattle pastures. Callaway, T. R., T. S. Edrington, et al. (2003). Ionophores: Their use as ruminant growth promotants and impact on food safety. Curr. Iss. Intest. Microbiol. 4:43-51. Callaway, T.R., Edrington, T.S., Rychik, J.L., Genovese, K.J., Poole, T.L., Jung, Y.S., Bischoff, K.M., Anderson, R.C., and Nisbet, D.J. 2003. Ionophores: Their use as ruminant growth promotants

Treatment of Bovine Respiratory Disease Complex. Virginia Cooperative Extension Publication. Publication 400-008. Dang, H.C. and W.J. Visek. 1960. Effects of urease injection on body weights of growing rats and chicks. Proc. Soc. Exp. Biol. Med. 105:164-167. Davey, R.J., W.W. Green, and J.W. Stevenson. 1955. The effect of aureomycin on growth and reproduction in swine. J. Anim. Sci. 14:507-512. Defoirdt, T., N. Boon, P. Sorgeloos, W. Verstraete, and P. Bossier. 2007. Alternatives to antibiotics to control bacterial infections: luminescent vibriosis in aquaculture as an example. Trends in Biotechnology. 25:472-479. Defoirdt, T., P. Sorgeloos, and P. Bossier. 2011. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr. Opin. Microbiol. 14:251-256. Dunlop, R.H. and P.B. Hammond. 1965. D-lactic acidosis of ruminants. Annals of the New York Academy of Science 119:1109-1132. Enemark, J.M.D. 2009. The monitoring, prevention and treatment of sub-acute ruminal acidosis (SARA): a review. The Veterinary Journal 176:32-43. Eyssen, H. 1962. The additive effects of nucleic acids and antibiotics as individual growth promo-

and impact on food safety. Curr. Issues Intest. Microbiol. 4:43-51. Castanon, J.I.R. 2007. History of the use of antibiotic as growth promoters in European poultry feeds. Poult. Sci. 86:2466-2471.

tants for chicks. Poult. Sci. 41:1822-1828. Eyssen, H. and P. deSomer. 1963. Effects of antibiotics on growth and nutrient absorption of chicks. Poult. Sci. 42:1373-1379. Eyssen, H. and P. deSomer. 1963b. The mode f ac-

Agric. Food Anal. Bacteriol. â&#x20AC;˘ AFABjournal.com â&#x20AC;˘ Vol. 4, Issue 2 - 2014

tion of antibiotics in stimulating growth of chicks. J. Exp. Med. 117:127-138. Eyssen, H. and P. deSomer. 1967. Effects of Streptococcus faecalis and a filterable agent on growth and nutrient absorption in gnotobiotic chicks. Poult. Sci. 46:323-333. Eyssen, H., and P. deSomer. 1963a. Effects of antibiotics on growth and nutrient absorption of chicks. Poult. Sci. 42:1373-1379. Feighner, S.D. and M.P. Dashkevicz. 1987. Subtherapeutic levels of antibiotics in poultry feeds and their effects on weight gain, feed efficiency, and bacterial cholytaurine hydrolase activity. Appl. Environ. Microbiol. 53:331-336.

gnotobiotic chickens mono-associated with Streptococcus faecium. Br. Poult. Sci. 24:111-114. Galyean, M.L., S.A. Gunter, and K.J. Malcom-Callis. 1995. Effects of arrival medication with tilmicosin phosphate on health and performance of newly received beef cattle. J. Anim. Sci. 73:1219-1226. Gilbert, P. and A.J. McBain. 2003. Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance. Clinical Microbiology Reviews. 16:189-208. Gordon, H.A., B.S. Wostmann, and E. Bruckner-Kardoss. 1963. Effects of microbial flora on cardiac output and other elements of blood circulation. Proc. Soc. Exp. Biol. Med. 114:301-304.

Ferket, P.R., C.W. Parks, and J.L. Grimes. 2002. Benefits of dietary antibiotic and mannanoligosaccharide supplementation for poultry. Multi-state poultry meeting. May 14-16, 2002. Flock, E. 2012. Pink slime removed from McDonald’s burgers- but other food additives remain. The Washington Post. Postted on 2/01/2012. Available: http://www.washingtonpost.com/blogs/ blogpost/post/pink-slime-removed-from-mcdonalds-burgers--but-other-weird-food-additives-remain/2012/02/01/gIQAdfvAiQ_blog.html Food Safety News. 2013. Pork producers agreet o phase out gestation crates across all of Canada. Published June, 4, 2013. Food Safety News. Available: http://www.foodsafetynews.com/2013/06/ gestation-crates-being-phased-out-across-canada/#.UrHN4NJDtVc Forbes, M. and J.T. Pank. 1959. Growth of germ-free and conventional chicks: effect of diet, dietary penicillin and bacterial environment. J. Nutr. 67:69-84. Ford, D.J., and M.E. Coates. 1971. Absorption of glucose and vitamins of the B complex by germfree and conventional chicks. Proc. Nutr. Soc. 30:10-11. Fuller, R., C.B. Cole, and M.E. Coates. 1984. The role of Streptococculs faecium in antibiotic-re-

Guillermo, F.G. and J.L. Berg. 1995. Efficacy of a feed-additive antibacterial combination for improving feedlot cattle performance and health. Canadian Vet. J. 36:223-229. Harbers, L.H., A.P. Alvares, A.I. Jacobson, and W.J. Visek. 1963. Effect of barbituric acid and choratetracycline upon growth, ammonia concentration and urease activit in the gastrointestinal tract of chicks. J. Nutr. 80:75-79. Hays, W.V., J.L. Krug, G.L. Cromwell, R.H. Dutt, and D.D. Kratzer. 1978. Effect of lactation length and dietary antibiotics on reproductive performance of sows. J. Anim. Sci. 46:884. Jensen, A.H., D.C. Aker, H.M. Maddock, G.C. Ashton, P.G. Homeyer, E.O. Heady, and D.V. Catron. 1955. Different protein levels with and without antibiotics for growing-finishing swine: effect on growth rate and feed efficiency. J. Anim. Sci. 14:69-81. Jukes, T.H., E.L.R. Stokstad, R.R. Taylor, T.J. Combs, H.M. Edwards, and G.B. Meadows. 1950. Growth promoting effect of auromycin on pigs. Arch. Biochem. 26:324-330. Lechtenberg, K.F., T.G. Nagaraja, and M.M. Chengappa. 1998. Antimicrobial susceptibility of Fusobacterium necrophorum isolated from bovine hepatic abscesses. Amer. J. Vet. Res. 59:44-47.

lieved growth depression in chickens, p. 395-403. In M. Woodbine (ed.), Antimicrobials and agriculture. Butterworths, London. Fuller, R., S.B. Houghton, and M.E. Coates. 1983. The effect of dietary penicillin on the growth of

Marseller, T., N Winkelman, C. Gebhart, W. Weldon, R. Muller, J. Weatherford, and L. Symanowski. 2001. Efficacy of intramuscular tylosin for the treatment and control of porcine proliferative enteropathy caused by Lawsonia intracellularis. Vet. Ther. 2:51-60.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

McCraken, V.J. and H.R. Gaskins. 1999. Probiotcs and the immune system. Pages 85-111 in Probiotics: A critical review. G.W. Tannock, ed. Horizon Scientific Press, Norfolk, UK. McCraken, V.J. and R.G. Lorenz. 2001. The gastrointestinal ecosystem: a precarious alliance among epithelium, immunity and microbiota. Cellu. Microb. 3:1-11. McOrist, S., J. Morgan, M.F. Veenhuizen, K. Lawrence, and H.W. Kroger. Oral administration of tylosin phosphate for treatment and prevention of proliferative enteropathy in pigs. Am. J. Vet. Res. 58:136-169. Monson, W.J., A.E. Harper, M.E. Winje, C.A. Elvehjem, R.A. Rhodes, and W.B. Sarles. 1954. A

Pradella, G. 2006. Antibiotic ban in the European Union: A pyrrhic victory? J. Vet. Pharmacol. Ther. 29:41. Raun, A. P., C. O. Cooley, E.L. Potter, R.P. Rathmacher, and L.F. Richardson. (1976). Effect of monensin on feed efficiency of feedlot cattle. J. Anim. Sci. 43:670-677. Rogers, J.A., M.E. Branine, C.R. Miller, M.I. Wray, S.J. Bartle, R.L. Preston, D.R. Gill, R.H. Pritchard, R.P. Stilborn, and D.T. Bechtol. 1995. Effects of dietary virginamycin on performance and liver abscess incidence in feedlot cattle. J. Anim. Sci. 73:9-20. Ruiz, M.E., V.C. Speer, V.W. Hays, and W.P. Switzer. 1968. Effect of feed intake and antibiotic on repro-

mechanism of the vitamin sparing effect of antibiotics. J. Nutr. 52:627-636. Moore, P.R., A. Evenson, T.D. Luckey, E. McCoy, E.A. Elvehjem, and E.B. Hart. 1946. Use of suophasuccidine, streptothriocin, and streptomycin in nutrition studies with the chick. J. Biol. Chem. 165:437-441. Muir, L. A., E. L. Rickes, P.F. Duquette, and G.E. Smith (1981). Prevention of induced lactic acidosis in cattle by thiopeptin. J. Anim. Sci. 52:635-643. Nagaraja, T. G., T. B. Avery, E.E. Barley, S.K. Roof, and A.D. Dayton. (1982). Effect of lasalocid, monensin or thiopeptin on lactic acidosis in cattle. J. Anim. Sci. 54:649-658. Nagaraja, T.G. and E.C. Titgemeyer. 2007. Ruminal acidosis in beef cattle: the current microbiological and nutritional outlook. J. Dairy Sci. 90:E17-E38. Nagaraja, T.G. and M.M. Chengappa. 1998. Liver abscesses in feedlot cattle: A review. J. Anim. Sci. 76:287-298. Norbrook Laboratories. 2013. Drug information sheet: available: http://www.norbrook.com/products/pen-strep-suspension-for-injection/ Owens, F.N., Secrist, D.S., Hill, W.J., and Gill, D.R. 1998. Acidosis in cattle: a review. J. Anim. Sci. 76:275-286. Perlman, D. Advances in Applied Microbiology.

duction in gilts. J. Anim. Sci. 27:1602. Russell, J.B. and H.J. Strobel. 1989. Effect of ionophores on ruminal fermentation. Appl. Environ. Microbiol. 55:1-6. Russell, J. B. and H. J. Strobel. 1988. Effects of additives on in vitro ruminal fermentation: a comparison of monensin and bacitracin, another grampositive antibiotitc. J. Anim. Sci. 66:552-558. Russell, J. B. and T. Hino 1985. Regulation of lactate production in Streptococcus bovis: a spiraling effect that contributes to rumen acidosis. J. Dairy Sci. 68:1712-1721. Salinas- Chavira, J., J. Lenin, E. Ponce, U. Sanchez, N. Torrentera, and R.A. Zinn. 2009. Comparative effects of virginamycin supplementation on characteristics of growth-performance, dietary energetic, and digestion of calf-fed Holstein steers. J. Anim. Sci. 87:4101-4108. Siddons, R.C. and M.E. Coates. 1972. The influence of the intestinal microflora on disaccharidase activities in the chick. Br. J. Nutr. 27:101-112. Sieburth, J.M., J. Gutierrrez, J. McGinnis, J.R. Stern, and B.H. Schneider. 1951. Effects of antibiotics on intestinal microflora and on growth of turkeys and pigs. Proc. Soc. Exp. Biol. Med. 76:15-18. Slyter, L. L. (1976). Influence of acidosis on rumen

1973. Vol. 16. Academic Press, Inc. New York, NY. Perry, T.W., W.M. Beeson, and B.W. Vosteen. 1953. The effect of an antibiotic or a surfactant on the growth and carcass composition of swine. J. Anim. Sci. 12:310-315.

function. J. Anim. Sci. 43:910-929. Smith, R.A. 1998. Impact of disease on feedlot performace: a review. J. Anim. Sci. 76:272-274. Smith, R.A., Gill, D.R., and M.T. Van Koevering. 1993. Effects of tilmicosin of ceftiofur on health and per-

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formance of stressed stocker catlle. Animal Science Research Report, Oklahoma Agriculture Experiment Station. pp. 308-311. Snowder, G.D., L.D. Van Vleck, L.V. Cundiff, and G.L. Bennett. 2006. Bovine respiratory disease in feedlot cattle: Environmental , genetic, and economic factors. J. Anim. Sci. 84:1999-2008. Subasinghe, R.P., P. Bueno, M.J. Phillips, C. Hough, S.E. McGladdery, and J.R. Arther (Eds.) 2001. Aquaculture development, health, and welfare. In Aquaculture in the Third Millenium Technical Proceedings of the Conference on Aquaculture in the Third Millenium (Subasinghe, R.P. et al., eds), pp. 167-191. Bangkok and FAO, NACA. Thompson, F.L., T. Iida, and J. Swings. 2004. Biodiveristy of Vibrios. Microbiol. Mol. Biol. Rev. 68, 403-431. Visek, W.J. 1978. The mode of growth promotion by antibiotics. J. Anim. Sci. 46:1147-1469. Yestal, C.M., W.M. Beeson, F.N. Andrews, L.M. Hutchings, and L.P. Doyle. 1952. Effect of aureomycin on the development and livability of newborn pigs. Purdue Mimeo. AH 87 Zimmerman, D.R. 1986. Role of subtherapeutic levels of antimicrobials in pig production. J. Anim. Sci. 62:6-16.

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REVIEW Effects of Co-nutrients in Foods and Bioremediation in the Environment on Methylmercury P. G. Crandall, and C. A. O’Bryan 1

Department of Food Science, University of Arkansas, 2650 Young Ave., Fayetteville, AR 72704

ABSTRACT Mercury, a potentially toxic metal, is present in the environment as a result of both natural processes and from man-made sources. The amount of mercury mobilized and released into the biosphere has increased significantly since the beginning of the industrial age. Inorganic mercury deposits in water and bottom sediments where it is subject to bacterial conversion to methylmercury, which bioaccumulates in the aquatic food chain with sometimes tragic consequences. This review discusses the production of methylmercury in the environment and exposure to and health effects for humans. We also discuss current knowledge of other nutrient interactions with methylmercury in the diet as well as possible methods for bioremediation of methylmercury in the environment. Keywords: Methylmercury, Minamata disease, mercury poisoning, biomagnification, bioaccumulation, bioavailability, bioremediation Agric. Food Anal. Bacteriol. 4: 86-95, 2014

INTRODUCTION The element mercury is a non-essential trace element that is toxic to humans and animals. At the fifty-third meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 2000) an update on the toxicity risks from methylmercury was summarized and a provisional tolerable weekly intake of methylmercury for the general population Correspondence: Philip G. Crandall, crandal@uark.edu Tel: +1 -479-575-7686 Fax: +1-479-575-6936

(3.3 µg/kg body weight) was reaffirmed with the admonition that pregnant women and nursing mothers may be in a higher risk category. The US Environmental Protection Agency has also calculated a reference dose (RfD) level for methylmercury, which is EPA’s estimate of the maximum acceptable daily exposure to humans that is not likely to cause harmful effects during a lifetime. The RfD for methylmercury was last revised by EPA 2001 and is currently 0.1 µg/ kg of body weight per day (Environmental Protection Agency, 2014), which is appreciably higher than the JECFA recommendations. In the environment,

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Figure 1. Mercury enters the food chain via manmade and natural emissions and is transformed into methylmercury in the lakes and oceans where it accumulates in fish (Environmental Protection Agency, 2014).

particularly lakes, waterways and wetlands, mercury can be converted from its elemental state to a highly toxic, organic compound called methylmercury through biogeochemical interactions. Once ingested methylmercury can easily cross the blood-brain and placental barriers and high levels of exposure may cause severe and irreversible damage, particularly to the fetal central nervous system (Clarkson and Magos, 2006). Methylmercury concentrations in water, soil, and sediments are usually very low especially when compared to the less toxic inorganic form (Zhang et al., 2010a; 2010b). However, methylmercury can accumulate (bioaccumulation) and be magnified (biomagnification) in aquatic food webs and even some terrestrial plants, for instance rice (Zhang et. al., 2010b), eventually posing a serious threat to humans through the consumption of fish and/or rice (Zhang et al., 2010a). See Figures 1 and 2 for additional information.

EXPOSURE TO METHYLMERCURY The main source of methylmercury contamination to humans is fish, a highly nutritious food with known benefits for human health. The Food and Drug Administration just completed and published a 10 year study on the levels of methylmercury contamination in the domestic fish supply (Tables 1 and 2; FDA, 2013). Fish are also a vital cultural and economic commodity for many communities around the world. All fish, however, do not have similar amounts of mercury because of bioaccumulation of methylmercury through the many levels of the aquatic food chain. Concentrations of total mercury vary widely across fish and shellfish species, with the mean values differing by as much as 100-fold (Keating et al., 1997). Methylmercury is bound to proteins, as well as to free amino acids, that are components of muscle tissues, and is not removed by any cooking or

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Figure 2. Mercury bioaccumulation. Methylmercury enters the base of the food web and is biomagnified at each successive level of the food chain. Highest levels are found in predators at the top of the aquatic food web (USGS, 2013).

Table 1. Fish and shellfish with the highest levels of mercury (FDA, 2013)

MERCURY CONCENTRATION (PPM) MEAN

MEDIAN

STDEV

MIN

MAX

NO. OF SAMPLES

MACKEREL KING

0.730

N/A

0.230

1.670

213

SHARK

0.979

0.811

0.626

4.540

356

SWORDFISH

0.995

0.870

0.539

3.220

636

TILEFISH (Gulf of Mexico)

1.450

N/A

0.650

3.730

SPECIES

Mercury was measured as Total Mercury N/A-data not available

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Table 2. Fish and shellfish with lower levels of mercury (FDA, 2013) SPECIES

MERCURY CONCENTRATION (PPM) MEAN

MEDIAN

STDEV

MIN

MAX

NO. OF SAMPLES

ANCHOVIES

0.017

0.014

0.015

0.049

BUTTERFISH

0.058

N/A

0.36

CATFISH

0.025

0.005

0.057

0.314

CLAM *

0.009

0.002

0.011

0.028

COD

0.111

0.066

0.152

0.989

115

CRAB 1

0.065

0.050

0.096

0.610

CRAWFISH

0.033

0.035

0.012

0.051

CROAKER ATLANTIC (Atlantic)

0.065

0.061

0.050

0.193

FLATFISH 2*

0.056

0.050

0.045

0.218

HADDOCK (Atlantic)

0.055

0.049

0.033

0.197

HAKE

0.079

0.067

0.064

0.378

HERRING

0.084

0.048

0.128

0.560

JACKSMELT

0.081

0.050

0.103

0.011

0.500

LOBSTER (Spiny)

0.093

0.062

0.097

0.270

MACKEREL ATLANTIC (N.Atlantic)

0.050

N/A

0.020

0.160

MACKEREL CHUB (Pacific)

0.088

N/A

0.030

0.190

MULLET

0.050

0.014

0.078

0.270

OYSTER

0.012

0.035

0.250

PERCH OCEAN *

0.121

0.102

0.125

0.578

POLLOCK

0.031

0.003

0.089

0.780

SALMON (CANNED) *

0.008

0.017

0.086

SALMON (FRESH/FROZEN) *

0.022

0.015

0.034

0.190

SARDINE

0.013

0.010

0.015

0.083

SCALLOP

0.003

0.007

0.033

SHAD AMERICAN

0.045

0.039

0.045

0.013

0.186

SHRIMP *

0.009

0.001

0.013

0.050

SQUID

0.023

0.016

0.022

0.070

TILAPIA *

0.013

0.004

0.023

0.084

TROUT (FRESHWATER)

0.071

0.025

0.141

0.678

TUNA (CANNED, LIGHT)

0.128

0.078

0.135

0.889

551

WHITEFISH

0.089

0.067

0.084

0.317

WHITING

0.051

0.052

0.030

0.096

Mercury was measured as Total Mercury except for species (*) when only methylmercury was analyzed. ND-mercury concentration below detection level (Level of detection = 0.01 ppm) 1 Includes: Blue, King, Snow 2 Includes: Flounder, Plaice, Sole Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

cleaning processes that do not destroy muscle tissues. In addition to fish, rice cultivated in areas contaminated with mercury can contain relatively high levels of methylmercury (Horvat et al., 2003; Zhang et al., 2010b). Other food sources of methylmercury have been reported including organ meats of terrestrial animals (Ysart et al., 2000), and chicken and pork (Lindberg et al., 2004), probably as a result of the use of fish meal as livestock feed. Persons living in certain communities also have higher methylmercury exposure because they consume the flesh of fish-eating marine mammals (Grandjean et al., 1995; Van Oostdam et al., 2005).

mammals, such as otters and whales, which consume fish from the top of the aquatic food chain receive the methylmercury that has bioaccumulated through this process (Mergler et al., 2007).

HEALTH EFFECTS OF METHYLMERCURY IN HUMANS When methylmercury is ingested it is readily and completely absorbed by the gastrointestinal tract. Methylmercury is complexed with the amino acid cysteine and with proteins and peptides containing cysteine; this complex is then recognized by

Mercury is found in the environment in three forms, elemental mercury, inorganic compounds and organic compounds; each form has specific solubility, chemical reaction, and toxicity characteristics (Clarkson, 2002; Goldman and Shannon, 2001). Elemental mercury is released via degassing from the crust and oceans of the earth, and the combustion of fossil fuels releases elemental mercury to the environment (ATSDR, 1999). Additional mercury is released from industrial waste; the total amount of mercury released each year from all sources may add up to as much as 9000 tons each year (ATSDR, 1999; Trasande et al., 2005). Mercury is deposited in surface waters from both industrial and naturally-occurring atmospheric sources where it can attach to particles suspended in the water. These particles eventually settle into the sediment where the mercury can be “methylated” during a complex chemical process facilitated by anaerobic organisms, thus forming methylmercury. Many factors dictate the occurrence rate of the methylation process. For example, studies have shown that water with a lower pH and higher dissolved organic carbon content generally results in higher levels of

the amino acid transporting system of the body as methionine, another essential amino acid (Kerper et al., 1992). Because this complex is recognized by the body as an essential amino acid, it is transported freely throughout the body including across the blood–brain barrier and across the placenta, where it is absorbed by the developing fetus. Since the methylmercury is so strongly bound to proteins and because the complex is recognized as an amino acid it is not readily removed from food or from the body (Carrier et al., 2001). There are several studies that suggest that methylmercury causes developmental delays in children exposed before birth, including attention deficits, loss of IQ points and decreased performance in tests of language skills and memory (Rice et al., 2003). There is insufficient data to make a causal link between pre-natal exposure to methylmercury from the mother’s diet and autism in spite of the expressed concerns of the public (van Wijngaarden et al., 2013). In adults, ingestion of methylmercury has been linked to increased risk of cardiovascular disease including heart attack (Salonen et al., 1995; Guallar et al., 2002; Choi et al., 2009), and there is some evidence that methylmercury can cause autoimmune diseases in sensitive individuals (Hultman

methylation (United States Geological Survey, 2009). Methylmercury is biomagnified in the aquatic food chain from bacteria, to plankton, through macroinvertebrates, to herbivorous fish, to fish-eating fish (Wiener et al., 2003). Humans and other fish eating

and Hansson-Georgiadis, 1999). In addition to chronic exposure to methylmercury there have been several episodes of acute exposure in which large numbers of people were severely poisoned by food contaminated with high levels of

MERCURY IN THE ENVIRONMENT

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methylmercury. The most widely known incident is probably the dumping of industrial waste that resulted in the pollution of water and fish and subsequent mass poisonings in Minamata and Niigata, Japan (Harada, 1995). Another such episode took place in Iraq in 1971; wheat treated with methylmercury was shipped to Iraq as seed grain not intended for human consumption. Due to a number of factors, including foreign-language labeling and late distribution within the growing cycle, this toxic grain was consumed as food by Iraqi residents in rural areas. The recorded death toll was 650 people, but figures at least ten times greater have been suggested, making this the largest mercury poisoning disaster

Khan and Wang, 2009; Li et al., 2012) . Shim et al. (2009) found that phytochemical rich foods, specifically green tea extract, black tea extract, and soy protein significantly reduced mercury bioaccessibility by 82 to 92%, 88 to 91%, and 44 to 87%, respectively. Wheat bran decreased bioaccessibility by 84%, oat bran by 59 to 75% and psyllium by 15 to 31% at amounts greater than 500 mg (Shim et al., 2009). Evidence also exists that suggests the developmental and cardiovascular toxicity of methylmercury may be mediated by co-exposures to omega-3 fatty acids, in particular docosahexaenoic acid (DHA) (Jin et al., 2007). Nøstbakken et al. (2012) found that omega-3 lessened methylmercury toxicity, either by decreas-

(Bakir et al., 1973). These episodes resulted in neurological symptoms including loss of feeling, loss of physical coordination, difficulty in speech, narrowing of the visual field, hearing impairment, blindness, and death. Children who had been exposed in-utero through their mothers’ ingestion were also affected with a range of symptoms including motor difficulties, sensory problems and mental retardation.

ing programmed cell death (eicosapentaenoic acid) or by reducing methylmercury uptake (DHA).

BIOREMEDIATION

The protective effect of selenium against methylmercury toxicity has been hypothesized for a number of years (Pařízek and Oštádalová, 1967; Skerfving, 1978; Cuvinaralar and Furness, 1991; Raymond and Ralston, 2004; Falnoga and Tusek-Znidaric, 2007; Yang et al., 2008; Khan and Wang, 2009). The protective effects of selenium against methylmercury toxicity in fetal brain development have now been confirmed but only in animal studies (Beyrouty and Chan, 2006; Sakamoto et al., 2013). Yang, et al. (2008) and Khan and Wang (2009) have summarized the several physiologic/biochemical mechanisms proposed to explain the antagonism between methylmercury and selenium. It seems likely that the molecular mecha-

Bioremediation is a waste management technique that involves the use of organisms to remove or neutralize pollutants from a contaminated site. The use of microbial biomass for bioremediation of toxic metals has been pursued for a number of years (Akthar et al., 1995, 1996; Akthar and Mohan, 1995; Gupta et al., 2000; Karna et al., 1999; Pethkar et al., 2001; Puranik and Paknikar, 1997; Volesky, 1987). Both live and non-living microbial biomass has been studied for removal of toxic metal ions but many researchers believe that non-living or processed biomass is a better choice. Non-living biomass does not have toxicity limitations as would living cells, nutrients are not needed for growth of biomass and since non-living biomass acts as an ion exchanger the process is rapid (Paknikar et al., 2003). The cell wall polymers of fungi are known to have functional groups such as amino, amide, hydroxyl, carboxyl, sulfhydryl and phosphate which have been implicated in metal binding (Akhtar et al., 1995; Gup-

nism involves the formation of insoluble, equimolar, and biologically unavailable mercury selenide precipitates, since approximately 1:1 molar ratios of selenium and mercury have been observed in marine mammals, sea birds and humans (Chen et al., 2006;

ta et al., 2000). Karunasagar et al. (2003) studied the effectiveness of a biosorbent prepared from biomass of Aspergillus niger for removal of methylmercury from dilute solutions. They determined that removal of methylmercury from spiked ground water samples

REDUCING BIOACESSABILITY

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was efficient and not influenced by other ions, and that the biosorbent was reusable for up to six cycles without appreciable loss of binding capacity. Live bacteria provide another means of methylmercury bioremediation (Barkay, et al., 2003; Barkay and Wagner-Dobler, 2005; Chen and Wilson, 1997; Miller, 1999; Nascimento and Chartone-Souza, 2003). Bacteria can break down mercury compounds through the acquisition of a transferable genetic element known as the mer operon (Omichinski2007). The mer operon is a dedicated set of mercury-resistant genes that are self-regulated by the DNA-binding protein MerR; bacteria resistant to methylmercury code for proteins that regulate mercury transport

cipitation, conventional coagulation, adsorption by activated carbons, adsorption by natural materials, ion exchange, or reverse osmosis. Mercury-resistant bacteria possess the mer operon enabling them to convert the toxic forms of mercury to nontoxic forms. Those possessing the merB gene are more valuable as they can detoxify methylmercury along with other organic mercurial compounds and inorganic mercury to nontoxic, volatile mercury. Bacteria harboring the merB gene and genetically modified organisms possessing the mer operon including merB are promising tools for use in bioremediation of methlymercury. However, the cons for the bacterial-based or plantbased processes may include production of large

(MerA, MerP, MerT) and mercury degradation (MerA and MerB) (Osborn et al., 1996; Sahlman et al., 1997; Silver and Phung, 1996; Wilson et al., 2000). Chien et al. (2010) made the point that there are substrate specificities among the MerB enzymes, elucidating the necessity for selecting the appropriate bacterial strains or MerB enzymes to apply them in bioremediation engineering for cleaning up specific mercury contaminants. Meagher (2000) engineered MerA and MerB into plants to remediate methylmercury contamination. Their theory was that remediation using plants is potentially more robust than bacterial remediation, because plants use solar energy, have roots that penetrate contaminated sediments, and accumulate a large aboveground biomass. There are actually a few well-characterized plant species used to clean up contaminated wetland ecosystems (Meagher, 2000). Plants such as cottonwood trees (Lyyra et al., 2007) and tobacco (Heaton et al., 2005) have been modified to express either MerB or both MerB and MerA; the plants converted the methylmercury to ionic mercury or elemental mercury, respectively; however, the elemental mercury was released into the atmosphere, where it may still pose a risk.

volumes of mercury-loaded biomass, the disposal of which is problematic.

CONCLUSIONS Bioremediation is considered to have advantages over conventional techniques such as chemical pre92

REFERENCES Akthar, N. and P. M. Mohan 1995. Bioremediation of toxic metal ions from polluted lake waters and industrial effluents by fungal biosorbent. Current Sci. 69: 1028–1030. Akthar, N., K. S. Sastry, and P. M. Mohan 1995. Biosorption of silver ions by processed Aspergillus niger biomass. Biotechnol. Letts. 17: 551–556. Akthar, N., K. S. Sastry, and P. M. Mohan 1996. Mechanism of metal biosorption by fungal biomass. Biometals 9: 21–28. ATSDR, Agency for Toxic Substances and Disease Registry. 1999. Toxicological profile for mercury: TP-93/10. Atlanta, Georgia: Centers for Disease Control. Bakir, F., S. F. Damluji, L. Amin-Zaki, M. Murtadha, A. Khalidi, N. Y. al-Rawi, S. Tikriti, H. I. Dahahir, T. W. Clarkson, J. C. Smith, and R. A. Doherty 1973. Methylmercury poisoning in Iraq. Science. 181:230-241. Barkay, T., S. M. Miller, A. O. Summers 2003. Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol. Rev. 27: 355-384. Barkay, T., and I. Wagner-Dobler 2005. Microbial transformations of mercury: potentials, challenges, and achievements in controlling mercury toxicity in the environment. Adv. Appl. Microbiol. 57: 1–52.

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Beyrouty, P. and H. M. Chan 2006.Co-consumption of selenium and vitamin E altered the reproductive and developmental toxicity of methylmercury in rats. Neurotoxicol. Teratol. 28: 49– 58. Carrier, G., M. Bouchard, R. C. Brunet, M. Caza 2001. A toxicokinetic model for predicting the tissue distribution and elimination of organic and inorganic mercury following exposure to methyl mercury in animals and humans. II. application and validation of the model in humans. Toxicol. Appl. Pharmacol. 171: 50–60. Chen, S., and D. B. Wilson 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2+-contami-

FDA. 2013. Mercury levels in commercial fish and shellfish (1990-2010). Available at: http://www.fda. gov/food/foodborneillnesscontaminants/metals/ ucm115644.htm Goldman, L. R., and M. W. Shannon 2001. Technical report: mercury in the environment: implication for pediatricians. American Academy of Pediatrics, Committee on Environmental Health. Pediatrics 108:197–205. Grandjean, P., P. Weihe, L.L. Needham, V.W. Burse, D.G. Patterson, Jr, E.J. Sampson, P.J. Jorgensen, and M. Vahter 1995. Relation of a seafood diet to mercury, selenium, arsenic, and polychlorinated biphenyl and other organochlorine concentrations in

nated environments. Appl. Environ. Microbiol. 63: 2442–2445. Chen, C. Y., H. W. Yu, J. J. Zhao, B. Li, L. Y. Qu, S. P. Liu, P. Q. Zhang, and Z. F. Chai, 2006. The roles of serum selenium and selenoproteins on mercury toxicity in environmental and occupational exposure Environ. Health Perspect. 114: 297– 301. Chien, M. F., M. Narita, K. H. Lin, K. Matsui, C. C. Huang, and G. Endo 2010. Organomercurials removal by heterogeneous merB genes harboring bacterial strains. J. Biosci. Bioeng. 110: 94-98. Choi, A.L., P. Weihe, E. Budtz-Jørgensen, P. J. Jørgensen, J. T. Salonen, T. P. Tuomainen, K. Murata, H. P. Nielsen, M. S. Petersen, J. Askham, and P. Grandjean 2009. Methylmercury exposure and adverse cardiovascular effects in Faroese whaling men. Environ. Health Perspect. 117: 367-372. Clarkson, T. W. 2002. The three modern faces of mercury. Environ. Health Perspect. 10:11–24. Clarkson, T. W. and L. Magos 2006. The toxicology of mercury and its chemical compounds Crit. Rev. Toxicol. 36: 609– 662. Cuvinaralar, M. L. A. and R. W. Furness 1991. Mercury and selenium interaction—A review Ecotox. Environ. Safe. 21: 348– 364. Environmental Protection Agency. 2014. Exposure to

human milk. Environ. Res. 71: 29–38. Guallar, E., I. Sanz-Gallardo, P. van’t Veer, P. Bode, A. Aro, J. Gómez-Aracena, J. D. Kark, R. A. Riemersma, J. M. Martín-Moreno, and F. J. Kok 2002/ Mercury, fish oils, and the risk of myocardial infarction. New England J. Med. 347: 1747-1754. Gupta, R., P. Ahuja, S. Khan, R. K. Saxena, and H. Mohapatra 2000. Microbial biosorbents: meeting challenges of heavy metal pollution in aqueous solutions. Current Sci. 78: 967–973. Harada, M. 1995. Minamata disease: methylmercury poisoning in Japan caused by environmental pollution. Crit. Rev. Toxicol. 25: 1-24. Heaton, A. C. P., C. L. Rugh, N.-J. Wang, R. B. Meagher 2005. Physiological responses of transgenic merA-TOBACCO (Nicotiana tabacum) to foliar and root mercury exposure. Water Air, Soil Pollut. 161:137-155. Horvat, M., N. Nolde, V. Fajon, V. Jereb, M. Logar, S. Lojen, R. Jacimovic, I. Falnoga, Q. Liya, J. Faganeli, and D. Drobne 2003. Total mercury methylmercury and selenium in mercury polluted areas in the province Guizhou, China. Sci. Total Environ. 304: 231–256. Hultman, P., and H. Hansson-Georgiadis 1999. Methyl mercury-induced autoimmunity in mice. Toxicol. Appl. Pharmacol. 154: 203–211.

methylmercury. Available at: http://www.epa.gov/ hg/exposure.htm Falnoga, I. and M. Tusek-Znidaric 2007. Selenium– mercury interactions in man and animals Biol. Trace Elem. Res. 119: 212– 220.

Jin, X. E. Lok, G. Bondy, D. Caldwell, R. Mueller, K. Kapal, C. Armstrong, M. Taylor, S. Kubow, R. Mehta, and H. M. Chan 2007. Modulating effects of dietary fats on methylmercury toxicity and distribution in rats. Toxicol. 230: 22–44.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

Karna, R.R., L. Uma, G. Subramanian, and P. M. Mohan 1999. Biosorption of toxic metal ions by alkaliextracted biomass of a marine cyanobacterium, Phormidium valderianum BDU 30501. World J. Microbiol. Biotechnol. 15: 729–732. Karunasagar, D., J. Arunachalam, K. Rashmi, J. N. L. Latha, and P. M. Mohan 2003. Biosorption of inorganic and methyl mercury by a biosorbent from Aspergillus niger. World J. Microbiol. Biotechnol.19: 291-295. Keating, M.H., K.R. Mahaffey, R. Schoeny, G.E. Rice, O.R. Bullock, R.B. Ambrose, J. Swartout and J.W. Nichols 1997. Mercury study report to Congress, Vol. III: fate and transport of mercury in the en-

Mergler, D., H. A. Anderson, L. H. M. Chan, K. R. Mahaffey, M. Murray, M. Sakamoto, and A. H. Stern 2007. Methylmercury exposure and health effects in humans: A worldwide concern. AMBIO 36: 3-11. Miller, S. M. 1999. Bacterial detoxification of Hg(II) and organomercurials. Essays Biochem. 34: 17–30. Nascimento, A. M., and E. Chartone-Souza 2003. Operon mer: Bacterial resistance to mercury and potential for bioremediation of contaminated environments. Genetics Molec. Res. 2: 92–101. Nøstbakken, O.J., L. Bredal, P. A. Olsvik, T. S. Huang, and B. E. Torstensen. 2012. Effect of marine Omega 3 fatty acids on methylmercury-induced toxicity in fish and mammalian cells in vitro. J. Biomed. Bio-

vironment. Office of Air Quality Planning and Standards and Office of Research and Development, U.S. Environmental Protection Agency, EPA452/R-97–005. EPA, Washington, D.C. Kerper, L.E., N. Ballatori, and T. W. Clarkson 1992. Methylmercury transport across the blood–brain barrier by an amino acid carrier. Am. J. Physiol. 262 (5 Pt. 2): R761–R765. Khan, M. A. K. and F. Y. Wang 2009. Mercury–selenium compounds and their toxicological significance: Toward a molecular understanding of the mercury–selenium antagonism Environ. Toxicol. Chem. 28: 1567– 1577. Li, Y.-F., Z. Dong, C. Chen, B. Li, Y. Gao, L. Qu, T. Wang, X. Fu, Y. Zhao, and Z. Chai, 2012. Organic selenium supplementation increases mercury excretion and decreases oxidative damage in longterm mercury-exposed residents from Wanshan, China Environ. Sci. Technol. 46: 11313– 11318. Lindberg, A., K.A. Bjornberg, M. Vahter, and M. Berglund 2004. Exposure to methylmercury in nonfish-eating people in Sweden. Environ. Res. 96: 28–33. Lyyra, S. R. B. Meagher, T. Kim, A. Heaton, P. Montello, R. S. Balish, and S. A. Merkle 2007. Coupling two mercury resistance genes in Eastern cotton-

technol. 2012: Article ID 417652, 13 pages, http:// dx.doi.org/10.1155/2012/417652 Osborn, A. M., K. D. Bruce, D. A. Ritchie, and P. Strike 1996. The mercury resistance operon of the IncJ plasmid pMERPH exhibits structural and regulatory divergence from other Gram-negative mer operons. Microbiol. 142: 337–345. Paknikar, K. M., A. V. Pethkar, and P. R. Puranik 2003. Bioremediation of metalliferous wastes and products using inactivated microbial biomass. Indian J. Biotechnol. 2: 426-443. Pařízek, J., and I. Oštádalová 1967.The protective effect of small amounts of selenite in sublimate intoxication Cell. Mol. Life Sci. 23: 142– 143. Pethkar, K.V., R. P. Gaikaiwari, and K. M. Paknikar 2001. Biosorptive removal of contaminating heavy metals from plant extracts of medicinal plants. Current Sci. 80: 1216–1218. Puranik P.R. and K. M. Paknikar 1997. Biosorption of lead and zinc from solutions using Streptoverticillium cinnamoneum waste biomass. J. Biotechnol. 55: 113–124. Raymond, L. J., and N. V.C. Ralston 2004. Mercury: selenium interactions and health implications Seychelles Med. Dent. J. 7 ( Special issue): 72– 77. Rice, D. C., R. Schoeny, and K. Mahaffey 2003. Meth-

wood enhances the processing of organomercury. Plant Biotechnol. J. 5: 254-262. Meagher, R. B. 2000. Phytoremediation of toxic elemental and organic pollutants. Curr. Opin. Plant Biol. 3: 153.

ods and rationale for derivation of a reference dose for methylmercury by the U.S. EPA. Risk Analysis 23: 107–115. Sahlman, L., W. Wong, and J. Powlowski 1997. A mercuric ion uptake role for the integral inner

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

membrane protein, MerC, involved in bacterial mercuric ion resistance. J. Biol. Chem. 272: 29518– 29526. Sakamoto, M., A. Yasutake, A. Kakita, M. Ryufuku, H. M. Chan, M. Yamamoto, S. Oumi, S. Kobayashi, and C. Watanabe 2013.Selenomethionine protects against neuronal degeneration by methylmercury in the developing rat cerebrum Environ. Sci. Technol. 47: 2862– 2868. Salonen, J. T., K. Seppänen, K. Nyyssönen, H. Korpela, J. Kauhanen, M. Kantola, J. Tuomilehto, H. Esterbauer, F. Tatzber, and R. Salonen 1995. Intake of mercury from fish, lipid peroxidation, and the risk of myocardial infarction and coronary, cardio-

son, G. Zareba, C. M. Burns, C. F. Shamlaye, and G. J. Myers 2013. Autism spectrum disorder phenotypes and prenatal exposure to methylmercury. Epidemiol. 24: 651-659. Volesky, B. 1987 Biosorbents for metal recovery. Trends Biotechnol. 5: 96–101. Wiener, J.G., D. P. Krabbenhoft, G. H. Heinz, and A. M. Scheuhammer 2003. Ecotoxicology of mercury in Hoffman, D.J., B.A. Rattner, G.A. Burton, Jr., and J. Cairns, Jr., eds., Handbook of Ecotoxicology, 2nd edition.: Boca Raton, Florida, CRC Press, p. 409-463. Wilson, J. R., C. Leang A. P. Morby, J. L. Hobman, and N. L. Brown 2000. MerF is a mercury transport

vascular, and any death in Eastern Finnish men”. Circulation 91: 645–655. Shim, S. M., M. G. Ferruzzi, Y. C. Kim, E. M. Janle, and C. R. Santerre 2009. Impact of phytochemicalrich foods on bioaccessibility of mercury from fish. Food Chem. 112: 46-50. Silver, S., and L. T. Phung 1996. Bacterial heavy metal resistance: New surprises. Ann. Rev. Microbiol. 50: 753–789. Skerfving, S. 1978. Interaction between selenium and methylmercury. Environ. Health Perspect. 25: 57–65. Trasande, L., P. J. Landrigan, and C. Schechter 2005. Public health and economic consequences of methyl mercury toxicity to the developing brain. Env. Health Persp. 113: 590-596. U.S. Geological Survey 2009. Mercury in the environment. http://www.usgs.gov/themes/factsheet/146-00/index.html Accessed 18 March 2014. USGS. 2013. The South Florida mercury science program. Available at: http://sofia.usgs.gov/publications/posters/merc_program/ Van Oostdam, J., S.G. Donaldson, M. Feeley, D. Arnold, P. Ayotte, G. Bondy, L. Chan, E. Dewaily, C. M. Furgal, H. Kuhnlein, E. Loring, G. Muckle, E. Myles, O. Receveur, B. Tracy, U. Gill, and S. Kalhok.

protein: Different structures but a common mechanism for mercuric ion transporters? FEBS Letts. 472: 78–82. Yang, D. Y., Y. W. Chen, J. M. Gunn, and N. Belzile 2008. Selenium, mercury in organisms: Interactions and mechanisms Environ. Rev. 16: 71– 92. Ysart, G., P. Miller, M. Croasdale, H. Crews, P. Robb, M. Baxter, C. de L’Argy, and N. Harrison 2000. 1997 UK Total Diet Study—dietary exposures to aluminium, arsenic, cadmium, chromium, copper, lead, mercury, nickel, selenium, tin, and zinc. Food Addit. Contam. 17: 775–786. Zhang, H., X. Feng, T. Larssen, G. Qiu, and R. D. Vogt 2010a. In inland China, rice, rather than fish, is the major pathway for methylmercury exposure Environ. Health Perspect. 118: 1183– 1188. Zhang, H. X. Feng, T. Larssen, L. Shang, and P. Li 2010b. Bioaccumulation of methylmercury versus inorganic mercury in rice (Oryza sativa L.) grain. Environ. Sci. Technol. 44: 4499– 4504.

2005. Human health implications of environmental contaminants in Arctic Canada: a review. Sci. Total. Environ. 351–352: 165–246. van Wijngaarden, E., P. W. Davidson, T. H. Smith, K. Evans, K. Yost, T. Love, S. W. Thurston, G. E. WatAgric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

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Contribution of Chemical and Physical Factors to Zoonotic Pathogen Inactivation during Chicken Manure Composting M.C. Erickson1*, J. Liao1, X. Jiang2, and M.P. Doyle1 Center for Food Safety and Department of Food Science and Technology, University of Georgia, Griffin, GA 2 Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC

ABSTRACT Land application is a common method for disposal of manure and litter that accumulate during poultry production; however, zoonotic pathogens residing in the manure may contaminate either directly or indirectly ready-to-eat produce crops. Aerobic composting of animal manure is a beneficial process treatment that inactivates these pathogens. Although heat is considered to be the primary contributing factor to inactivation, ammonia and volatile acids may also serve antimicrobial roles during composting. This study was designed to determine the relative contributions of chemicals and heat to the inactivation of Salmonella and Listeria monocytogenes in chicken manure-based compost mixtures formulated to give initial carbon:nitrogen (C:N) ratios of 20:1, 30:1 and 40:1. The different initial C:N ratio formulations of the compost mixtures had no effect on pH or the cumulative heat generated. In general, there was within all compost mixtures an initial decline in pH followed by an increase in pH that coincided with an increase in temperature. Levels of ammonia and volatile acids were higher in compost mixtures formulated to an initial C:N ratio of 20:1 than in other C:N formulations. The inactivation rates of Salmonella and L. monocytogenes within 20:1 C:N formulations were higher than in other formulations. Regression models derived from the data revealed that volatile acid levels, in addition to heat, played a major role in pathogen inactivation. Therefore, it may be advantageous to formulate compost mixtures containing chicken litter to an initial C:N of 20:1 to take advantage of the antimicrobial activity of volatile acids generated when sub-lethal temperatures occur. Keywords: manure, litter, composting, chicken, heat, ammonia, volatile acids, pH, Listeria monocytogenes, Salmonella Agric. Food Anal. Bacteriol. 4: 96-108, 2014

Correspondence: M.C. Erickson, mericks@uga.edu Tel: +1 -770-412-4742 Fax: +1-770-229-3216

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INTRODUCTION Poultry production in the United States is a major enterprise having had a combined value in broiler, egg, and turkey production of $38 billion in 2012 (USDA, 2012). Although meat and eggs are the major outputs from this enterprise, a substantial amount of manure (generated from layer and turkey operations) and litter (mixture of manure, bedding material, wasted feed, feathers, and soil generated from broiler operations) is also produced. For example, the estimated tons of manure produced from poultry operations in the U.S. in 2007 was 81 million tons (US EPA, 2013).

sources are frequently used to irrigate fields growing ready-to-eat produce (Gu et al., 2013a), hence animal manure should be treated to inactivate pathogens prior to land application. A treatment that is often recommended to inactivate vegetative bacterial pathogens in manures is thermophilic aerobic composting. In this process, manure is mixed with one or more carbon amendments to produce a nutrient-rich environment favorable for the metabolism of thermophilic microorganisms. The major factor responsible for inactivating pathogens in such systems is heat generated by the metabolic activity of these thermophilic microorganisms (Erickson et al., 2010; Wichuk and

To dispose of this waste, land application has offered the best solution (Moore et al., 1998; Ritz and Merka, 2013). Poultry manure can harbor zoonotic pathogens such as Salmonella, Listeria monocytogenes, and Campylobacter (Chinivasagam et al., 2010; Hutchison et al., 2004, 2005), and if applied to fields growing ready-to-eat produce, these pathogens may contaminate those crops. Once excreted from the animal, pathogen survival is dependent on storage conditions (Goss et al., 2013; Leifert et al., 2008; Ziemer et al., 2010). If left undisturbed, Williams and Benson (1978) determined that Salmonella Typhimurium survived for at least 18 months in chicken litter at 11 or 25°C, and 13 days at 38°C. Decimal reduction times for S. Typhimurium in poultry manure are not only affected by storage temperature, but also by the type of matrix, being greater in manure slurries compared to manure piles (Himathongkham et al., 2000). According to USDA, only 5% of all U.S. cropland in 2006 was fertilized with manure, with most chicken manure being applied to peanut and cotton fields (MacDonald et al., 2009). Although this mode of disposal would appear to have a minimal food safety risk, natural waterways and irrigation ponds in the Southeastern U.S. have been found contami-

McCartney, 2007)). As a result, process conditions that are based on time and temperature have been promulgated in regulations or guidelines worldwide (Hogg et al., 2002). For example, guidelines within the U.S. include either a minimum temperature of 55°C for 3 days in aerated static piles or in-vessel systems or 55°C for 15 days in windrow systems (narrow trapezoidal elongated rows) during which time the piles must be turned a minimum of 5 times to ensure that all material is subjected to the necessary thermal conditions (US EPA, 1999). Although heat is the primary mechanism for inactivating pathogens during aerobic composting, temperature stratification within static piles can result in extended survival of pathogens at the surface as well as extended survival of pathogens at interior sites of piles composted during the winter (Berry et al., 2013; Erickson et al., 2010; Shepherd et al., 2007). In addition, exposure of the pathogen to nonlethal heat or selected moisture conditions could lead to metabolic alterations in the pathogen that makes them more resistant to the thermal conditions encountered during the thermophilic phase of composting (Chen et al., 2013; Shepherd et al., 2010; Singh et al., 2011, 2012). As evidence of this potential activity, Salmonella, Escherichia coli

nated with Salmonella and Campylobacter, especially after precipitation events (Gu et al., 2013b; Haley et al., 2009; Luo et al., 2013) and likely occurred from pathogen runoff of peanut and cotton fields amended with poultry manure. These water

O157:H7, and Listeria survived in poultry manurebased compost piles when exposed to temperatures above 55°C for more than 8 days (Hutchison et al., 2005). Hence, other factors, either chemical or biological, may provide a greater contribution

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to pathogen inactivation under those conditions. For example, accumulation of free ammonia in poultry manure has been reported to contribute to inactivation of S. Typhimurium and E. coli O157:H7 in poultry manure (Himathongkham et al., 2000). Alternatively, volatile acids generated during the early phase of composting in cow manure systems formulated to have an initial carbon:nitrogen (C:N) ratio of 20:1 were suggested to be bactericidal agents effective against Salmonella but not Listeria (Erickson et al., 2009a,b). Given that there are differences in microbial and raw material composition between cow and chicken manure (De Bertoldi et al., 1987; Lynch, 1987; Wang et al., 2007), it was

monella were thawed and individually streaked onto plates containing brain heart infusion agar (Becton Dickinson, Sparks, MD) with 8 µg/mL of erythromycin (BHIA-E) and tryptic soy agar (Difco Laboratories, Detroit, MI) with 100 µg/mL ampicillin (TSA-A), respectively. Following incubation of plates at 37°C for ca. 24 h, individual colonies were removed and subsequently streaked onto a second plate and held at 37°C for an additional 24 h. From this second set of plates, individual colonies of L. monocytogenes and Salmonella were removed and inoculated into 10 ml of brain heart infusion broth (Becton Dickinson) containing 8 µg/mL erythromycin (BHIB-E) and tryptic soy broth containing 100 µg/mL ampicillin (TSB-

the objective of this study to determine the relative contributions of heat, volatile acids, and ammonia to the inactivation of Salmonella and L. monocytogenes in chicken manure-based compost mixtures formulated to C:N ratios ranging from 20:1 to 40:1.

A), respectively. These suspensions were incubated for ca. 24 h at 37°C with agitation (150 rpm) before harvesting the bacteria by centrifugation (4,050 x g, 15 min, 4°C). The pelleted cells were washed three times in 0.1% peptone water (Difco) and resedimented by centrifugation before reconstituting in 0.1% peptone water to an optical density at 630 nm of ca. 0.5 that corresponded to a concentration of ca. 109 CFU/mL. The five strains of L. monocytogenes were then combined in equal proportions to make one 5-strain stock culture mixture, whereas the four strains of Salmonella were combined for one 4-strain stock culture mixture. Each of these stock culture mixtures was then diluted 10-fold with deionized water for mixtures of 108 CFU/mL that were used to spray chicken litter. Immediately after preparation of the spray mixtures, L. monocytogenes and Salmonella was enumerated by plating serial dilutions (1:10) on modified oxford medium (Acumedia Manufacturers, Lansing, MI) containing 10 mg/mL buffered colistin methanesulfonate, 20 mg/mL buffered moxalactam solution, and 8 µg/mL erythromycin (MOX-E) and TSA-A, respectively. Salmonella colonies emitted a bright green fluorescence when plates were held under a handheld UV light (365 nm) and the fluorescent colonies were counted as Salmonella. Fluorescent L.

MATERIALS AND METHODS Pathogen Strains and Their Preparation for Experimental Trials Five strains of Listeria monocytogenes (101M, 12443, F6854, G3982, and H7550) from the culture collection housed at the Center for Food Safety, University of Georgia were used for these studies. In addition, three strains of Salmonella enterica serovar Enteritidis (ME-18, H4639, and H3353) and one strain of S. enterica serovar Newport (11590) were also used from the culture collection. All strains had been labeled with the green-fluorescent plasmid (GFP), but for L. monocytogenes strains, the plasmid also contained an erythromycin-resistant marker, whereas Salmonella strains contained an ampicillin-resistant marker. Previously, plasmid stability of these GFPlabeled strains was reported to range from 8 to 52% and 15 to 77% plasmid loss after 20 generations for the L. monocytogenes and Salmonella strains, respectively (Ma et al., 2011). To prepare the pathogen strains for challenge studies, frozen cultures of L. monocytogenes and Sal98

monocytogenes colonies were smaller than Salmonella colonies and required a Leica MZ16 FA stereo fluorescence microscope (Bannockburn, IL) for visualization and counting.

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Compost Ingredients, Preparation, and Experimental Design Fresh chicken litter was collected from a broiler production facility located in Orchard Hill, GA. Batches were collected at different times for each independent replicate trial. Following transport, the litter was mixed thoroughly and a portion of the litter was removed for compositional analysis. The remainder of the litter was frozen to kill insect eggs and then held at -20°C until which time it was ready to be composted. Wheat straw and cottonseed meal were purchased from a local feed store and served as the major carbon sources for the compost mixtures. Chicken litter was added to a 28-L sanitized bowl and sprayed manually using a spray bottle with both the Salmonella and L. monocytogenes inocula for populations approximating 107 CFU/g. This inoculated mixture was then mixed with a Hobart mixer (model D320:0.75 h.p.). Wheat straw, cottonseed meal, and water were then added in such quantities that compost mixtures had an initial moisture content of 60% and a C:N ratio of either 20:1, 30:1, or 40:1. Immediately after mixing, the compost mixtures were sampled for chemical and microbiological analysis. The remainder of the compost mixture was then placed in one of three bioreactors. In this experimental study, three independent trials were conducted wherein each trial consisted of three bioreactor systems containing one compost mixture each of the 20:1, 30:1 and 40:1 C:N ratio mixtures. The compost mixtures were composted for up to 6 days and were sampled on days 1, 2, 3, and 6 to measure microbiological and chemical parameters.

shelf was supported 5 cm above the bottom. Two sampling ports (3 cm diameter) at heights of 6 to 9 cm and 10 to 13 cm above the PVC shelf and a hole (0.5 cm diameter) at a height of 6.5 cm above the shelf for insertion of a thermocouple wire were drilled into the sides of the bioreactors. Bioreactors were housed within a Precision 30 Mechanical Convection incubator (Thermo Fisher Scientific, Waltham, MA) that was maintained at a temperature of 40°C. Trapped air in the incubator was vented to a filtered exhaust system. Compost material (ca. 5 kg) was placed into each bioreactor after which a type T thermocouple wire was inserted through the small hole to a site designated as the bottom center (16 cm from bioreactor wall). An additional thermocouple was inserted to a depth of 10 cm into the top center of the compost mixture. All thermocouples (two per bioreactor) were connected to a multi-channel HotMux data logger (DCC Corp., Pennsauken, NJ) that was programmed to record temperatures at the 6 locations at 30-min intervals. Cumulative heat > 40°C (degree-days) was calculated as the product of time (days) and temperature (°C above the ambient incubator temperature of 40°C). Oxygen levels in the bioreactor system were measured on all sampling days using a Demista OT-21 oxygen probe (Arlington Heights, IL) prior to removing duplicate samples (25 g) with a sanitized grabbing tool at both the bottom center and top center locations.

Chemical and Microbiological Analyses

Bioreactors (46 cm high x 32 cm diameter) were constructed from PVC plastic pipe. Tightly fitting

All compost ingredients (chicken litter, wheat straw, and cottonseed meal) as well as the initial compost mixtures were analyzed for carbon, nitrogen, and moisture contents. Carbon content was determined on the basis of ash content obtained after combustion of samples at 550°C. The University

PVC covers had holes drilled into their center such that the bottom cover hole allowed condensate to drip into an attached bottle and the top cover hole allowed compressed air (155 ml/min) to be delivered to the system. Within the biochamber, a perforated

of Georgia’s Soil Testing Laboratory (Athens, Georgia) was used for analysis of nitrogen content via a macro-Kjeldahl method. Moisture levels were based on residual weights of vacuum dried samples. Ammonia concentrations in compost samples

Composting Apparatus and Sampling

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(5 g) was determined with a phenol-hypochlorite spectrophotometric procedure (Weatherburn, 1967), whereas the Hach spectrophotometric Method 8196 test kit (Loveland, CO) as adapted by Montgomery et al. (1962) was used to measure volatile acid concentrations in compost samples. Measurement of pH was made with an Acumet Basic pH meter (Fisher Scientific, Pittsburgh, PA) on compost samples (5 g) dispersed in 250 ml of deionized water. Salmonella and L. monocytogenes were enumerated by direct plate counts (limit of detection was 2 log CFU/g) or detected by selective enrichment culture (limit of detection was 1 log CFU/g). In either case, compost samples (5 g) placed in a Whirl-Pak

CFU/g. After conversion of enrichment culture data, all data were subjected to general linear models analysis of variance (GLM ANOVA) to determine the significance of experimental variables over all sampling times examined in the study. To differentiate treatments at individual sampling times, the data were subjected to one-way ANOVA and when statistical differences were observed (P < 0.05), sample means were differentiated using the least significant difference test. Multiple linear regression analysis was also conducted on data from each sampling day and treatment in an attempt to relate the total pathogen loss in the mixtures to the independent variables of pH, cumulative heat, and concentrations

bag were first pummeled in a Stomacher 400 Circulator (Seward Ltd., West Sussex, UK) for 1 min after adding 45 mL of 0.1% peptone water. Diluted (1:10) aliquots of this homogenate were applied to either TSA-A plates to enumerate Salmonella or MOX-E plates to enumerate L. monocytogenes. Enrichment cultures of Salmonella and L. monocytogenes consisted of adding 1 mL of the homogenate to 9 ml of selective enrichment medium (TSB-A or BHIB-E, respectively) and incubating this mixture for 24 h at 37°C. Aliquots of these enriched samples were then streaked onto TSA-A or MOX-E plates to determine the presence or absence of fluorescent Salmonella or L. monocytogenes colonies, respectively.

of volatile acids and ammonia.

Statistical Analyses The StatGraphics Centurion XVI software, version 16.1.03 (StatPoint Technologies, Inc., Herndon, VA) was used for statistical analysis of the collected data; however, pathogen populations were first converted to logarithmic values prior to conducting these operations. When samples did not yield any colonies during plate count enumeration but did have fluorescent colonies on plates streaked from enrichment cultures, a value of 1.0 log CFU/g, corresponding to the limit of detection by enrichment culture, was assigned to that sample. Otherwise, samples yielding negative results for both plate counts and enrichment cultures were assigned a value of 0.0 log 100

RESULTS AND DISCUSSION Chicken litter, collected from broiler houses, was mixed with wheat straw, cottonseed meal, and water in combinations to give mixture treatments varying in their initial C:N ratio. Following analysis of these initial compost mixtures, the C:N ratios that were measured for the 3 independent replicate trials averaged 20.6 ± 1.7, 32.4 ± 2.4, and 43.6 ±1.7, respectively. Initial moisture contents in the 20:1, 30:1, and 40:1 C:N ratio formulations were 62.7 ± 1.9, 60.8 ± 1.7, and 60.5 ± 2.8%, respectively. Continued monitoring of moisture contents on days 2 and 6 revealed that compost mixtures were generally above 40% moisture during this time and thus aerobic microbial activity would not have been inhibited (Rynk, 1992). Oyxgen concentrations during composting were also well above the 5% level that is considered to limit aerobic microbial activity (Rynk, 1992). All compost mixtures were initially characterized as slightly alkaline (Table 1). After one day of composting, the pH of all mixtures had decreased from 1.5 to 2.2 units and declines were greater as the C:N ratio of the compost formulation decreased. After this point in time, the pH of all mixtures increased. Overall, there were no significant differences in pH with the different C:N ratio treatments throughout the composting period (P < 0.05).

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Table 1. pH (mean ± S.D.) in compost mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

Initial C:N ratio Days

20:1

30:1

40:1

7.69 ± 0.48 c-e1

7.89 ± 0.19 d-f

7.79 ± 0.22 d-f

5.50 ± 0.39 a

5.96 ± 1.36 a

6.30 ± 1.37 ab

6.47 ± 1.68 a-c

7.52 ± 2.00 d

7.40 ± 1.80 cd

8.09 ± 0.95 d-f

7.37 ± 1.48 cd

7.26 ± 1.58 b-d

9.00 ± 0.22 f

8.55 ± 0.78 ef

8.12 ± 1.05 d-f

Levels followed by a different letter are significantly different (P < 0.05)

Table 2. Volatile acid concentrations (mg/g, mean ± S.D.) in compost mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

Initial C:N ratio Days

20:1

30:1

40:1

5.49 ± 2.37 a-c1

4.27 ± 1.32 a

3.68 ± 1.32 a

11.68 ± 3.52 d-g

12.42 ± 3.26 fg

9.08 ± 4.04 c-e

12.08 ± 3.59 e-g

11.34 ± 4.91 d-g

8.79 ± 3.88 b-d

13.26 ± 6.88 g

9.76 ± 4.49 d-f

5.79 ± 4.28 ab

8.80 ± 4.20 b-d

4.79 ± 2.67 a

4.85 ± 2.99 a

Levels followed by a different letter are significantly different (P < 0.05)

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Table 3. Ammonia concentrations (µg/g, mean ± S.D.) in compost mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

Initial C:N ratio Days

20:1

30:1

40:1

131.6 ± 24.7 a-c1

114.2 ± 23.8 ab

99.7 ± 30.2 ab

218.6 ± 94.3 c-e

215.9 ± 74.2 c-e

132.5 ± 23.4 b

271.0 ± 98.9 e

217.0 ± 56.4 c-e

145.3 ± 19.9 bc

379.4 ± 158.6 f

185.8 ± 47.1 b-d

123.9 ± 73.8 ab

246.6 ± 37.9 de

168.8 ± 216.7 bc

54.3 ± 57.3 a

Levels followed by a different letter are significantly different (P < 0.05)

These results were in contrast to those that were observed when compost mixtures were formulated to different C:N ratios with dairy manure as the nitrogen source (Erickson et al., 2009a, b). In those studies, compost mixtures formulated to a C:N ratio of 40:1 did not decline in pH during the first day of composting. Volatile acids, including acetate, butyrate, and propionate, are produced during the early phases of aerobic composting and digestion (Beck-Friis et al., 2003; Ugwuanyi et al., 2005a,b) and may be potential contributors to the pH declines observed in this study during the first day of composting of chicken litter. This suggestion was corroborated by the observed increase in volatile acid levels that occurred in the chicken litter compost mixtures during the first day (Table 2). The greatest increase in volatile acid

in 20:1 C:N compost mixtures than in the 40:1 C:N compost mixtures. Generally, facultative anaerobic microorganisms produce volatile acids in response to low oxygen concentrations (Brinton, 1998); however, it would appear that the nutrient conditions provided in the 20:1 C:N compost mixtures were more conducive than the other compost mixture formulations for generating such compounds. Ammonia is another common byproduct produced during the degradation of chicken manure or chicken litter (Bush et al., 2007; Himathongkham et al., 2000). There were significant differences in the ammonia concentrations of the different formulations of the chicken compost mixtures (Table 3). Specifically, when all sampling days were taken into account, the 20:1 C:N ratio compost mixture had the highest ammonia concentrations, whereas the low-

concentrations was observed in the 20:1 C:N compost mixtures, whereas the least increase occurred in the 40:1 C:N compost mixture. Furthermore, as composting continued, volatile acid levels declined in all compost mixtures, but the decrease was slower

est levels were in the 40:1 C:N ratio compost mixtures (P < 0.05). In addition, during the composting process, the ammonia concentrations were continuously shifting, with maximal levels found in the 20:1 C:N mixtures on day 3, whereas maximal levels in

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Table 4. Cumulative metabolic heat > 40°C (degree-days1, mean ± S.D. )during composting of mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

Initial C:N ratio Days

20:1

30:1

40:1

2.90 ± 2.00 a2

2.84 ± 2.36 a

3.48 ± 3.17 a

6.42 ± 4.53 ab

9.14 ± 10.67 a-c

9.16 ± 7.79 a-c

15.96 ± 10.02 cd

16.72 ± 16.09 b-d

19.05 ± 11.17 d

Accumulated product of temperature (°C above the ambient incubator temperature of 40°C) and composting time (days) 2 Levels followed by a different letter are significantly different (P < 0.05) 1

Table 5. Fate of Salmonella and L. monocytogenes populations (log CFU/g, mean ± S.D.) in compost mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

Salmonella Day

20:1 C:N

30:1 C:N

L. monocytogenes 40:1 C:N

20:1 C:N

30:1 C:N

40:1 C:N

7.43 ± 0.25 f

7.22 ± 0.26 f

7.38 ± 0.44 f

7.53 ± 0.17 g

7.21 ± 0.12 g

7.14 ± 0.13 g

3.64 ± 0.83 d

3.73 ± 1.96 d

5.31 ± 1.16 e

3.49 ± 1.22 e

3.79 ± 1.09 e

4.90 ± 0.97 f

1.58 ± 1.82 bc

0.48 ± 0.89 a

2.57 ± 2.33 c

1.21 ± 1.48 c

0.99 ± 0.88 bc

2.40 ± 1.83 d

0.28 ± 0.66 a

1.82 ± 2.42 c

0.00 ± 0.00 a

0.28 ± 0.66 ab

1.12 ± 1.05 c

0.00 ± 0.00 a

0.57 ± 0.84 ab

0.00 ± 0.00 a

Populations for each pathogen followed by a different letter are significantly different (P < 0.05).

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both the 30:1 and 40:1 C:N mixtures were detected on day 2. The increased generation of both ammonia and volatile acids in compost mixtures initially formulated to low C:N ratios agrees with the models presented by Delgado-Rodríguez et al. (2010) that demonstrated a higher level of volatile compounds present during municipal solid waste composting at low C:N ratios. Temperatures during composting of the mixtures were monitored throughout the period when samples were collected from the bioreactors. To assess the cumulative heat exposure above the ambient incubator temperature of 40°C, time-temperature curves were integrated using 40°C as the baseline.

statistically different for the 30:1 C:N formulations. During composting of chicken litter with different C:N ratio mixtures, pathogen levels were monitored (Table 5). Using ANOVA on data collected shortly after composting was initiated (days 1 and 2 only), it was revealed that the C:N ratio had a significant effect on inactivation of L. monocytogenes and Salmonella (P < 0.05). For both pathogens, the levels of the pathogen were higher in mixtures formulated to a C:N ratio of 40:1 than in those mixtures formulated to either 20:1 or 30:1. Slower inactivation had been observed previously for Salmonella in dairy manure compost mixtures of formulations having a C:N ratio of 40:1 compared to ratios of 20:1 and 30:1

Results for the first 3 days of composting, expressed as cumulative heat > 40°C (degree days), are presented in Table 4 and, although heat generation in compost mixtures was slightly greater as the C:N ratio increased, it was not significantly different (P > 0.05). Heat generation within each bioreactor was fairly homogeneous, as location was not a significant factor affecting the cumulative levels (P > 0.05). In contrast, over the three independent replicate trials, the level of heat accumulated in the compost mixtures was significantly different from each other (P < 0.05). As the manure source for each of these independent trials was collected at separate times from the broiler houses, a plausible explanation is that the chicken litter had been collected in the houses at different periods of time before being removed for composting. Aged manure used in composting mixtures produces less heat than fresh manure (Berry et al., 2013; Li et al., 2008). Such variability in manure age and subsequent variability in heat generation in this study would likely have contributed to an inability to detect a significant effect of C:N ratio on heat generation. A similar situation was also likely responsible for the inconsistent response of heat generation in compost mixtures formulated to different C:N ratios when using dairy manure (Erickson

(Erickson, 2009a), whereas the C:N ratio in dairy manure formulations did not affect the inactivation of L. monocytogenes (Erickson et al., 2009b). Pathogen inactivation, however, was not log-linear, but was characterized as biphasic. Hence, to determine if the C:N treatment affected inactivation during tailing, the number of days to complete inactivation of the pathogen was recorded for each replicate trial. For Salmonella, the days to complete inactivation ranged from 2 to 4, 2 to 5, and 3 to 8 for 20:1, 30:1, and 40:1 C:N formulations, respectively, whereas the days to complete inactivation of L. monocytogenes ranged from 2 to 3, 2 to 4, and 4 to 5, respectively. Given that only three values for each treatment were available, ANOVA applied to the days to inactivation data failed to reveal any significant effect by the C:N ratio of the mixture for either pathogen (P > 0.05). Despite this negative response, there is a trend of increasing days to inactivation with an increasing initial C:N ratio of the compost mixture and if explored in the future with a larger number of trial replicates, could prove to be significant. Comparison of Salmonella and L. monocytogenes responses in the composting mixtures revealed no significant differences in the rate of inactivation or days to inactivation (Table 5, P > 0.05). The similar-

et al., 2009b). In that study, no statistical differences occurred in the heat generated for the different C:N formulations in the bioreactor trials inoculated with E. coli O157:H7, whereas in bioreactor trials inoculated with L. monocytogenes, 20:1 formulations were

ity in responses contrast to those reported for composting of rural sewage sludge with straw (Pourcher et al., 2005) and composting of swine manure (Grewal et al., 2007), in which L. monocytogenes persisted for longer periods of time than Salmonella. Dif-

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ferences in comparative pathogen response in this study and others may have arisen due the different isolates used or to the different formulations used for composting. To understand the contribution of potential chemical and physical factors to pathogen losses during composting on any one sampling day, models were derived using backward stepwise regression. Both cumulative heat and levels of volatile acids were factors included in those models, described below, and explained 19.7% and 28.9 % of the variability in the data for Salmonella and L. monocytogenes, respectively.

least in the 40:1 C:N compost mixtures (P < 0.05). Moreover, in the 20:1 C:N compost mixtures, the inactivation rates of both Salmonella and L. monocytogenes were higher as well as the days required to achieve complete inactivation were in general sooner, than in compost mixtures formulated to either 30:1 or 40:1. Multiple linear regression models that were derived from fitting pathogen losses to cumulative heat and volatile acid levels were significant and explained 20 to 29% of the variability in the data. Hence, in conditions where heat may be insufficient to inactivate pathogens (winter composting or at the surface of unturned static compost piles), it may be advantageous to formulate the initial C:N ratio of

Salmonella losses = 1.455 + (0.121*cumulative heat>40°C) + (0.221* volatile acid concentration) (P = 0.0123)

chicken litter compost mixtures to values approaching 20:1, as higher volatile acid concentrations in these mixtures provide additional antimicrobial activity.

L. monocytogenes losses = 0.076 + (0.215*cumulative heat>40°C) + (0.317* volatile acid concentration) (P = 0.0018) These models reveal that volatile acids, in addition to heat, have a bactericidal role in chicken litter compost mixtures, particularly in those formulations (i.e. 20:1 C:N compost mixtures) in which high concentrations of volatile acids are produced.

ACKNOWLEDGEMENTS The project was supported by the National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service, grant # 2008-35201-18658.

REFERENCES CONCLUSIONS In summary, chicken litter was mixed with wheat straw and cottonseed meal to give formulations having initial C:N ratios of 20:1, 30:1 or 40:1. Although the pH decreased in all formulations during the first day of composting, the C:N ratio of the formulation did not have a significant effect on pH (P > 0.05) nor did it have a significant effect on the cumulated heat generated in the mixtures during composting (P >

Beck-Friis, B., S. Smårs, H. Jönsson, Y. Eklind, and H. Kirchmann. 2003. Composting of source-separated household organics at different oxygen levels: Gaining an understanding of the emission dynamics. Compost Sci. Util. 11:41-50. Berry, E.D., P.D. Millner, J.E. Wells, N. Kalchayanand, and M.N. Guerini. 2013. Fate of naturally occurring Escherichia coli O157:H7 and other zoonotic pathogens during minimally managed bovine feedlot manure composting processes. J. Food

0.05). In contrast, the different C:N formulations did have a significant effect on ammonia concentrations and volatile acids produced during composting, with the greatest amounts of these antimicrobials being generated in the 20:1 C:N compost mixtures and the

Prot. 76:1308-1321. Brinton, W.F. 1998. Volatile organic acids in compost: Production and odorant aspects. Compost Sci. Util. 6(1):75-82. Bush, D.J., M.H. Poore, G.M. Rogers, and C. Altier.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

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2007. Effect of stacking method on Salmonella elimination from recycled poultry bedding. Bioresource Technol. 98:571-578. Chen, Z., J. Diao, C. Ionita, and X. Jiang. 2013. Thermal inactivation of desiccation-adapted Salmonella spp. in aged chicken litter. Appl. Environ. Microbiol. 79:7013-7020. Chinivasagam, H.N., M. Redding, G. Runge, and P.J. Blackall. 2010. Presence and incidence of foodborne pathogens in Australian chicken litter. Br. Poultry Sci. 51:311-318. De Bertoldi, M., M.P. Ferranti, P. L’Hermite, and F. Zucconi. 1987. Compost: Production, Quality and Use. Elsevier Applied Science, New York, NY.

manure treatment. Compost Sci. Util. 15:53-62. Gu, G.Y., Z.Y. Luo, J.M. Cevallos-Cevallos, P. Adams, G. Vellidis, A. Wright, and A.H.C. Van Bruggen. 2013a. Factors affecting the occurrence of Escherichia coli O157 contamination in irrigation ponds on produce farms in the Suwanee River watershed. Can. J. Microbiol. 59:175-182. Gu, G.Y., Z.Y. Luo, J.M. Cevallos-Cevallos, P. Adams, A. Wright, and A.H.C. van Bruggen. 2013b. Occurrence and population density of Campylobacter jejuni in irrigation ponds on produce farms in the Suwannee River watershed. Can. J. Microbiol. 59:339-346. Haley, B.J., D.J. Cole, and E.K. Lipp. 2009. Distribu-

Delgado-Rodríguez, M., M. Ruiz-Montoya, I. Giraldez, I.O. Cabeza, R. López, and M.J. Díaz. 2010. Effect of control parameters on emitted volatile compounds in municipal solid waste and pine trimmings composting. J. Environ. Sci. Health Part A 45:855-862. Erickson, M.C., J. Liao, L. Ma., X. Jiang, and M.P. Doyle. 2009a. Inactivation of Salmonella spp. in cow manure composts formulated to different initial C:N ratios. Bioresource Technol. 101:10141020. Erickson, M.C., J. Liao, L. Ma., X. Jiang, and M.P. Doyle. 2009b. Pathogen inactivation in cow manure compost. Compost Sci. Util. 17:229-236. Erickson, M.C., J. Liao, G. Boyhan, C. Smith, L. Ma, X. Jiang, and M.P. Doyle. 2010. Fate of manure-borne pathogen surrogates in static composting piles of chicken litter and peanut hulls. Bioresource Technol. 101:1014-1020. Erickson, M.C., F. Critzer, and M.P. Doyle. 2010. Composting criteria for animal manure. Georgetown University Pew Charitable Trusts, Produce Safety Project, Issue Brief. Available at: http://www.pewhealth.org/uploadedFiles/PHG/Content_Level_ Pages/Reports/PSP%20Issue%20Brief%20Series. pdf. Accessed April, 2014.

tion, diversity, and seasonality of waterborne Salmonellae in a rural watershed. Appl. Environ. Microbiol. 75:1248-1255. Himathongkham, S., H. Riemann, S. Bahari, S. Nuanualsuwan, P. Kass, and D.O. Cliver. 2000. Survival of Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure and manure slurry at sublethal temperatures. Avian Dis. 44:853-860. Hogg, D., J. Barth, E. Faviono, M. Centemero, V. Caimi, F. Amlinger, W. Devliegher, W. Brinton, and S. Antler. 2002. Comparison of compost standards within the EU, North America, and Australasia. Main Report, The Waste and Resources Action Programme, Banbury, Oxon, U.K. Available at: http://www.compostingvermont.org/pdf/WRAP_ Comparison_of_Compost_Standards_2002.pdf. Accessed April, 2014. Hutchison, M.L., L.D. Walters, S.M. Avery, B.A. Synge, and A. Moore. 2004. Levels of zoonotic agents in British livestock manures. Lett. Appl. Microbiol. 39:207-214. Hutchison, M.L., L.D. Walters, S.M. Avery, F. Munro, and A. Moore. 2005. Analyses of livestock production, waste storage, and pathogen levels and prevalences in farm manures. Appl. Environ. Microbiol. 71:1231-1236.

Goss, M.J., A. Tubeileh, and D. Goorahoo. 2013. A review of the use of organic amendments and the risk to human health. Adv. Agron. 120:275-379. Grewal, S., S. Sreevatsan, and F.C. Michel, Jr. 2007. Persistence of Listeria and Salmonella during swine

Leifert, C., K. Ball, N. Volakakis, and J.M. Cooper. 2008. Control of enteric pathogens in ready-to-eat vegetable crops in organic and ‘low-input’ production systems: a HACCP-based approach. J. Appl. Microbiol. 105:931-950.

106

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

Li, X.J., R.H. Zhang, and Y.Z. Pang. 2008. Characteristics of dairy manure composting with rice straw. Bioresource Technol. 99:359-367. Luo, Z., G. Gu, M.P. Adams, G. Vellidis, A. VanBruggen, M. Danyluk, and A. Wright. 2013. Distribution and genetic diversity of Salmonella enterica isolated from irrigation water in the Suwanee River Watershed. J. Food Prot. Online Supplement 76S:104. (Abstr.) Lynch, J.M. 1987. Lignocellulolysis in composts. In: M. De Bertoldi, M.P. Ferranti, P. L’Hermite, and F. Zucconi, Eds. Compost: Production, Quality and Use. Elsevier, New York. pp. 178-179. Ma, L, G. Zhang, and M.P. Doyle. 2011. Green fluo-

Shepherd, M.W., Jr., P. Liang, X. Jiang, M.P. Doyle, and M.C. Erickson. 2007. Fate of Escherichia coli O157:H7 during on-farm dairy manure-based composting. J. Food Prot. 70:2708-2716. Shepherd, M.W., Jr., R. Singh, J. Kim, and X.P. Jiang. 2010. Effect of heat-shock treatment on the survival of Escherichia coli O157:H7 and Salmonella enterica Typhimurium in dairy manure co-composted with vegetable wastes under field conditions. Bioresource Technol. 101:5407-5413. Singh, R., J. Kim, M.W. Shepherd, Jr., F. Luo, and X. Jiang. 2011. Determining thermal inactivation of Escherichia coli O157:H7 in fresh compost by simulating early phases of the composting process.

rescent protein labeling of Listeria, Salmonella, and Escherichia coli O157:H7 for safety-related studies. PLoS One 6:e18083. MacDonald, J.M., M.O. Ribaudo, M.J. Livingston, J. Beckman, and W. Huang. 2009. Manure use for fertilizer and for energy: Report to Congress. USDAERS Publication No. AP-037. 53 pp. http://www. ers.usda.gov/ersDownloadHandler.ashx?file=/media/156155/ap037_1_.pdf. Accessed Jan, 2014. Montgomery, H.A.C., J.F. Dymock, and N.S. Thom. 1962. The rapid colorimetric determination of organic acids and their salts in sewage-sludge liquor. Analyst 87:949-955. Moore, P.A., Jr., T.C. Daniel, A.N. Sharpley, and C.W. Wood. 1998. In: Wright, R.J., W.D. Kemper, P.D. Millner, J.F. Power, and R.K. Korcak, Eds. Poultry manure management. Agricultural uses of municipal, animal, and industrial byproducts. USDA-ARS: Conservation Research Report #44. pp. 60-77. Pourcher, A.-M., P. Morand, F. Picard-Bonnaud, S. Billaudel, S. Monpoeho, M. Federighi, V. Ferré, and G. Moguedet. 2005. Decrease of enteric microorganisms from rural sewage sludge during their composting in straw mixture. J. Appl. Microbiol. 99:528-539. Ritz, C.W., and W.C. Merka. 2013. Maximizing poultry

Appl. Environ. Microbiol. 77:4126-4135. Singh, R., J. Kim and X. Jiang. 2012. Heat inactivation of Salmonella spp. in fresh poultry compost by simulating early phase of composting process. J. Appl. Microbiol. 112:927-935. Ugwuanyi, J.O., L.M. Harvey, and B. McNeil. 2005a. Effect of digestion temperature and pH on treatment efficiency and evolution of volatile fatty acids during thermophilic aerobic digestion of model high strength agricultural waste. Bioresource Technol. 96:707-719. Ugwuanyi, J.O., L.M. Harvey, and B. McNeil. 2005b. Effect of aeration rate and waste load on evolution of volatile fatty acids and waste stabilization during thermophilic aerobic digestion of a model high strength agricultural waste. Bioresource Technol. 96:721-730. United States Department of Agriculture [USDA]. 2013. Poultry – Production and Value. 2012 Summary. Available at: http://usda.mannlib.cornell.edu/ usda/current/PoulProdVa/PoulProdVa-04-29-2013. pdf. Accessed April, 2014 United States Environmental Protection Agency [US EPA]. 1999. Standards for the use or disposal of sewage sludge (40 CFR parts 403 and 503). Revised August 4, 1999. http://www.epa.gov/EPA-WA-

manure use through nutrient management planning. UGA Extension Bulletin 1245. Rynk, R. 1992. On-farm Composting Handbook. Northeast Regional Agricultural Engineering Service, Ithaca, NY.

TER/1999/August/Day-04/w18604.htm. Accessed Jan, 2014. United States Environmental Protection Agency [US EPA]. 2013. Literature review of contaminants in livestock and poultry manure and implications for

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

107

water quality. http://water.epa.gov/scitech/cec/ upload/Literature-Review-of-Contaminants-inLivestock-and-Poultry-Manure-and-Implicationsfor-Water-Quality.pdf. Accessed Jan, 2014. Wang, C.-M., C.-L. Shyu, S.-P. Ho, and S.-H. Chiou. 2007. Species diversity and substrate utilization patterns of thermophilic bacterial communities in hot aerobic poultry and cattle manure composts. Microbial Ecol. 54:1-9. Weatherburn, M.W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39:971-974. Wichuk, K.M. and D. McCartney. 2007. A review of the effectiveness of current time-temperature regulations on pathogen inactivation during composting. J. Environ. Engr. Sci. 6:573-586. Williams, J.E., and S.T. Benson. 1978. Survival of Salmonella Typhimurium in poultry feed and litter at 3 temperatures. Avian Dis. 22:742-747. Ziemer, C.J., J.M. Bonner, D. Cole, J. Vinjé, V. Constantini, S. Goyal, M. Gramer, R. Mackie, X.J. Meng, G. Myers, and L.S. Saif. 2010. Fate and transport of zoonotic, bacterial, viral, and parasitic pathogens during swine manure treatment, storage, and land application. J. Anim. Sci. 88:E84-E94.

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REVIEW Alternative Antimicrobial Supplements That Positively Impact Animal Health and Food Safety P. R. Broadway1, J. A. Carroll2, and T. R. Callaway3 Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX Livestock Issues Research Unit, Agricultural Research Service, USDA, Lubbock, TX 3 Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, USDA, College Station, TX 1

ABSTRACT Recently, a vast array of potential antibiotic alternatives have been introduced and researched in the livestock industry as a means to provide livestock producers with products that will positively impact animal health and performance. Some of these products may be used in conjunction with current antibiotic usage strategies, and some of these products may be used to completely replace some antibiotics in livestock production. These innovative antibiotic alternatives include direct fed microbials (DFM), yeast extracts, bacteriocins, bacteriophages, phytochemicals, and various acids. Many of these products have the ability to promote animal health and improve growth performance simultaneously, and some of these compounds may additionally enhance food safety through pre-harvest pathogen reduction. Antibiotic alternatives may be essential tools for livestock production in the future should legislation arise that inhibits prophylactic usage of conventional antibiotics and as a means to appeal to shifting consumer demands. Furthermore, it is also possible that these alternatives can be used as an additional supplement to incorporate into current practices and strategies in livestock production to maximize the potential to enhance both animal health and growth performance. This review will discuss potential alternative antimicrobial supplements in animal agriculture and their impact on animal health, performance and pathogen reduction. Keywords: Antibiotic, livestock, animal health, review Agric. Food Anal. Bacteriol. 4: 109-121, 2014

Correspondence: Todd Callaway, todd.callaway@ars.usda.gov Tel: +1-979-260-9374 Fax: +1-979-260-9332.

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INTRODUCTION Internet news sources and social media play increasing roles in publicizing livestock production practices and influence public opinion and perception, both fairly and unfairly. Recently, internet media sources have published propaganda to condemn the use of antibiotics in meat and poultry (Consumer Reports, 2012, 2013). Consumer-driven changes to the market due to shifting consumer demands mean that the beef, pork and poultry industries are faced with increasing challenges to profitability and must search for alternatives to remain profitable while still remaining environmentally friendly. Thus, the

tive effects antibiotics exert on growth promotion, growth rates, and overall animal health, thus producers have adopted the use of these pharmaceuticals over the years as a strategy to increase profitability, performance and animal health. Antibiotics are typically defined as compounds that inhibit bacteria (antibacterials), while antimicrobials are compounds that inhibit microorganisms. Many livestock producers currently utilize antibiotic/antimicrobial feeding and production strategies that alter the microbial ecology of the GI tract of the animal to benefit the overall production efficiency of their animals, as well as strategies that can eliminate or reduce foodborne pathogens that may contaminate the food supply

animal production industry needs potential alternative strategies to supplement or replace current antibiotic implementation practices. Furthermore, as consumer purchasing trends continue to gravitate towards “natural” and “organic” products, the livestock industry needs to be equipped to survive in an environment where sub-therapeutic concentrations of antibiotics supplemented as feed additives are absent. Consumer demand and public concern have previously been shown to influence antibiotic legislation. For example, the European Union (EU) banned sub-therapeutic supplementation of animal feeds with antibiotics (Pradella, 2006). Recently, the U.S. Food and Drug Administration (FDA) issued a guidance to further regulate antibiotic usage in food animals. While this directive does not eliminate the use of antibiotics, some believe this is the beginning of the end for antibiotic usage in food animals. Fortunately, substantial research has been conducted regarding the use of antibiotics in food animals, and many alternatives have been proposed and evaluated in regard to meeting the shifting consumer demands without impinging on the health, welfare, profitability, or wholesomeness of the food supply. The gastrointestinal (GI) tract of animals is populated with a complex microbial ecosystem that is es-

(Perlman 1973). However, these positive effects must be replicated by strategies that fill in the gap left if and when antibiotic use in food animals in banned in the U.S. While strategies such as genetic selection, selective breeding, and other management practices are utilized to promote health and profitability along with antibiotics, this review will focus primarily on feed additives.

sential for the function, growth, and overall health of the animal (Chaucheyras-Durand and Durand, 2010); therefore, any potential alternatives to antibiotics must support this symbiotic relationship. Evidence across multiple species generally supports the posi-

cies to species. Additionally, most alternatives have been evaluated as potential prophylactic treatments, and they may not be effective in treating actual illness or disease. Often times, the cost of antibiotic alternatives are offset by increases in performance; how-

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ALTERNATIVES TO ANTIBIOTICS IN LIVESTOCK PRODUCTION Due to recent developments in feed additives, direct fed microbials (DFM), and pharmaceuticals, producers now have multiple options available to enhance the natural microbial ecology of the animal, to prevent illness and improve production efficiency. While numerous alternatives to antibiotic use have been researched in animal production systems (Table 1), to date, there has been no “silver bullet” identified, nor is one likely to be found. While it has been demonstrated that many of the currently used antibiotics work across species, the results associated with the use of antibiotic alternatives have been inconsistent at times with varied results generated from spe-

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Table 1. Alternatives to antibiotics that may be used in food animals that may be used to promote overall animal health and/or impact pathogen colonization and shedding

DFM1

Bacteriophages

Phytochemicals

Acids

Bacillus

Finylase

Citrullene

Acetic acid

Lactobacillus

Citrus pulp

Caproic acid

Lactococcus

Curcumin

Formic acid

Streptococcus

Eugenol

Fumarate

Yeast

Flavonoids

Malate

Yeast cell wall

Limonene

Propionic acid

Linalool

Sodium Chlorate

Piperin Thymol Direct-Fed Microbials

ever, depending on different management and production strategies, these alternatives have not been demonstrated to be as effective as antibiotics. Each strategy has specific advantages and limitations including: effectiveness, production stage/system, animal age, animal type, changes in performance variables, cost, and labor. All of these variables must be explored before implementing antibiotic alterative(s) in a livestock production setting.

Direct-Fed Microbials (DFM) One such strategy that may be a potential alternative to antibiotics is known as competitive enhancement. Callaway et al. (2008) defines competitive

enhancement strategies as introducing live cultures of bacteria or fungi into the GI tract that provide a competitive advantage to commensal organisms that can, in turn, exclude pathogenic bacteria (e.g., supplementing with a probiotic, or addition of a prebiotic). Probiotic supplements fed to livestock are defined by Chaucheyras-Durand and Durand (2010) as “live microorganisms that possess the ability to evoke positive health benefits (at appropriate dosage concentrations) in the animal to which the microorganism was administered”; however, when used to treat or prevent disease, this definition is not identical to the definition provided by the Food and Drug Administration (FDA). Prebiotics are defined as feed ingredients that benefit the host by selectively stimulating the growth or activity of bacteria (Gibson and

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Roberfroid, 1995). A combination of prebiotic ingredients is referred to as synbiotics (Patterson and Burkholder, 2003). Fuller et al. (1989) explained that the fully developed GI tracts use its microbial ecosystem symbiotically to fill voids in microbial populations and thereby inhibit the colonization of pathogens. However, even when the GI tract is fully developed and contains a healthy microflora, some pathogens may be able to attach and cause illness. When a GI flora ecosystem is fully developed and the system is in a well-balanced state, the bacteria attach to the intestinal epithelium and can possibly physically inhibit the binding and colonization of pathogens that may be introduced into the gut (Collins and Gibson,

ria have the ability to inhibit pathogens such as E. coli (Russell and Mantovani, 2002) that pose a risk to human health. Brashears et al. (2003) reported that supplementing feedlot cattle with a Lactobacillus DFM was effective in reducing E. coli O157:H7 in fecal samples as well as reducing prevalence at slaughter. As in cattle, Lactobacillis has been shown to inhibit multiple strains of Salmonella in poultry (Jin et al., 1996). A combination of L. acidophilus and Streptococcus faecium cultures were shown to reduce colonization of Campylobacter jejuni (Morishita et al., 1997). Reductions in E. coli and Salmonella strains have also been observed when poultry were supple-

1999; Lloyd et al., 1977). Numerous compounds are produced by bacteria in the gut such as volatile fatty acids (VFA) that may inhibit the growth of and/or kill pathogens introduced to the animal (Wolin, 1969; Walsh et al., 2008). Direct-fed microbials have recently gained interest from the livestock feed industry as a tool to increase performance while simultaneously serving as a replacement for antibiotics (Ghorbani et al., 2002). These DFM products have been shown to enhance the formation of a healthy microbial community within the GI tract of the animal (Fuller, 1999). As with antibiotics, addition of DFM in the diet of dairy cows has been shown to reduce the risk of ruminal acidosis (Nocek et al., 2000). While the mode of action that reduces acidosis with the inclusion of lactate producing bacteria is not fully understood, the phenomena may be the result of changes in fermentation and microbial populations (Ghorbani et al., 2002). Enhanced performance parameters such as feed efficiency and ADG have also been reported in feedlot cattle supplemented with DFM (SwinneyFloyd et al., 1999; Rust et al., 2000). Some bacteria and DFM feed additives produce compounds called bacteriocins which are proteins synthesized by bacteria that inhibit the growth of

mented with Bacillus subtilis spores (LaRagione et al., 2001; Laragione and Woodward, 2003). While bacteriocins may be beneficial in most cases, some bacteria may exhibit resistance to specific bacteriocins (Russell and Mantovani, 2002). Colicin E1, a bacteriocin produced by E. coli, has been shown to be effective against pathogens that may be present in young swine that contribute to diarrheal symptoms (Cutler et al., 2007). Reduction/elimination of these pathogens through the use of Colicin E1 may be an effective tool to improve health and performance (Cutler et al., 2007). In addition to pre-harvest pathogen reduction applications, Colicin E1 has also been used successfully when applied directly to beef carcasses to inhibit E. coli O157:H7 (Patton et al., 2008). Researching probiotic supplementation in swine has yielded inconsistent results (Turner et al., 2001). Some studies have shown no differences in the growth and performance of swine fed Lactobacilli (Harper et al., 1983), while other studies reported enhanced growth and profitiability (Jasek et al., 1992; Gombo et al., 1995). Bacillus spp. feed supplementation has also been shown to decrease incidence of disease, reduce E. coli shedding and improve feed efficiency in swine (Bonomi, 1992; Kyriakis et al., 1999; Succi et al., 1995). Other probiotics that

other bacteria occupying the same environmental niche (Jack et al., 1995). Such bacteriocins have been isolated from the rumen of cattle (Wells et al., 1997; Russell and Mantovani, 2002). Bacteriocins, such as those commonly produced by Lactococcus bacte-

have exhibited positive effects in growing and finishing swine are Streptococcus spp. (Turner et al., 2001). Multiple studies with Streptococcus cultures have suggested that supplementation with these probiotics enhance growth and feed conversion in swine

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(Kumpercht and Zobac, 1998; Roth and Kirchgessner, 1986; Underahl, 1983). Another probiotic that may be utilized in swine is a yeast culture. Yeast cultures fed to swine have been reported to increase growth performance (Bertin et al., 1997; Maloney et al. 1998; Matthew et al., 1998). Yeast DFM may enhance digestion and maintain the microbial ecosystem of the GI tract in swine (Van Heugten et al., 2003), thereby making yeast DFM a possible antibiotic alternative during the weaning phase of young swine by inhibiting colonization of pathogens and improving performance (Anderson et al., 1999). Yeasts have also been used in cattle as antibiotic alternatives (Jouany and Margavi, 2007).

help alleviate infections caused by E. coli (Buts et al., 2006), Salmonella (Mahzounieh et al., 2006), and Clostridium (Katz, 2006) in lab animals. In poultry, supplementation with yeast cell wall products has yielded inconsistent results with respect to growth performance and pathogen reduction (Griggs and Jacob, 2005).

For example, supplementation of live yeast probiotics in dairy cattle has been reported to increase milk production and dry matter intake (Jouany, 2006; Sniffen et al., 2004; Stella et al., 2007). Yeast cell wall products are another type of feed supplement that have been fed to livestock as a means to improve animal performance, and to eliminate the pathogenicity of certain bacteria. Yeast cell wall components have been reported to function as immunomodulators that activate immune components such as macrophages, neutrophils, and other immunocompetent cells (Eicher et al., 2006; Onderdonk et al., 1992; Seljelid et al., 1987). Approximately half of the yeast cell wall is composed of biological response modifiers (Bohn and BeMiller, 1995), and these components have antibacterial (Kogan et al., 1989), antimutagenic (Kogan et al., 2005), antioxidant, and antitumor (Khalikova et al., 2005) activities that may promote animal health. Kogan and Kocher (2007) suggested that yeast cell wall polysaccharides may prevent bacterial attachment of pathogens to the mucosal epithelium in swine. Multiple studies conclude that yeast products may protect swine from bacterial infections while improving performance parameters such as weight gain (Lemieux et al., 2003; Rozeboom et al., 2005). Yeast cell wall prod-

ject phage DNA, take control of that cell, reproduce, and release new phages that lyse (or rupture) the host bacterial cell, thus resulting in bacterial cell death (Guttman et al., 2004 Kutter and Sulakvelidze, 2005). However, there are limitations to the use of bacteriophages. Just as bacteria can become resistant to antibiotics, bacteria may also become resistant to bacteriophages (Sklar and Joerger, 2000; Smith and Huggins, 1982; Smith et al., 1987). Another concern with the use of a bacteriophage is the passage of the supplement through the GI tract. Factors such as pH, viscosity, and microbial populations may influence the survivability and effectiveness of bacteriophage therapies (Hurley et al., 2008). Hurley et al. (2008) reported no reduction in fecal Salmonella shedding when fed to 28-day old chickens. Sklar and Joerger, (2000) reported minimal reductions in Salmonella populations of chickens when fed bacteriophages, and these researchers hypothesized that the intracellular nature of Salmonella may prevent phage attachment. When isolated and fed to feedlot cattle, researchers concluded that bacteriophages could be beneficial in a pre-harvest pathogen reduction intervention strategy to combat food pathogens such as E. coli (Callaway et al., 2008; Johnson et al., 2008). Additionally, phage therapy has also been reported

ucts have also been shown to improve metabolism in heifers during an endotoxin immune challenge without degradation of carcass tissues (Burdick Sanchez et al., 2013). With respect to animal health and food safety, yeast products have been reported to

successful in pathogen reduction in swine (Wall et al., 2010) and sheep (Bach et al., 2003). In fact, Rozema et al. (2009) reported that supplementation of feedlot cattle with bacteriophages for the control of E. coli O157:H7 shedding may be an alternative

Bacteriophages Bacteriophages are viruses found commonly in the GI tract and environment that prey specifically on bacteria, including pathogenic bacteria. Bacteriophages can bind to specific bacterial receptors, in-

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pre-harvest intervention that could be utilized to promote food safety in finishing cattle (Dini and De Urraza 2010). Also a product called FinalyseÂŽ has been developed for use as a hide spray to reduce E. coli O157:H7 on hides of cattle before they enter the slaughter plant, and FDA has issued no objection order to the use of phage (Coffey et al., 2011).

Phytochemicals Phytochemicals, such as citrus pulp, are being explored as replacements for sub-therapeutic antibiotics supplementation in animal feeds. Citrus pulp and peel are low cost byproduct feedstuffs with good

phytochemicals including garlic (Singh and Shukla, 1984), cinnamon (Hernandez et al., 2004) and black pepper (Dorman et al., 2000) have been reported to reduce pathogen populations and enhance metabolism in some cases. Phytochemicals found in the seaweed such as A. nodosum have been reported to reduce E. coli prevalence of feces and on the hides of cattle at harvest (Behrends et al., 2000). Feeding this same compound to swine was reported to enhance growth parameters but was unsuccessful in treating Salmonella infections (Turner, 2001). Other plant extracts called Saponins have been reported to alter ruminal microflora (Killeen et al., 1998), and have been reported to

nutritive values (high TDN) that have been used in beef and dairy production for many years (Arthington et al., 2002). Citrus pulp and peel contain essentials oils, including but not limited to citrullene, linalool, and limonene, that are bactericidal and can alter the microbial ecology of the GI tract (Lota et al., 2002; Fisher and Phillips, 2006; Viuda-Martos et al., 2008). Recent studies have demonstrated that feeding citrus pulp and peel to cattle, sheep, and swine can reduce populations of E. coli O157:H7 and Salmonella Typhimurium (Nannapaneni et al., 2008; Callaway et al., 2008; Callaway et al., 2011). While citrus pulp may have antipathogenic effects in cattle, Broadway et al. (2013) reported that there were only minimal changes to the bacterial ecology in the rumen of cattle supplemented with dried citrus pulp, thereby concluding that citrus pulp may be used an alternative agent to prevent colonization and shedding of foodborne pathogens without significantly altering ruminal microbial ecology and digestibility. Essential oils have also been researched in poultry to control pathogens (Griggs and Jacob, 2005). Thymol, eugenol, curcumin, and piperin are some of the essential oils found in thyme, clove, turmeric, and black pepper, and these products have been shown to inhibit enteridis causing Clostridium perfringens

prevent the growth of E. coli (Sen et al., 1998). Other naturally occurring phytochemicals, such as flavonoids, may be included in a production strategy to promote overall animal health and decrease pathogen shedding into the food supply (Holiman et al., 1996; Mandalari et al., 2007). Flavonoids are found in plant tissues and bark, and display some antioxidant capability (Pietta, 2000). Flavonoids have also been reported to decrease the viability of pathogenic bacteria such as E. coli and Salmonella, as well as Candida albicans and Sacchromyces species (Mandalari et al., 2007; Sohn et al., 2004; Friedman, 2007). Therefore, flavonoids may be an alternative to antibiotics that could positively impact the health of the animal while promoting food safety through the reduction of pathogenic microorganisms.

in poultry (Mitsch, et al., 2004). Other pathogens such as Salmonella and E. coli have been reduced, in vitro, when cultured with thymol (Marino et al., 1999; Karapinar and Aktug, 1987; Helander et al., 1998) and thyme (Aktug and Karapinar, 1986). Other

ganic acids may target the cell wall and membrance and interfere with bacterial metabolism, and internalization of dissociated acid components into the cytoplasm also alters pH and interferes with cellular metabolism (Ricke, 2003). However, the mechanisms

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Acids Inorganic and organic acids, as well as inorganic compounds, are another potential natural alternative that could be incorporated into livestock production. Acids are used to eliminate foodborne pathogens in food production, and they also may be beneficial in live animals to decrease or eliminate the presence of pathogens and improve digestion. Or-

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by which acids are bacteriocidal have not been fully elucidated and may target a variety of bacterial components and metabolic processes (Ricke, 2003). Organic acids such as lactic acid in diets have been reported to decrease the incidence of pathogens in livestock (Byrd et al. 2001). Other acids such as propionate have been used to improve the ruminal fermentation, and research pertaining to malate/fumarate reported increased lactic acid utilization by Megasphaera and Selenomonas ruminantium which resulted in similar impacts as those seen when utilizing ionophores (Martin and Nisbet, 1990; Nisbet and Martin, 1990; Nisbet and Martin, 1993; Waldrip and Martin, 1993). Propionic acid and formic acid fed

CONCLUSION As more increasingly negative attention surrounds the use of antibiotics in livestock production, and as consumers increase the pressure for future legislation regarding livestock production practices, more research and product development is needed to find suitable alternatives to the use of antibiotic supplemented feed for growth promotion and health benefits to meet the demand of an ever growing and evolving consumer population. There are many currently available or near-market ready products that can assist livestock producers in the control of

in combination has also been researched as a feed supplement in broilers, and the acid was reported to decrease populations of Salmonella in experimentally-infected birds (Hinton and Linton, 1988). Similarly, caproic acid was also shown to decrease Salmonella Enteridis in chickens (Van Immerseel et al. 2004). In addition to Salmonella inhibition, supplementing a combination of organic acids such as formic, acetic, and propionic acids were reported to reduce the growth of Campylobacter (Chaveerach et al., 2004). Another antibiotic alternative that may reduce the prevalence of foodborne pathogens is sodium chlorate, an organic product shown to inhibit nitrate reductase positive bacteria. Callaway et al. (2002), and Anderson et al. (2000, 2002) reported that E. coli populations could be reduced without significant changes in the microbial ecology of the rumen of cattle. Byrd et al. (2003) reported that water treatment with sodium chlorate was able to decrease the prevalence of S. Typhimurium in broilers. Additionally, sodium chlorate has been reported to decrease E. coli O157:H7 populations in the GI tract of inoculated swine (Anderson et al., 2001a), Sodium chlorate has also been reported to decrease Salmonella in swine prior to harvest (Anderson et al., 2001b). Other studies have reported chlorate to inhibit the

bacterial populations within their animals to assist in the maintenance of growth and production performance parameters while simultaneously improving the safety of food products for the consumer. However, many factors must be taken into consideration when selecting a particular product as an alternative to antibiotics, and there are advantages and limitations to each product. Therefore, producers must select product(s) that best suit their specific operational needs and are economically feasible.

survival of Salmonella while not interfering with potentially beneficial species in the GI tract (Anderson et al., 2001a,b; Byrd et al., 2003; Jung et al., 2003); however, chlorate is still awaiting FDA approval for use in food animals.

nella Typhimurium concentrations in the weaned pig gut. J. Food Prot. 64:255â&#x20AC;&#x201C;258. Anderson, R. C., S. A. Buckley, L. F. Kubena, L. H. Stanker, R. B. Harvey, and D. J. Nisbet, 2000. Bactericidal effect of sodium chlorate on Escherichia coli

REFERENCES Aktug, S.E. and M. Karpinar. 1986. Sensitivity of some common food-poisoning bacteria to thyme, mint, and bay leaves. Int. J. Food Microbiol. 3:349354. Anderson, D.B., V.J. McCracken, R.I. Aminov, J.M. Simpson, R.I. Mackie, M.W.A. Verstegen, and H.R. Gaskins. 1999. Gut microbiology and growthpromoting antibiotics in swine. Pig News Info. 20:115-122. Anderson, R. C, S. A. Buckley, T. R. Callaway, K. J. Genovese, L. F. Kubena, R. B. Harvey, and D. J. Nisbet. 2001a. Effect of sodium chlorate on Salmo-

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O157:H7 and Salmonella typhimurium DT104 in rumen contents in vitro. J. Food Prot. 63:1038-1042. Anderson, R. C., T. R. Callaway, T. J. Anderson, L. F. Kubena, N. K. Keith, and D. J. Nisbet. 2002. Bactericidal effect of sodium chlorate on Escherichia coli concentrations in bovine ruminal and fecal contents in vitro. Microb. Ecol. Health D. 14:24–29. Anderson, R.C., S.A. Buckley, T.R. Callaway, K.J. Genovese, L.F. Kubena, R.B. Harvey, and D.J. Nisbet. 2001. Effect of sodium chlorate on Salmonella Typhimurium concentrations in the weaned pig gut. J. Food Prot. 64:255-258. Anderson, R.C., T.R. Callaway, S.A. Buckley, T.J. Anderson, K.J. Genovese, C.L. Sheffield, and D.J.

Brashears, M.M, M.L. Galyean, G.H. Loneragan, J.E. Mann, and K. Killinger-Mann. 2003. Prevalence of Escherichia coli O157:H7 and performance by beef feedlot cattle given Lactobacillus Direct-Fed Microbials. J. Food Prot. 66:748-754. Broadway, P.R., T.R. Callaway, J.A. Carroll, N.C. Burdick, J.R. Donaldson, R.J. Rathmann, B.J. Johnson, J.T. Cribbs, L.M. Durso, D.N. Miller, D.J. Nisbet, and T.B. Schmidt. Evaluation of the ruminal bacterial diversity of cattle fed diets containing citrus pulp pellets (CPP) using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Agri. Food & Anal. Bacteriol. 2:297-308. Burdick Sanchez, N.C., T.R. Young, J.A. Carroll, J.R.

Nisbet. 2001. Effect of oral sodium chlorate administration on Escherchia coli O157:H7 in the gut of experimentally infected pigs. Int. J. Food Microbiol. 71:125-130. Arthington, J.D., W.E. Kunkle, and A.M. Martin. 2002. Citrus pulp for cattle, in the Veterinary Clinics of North America – Food Animal Practice, G. Rogers and M. Poore, Editors. W.B. Saunders Company: Philidelphia, PA, p. 317-328. Bach, S.J., T.A. McAllister, D.M. Veira, V.P.J. Gannon, and R.A. Holley. 2003. Effect of bacteriophage DC22 on Escherichia coli O157:H7 in an artificial rumen system (Rusitec) and inoculated sheep. Anim.Res. 52:89-101. Behrends, L.L., J.R. Blanton, M.F. Miller, K.R. Pond, and V.G. Allen. 2000. Tasco supplementation in feedlot cattle: Effects on pathogen loads. J. Anim. Sci. 78:106 (Abs.). Bertin, G., M. Brault, M. Baud, M. Mercier, and J. Tournot. 1997. Saccharomyces cervisiae I-1079, microbial feed additive: Zootechnical effects on piglets. In Digestive Physiology in Pigs. EAAP Publ. No 88. P. 446. EEAP, Paris, France. Bohn, J.A. and J.N. BeMiller. 1995. (1-3) β-D-Glucans as biological response modifiers: a review of structure-functional activity relationships. Carbohydr.

Corley, R.J. Rathmann, and B.J. Johnson. Yeast cell wall supplementation alters the metabolic responses of crossbred heifers to an endotoxin challenge. Innate Immun. Accepted 29 Jan. 2013. Buts, J.P., N. Dekeyser, C. Stilmant, E. Delem, F. Smets, and E. Sokal. 2006. Saccharomyces boulardii produces in rat small intestine a novel protein phosphatase that inhibits E. coli endotoxin by dephosphorylation. Pediatric Research. 60:24-29. Byrd, J. A., B. M. Hargis, D.J. Caldwell, R.H. Bailey, K.L. Herron, J.L. McReynolds, R.L. Brewer, R.C. Anderson, K.M. Bischoff, T.R. Callaway, and L.F. Kubena. 2001. Effect of lactic acid administration in the drinking water during preslaughter feed withdrawal on Salmonella and Campylobacter contamination of broilers. Poultry Sci. 80:278-283. Byrd, J.A., R.C. Anderson, T.R. Callway, R.W. More, K.D. Knape, L.F. Kubena, R.L. Ziprin, and D.J. Nisbet. 2003. Effect of experimental chlorate product administration in the drinking water on Salmonella typhimurium contamination of broilers. Poultry Sci. 82:1403-1406. Callaway, T. R., J. A. Carroll, J. D. Arthington, C. Pratt, T. S. Edrington, R. C. Anderson, M. L. Galyean, S. C. Ricke, P. Crandall, and D. J. Nisbet. 2008. Citrus products decrease growth of E. coli O157:H7

Polym. 28:3-14. Bonomi, A. 1992. Probiotics in pig breeding. Results from the use of Bacillus subtilis and Bacillus licheniformis. Experimental contribution. Riv. Soc. Ital. Sci. Aliment. 21:481.

and Salmonella Typhimurium in pure culture and in fer¬mentation with mixed ruminal microorganisms in vitro. Foodborne Path. Dis. 5:621-627. Callaway, T. R., J. A. Carroll, J. D. Arthington, T. S. Edrington, M. L. Rossman, M. A. Carr, N. A.

116

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Krueger, S. C. Ricke, P. Crandall, and D. J. Nisbet. 2011a. Escherichia coli O157:H7 populations in ruminants can be reduced by orange peel product feeding. J. Food Prot. 74:1917-1921. Callaway, T. R., R. C. Anderson, K. J. Genovese, T. L. Poole, T. J. Anderson, J. A. Byrd, L. F. Kubena, and D. J. Nisbet. 2002. Sodium chlorate supplementation reduces E. coli O157:H7 populations in cattle. J. Anim. Sci. 80:1683–1689. Callaway, T.R. , T.S. Edrington, A.D. Brabban, R.C. Anderson, M.L. Rossman, M.J. Engler, M.A. Carr, K.J. Genovese, J.E. Keen, M.L. Looper, E.M. Kutter, and D.J. Nisbet. 2008. Bacteriophage isolated from feedlot cattle can reduce Escherichia coli

E1 is effective in preventing postweaning diarrhea caused by F18-positive Escherichia coli in pigs. Antimicrobial Agents and Chemotherapy. 51:38303835. Dini, C. and P. J. De Urraza. 2010. Isolation and selection of coliphages as potential biocontrol agents of enterohemorrhagic and Shiga toxin-producing E. coli(EHEC and STEC) in cattle. J. Appl. Microbiol. 109:873-887. Dorman, H.J.D. and S.G. Deans. 2000. Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88:308-316. Eicher, S.D., C.A. McKee, J.A. Carroll, and E.A. Pajor. 2006. Supplemental vitamin C and yeast cell wall

O157:H7 populations in ruminant gastrointestinal tracts. Foodborne Path. Dis. 5:183-191. Callaway, T.R., T.S. Edrington, R.C. Anderson, J.A. Byrd, M.H. Kogut, R.B. Harvey, and D. J. Nisbet. 2008. Using antimicrobial cultures, bacteriocins, and bacteriophages to reduce carriage of foodborne pathogens in cattle and swine. Foodborne Path. and Dis. 5:205-224. Chaveerach, P.L., J.A. Lipman, and F. van Knapen. 2004. Antagonistic activities of several bacteria on in vitro growth of 10 strains of Campylobacter jejuni/coli. Int. J. Food Microbiol. 90:43-50. Coffey, B. L. Rivas, G. Duffy, A. Coffey, R.P. Ross, and O. McAuliffe. 2011. Assessment of Escherichia coli O157:H7-specific bacteriophages e11/2 and e4/1c in model broth and hide environments. Int. J. Food Microbiol. 147:188-194. Collins, D.M. and G.R. Gibson. 1999. Probiotics, prebiotics, and synbiotics: approaches for modulating the microbial ecology of the gut. Am. J. Clin. Nutr. 69:1052S-1057S. Consumer Reports. 2012. Antibiotics are widely used by U.S. meat industry. Available: http://www. consumerreports.org/cro/2012/06/antibiotics-arewidely-used-by-u-s-meat-industry/index.htm Consumer reports. 2013. Use of antibiotics on

β-glucan as growth enhancers in newborn pigs and as immunomodulators after an endotoxin challenge after weaning. J. Anim. Sci. 84:2352-2360. Fisher, K., and C.A. Phillips. 2006. The effect of lemon, orange and bergamot essential oils and their components on the survival of Campylobacter jejuni, Escherichia coli O157, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus in vitro and in food systems. J. Appl. Microbiol. 101:1232-1240. Friedman, M. 2007. Overview of antibacterial, antitoxin, antiviral, and antifungal activities of tea flavonoids and teas. Mol. Nutr. Food Res. 51:116-134. Fuller, R. 1989. Competitive exclusion and microlfora management: strategy for the swine industry. J. Swine Health Prod. 7:229-232. Fuller, R. 1999. Probiotics for farm animals. In: G.W. Tannock (ed.) Probiotics – A Critical Review. P. 15. Horizon Scientific Press, Wymondham, England. Ghorbani, G.R., D.P. Morgavi, K.A. Beauchemin, and J.A.Z. Leedle. 2002. Effects of bacterial direct-fed microbials on ruminal fermentation, blood variables, and the microbial populations of feedlot cattle. J. Anim. Sci. 80:1977-1985. Gibson, G.R. and M.B. Roberfoid. 1995. Dietary modulation of the human colonic microbiota:

healthy animals must stop. Available: http://www. consumerreports.org/cro/2013/10/antibiotic-resistant-superbugs-salmonella-outbreak/index.htm Cutler, S.A., S.M. Loneragan, N.Cornick, A.K. Johnson, and C.H. Stahl. Dietary inclusion of Colicin

introducing the concept of prebiotics. J. Nutr. 125:1401-1412. Gombo, S., J. Tossenberger, and C. Szabo. 1995. Effects of probiotics and yeast culture on the performance of pigs and dairy cows. Krmiva. 37:13.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

117

Griggs, J.P. and J.P. Jacob. 2005. Alternatives to antibiotics for organic poultry production. J. Appl. Poult. Res. 14:750-756. Guttman, B., R. Raya, and E. Kutter. Basic phage biology. In: Bacteriophages: Biology and Applications. Kutter E and Sulakvelidze A (eds). New York: CRC Press, 2004, pp. 29–66. Harper, A.F., E.T. Kornegay, K.L. Bryant, and H.R. Thomas. 1983. Efficacy of virginamycin and a commercially-available Lactobacillus probiotic in swine diets. Anim. Feed Sci. Technol. 8:69-76. Helander, I.M., H.L. Alakomi, K. Latva-Kala, T. MattilaSandholm, I. Pol, E.J. Smid, L.G.M. Gorris, and A. von Wright. 1998. Characterization of the action o

N.C. Rath, and A.M. Donoghue. 2008. Bacteriophages for prophylaxis and therapy in cattle, poultry, and pigs. Anim. Health Res. Rev. 9:201-215. Jouany, J.P., 2006. Optimizing rumen functions in the close-up transition period and early lactation to dry matter intake and energy balance in cows. Anim. Repro. Sci. 96:250-264. Jouany, J.P and D.P Morgavi. 2007. Use of ‘natural’ products as alternative to antibiotic feed additives in ruminant production. Animal. 1:1143-1466. Jung, Y.S., R.C. Anderson, J.A. Byrd, T.S. Edrington, R.W. Moore, T.R. Callaway, J. McReynolds, and D.J. Nisbet. 2003. Reduction of Salmonella Typhymurium in experimentally challenged broilers by nitrate

fselected essential oil components on gram-negative bacteria. J. Agric. Food Chem. 46:3590-3595. Hernandez, F., J. Madrid, V. Garcia, J. Orengo, and M.D. Megias. 2004. Influence of two plant extracts on broilers performance, digestibility, and digestive organ size. Poult. Sci. 83:169-174. Hinton, M., and A.H. Linton. 1988. Control of Salmonella infections in broiler chickens by the acid treatment of their feed. Vet. Rec. 123:416-421. Holiman, P.C.H., M.G.L. Hertog, and M.B. Katan. 1996. Analysis and health effects of flavonoids. Food Chem. 57:43-46. Hurley, A., J.J. Maurer, and M.D. Lee. 2008. Using bacteriophages to modulate Salmonella colonization of the chicken’s gastrointestinal tract: lessons learned from in silico and in vivo modeling. Avian Diseases. 52:599-607. Jack, R. W., J. R. Tagg, and B. Ray. (1995). Bacteriocins of gram-positive bacteria. Microbiol. Mol. Biol. Rev. 59:171-200. Jasek, S., R. Kalinowska, D. Knecht, and R. Pawiak. 1992. Effect of Biogen probiotic additon on reproduction results and physiological indices in pigs. Effect of Biogen T dietary additive on fattening performance and slaughter value. Roczniki Naukowe Zootechniki. 31:239.

adaptation and chlorate supplementation in drinking water. J. Food Prot. 66:660-663. Karapinar, M. and S.E. Aktug. 1987. Inhibition of foodborne pathogens by thymol, eugenol, menthol and anethole. Int. J. Food Microbiol. 4:161-166. Katz, J.A. 2006. Probiotics for the prevention of antibiotic associated diarrhea and Clostridium difficile diarrhea. J. Clin. Gastroenterology. 40:249-255. Khalikova, T.A., S.Ya., Zhanaeva, T.A. Korolenko, V.I. Kaledin, and G. Kogan. 2005. Regulation of activity of cathepsins B, L, and D in murine lymphosarcoma model at a combined treatment with cyclophosphamide and yeast polysaccharide. Cancer Lett. 223:77-83. Killeen, G.F., C.A. Madigan, C.R. Connolly, G.A. Walsh, C.Clark, M.J. Hynes, B.F. Timmins, P. James, D.R. Headon, and R.F. Power. 1998. Antimicrobial saponins of Yucca schidigera and the implications of the invitro properties for their in vivo impact. J. Agric. Food Chem. 46:3178-3186. Kogan, G., L. Masler, J. Sandula, J. Navarova, T. Trnovec. 1989. Recent results on the structure and immunomodulating activities of yeast glucan. In: Crescenzi, V., I.C.M. Dea, S. Paoletti, SS. STvala, and I.W. Sutherland (Eds.). Biomedical and Biotechnological Advances in Industrial Polysaccha-

Jin, L.Z., Y.W. Ho, N. Abdullah, M.A. Ali, and S. Jalaludin. 1996. Antagonistic effects on intestinal Lactobacillus isolates on pathogens of chicken. Lett. Appl. Microbiol. 23:67-71. Johnson, R.P., C.L. Gyles, W.E. Huff, S. Ojha, G.R. Huff,

rides. Gordon and Breach Science Publishers. New York. Pp. 251-258. Kogan, G. A Stasko, K. Bauerova, M. Polovka, L. Soltes, V. Brezova, J. Navarova, and D. Mihalova. 2005. Antioxidant properties of yeast (1-3) β-D-

118

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

glucan studied by electron paramagnetic resonance spectroscopy and its activity in the adjuvant arthritis. Carbohydr. Polym. 61:L 18-28. Kogan, G. and A. Kocher. 2007. Role of yeast cell wall polysaccharides in pig nutrition and health protection. Livestock Sci. 109:1-3. Kumprecht, I. and P. Zobac. 1998. Study of the effect of a combined preparation containing Enterococcus faecium M-74 (Streptococcus faecium) and mannan-oligosaccharides in diets for weanling piglets. Czech J. Anim. Sci. 43:477-481. Kutter, E. ,and A. Sulakvelidze. Bacteriophages: Biology and Applications. New York: CRC Press, 2005. Kyriakis, S.C., V.K. Tsiloyiannis, J. Vlemmas, K. Sar-

stable yeast product in pelleted diets for weanling pigs. Kansas Agric. Exp. Stn. Rep. Prog. No. 819. P. 62. Kansas State Swine Day Report. Mandalari, G, R. N. Bennet, G.Bisignano, D. Trombetta, A. Saija, C. B. Faulds, M.J. Gasson, A. Narbad. 2007. Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry. J. App. Microbiol. 103:2056-2064. Marino, M., C. Bersani, and G. Comi. 1999. Antimicrobial activity of the essential oils of Tymus vulgaris L. measured using a bioimpedometric method. J. Food Prot. 62:1017-1023. Martin, S.A., and D.J. Nisbet. 1990. Effects of Asper-

ris, A.C. Tsinas, C. Alexopoulos, and L. Jansegers. 1999. The effect of probiotic LSP 122 on the control of post-weaning diarrhea syndrome of piglets. Res. Vet. Sci. 67:223-228. La Ragione, R.M., G. Casula, S.M. Cutting, and M.J. Woodward. 2001. Bacillus subtilis spores competitively exclude Escherchia coli O78:K80 in poultry. Vet. Microbiol. 79:133-142. La Ragione, R.M. and M.J. Woodward. 2003. Competitive exclusion by Bacillus subtillis spores of Salmonella enteric serotype Enteridis and Clostridium perfringens in young chickens. Vet. Microbiol. 94:245-256. LeMieux, M., L.L. Southern, T.D. Bidner. 2003. Effect of mannan oligosaccharides on growth performance of weanling pigs. J. Anim. Sci. 81:2482– 2487. Lloyd, A.B., R.B. Cumming, and R.D. Kent. 1977. Prevention of Salmonella typhimurium infection in poultry by pre-treatment of chickens and pults with intestinal extracts. Aust. Vet. J. 53:82-87. Lota, M.L., D. de Rocca Serra, F. Tomi, C. Jacquemond, J. Casanova. 2002. Volatile components of peel and leaf oils of lemon and lime species. J. Agric. Food Chem. 50:796-805. Mahzounieh, M., I. Karimi, T.Z. Salehi, and R. Mar-

gillus oryzae fermentation extract on fermentation of amino acids, bermudagrass and starch by mixing ruminal microorganisms in vitro. J. Anim. Sci. 68:2142-2149. Mathew, A.G., S.E. Chattin, C.M. Robbins, and D.A. Golden. 1998. Effects of a direct-fed yeast culture on enteric microbial populations, fermentation acids, and performance of weanling pigs. J. Anim. Sci. 76:2138-2145. Mitsch, P., K. Zitterl-Eglseer, B. Kohler, C. Gabler, R. Losa, and I. Zimpernik. 2004. The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens. Poult. Sci. 83:669-675. Morishita, T.Y., P.P. Aye, B.S. Harr, C.W. Cobb, and J.R. Clifford. 1997. Evaluation of an avian specific probiotic to reduct the colonization and shedding of Campylobacter jejuni in broilers. Avian Dis. 41:850-855. Nannapaneni, R., A. Muthaiyan, P. G. Crandall, M. G. Johnson, C.A. O’Bryan, V. I. Chalova, T. R. Cal¬laway, J. A. Carroll, J. D. Arthington, D. J. Nisbet, and S. C. Ricke. 2008. Antimicrobial activity of commercial citrus-based natural extracts against Escherichia coli O157:H7 isolates and mutant strains. Foodborne Path. Dis. 5:695-699.

janian. 2006. The preventive effect of Saccharomyces bouldarii in pathogenesis of Salmonella typhimurium in experimentally infected rats. Maloney, C.A., J.D. Hancock, R.H. Hines, H. Cao, C.S. Nemecek, and J.S. Park. 1998. Effects of a heat-

Nisbet, D.J. and S.A. Martin. 1990. Effect of dicarboxylic acids and Aspergillus oryzae fermentation extract on lactate uptake by the ruminal bacterium Selenomonas ruminantium. Appl. Environ. Microbiol. 56:3515-3518.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

119

Nisbet, D.J. and S.A. Martin. 1993. Effects of fumarate, L-malate, and Aspergillus oryzae fermentation extract on D-lactate utilization by the ruminal bacterium Selenomonas ruminantium. Curr. Microbiol. 26:133-136. Nocek, J.E., W.P. Kautz, J.A.Z. Leedle, and J.G. Allman. 2000. Altering diurinal pH and in situ digestion in dairy cows with ruminal supplementatioin of direct-fed microbials (DFM) and yeast. J. Dairy Sci. 83(Suppl. 1):1242 (Absr.). Onderdonk, A.B., R.L. Cisneros, P. Hinkson, and G. Ostroff. 1992. Anti-infective effect of polybeta 1-6 glucotriosyl-beta 1-3-glucopyranose glucan in vivo. Infect. Immun. 60:1642-1647.

Bovamine rumen culture on the performance and carcass characteristics of feedlot steers. p 22–26. Mich. Agric. Exp. Sta. Beef Cattle, Sheep and Forage Sys. Res. Dem. Rep. No. 569, East Lansing. Seljelid, R.L., T. Rassmussen, O. Larm, and J. Hoffman. 1987. The protective effect of β1-3D-glucanderivated platic beads against Escherichia coli infection in mice. Scan. J. Immunol. 25:55-60. Sen, S., H.P.S. Makkar, S. Muetzel, and K. Becker. 1998. Effect of Quillaja saponaria saponins and Yucca schidigera plant extract on growth of Escherichia coli. Lett. Appl. Microbiol. 27:35. Singh, K.V. and N.P. Shulka. 1984. Activity on multiple resistant bacteria of garlic (Allium Stivum) ex-

Patterson, J.A. and K.M. Burkholder. 2003. Application of prebiotics and probiotics in poultry production. J. Poult. Sci. 82:627-361. Patton, B.S., S.M. Lonergan, S.A. Culter, C.H. Stahl, and J.S. Dickinson. 2008. Application of Colicin E1 as a prefabrication intervention strategy. J. Food Prot. 71:2519-2522. Pietta, P.G. 2000. Flavonoids as antioxidants. J. Nat. Prod. 63:1035-1042. Ricke, S.C. 2003. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Pout. Sci. 82:632-639. Roth, F.X., and M. Kirchgessner. 1986. Nutritive effects of Streptococcus faecium (Strain M 74) in starter pigs. Landwirtsch. Forsch. 39:198. Rozeboom, D.W., D.T. Shaw, R.J. Tempelman, J.C. Miguel, J.E. Pettigrew, and A. Connolly. 2005. Effects of mannan oligosaccharide and an antimicrobial product in nursery diets on performance of pigs reared on three different farms. J. Anim. Sci. 83:2637–2644. Rozema, E., T. Stephens, S.J. Bach, E.K. Okine, R.P. Johsnon, K. Stanford, and T.A. McAllister. 2009. Oral and rectal administration of bacteriophages for control of Escherichia coli O157:H7 in feedlot cattle. J. Food Prot. 72:241-250.

tract. Fitoterapia. 55:313-315. Sklar, I.B. and R.D. Joerger. 2000. Attempts to utilize bacteriophage to combat Salmonella enteric enteridis infection in chickens. J. Food Safety. 21:15-29. Smith, H.W. and M.B. Huggins. 1982. Successful treatment of experimental Eschercichia coli infection in mice using phage: its general superiority over antibiotics. J. Gen. Microbiol. 128:307-318. Smith, H.W., M.B. Huggins, and K.M. Shaw. 1987. The control of experimental Escherichia coli diarrhea in calves by means of bacteriophages. J. Gen. Microbiol. 133:1111-1126. Sniffen, C.J., Chaucheryas-Durand, F., De Ondarza, M.B., and Donaldson, G. 2004. Predicting the impact of a live yeast strain on rumen kinetics and ration formulation. In: proceeding of the 19th Annual Southwest Nutrition and Management conference, 24-25 Februrary 2004, Tempe, AZ, pp. 53-59. Sohn, H-Y, Son, KH, Kwon, C-S, Kang, SS. 2004. Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Morus alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sorphora flavescens Ait and Echinosophora koreensis Nakai. Phytomedicine. 11:666-672.

Russell, J.B. and H.C. Mantovani. 2002. The bacteriocins of ruminal bacteria and their potential as an alternative to antibiotics. J. Mol. Microbiol. Biotechnol. 4:347-355. Rust, S. R., K. Metz, and D. R. Ware. 2000. Effect of

Stella, A.V., R. Paratte, L. Valnegri, G. Cigalino, G. Soncini, E.Chevaux, V. Dell’Orto, and G. Savoini. 2007. Effect of the administration of live Saccharomyces cerevisiae on milk production, milk composition, blood metabolites, and fecal flora in early

120

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

lactating dairy goats. Small Ruminant Res. 67:7-13. Succi, G., A. Sandrucci, A. Tamburini, A. Adami, and V. Cavazzoni. 1995. Effects of using a new strain of Bacillus coagulans as a probiotic on the performance of piglets. Riv. Suinicol. 36:59. Swinney-Floyd, D., B.A. Gardiner, F.N. Owens, T. Rehberger, and T. Parrott. 1999. Effects of inoculation with strain P-63 alone or in combination with Lactobacillus acidophilus LA53545 on performance of feedlot cattle. J. Anim. Sci. 77:77 (Abstr.). Turner, J.L., S.S. Dritz, and J.E. Minton. 2001. Review: Alternatives to conventional antimicrobials in swine diets. The Professional Animal Scientist. 17:217-226.

2010. Phage therapy to reduce preprocessing Salmonella infections in market-weight swine. Appl. Environ. Microbiol. 76:48-53. Walsh, M.C., G.E. Gardiner, O.M. Hart, P.G. Lawlor, M. Daly, B. Lynch, B.T. Richert, S. Radcliffe, L Giblin, C. Hill, G.F. Fitzgerald, C. Stranton, and P. Ross. 2008. Predominace of a bacteriocin-producing Lactobacillus salivarius component of a fivestrain probiotic in the porcine ileum and effects on host immune phenotype. FEMS Microbiol. Ecol. 64:317-327. Wells, J. E., D. O. Krause, et al. 1997. A bacteriocin-mediated antagonism by ruminal lactobacilli against Streptococcus bovis. FEMS Microbiol.

Turner, J.L. 2001. Effects of natural alternatives to conventional antimicrobials on growth performance and immune function of nursery pigs during n acute enteric disease challenge with Salmonella typhimurium, Ph.D. dissertation. Kansas State University, Manhattan, KS. Underahl, N.R. 1983. The effect of feeding Streptococcus faecium upon Escherichia coli induced diarrhea in gnotobiotic pigs. Prog. Food Nutr. Sci. 7:5. Van Heugeten, E., D.W. Funderburke, and K.L. Dorton. 2003. Growth performance, nutrient digestibility, and fecal microflora in weanling pigs fed live yeast. J. Anim. Sci. 81:1004-1012. Van Immerseel, F., J. de Buck, F. Boyen, L. Bohez, F. Pasmans, J. Volf, M. Sevcik, I. Rychlik, F. Haesebrouck, and R. Ducatelle. 2004. Medium-chain fatty acids decrease colonization and invasion through hilA suppression shortly after infection of chickens with Salmonella enteric serovar Enteridis. Appl. Environ. Microbiol. 70:3582-3587. Viuda-Martos, M., Y. Ruiz-Navajas, J. FernandezLopez, and J. Perez-Alvarez. 2008. Antibacterial activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils. J. Food Safety. 28:567-576.

Ecol. 22:237-243. Wolin, M.J. 1969. Volatile fatty acids and the inhibition of Escherichia coli growth by rumen fluid. Appl. Microbiol. 17:83-87.

Waldrip, H.M. and S.A. Martin. 1993. Effects of an Aspergillus oryzae fermentation extract and other factors on lactate utilization by the ruminal bacterium Megasphaera elsdenii. J. Anim. Sci. 71:2770-2776. Wall, S.K., J. Zhang, M.H. Rostango, and P.D. Ebner. Agric. Food Anal. Bacteriol. â&#x20AC;˘ AFABjournal.com â&#x20AC;˘ Vol. 4, Issue 2 - 2014

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REVIEW Human Health Benefits of Isoflavones From Soybeans K. Kushwaha1, C. A. O’Bryan1,3, Dinesh Babu1§, P. G. Crandall1,3, P. Chen2, and S.-O. Lee1 Department of Food Science, University of Arkansas, 2650 Young Ave., Fayetteville, AR 72704 2 Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701 3 Center for Food Safety, University of Arkansas, Fayetteville, AR 72704 § Present address: Food Safety Toxicology, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71209 1

ABSTRACT Isoflavones are a group of chemicals that are found in legumes, predominantly in soybean and soy products. Soy isoflavones have been a component of the diet of certain populations for centuries. Many health claims have been made for isoflavones including: cancer prevention, alleviation of menopausal symptoms, positive effects on bone health and a lowering of blood lipids leading to lowered susceptibility to cardiovascular disease. However, because of their estrogenic activity some negative effects of isoflavones have been postulated. This review examines the literature associated with benefits as well as the negative effects of consumption of soy isoflavones. Results in some studies are limited or conflicting, but when viewed in its entirety, the current literature supports the safety of isoflavones as typically consumed in diets based on soy containing products. Keywords: Isoflavones, soybeans, soy products, health benefits, cancer prevention, bone metabolism, blood lipids Agric. Food Anal. Bacteriol. 4: 122-142, 2014

INTRODUCTION Soybeans are legumes, plants that form root nodules containing nitrogen-fixing soil bacteria (Rhizobia) in a symbiotic relationship. The soybean plant releases chemical signals, called isoflavonoids, to attract the nitrogen fixing bacteria (Rolfe, 1988). Isoflavonoids, also known as isoflavones, are produced by the same pathway that produces flavonoids, the Correspondence: Philip G. Crandall, crandal@uark.edu Tel: +1 -479-575-7686 Fax: +1-479-575-6936

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phenylpropanoid pathway. The phenylpropanoid pathway begins with phenylalanine and naringenin being converted into the isoflavone genistein by two enzymes, isoflavone synthase and a dehydratase, that are found only in legumes (Deavours and Dixon, 2005). Naringenin chalcone, another intermediate is converted to daidzein by the sequential action of two other legume-specific enzymes, chalcone reductase and type II chalcone isomerase as well as isoflavone synthase (Deavours and Dixon, 2005). Within the soybean, isoflavones are bound to a sugar molecule (glycosidic form) but fermentation or digestion

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Table 1. Total isoflavones. daidzein and genistein of selected soyfoods. expressed in mg/100g. Values have been taken from USDA database (USDA. 2008).

Food product

Total isoflavones

Daidzein

Genistein

Soy flour, full-fat

177.89

71.19

96.83

Soy flour, textured

148.61

59.62

78.90

Soy flour, defatted

131.19

57.47

71.21

Soybeans

128.34

46.46

73.76

Soy protein concentrate, aqueous washed

102.07

43.04

55.59

Soy protein isolate

97.43

33.59

59.62

Natto

58.93

21.85

29.04

Soybean chips

54.16

26.71

27.45

Tofu, fried

48.35

17.83

28.00

Tempeh

43.52

17.59

24.85

Miso

42.55

16.13

24.56

Soybean sprouts

40.71

19.12

21.60

Tofu, soft

29.24

8.59

20.65

Tofu, silken

27.91

11.13

15.58

Soy infant formula. powder

25.00

7.23

14.75

Tofu, firm

22.70

8.00

12.75

Soy hot dog

15.00

3.40

8.20

Okara

13.51

5.39

6.48

Soy protein concentrate, alcohol extracted

12.47

6.83

5.33

Bacon, meatless

12.10

2.80

6.90

Soy milk

9.65

4.45

6.06

Vegetarian burger

9.30

2.95

5.28

Soy cheese, Mozzarella

7.70

1.10

3.60

Soy cheese, Cheddar

7.15

1.80

2.25

Soy drink

7.01

2.41

4.60

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Figure 1. Chemical structures of soybean isoflavones (Naya and Imai, 2013).

Reproduced with permission of Nayga and Imai (2013), available from http://www.intechopen.com/books/ soybean-bio-active-compounds/recent-advances-on-soybean-isoflavone-extraction-and-enzymatic-modification-of-soybean-oil

results in the release of the sugar molecule from the isoflavone, leaving the isoflavone as an aglycone (Zubik and Meydani, 2003). Soy isoflavone glycosides include genistin, daidzin, and glycitin, while the aglycones include genistein, daidzein, and glycitein (Figure 1). The typical composition for soybeans is 40% diadzin/diadzein, 50% genistin/genestein and 10% glycetin/glycitein (Murphy et al., 1999). People from countries that consume large amounts of soy foods are reported to have an improved chronic disease burden compared with countries consuming very little soy (Boring et al., 1994; Thom et al., 1992). The average isoflavone intake from soy food by Asian women ranges from 25 to 50 mg/day (Messina et al., 2006) compared to nonAsian women who take in less than 2 mg/day (Van Erp-Baart et al., 2003; de Kleijn et al., 2001). Health

1999) relief of menopausal symptoms (Messina,1999) and decreased risk of certain types of cancer such as breast and prostate (Severson et al., 1989; Jacobsen et al., 1998; Lee et al., 1991; Wakai et al., 1999). Since isoflavones are capable of exerting estrogen-like effects they are often referred to as phytoestrogens (Lampe, 2003). Since isoflavones structurally closely resemble esterogenic steroids of animals they are able to bind to both estrogen receptors alpha (ERα) and beta (ERβ) (Figure 2) (Kuiper et al., 1997; Kuiper et al., 1998). This review will look at the evidence, pro and con, regarding the health benefits of isoflavones derived from soy.

benefits attributed to soy isoflavones include lowering blood pressure ( Hooper et al., 2008), prevention of coronary heart disease (Nagata et al., 1998; Smit et al., 1999), better bone health ( Ho et al., 2001; Mei et al., 2001; Horiuchi et al., 2000; Tsuchida et al.,

Soybeans and soy products represent one of the richest and cheapest sources of protein (Codina et al., 2003). Soybeans have been used as a major part of the diet in Asian countries and some other parts of the world for more than 5,000 years. Soy foods

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Figure 2. Comparison between isoflavone and estrogen molecule showing similarity in conformation (Setchell and Cassidy, 1999).

Reproduced with permission of American Society for Nutrition

eaten in the Asian countries are often fermented by microorganisms; for example miso which is added to soups and stews in Japan, soy paste used in Korea and tempeh, with a meat-like texture in Indonesia (Synder and Kwon, 1987a). Soy sauce is perhaps the most familiar soy product which is made either by a long fermentation process or by acid hydrolysis; this acid hydrolyzed product contains no isoflavones (Luh, 1995). Soy milk is made by extracting the proteins and lipids in soybeans with boiling water; soymilk may then be curdled to prepare tofu, which is pressed to remove water and can be fried or added to numerous other dishes (Snyder and Kwon, 1987a). Soybean-containing foods have become more popular in the United States, especially after October 1999 when the Food and Drug Administration (FDA) approved a health claim for soy proteins for reducing heart disease (FDA, 1999). However, soy foods in the United States are generally quite different from the forms of soy consumed in Asia. Soybeans grown in the United States are utilized mostly as a source

soy protein concentrate, which is even higher in protein. When the soy flour is extracted with hot, aqueous 65% alcohol it forms a different type of soy protein concentrate which contains no isoflavones. Both of these soy protein concentrates can be extruded to form textured soy protein, a meat-like product. The proteins in soy flour may be solubilized with a mild alkaline extraction followed by a precipitation at a low pH to produce soy protein isolate (SPI). This SPI is widely found in canned foods or is used by athletes as a source of protein. There are also new “soy” foods such as soy cheese, soy ice cream and soy yogurt.

of edible oil. After extraction the defatted soy flour, which is high in protein, is used in many bread and cake products, particularly in doughnuts (Snyder and Kwon, 1987b). Alternatively the soy flour is washed with water to remove soluble carbohydrates creating

may be lost and the chemical composition may also change. Soybeans for oil production have the hulls removed and the remainder of the beans after the fat is removed is pressed into flakes and then ground into soy flour. The isoflavone content of the soy flour

EFFECT OF PROCESSING ON ISOFLAVONE CONTENT OF FOODS Processing has a substantial influence on the amount and form of isoflavones in soyfood products. During the course of processing, some isoflavones

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is about the same as whole soybeans, indicating that defatting and milling causes little loss or transformation of isoflavones; soy oils contain only traces of isoflavones because of the highly polar nature of the isoflavones leading to an inability to partition into the lipophilic oil during soy oil extraction (Coward et al., 1998; Setchell, 1998). Production of soy protein concentrate using alcohol washing causes the loss of most of the isoflavones, whereas a substantial amount of isoflavones are retained after water washing (Wang and Murphy, 1996). More isoflavones are retained in SPI than in soy protein concentrate (Wang and Murphy, 1996). Fermented soybean products such as miso and natto are reported to have higher

both ERs than does the precursor daidzein (Muthyala et al., 2004). Human gut microflora metabolize daidzein to produce only the S isomer (Setchell et al., 2005). Studies that measured urinary equol excretion after soy consumption indicated that only about 33% of individuals from Western populations metabolize daidzein to equol (Setchell et al., 2002). The prevalence of equol producers appears to be higher in Asian populations than in non-Asian (Arai et al., 2000; Wu et al., 2006; Cassidy et al., 2006) and appears to be linked to the gastrointestinal normal flora in these individuals (Setchell et al., 2002). The role of gut microflora in the production of equol was elucidated in experiments with germ-

isoflavone content than in products such as soymilk and tofu, which is thought to be due to the action of bacteria during fermentation (Fukutake et al., 1996). Extruded soy products such as cereals have a lower amount of isoflavones due primarily to the heat and loss of moisture during the extrusion (Mahungu et al., 1999).

free rats which, when fed daidzein, did not produce equol; when the rats were inoculated with fecal flora from equol producers they were able to produce equol from daidzein (Bowey et al., 2003; Axelson and Setchell, 1981). A number of bacterial species capable of converting daidzein to the S isomer of equol in vitro have been isolated from both food and human gut flora including, Lactococcus garviae from Italian cheese (Fortina et al., 2007), 6 strains of bacteria belonging to the Coriobacteriaceae family from tofu brine (Abiru et al., 2013), Eggerthella spp strain YY791 (Yokoyama and Suzuki, 2008) and YY7918 (Yokoyama et al., 2011), and Slackia isoflavoniconvertens (Schroder et al., 2013) from human gut flora. Many researchers have proposed that equol producers may have improved disease risk patterns as compared with non-producers (Fujioka et al., 2004; Kurahashi et al., 2008; Lampe, 2009; Magee, 2011; Setchell et al., 2002; Wu et al., 2007). There is much evidence that suggests that equol producers have a lower breast cancer risk as compared with non-producers (Atkinson et al., 2003; Duncan et al., 2000; Falk et al., 2005; Ursin et al., 1999).

ISOFLAVONE METABOLISM After the soybean is eaten, the glycosidic forms of the isoflavones undergo hydrolysis due to the action of the brush border and bacterial β-glucosidases to remove the sugar moiety; the aglycone form is then either absorbed or undergoes further metabolism by intestinal bacteria in the large bowel (Chen et al., 2003; Setchell et al., 2003). The isoflavone daidzein is usually metabolized to dihydrodaidzein or Odesmethylangolensin (Bowey et al., 2003; Setchell, 1998; Yuan et al., 1995; Zubik and Meydani, 2003). In a small number of persons daidzein may also be metabolized in the intestine to equol, a metabolite that has greater estrogenic activity than daidzein (Muthyala et al., 2004). Equol exists in 2 stereoisomers, R or S, which differ significantly from each other in terms of their binding affinities with estrogen receptor (ER) (Muthyala et al., 2004). The S isomer has a high binding affinity for both receptors but prefers ERβ, whereas the R isomer binds weakly and prefers ERα; however, both isomers have a higher affinity for 126

DISEASE PREVENTION ACTIVITIES Isoflavones and Bone Health Loss of bone mass, known as osteoporosis, poses a major human health threat by contributing to bone

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fractures in those afflicted. Worldwide, osteoporosis causes more than 8.9 million fractures annually, resulting in an osteoporotic fracture every 3 seconds (Johnell and Kanis, 2006). Osteoporosis is estimated to affect 200 million women worldwide; approximately 10% of women aged 60, 20% of women aged 70, 40% of women aged 80 and 67% of women aged 90 are afflicted (Kanis, 2007). One of the means by which isoflavones promote bone health is hypothesized to be because of their affinity for ERβ since bone tissue contains large amounts of ERβ (Messina, 1999). Arjmandi et al. (1998) found that there was a significant increase in insulin-like growth factor 1 (IGF-1) mRNA in isoflavone treated groups

ach (International Agency for Research on Cancer, 2013). It is estimated that more than two-thirds of human cancers could be prevented by modification of lifestyle including dietary modification (Haque et al., 2010). The U.S. National Cancer Institute has been actively investigating the anticancer effects of soybeans since 1991 (Messina and Barnes, 1991). Akiyama et al., (1987) demonstrated that genistein was a specific inhibitor of a tyrosine-specific protein kinase, an enzyme that is often overexpressed in cancer cells. Constantinou et al. (1990) found that genistein suppressed growth and induced differentiation in leukemia cells. Later, genistein was found to inhibit multiple protein tyrosine kinases relevant

compared to control groups; since IGF-1 mRNA stimulates bone formation, this is another possible mechanism for the positive role of isoflavone in bone health. Isoflavones have also been postulated to prevent osteoporosis because of their similar structure to ipriflavones, which inhibit bone resorption in humans (Tsuda et al., 1986; Brandi, 1997). There is inconsistent data available on the beneficial effect of soy isoflavones on bone density in human studies due to the small subject number and short duration of the soy consumption, a larger effect on bone density was observed in animal models using higher doses (Arjmandi et al. 1998). From epidemiological studies as well as clinical trials, Messina et al., (2004) showed that Asian women who take in more soy isoflavones have higher bone mineral density and have a low rate of hip fracture compared to non-Asians, and concluded that isoflavones reduce bone loss in postmenopausal women.

Cancer affects persons of every socioeconomic level and every area of the world; cancer accounts for one in every eight deaths worldwide – more than HIV/ AIDS, tuberculosis, and malaria combined (American

to cancer cell proliferation (Bektic et al., 2005). Rabiau et al. (2010) treated human prostate cancer cells with genistein or daidzein and found that they down regulated growth factors involved in proliferation of new blood vessels in tumors. In a study reported in 2011, prostate cancer patients scheduled for radical prostatectomy were randomly assigned to receive a placebo or 30 mg genistein daily for 3 to 6 weeks before surgery. Among the patients who received genistein, serum prostate specific antigen (PSA) levels decreased by 7.8%, whereas serum PSA levels increased by 4.4% in patients who received the placebo (Lazarevic et al., 2011). Consumption of soy isoflavones is higher in Asian diets as compared to Western; daily consumption of soy isoflavones in Japan ranges from 26 to 54 mg, compared to 0.5 to 3 mg in the United States (Nagata, 2010). Breast cancer incidence increased by more than 20% between 2008 and 2012 and mortality from breast cancer increased by 14%; it is the most common cause of cancer deaths among women and the most frequently diagnosed cancer in women in 140 of 184 countries (International Agency for Research on Cancer, 2013). Some epidemiologic evidence suggests that soy consumption early in life and through puberty reduces breast cancer risk (Mes-

Cancer Society, 2013). An estimated 14.1 million new cancer cases and 8.2 million cancer-related deaths occurred in 2012 (International Agency for Research on Cancer, 2013). The most common causes of cancer death were cancers of the lung, liver, and stom-

sina and Hilakivi, 2009) and this has been supported by animal studies which suggest that soy intake is protective at specific stages of development but not at other points (Warri et al., 2008). Lamartiniere et al., (2000) briefly exposed young rodents to dietary

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supplements of genistein and found that it reduced mammary cancers indicating early intake of soy isoflavones protects against breast cancer. Animal studies demonstrated that when carcinogenic rodents were fed with isoflavone-rich soy protein or isolated isoflavones, mammary carcinogenesis was inhibited by 25 to 50% (Magee and Rowland, 2004; Messina and Loprinzi, 2001) indicating that isoflavones do have an antiesterogenic effect. Pisani et al., (1999) and Kitamura et al. (2002) reported low risk of breast cancer in the Asian countries where soy is commonly consumed as isoflavone might exert an antiesterogenic effect on breast tissue. Adults who consumed large amounts of soy as ad-

multiple trials no effects on breast proliferation or mammographic density were observed for isoflavones and considerable epidemiologic data shows either no effect or only a modest protective role of soy/isoflavone intake on breast cancer risk (Messina and Wood, 2008). Studies conducted by Caan et al., (2011) and Guha et al., (2009) reported that soy consumption had no adverse effects on breast cancer survivors. Furthermore, they suggest that soy consumption at levels comparable to those among Asian populations does not detract from the benefits of tamoxifen therapy, and may even offer some protection against recurrence and cancer-related death. The American Cancer Society stated that con-

olescents have been determined to have a lower risk for breast cancer as compared to adults who did not consume large amounts of soy in adolescence (Shu et al., 2001). Wu et al. (1996) found that among Asian Americans, tofu consumption protected against both pre- and postmenopausal breast cancer. Another study reported by Chinese epidemiologists wherein adolescents had high soy consumption which resulted in a 50% reduction in adult breast cancer risk whereas adult intake did not impact these findings (Shu et al., 2001). Similarly, a U.S. case-control study involving Asian Americans reported that high soy consumption during both adolescence and adulthood was associated with a one-third reduction in risk whereas high adult intake alone was not protective (Wu et al., 2002). Both dosage and timing of exposure to soy isoflavones appear relevant to their potential chemopreventive effect. Two meta-analyses (Dong and Qin, 2011; Wu et al., 2008) found soy intake to be significantly associated with reduced risk of breast cancer in Asian but not Western human populations, which may be explained by both higher soy intake among Asians and their tendency to consume soy from an early age. Several studies have shown mixed results regarding the effect of isoflavones supplements on the

sumption of soy foods would not decrease survival nor increase recurrence of cancers, but there was not enough evidence to make a statement about isoflavone supplements (Rock et al., 2012). However, in another study conducted by a U.S.- Chinese research team, researchers monitored and measured intake of soy isoflavones over the course of seven years; they determined that soy isoflavones significantly reduced risk of cancer recurrence in patients who consumed at least 10 mg of isoflavones (approximately 3 g of soy protein) per day (Nechuta et al., 2012). Bloedon et al. (2002) and Allred et al. (2001) found that soy protein and isoflavones stimulated the growth of mammary tumors in ovariectomized mice implanted with estrogen-sensitive breast cancer cells. In contrast Zhou et al., (2004) demonstrated that isoflavones could inhibit the growth of tumors in mice when intact ovaries were implanted with these same types of cells. Messina et al., (2006) suggested that a daily dose of 120 mg isoflavones may be useful in prostate cancer prevention, but recommended consumption of soy foods rather than isolated isoflavones supplements, because other soy components such as soy protein, fiber and saponins may offer additional health benefits. Nagata et al., (2007) studied the ef-

proliferation of breast cells in breast cancer patients. In 2007, a Japanese collaborative cohort study suggested that consumption of soy foods such as tofu, boiled beans, and miso soup has no protective effects against breast cancer (Nishio et al., 2007). In

fect of dietary isoflavone against prostate cancer in Japanese males and found that inclusion of dietary isoflavone might be an effective dietary protective factor against prostate cancer in Japanese men. In this study male subjects in the highest category of

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isoflavones intake (greater than 90 mg/day) exhibited a 58% lower prostate cancer risk than male subjects in the lowest category (less than 30 mg/day). In one study, early-stage prostate cancer patients were randomly assigned to receive a soy protein supplement (60 mg/day isoflavones) or a placebo daily for 12 weeks. Patients who received the soy protein supplement exhibited greater decreases in total serum PSA and free testosterone than did patients who received the placebo, but these differences were not statistically significant (Kumar et al., 2004). In two small studies prostate cancer patients were fed soy isoflavone and in these studies there appeared to be a decrease in the rate of the rising serum PSA con-

yanaga et al., (2012) wherein isoflavones had no influence on the level of PSA, but biopsies showed that isoflavone intake reduced the incidence of prostate cancer, but that this difference was not statistically significant. Several epidemiologic studies have also shown no association between high consumption of fermented soy foods and prostate cancer (Hwang et al., 2009). Isoflavones are purported to slow prostate cancer growth and cause cancer cells to die (Fotsis et al., 1993). Supplementation with soy protein or soy isoflavone decreased the markers of cancer development and progression in prostate cells including PSA, testosterone, and androgen receptor in patients with prostate cancer (Kumar et al., 2004;

centration associated with prostate tumor growth (Fischer et al., 2004; Hussain et al., 2003). A trial of soy milk supplementation (141 mg/day isoflavones) in men with PSA recurrent prostate cancer found that PSA levels increased by an average of 20% over a 12-month period compared to a 56% yearly increase prior to the study (Pendleton et al., 2008). Hamilton-Reeves and coworkers at the University of Minnesota, (2007) examined the potential protective effect of soy protein isolate, with low and high levels of isoflavones, on prostate cancer risk in men at high risk for developing the advanced form of prostate cancer. They found that soy protein isolate consumption suppressed androgen receptor expression in the prostate and could be beneficial in preventing prostate cancer. Kumar et al., (2007) treated 53 prostate cancer patients with 80 mg purified isoflavones or a placebo for 12 weeks. Although plasma isoflavones increased with no observed clinical toxicity, there was no modulation of serum sex hormone binding globulin, total estradiol, or testosterone in the isoflavone-treated group compared to placebo. The study establishes the need to explore other potential mechanisms by which prolonged and consistent purified isoflavone consumption may modulate prostate cancer risk.

Dalais et al., 2004) or in men at high risk for developing advanced prostate cancer (Hamilton-Reeves et al., 2007).

A meta-analysis of eight studies performed by Yan and Spitznagel (2009) found that isoflavone consumption was associated with a reduction in risk of prostate cancer, but the association was not statistically significant. Similar results were reported by Mi-

that followed a standard low saturated fat diet. Other epidemiological studies have also suggested that Asian populations consuming large amounts of soy have lower rates of cardiovascular disease than Western populations (Zhang et al. 2003).

Isoflavones and Cardiovascular Disease In 2010, cardiovascular disease (CVD) was the leading cause of death responsible for 597,689 deaths in the U.S. (CDC, 2013). An estimated 30% of all global deaths in 2008 were from CVD (WHO, 2011a); of these deaths, an estimated 7.3 million were due to coronary heart disease and 6.2 million were due to stroke (WHO, 2011b). In order to reduce coronary heart disease it is recommended that saturated fat should be replaced with polyunsaturated fatty acids. Soy foods are ideal for this replacement since they contain the omega-6 polyunsaturated fatty acid (PUFA) linoleic acid, which comprises about 55 percent of the total fat in soybeans and which reduces blood cholesterol levels (Slavin et al., 2009; Jenkins et al., 2002). Jenkins et al. (2011) recommended a diet supplemented with cholesterol-lowering foods including soyfoods like soymilk and soy meat alternatives, oats, nuts and plant sterols for adults with high cholesterol. This diet lowered low density lipoprotein (LDL) cholesterol by 13.8% compared with a decrease of only 3% in those

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Research suggests that soy protects against atherosclerosis by lowering cholesterol, or by increasing blood levels of nitric oxide which helps in blood vessel dilation, inhibits oxidative damage caused by cholesterol and prevents the adhesion of white cells to the vascular wall. (Cena and Steinberg, 2011). A combination of soy protein and isoflavones appear to exhibit the strongest hypocholesterolemic effects compared to isolated soy protein or soy isoflavones alone (Mortensen et al., 2009). Research studies suggest that soy protein decreases postprandial triglyceride levels, which is increasingly viewed as important for reducing coronary heart disease risk (Zhan and Ho, 2005). Whole soy can promote a 3%

29 clinical trials found that compared to animal protein, soy protein significantly reduced blood levels of several lipids (total cholesterol, LDL cholesterol and triglycerides) (Anderson et al. 1995). There is additional data suggesting isoflavones have independent coronary benefits. In several studies isoflavones have been shown to enhance endothelial function (Walker et al., 2001; Squadrito et al., 2002, 2003) and systemic arterial elasticity (Nestel et al., 1997, 1999); both of these measures are considered to be indicators of coronary health (Bonetti et. al., 2003; Herrington et al., 2004). In several research studies it has been shown that on average, soy protein lowers LDL cholesterol ap-

to 5% reduction in blood cholesterol (Lichtenstein et al. 2006; Zhan and Ho 2005). Henrotin et al., (2003) concluded that soy protein led to increased blood levels of L-arginine (the amino acid that the human body uses to produce nitric oxide) and nitric oxide metabolites. Isoflavones increase endothelial nitric oxide production, enhancing vasodilation and improving blood flow (Taku et al., 2007; Zhan and Ho, 2005). Specific findings include beneficial effects on lipids and lipoproteins, with a decline in total cholesterol (9%), LDL cholesterol (13%), and triglycerides (11%) and an increase in high density lipoprotein (HDL) cholesterol (2.4%) (Anderson et al., 1995). Isoflavones found in soybean and soy foods provide cardiovascular health benefits by neutralizing free radicals that cause oxidative damage to cells thus improving arterial elasticity, a vascular function that normally decreases with age, helping to reduce LDL cholesterol levels (Steinberg et al. 2003). Otherwise, these free radicals within blood vessels can oxidize circulating LDL cholesterol, starting a cascade of inflammatory events that ultimately increases the risk of developing heart disease. In a study conducted by Candy (1996) 61 middle-aged men that had been diagnosed as having a high risk of developing coronary disease were asked to consume soy protein

proximately 4% (Sacks et al., 2006; Zhan and Ho, 2005). Each one percent reduction in cholesterol lowers coronary heart disease risk at least 2% (Law et al., 1994). A meta-analysis conducted by Weggemans and Trautwein (2003) found that soy protein slightly raised HDL cholesterol levels leading tham to conclude that as a result of the changes in lipid levels, soy could reduce heart disease risk by as much as 20 percent. Furthermore, there is evidence to suggest that soyfoods may decrease blood pressure (Rivas et al., 2000) and increase LDL cholesterol particle size (Desroches et al., 2004). Dong et al., (2011) analyzed 27 clinical (human intervention) studies and found that on average, soy lowered blood pressure about 2 Â˝ points. Li et al. (2010) studied the effect of oral isoflavone supplementation on vascular endothelial function in postmenopausal women and concluded that isoflavone helps to improve endothelial function. Zhan and Ho (2005) reported that the inclusion of soy in the diet can decrease blood levels of LDL cholesterol. A meta-analysis of 7 studies found that soy protein that contained enriched isoflavones, and in comparison with animal protein without isoflavones, were associated with a significant decrease in serum total cholesterol (0.32 mmol/L or 5.69%) in the hyper-

(20 g) and soy isoflavone (80 mg) for five weeks; those consuming soy showed significant reductions in both diastolic and systolic blood pressure compared to those who were given a placebo diet containing olive oil. In the mid-1990s, a meta-analysis of

cholesterolemic subcategory and LDL cholesterol (0.18 mmol/L or 4.98%) in the total human population. It was also reported in this study that a significant increase in serum HDL cholesterol (0.04 mmol/L or 3.00%) occurred in the total population. Similarly

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a study by Steinberg et al., (2003) demonstrated that consuming intact soy protein and isoflavone may improve vascular function better than the consumption of either component alone. Gardner et al., (2007) compared cholesterol levels between those who drank soymilk and those who drank 1% dairy milk as part of an overall diet containing moderate fat (35% of calories). In only four weeks, the soymilk group exhibited a 5% reduction in LDL cholesterol, a statistically significant advantage over those who consumed dairy milk.

ROLE OF ISOFLAVONES IN REDUCING MENOPAUSAL SYMPTOMS AND HOT FLASHES Hot flashes are the primary reason that women seek medical attention for menopausal symptoms (Tice et al., 2003). A low incidence of hot flashes and other menopausal symptoms in Japanese women is believed to be due to the estrogen-like effects of soy isoflavones (Kuiper et al., 1998; Lock, 1992; 1994). Women fed with soy flour (45 g) daily demonstrated a reduction (40%) in menopause symptoms (Allaoua et al., 2005). Conversely, other studies have reported no beneficial effect of soy intake on menopausal symptoms. A meta-analysis of 25 trials involving 2,348 participants published between 1966 and 2004 concluded that soy phytoestrogens did not improve hot flashes or other menopausal symptoms (Krebs et al., 2004). The isoflavone daidzein has been reported to reduce hot flashes in menopausal women. The chemical structure of daidzein is very similar to the human body’s own estrogen. A study was conducted by Khaodhiar et al., (2008) on 190 women ranging in age from 38 to 60 years in various stages of menopause, who had 4 to 14 hot flashes daily. The women were given either one or two concentrations of diadzein rich isoflavone-aglycone. The number of hot flashes in the diadzein rich isoflavone groups was reduced by 52% and 51% at the end of 12 weeks, while the placebo group experienced a 39% reduction. A few studies using higher doses of isoflavone (50 to 80 mg/day), enrolling women with more vaso-

motor symptoms at baseline (4 to 7 symptoms/day) and with larger sample sizes, have exhibited mildly beneficial effects on self-reported frequency and severity of vasomotor symptoms (Albertazzi et al. 1998, Washburn et al. 1999). Han et al., (2002) reported a 26% decrease in hot flash frequency in a group who consumed 100 mg/day of isoflavone as compared to a group who receive a placebo. Isoflavones from soy have also received attention as a possible alternative to conventional hormone replacement therapy (HRT) (Brandi, 1999; Eden, 2001; Elkind-Hirsch, 2001; Glazier and Bowman, 2001; Vincent and Fitzpatrick, 2000). Since there is a chemical similarity to the female sex hormone estrogen, isoflavones have been used in studies for relief of menopausal symptoms (Adlercreutz et al., 1992; North and Sharples, 2001).

A ROLE FOR ISOFLAVONES IN OBESITY Obesity is a state of excessive fat accumulation in the body, especially in abdominal adipose tissue, and is closely linked to metabolic disorders, which include diabetes, cardiovascular disease, nonalcoholic fatty liver disease, dyslipidemia, and other health problems (Després et al., 2008; Lois et al., 2008;). Nearly 20% of women aged 55 to 65 years suffer an increase in glucose tolerance and insulin resistance, thought to be due to estrogen defciency (Gaspard et al., 1995; Tufano et al., 2004). The effect of soy isoflavone supplementation on postmenopausal women has yielded mixed responses in several clinical trials due to differences in dose, duration of isoflavone supplementation, body weight, physical status of individual, and variability of experimental designs (Zhang et al., 2013). Some researchers have suggested a reduction in body weight (Sites et al., 2007; Gardner et al., 2001), fasting blood glucose (Villa et al., 2009; Crisafulli et al., 2005), blood fat levels (Reynolds et al, 2006) and insulin level (Villa et al., 2009; Crisafulli et al., 2005) as possible causes. However, other researchers did not reach this same conclusion (Charles et al., 2009; Khaodhiar et al., 2008).

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EFFECT OF ISOFLAVONES ON THYROID FUNCTION Concerns have been expressed that soy isoflavone intake adversely affects thyroid function (Messina and Redmond, 2006). The thyroid gland releases two primary hormones T4 and T3 (thyroxine and trio-iodothyronine, respectively) in a ratio of roughly 80:20 in response to the signal sent by thyroid stimulating hormone (TSH). Several studies have reported the interaction of soy isoflavones with thyroid function. Isoflavones lead to immune dysfunction by causing potent stimulation of T cell and B cell mediated immunity due to induced structural

target genes. Similarly, in vitro studies by Divi et al., (1996; 1997) have shown that isoflavones inhibit thyroid peroxidase. Genistein and daidzein block thyroid peroxidase-catalyzed tyrosine iodination by acting as alternate substrates (Divi et al., 1997). Chang and Doerge (2000) reported that consumption of soy could cause goiter only in animals or humans consuming diets marginally adequate in iodine or who were predisposed to develop goiter and in most cases dietary supplementation with adequate iodine can reverse the disorders (Schone et al., 1990). However, study by Poirier et al., (1999) reported that even the feeding of genistein to rats, which as an animal species are very sensitive to goitrogenic agents,

changes in thyroid peroxidase (Chen and Rogan, 2004). A study in premenopausal women (Duncan et al., 1999) demonstrated a decline in free T3 levels with a high isoflavone diet. Another study conducted with post-menopausal women demonstrated a rise in T4 with 56 mg isoflavones/day and a rise in T3 and TSH with 90 mg isoflavones/day at 6 months compared with controls, but these alterations were considered clinically non-significant (Persky et al., 2002). Several other studies revealed non-significant changes in thyroid profile with isoflavones in menopausal women (Borchers et al., 2008; Duncan et al., 1999; Teas et al., 2007). The precise reason for such reported variations is unclear although differences in doses, dosage forms, isoflavone composition or the duration of treatment may all be considered important factors. Milerová et. al., (2006) reported on a study that looked at thyroid hormones and thyroid autoantibodies, along with blood levels of daidzein and genistein. The study focused on children without overt thyroid disease, who were not iodine deficient. They found a significant positive association of genistein with thyroglobulin autoantibodies and a negative correlation with thyroid volume. They concluded that even small differences in soy phytoestrogen intake may influence thyroid function, which

does not disrupt normal thyroid functioning.

could be important when iodine intake is insufficient. Studies by Huang et al., (2005) and Xiao et al., (2004) have shown that isoflavones suppress the binding ability of hepatic thyroid hormone receptor to the thyroid hormone response element of the

the production of equol from daidzein, since equol may actually confer more health benefits than daidzein. Much more research needs to be carried out in this area, in order to understand how soy can have health benefits in the broader population.

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CONCLUSIONS Consumers in the U. S. have become aware of the potential health benefits of soy, and as a result consumption in the U. S. has increased although it still remains far below that in Asia. There is a great deal of evidence to support the beneficial effects of soy isoflavones in the prevention of bone loss in postmenopausal women. Although isoflavones have been demonstrated to positively impact the biomarkers of prostate cancer, their potential benefits have not been substantiated in clinical trials. Beneficial effects of isoflavones for relieving menopause symptoms and prevention of breast cancer have not been proven and the antithyroid actions of soyfoods appear to be consistent in both animals and humans. The inconsistency of results from animal and human studies may be partially due to variation in the bioavailability of the isoflavones. Varying levels and duration of isoflavone consumption have also been shown to be of importance in whether soyfoods have a beneficial role. In addition, the role of the gut microflora may be particularly important in

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ACKNOWLEDGEMENTS

Abiru, Y., T. Ueno, and S. Uchiyama. 2013. Isolation and characterization of novel S-equol–producing bacteria from brines of stinky tofu, a traditional fermented soy food in Taiwan. Int. J. Food Sci. Nutr. 64:936–943. Adlercreutz, H., E. Hamalainen, S. Gorbach, and

women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration. J. Nutr.130:2243–2250. Arjmandi, B. H., R. Birnbaum, N. V. Goyal, M. J. Getlinger, S. Juma, L. Alekel, C. M. Hasler, M. L. Drum, B. W. Hollis, and S. C. Kukreja. 1998. Bonesparing effect of soy protein in ovarian hormonedeficient rats is related to its isoflavone content. Am. J. Clin. Nutr. 68:1364S-1368S. Atkinson, C., H.E. Skor, F.E. Dawn, D. Scholes, C. Chen, K. Wahala, S. M. Schwartz, and J. W. Lampe 2003. Urinary equol excretion in relation to 2-hydroxyestrone and 16alpha-hydroxyestrone concentrations: an observational study of young to

B. Goldin 1992. Dietary phtyoestrogens and the menopause in Japan. Lancet. 339:1233-1233. Akiyama, T., J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, and Y. Fukami 1987. Genistein, a speciﬁc inhibitor of tyrosinespeciﬁc protein kinases. J. Biol. Chem. 262:5592–5595. Albertazzi, P., F. Pansini, G. Bonaccorsi, L. Zanotti, E. Forini, and D. De Aloysia, 1998. The effect of dietary soy supplementation on hot flushes. Obstet. Gynecol. 91:6-11. Allaoua, A., I. B. Joyce, and B. Denis 2005. Soybean isoflavones efficacy of extraction conditions and effect of food type on extractability. Food Res. Int. 38:1199-1204. Allred, C. D., K. F. Allred, Y. H. Ju, S. M. Virant and W. G. Helferich 2001. Soy diets containing varying amounts of genistein stimulate growth of oestrogen-dependent (MCF-7) tumors in a dose-dependent manner. Cancer Res. 61:5045–5050. American Cancer Society. 2013. Cancer facts and figures, 2013. http://www.cancer.org/acs/groups/ content/@epidemiologysurveilance/documents/ document/acspc-036845.pdf Accessed 11 March 2014. Anderson, J. W., B. M. Johnstone, and M. E. CookNewell 1995. Meta-analysis of the effects of soy

middle-aged women. J. Steroid Biochem. Mol. Biol. 86:71–77. Axelson, M. and K.D. Setchell. 1981. The excretion of lignans in rats—evidence for an intestinal bacterial source for this new group of compounds. FEBS Lett. 123:337–342. Bektic, J., R. Guggenberger, I. E. Eder, A. E. Pelzer, A. P. Berger, G. Bartsch, and H. Klocker 2005. Molecular effects of the isoflavonoid genistein in prostate cancer. Clin. Prostate Cancer 4:124-129. Bloedon, L. T., A. R. Jeffcoat, W. Lopaczynski W, M. J. Schell, T. M. Black, K. J. Dix, B. F. Thomas, C. Albright, M. G. Busby, J. A. Crowell, and S. H. Zeisel 2002. Safety and pharmacokinetics of purified soy isoflavones:single-dose administration to postmenopausal women. Am. J. Clin. Nutr. 76:11261137. Bonetti, P. O., L. O. Lerman, and A. Lerman 2003. Endothelial dysfunction: a marker of atherosclerotic risk. Arterioscler. Thromb. Vasc. Biol. 23:168-175. Borchers, T. R., B. Chew, J. S. Park, M. McGuire, L. Fournier, and K. Beerman 2008. Effects of dietary and supplemental forms of isoflavones on thyroid function in healthy postmenopausal women. Top. Clin. Nutr. 23:13–22. Boring, C. C., T. S. Squires, T. Tong, and S. Mont-

protein intake on serum lipids. N. Engl. J. Med. 333:276–282. Arai, Y., S. Watanabe, M. Kimira, K. Shimoi, R. Mochizuki, and N. Kinae. 2000. Dietary intakes of flavonols, flavones and isoflavones by Japanese

gomery 1994. Cancer statistics 1994. CA - Cancer J. Clin. 44:7-26. Bowey, E., H. Adlercreutz, and I. Rowland. 2003. Metabolism of isoflavones and lignans by the gut microflora: a study in germ-free and human flora

The writing of this review was supported in part by a grant from the Arkansas Soybean Board.

REFERENCES

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

133

associated rats. Food Chem. Toxicol. 41: 631–636 Brandi, M. L. 1999. Phytoestrogens and menopause. Environ. Toxicol. Pharmacol. 7:213-216. Caan, B. J., L. Natarajan, B. Parker, E. B. Gold, C. Thomson, V. Newman, C. L. Rock, M. Pu, W. Al-Delaimy, and J. P. Pierce 2011. Soy food consumption and breast cancer prognosis. Cancer Epidemiol. Biomarkers Prev. 20:854-858. Candy, O. 1996. The role of soy in medicine, Part 1. Palais des Congress, 15-17 Sept., 1996. Cassidy, A., J. E. Brown, A. Hawdon, M. S. Faughnan, L. J. King J. Millward J, L. Zimmer-Nechemias, B. Wolfe, K. D. Setchell. 2006. Factors affecting the bioavailability of soy isoﬂavones in humans after ingestion of physiologically relevant levels from different soy foods. J. Nutr. 136:45–51. CDC, Centers for Disease Control and Prevention. 2013. Leading causes of death. http://www.cdc. gov/nchs/fastats/lcod.htm Accessed 11 March 2014. Cena, E. R. and F. M. Steinberg. 2011. Soy may help protect against cardiovascular disease. California Agric. 65:118-123. Chang, H. C., and D. R. Doerge 2000. Dietary genistein inactivates rat thyroid peroxidase in vivo without an apparent hypothyroid effect. Toxicol. Appl. Pharmacol. 168:244–252. Charles, C., J. Yuskavage, O. Carlson, M. John, A. S. Tagalicud, M. Maggio, D. C. Muller, J. Egan, and S. Basaria 2009. Effects of high-dose isoflavones on metabolic and inflammatory markers in healthy postmenopausal women. Menopause. 16:395–400. Chen, A., and W. J. Rogan 2004. Isoflavones in soy infant formula: a review of evidence for endocrine and other activity in infants. Ann. Rev. Nutr. 24:33–54. Chen, J., H. Lin, and M. Hu. 2003. Metabolism of flavonoids via enteric recycling: Role of intestinal disposition. J. Pharmacol. Exp. Ther. 304:12281235. Codina, R., Ardusso, L., Lockey, R. F., Crisci, C. and Medina I. 2003. Allergenicity of varieties of soybean. Allergy 58:1293-1298. Constantinou, A., K. Kiguchi, and E. Huberman 1990. 134

Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein. Cancer Res. 50:2618–2624. Coward, L., M. Smith, M. Kirk, and S. Barnes. 1998. Chemical modification of isoflavones in soy foods during cooking and processing. Am. J. Clin. Nutr. 68(suppl):1486S–1491S. Crisafulli, A., D. Altavilla, H. Marini, A. Bitto, D. Cucinotta, N. Frisina, F. Corrado, R. D’Anna, G. Squadrito, E. B. Adamo, R. Marini, A. Romeo, F. Cancellieri, M. Buemi, and F. Squadrito 2005. Effects of the phytoestrogen genistein on cardiovascular risk factors in postmenopausal women. Menopause. 12:186–192. Dalais, F. S., A. Meliala, N. Wattanapenpaiboon, M. Frydenberg, D. A. Suter, W. K. Thomson, and M. L. Wahlqvist 2004. Effects of a diet rich in phytoestrogens on prostate-specific antigen and sex hormones in men diagnosed with prostate cancer. Urology. 64:510–515. de Kleijn, M. J., Y. T. van der Schouw, P. W. Wilson, H. Adlercreutz, W. Mazur, D. E. Grobbee and P. F. Jacques 2001. Intake of dietary phytoestrogens is low in postmenopausal women in the United States: the Framingham study. J. Nutr.131:1826–1832. Deavours, B.E. and R. A. Dixon. 2005. Metabolic engineering of isoflavonoid biosynthesis in alfalfa. Plant Physiol. 138:2245–2259. Després, J. P., I. Lemieux, J. Bergeron, P. Pibarot, P. Mathieu, E. Larose, J. Rodés-Cabau, O. F. Bertrand, and P. Poirier 2008. Abdominal obesity and the metabolic syndrome: contribution to global cardiometabolic risk. Arterioscler. Thromb. Vasc. Biol. 28:1039-1049. Desroches, S., J. F. Mauger, L. M. Ausman, A. H. Lichtenstein, and B. Lamarche 2004. Soy protein favorably affects LDL size independently of isoflavones in hypercholesterolemic men and women. J. Nutr. 134:574-579. Divi, R. L., H. C. Chang and D. R. Doerge 1997. Antithyroid isoflavones from soybean: isolation, characterization , and mechanisms of action. Biochem. Pharmacol. 54:1087-1096. Divi, R. L. and D. R. Doerge 1996 .Inhibition of thyroid peroxidase by dietary flavonoids. Chem. Res.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

Toxicol. 96:16-23. Dong, J. Y., X. Tong, Z. W. Wu, P. C. Xun, K. He, and L. Q. Qin 2011. Effect of soya protein on blood pressure: a meta-analysis of randomised controlled trials. Br. J. Nutr. 106:317-326. Dong, J. Y. and L. Q. Qin 2011. Soy isoflavones consumption and risk of breast cancer incidence or recurrence: a meta-analysis of prospective studies. Breast Cancer Res. Treat. 125:315-323. Duncan, A. M., B. E. Merz, X. Xu, T. C. Nagel, W. R. Phipps, and M. S. Kurzer 1999. Soy isoflavones exert modest hormonal effects in premenopausal women. J. Clin. Endocrinol. Metab. 84:192–197. Duncan, A.M., B.E. Merz-Demlow, X. Xu, W.R. Phipps, and M.S. Kurzer 2000. Premenopausal equol excretors show plasma hormone profiles associated with lowered risk of breast cancer. Cancer Epidemiol. Biomarker. Prev. 9:581–586. Eden, J. A. 2001. Managing the menopause: phytooestrogens or hormone replacement therapy? Ann. Med. 33:4-6. Elkind-Hirsch, K. 2001. Effect of dietary phytoestrogens on hot flushes: Can soy-based proteins substitute for traditional estrogen replacement therapy? Menopause 8:154-156. Falk, R.T., T.R. Fears, X. Xu, R.N. Hoover, M.C. Pike, A.H. Wu, A. M. Nomura, L. N. Kolonel, D. W. West, D. W. Sepkovic, H. L. Bradlow and R. G. Ziegler 2005. Urinary estrogen metabolites and their ratio among Asian American women. Cancer Epidemiol. Biomarkers Prev. 14:221–226. FDA, Food and Drug Administration. 1999. Food labeling: Health claims; soy protein and coronary heart disease. Fed Regist. 64(206):57700-57733. Fischer, L., C. Mahoney, A. R. Jeffcoat, M. A. Koch, B. E. Thomas, J. L. Valentine, T. Fortina, M.G., G. Ricci, R. Foschino, C. Picozzi, P. Dolci, G. Zeppa, L. Cocolin, and P. L. Manachini 2007. Phenotypic typing, technological properties and safety aspects of Lactococcus garvieae strains from dairy environments. J. Appl. Microbiol. 103:445–453. Fotsis, T., M. Pepper, H. Adlercreutz, G. Fleischmann, T. Hase, R. Montesano, and L. Schweigerer (1993). Genistein, a dietary –derived inhibitor of in vitro angiogenesis. Proc. Natl. Acad. Sci. 90:2690-2694.

Fujioka, M., M. Uehara, J. Wu, H. Adlercreutz, K. Suzuki, K. Kanazawa, K. Takeda, K. Yamada, and Y. Ishimi 2004. Equol, a metabolite of daidzein, inhibits bone loss in ovariectomized mice. J. Nutr. 134:2623–2627. Fukutake, M., M. Takahashi, K. Ishida, H. Kawamura, T. Sugimura, and K. Wakabayashi. 1996. Quantification of genistein and genistin in soybeans and soybean products. Food Chem. Toxicol. 34:457– 461. Gardner, C. D., M. Messina, A. Kiazand, J. L. Morris, and A. A. Franke 2007. Effect of two types of soy milk and dairy milk on plasma lipids in hypercholesterolemic adults: a randomized trial. J. Am. Coll. Nutr. 26:669-677. Gardner, C. D., K. A. Newell, R. Cherin, and W. L. Haskell 2001. The effect of soy protein with or without isoflavones relative to milk protein on plasma lipids in hypercholesterolemic postmenopausal women. Am. J. Clin. Nutr. 73:728–735. Gaspard, U. J., J. M. Gottal, and F. A. van den Brule 1995. Postmenopausal changes of lipid and glucose metabolism: a review of their main aspects. Maturitas. 21:171–178. Glazier, M.G. and M. A. Bowman 2001. A review of the evidence for the use of phytoestrogens as a replacement for traditional estrogen replacement therapy. Arch. Intern. Med. 161:1161-1172. Guha, N., M. L. Kwan, Q. P. Quesenberry Jr, E. K. Weltzien, A. L. Castillo, and B. J. Caan 2009. Soy isoflavones and risk of cancer recurrence in a cohort of breast cancer survivors: The Life after Cancer Epidemiology study. Breast Cancer Res. Treat. 118:395-405. Hamilton-Reeves, J. M., S. A. Rebello, W. Thomas, J. W. Slaton, M. S. Kurzer 2007. Isoflavone-rich soy protein isolate suppresses androgen receptor expression without altering estrogen receptor-beta expression or serum hormonal profiles in men at high risk of prostate cancer. J. Nutr. 137:1769–1775. Han, K. K., J. M. Soares Jr, M. A. Haidar, G. R. de Lima, and E. C. Baracat 2002. Benefits of soy isoflavone therapeutic regimen on menopausal symptoms. Obstet. Gynecol. 99:389–394.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

135

Haque, N., U. Salma, T. R. Nurunnabi, A. K. Haque, I. J. Mukti, S. Pervin, and R. Nahar R. 2010. Lifestyle related causes of cancer and chemoprevention through phytonutrients. Pak. J. Biol. Sci. 13:916-26. Henrotin, Y. E., C. Sanchez, M. A. Deberg, N. Piccardi, G. B. Guillou, P. Msika, and J. Y. Reginster 2003. Avocado/soybean unsaponifiables increase aggrecan synthesis and reduce catabolic and proinflammatory mediator production by human osteoarthritic chondrocytes. J. Rheumatol. 8:1825-1834. Herrington, D. M., W. V. Brown, L. Mosca, W. Davis, B. Eggleston, W. G. Hundley, and J. Raines

Latest world cancer statistics. http://www.iarc.fr/ en/media-centre/pr/2013/pdfs/pr223_E.pdf Accessed 11 March 2014. Jacobsen, B. K., S. F. Knutsen, and G. E. Fraser 1998. Does high soy milk intake reduce prostate cancer incidence? The Adventist Health Study (United States). Cancer Causes Control 9:553-557. Jenkins, D. J., C. W. Kendall, D. Faulkner, E. Vidgen, E. A. Trautwein, T. L. Parker, and A. Marchie. 2011. Effect of a dietary portfolio of cholesterol-lowering foods given at 2 levels of intensity of dietary advice on serum lipids in hyperlipidemia: epidemiology of soy exposures and breast cancer risk. JAMA 306:831-839.

2004. Relationship between arterial stiffness and subclinical aortic atherosclerosis. Circulation 110:432-437. Ho, S. C., S. G. Chan, Q. Yi, E. Wong, and P. C. Leung 2001. Soy intake and the maintenance of peak bone mass in Hong Kong Chinese women. J. Bone Miner. Res. 16:1363-1369. Hooper, L., P.A. Kroon, E.B. Rimm, J. S. Cohn, I. Harvey, K. A. Le Cornu, J. J. Ryder, W. L. Hall, and A. Cassidy 2008. Flavonoids, flavonoid-rich foods, and cardiovascular risk: a meta-analysis of randomized controlled trials. Am. J. Clin. Nutr. 88:38–50. Horiuchi, T., T. Onouchi, M. Takahashi, H. Ito, and H. Orimo 2000. Effect of soy protein on bone metabolism in postmenopausal Japanese women. Osteoporos. Int. 11:721-724. Huang, W., C. Wood, M. R. L’Abbé, S. Gilani, K. A. Cockell and C. W., Xiao 2005. Soy protein isolate increases hepatic thyroid hormone receptor content and inhibits its binding to the target genes in rats. J. Nutr. 135:1631–1635. Hussain, M., M. Banerjee, F. H. Sarkar, Z. Djuric, M. N. Pollak, D. Doerge, J. Fontana, S. Chinni, J. Davis, J. Forman, D. P. Wood & O. Kucuk 2003. Soy isoflavones in the treatment of prostate cancer. Nutr. Cancer 47:111-117.

Johnell, O. and J. A. Kanis 2006. An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporos Int. 17:1726. Kanis, J. A. 2007. WHO Technical Report, University of Sheffield, UK: 66. Khaodhiar, L., H. A. Ricciotti, L. Li, W. Pan, M. Schickel, J. Zhou, G. L. Blackburn 2008. Daidzein-rich isoflavone aglycones are potentially effective in reducing hot flashes in menopausal women. Menopause. 15:125–132. Kitamura, A., H. Iso, M. Iida, Y. Naito, S. Sato, D. R. Jacobs, M. Nakamura, T. Shimamoto, and Y. Komachi 2002. Trends in the incidence of coronary heart disease and stroke and the prevalence of cardiovascular risk factors among Japanese men from 1963 to 1994. Am. J. Med. 112:104-109. Krebs, E. E., K. E. Ensrud, R. MacDonald, and T. J. Wilt 2004. Phytoestrogens for treatment of menopausal symptoms: a systematic review. Obstet. Gynecol. 104:824–836. Kuiper, G. G., B. Carlsson, K. Grandien, E. Enmark, J. Haggblad, S. Nilsson, J. A. Gustafsson 1997. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinol. 138:863-870. Kuiper, G. G., J. G. Lemmen, B. Carlsson, J. C. Cor-

Hwang, Y. W., S. Y. Kim, S. H. Jee, Y. N. Kim, and C. M. Nam 2009. Soy food consumption and risk of prostate cancer: a meta-analysis of observational studies. Nutr. Cancer 61:598-606. International Agency for Research on Cancer. 2013.

ton, S. H. Safe, P. T. van der Saag, B. van der Burg, and J. A. Gustafsson 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinol. 139:4252-4263. Kumar, N. B., A. Cantor, K. Allen, D. Riccardi, K.

136

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

Besterman-Dahan, J. Seigne, M. Helal, R. Salup, and J. Pow-Sang 2004. The specific role of isoflavones in reducing prostate cancer risk. Prostate 59:141-147. Kumar, N. B., J. P. Krischer, K. Allen, D. Riccardi, K. Besterman-Dahan, R. Salup, L. Kang, P. Xu, and J. Pow-Sang 2007. Phase II randomized, placebocontrolled clinical trial of purified isoflavones in modulating steroid hormones in men diagnosed with localized prostate cancer. Nutr. Cancer 59:163-168. Kurahashi, N., M. Iwasaki, M. Inoue, S. Sasazuki, and S. Tsugane 2008. Plasma isoflavones and subsequent risk of prostate cancer in a nested case-con-

flavone supplementation on vascular endothelial function in postmenopausal women: a meta-analysis of randomized placebo-controlled trials. Am. J. Clin. Nutr. 91:480-486. Lichtenstein, A. H., L. J. Appel, M. Brands M, et al. 2006. Diet and lifestyle recommendations revision 2006: A scientific statement from the American Heart Association Nutrition Committee. Circulation 114:82–96. Lock M. 1992. Contested meanings of the menopause. Lancet 337:1270-1272. Lock, M. 1994. Menopause in cultural context. Exp. Gerontol. 29:307-317. Lois, K., J. Young, and S. Kumar 2008. Obesity; epi-

trol study: the Japan Public Health Center. J. Clin. Oncol. 26:5923–5929. Lampe, J. W. 2003. Isoflavonoid and lignan phytoestrogens as dietary biomarkers. J. Nutr. 133 Suppl 3:956S-964S. Lampe, J.W. 2009. Is equol the key to the efficacy of soy foods? Am. J. Clin. Nutr. 89:1664S–1667S. Law, M. R., N. J. Wald, and S. G. Thompson 1994. By how much and how quickly does reduction in serum cholesterol concentration lower risk of ischaemic heart disease? BMJ. 308:367-372. Lazarevic, B., G. Boezelijn, L. M. Diep, K. Kvernrod, O. Ogren, H. Ramberg, A. Moen, N. Wessel, R. E. Berg, W. Egge-Jacobsen, C. Hammarstrom, A. Svindland, O. Kucuk, F. Saatcioglu, K. A. Taskèn, and S. J. Karlsen 2011. Efficacy and safety of short-term genistein intervention in patients with localized prostate cancer prior to radical prostatectomy: a randomized, placebocontrolled, double-blind Phase 2 clinical trial. Nutr. Cancer 63:889-898. Lamartiniere, C. A., Y. X. Zhao, and W. A. Fritz WA. Genistein: mammary cancer chemoprevention, in vivo mechanisms of action, potential for toxicity and bioavailability in rats. J. Women’s Cancer 2000; 2:11-9.

phenomenon or cause of metabolic syndrome? Int. J. Clin. Pract. 62:932–938. Luh, B. S. 1995. Industrial production of soy sauce. J. Indust. Microbiol. 14:467-471. Magee, P.J. 2011. Is equol production beneficial to health? Proc. Nutr. Soc. 70:10–18. Magee, P. J., and I. T. Rowland 2004. Phyto-oestrogens, their mechanism of action: current evidence for a role in breast and prostate cancer. Br. J. Nutr. 91:513-531. Naya, M. and M. Imai. 2013. Recent advances on soybean isoflavone extraction and enzymatic modification of soybean oil in Soybean - Bio-Active Compounds, Prof. Hany El-Shemy (Ed.), ISBN: 978-953-51-0977-8, InTech, DOI: 10.5772/52603. Available from: http://www. intechopen.com/books/soybean-bio-activecompounds/recent-advances-on-soybean-isoflavone-extraction-and-enzymatic-modificationof-soybean-oil Mahungu, S. M., S. Diaz-Mercado, J. Li, M. Schwenk, K. Singletary, and J. Faller. 1999. Stability of isoflavones during extrusion processing of corn/soy mixture. J. Agric. Food Chem. 47:279–284. Mei, J., S. S. Yeung, and A. W. Kung 2001. High dietary phytoestrogen intake is associated with high-

Lee, H. P., L. Gourley, S. W. Duffy, J. Esteve, J. Lee, and N. E. Day 1991. Dietary effects on breast-cancer risk in Singapore. Lancet 337:1197-1200. Li, S. H., X. X. Liu, Y. Y. Bai, X. J. Wang, K. Sun, J. Z. Chen, and R. T. Hui RT 2010. Effect of oral iso-

er bone mineral density in postmenopausal but not premenopausal women. J. Clin. Endocrinol. Metab. 86:5217-5221. Messina, M. 1999. Legumes and soybean overview of their nutritional profiles and health effects. Am.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

137

J. Clinical Nutr. 70(suppl.):439-450. Messina, M., and S. Barnes. 1991. The role of soy products in reducing risk of cancer. J. Natl. Cancer Inst. 83:541-546. Messina, M., and L. Hilakivi-Clarke 2009. Early intake appears to be the key to the proposed protective effects of soy intake against breast cancer. Nutr. Cancer 61:792-728. Messina, M., S. Ho, and D. L. Alekel 2004. Skeletal benefits of soy isoflavones: a review of theclinical trial and epidemiologic data. Curr. Opin. Clin. Nutr. Metab. Care 7:649-658. Messina, M., O. Kucuk, and J. W. Lampe 2006. An overview of the health effects of isoflavones

Millar, W. L. Hall, F. Kramer Birkved, I. K. Sorensen, and G. Sontag 2009. Analytical and compositional aspects of isoflavones in food and their biological effects. Mol. Nutr. Food Res. 53(Suppl 2):S266–309. Murphy, P. A., T. Song, G. Buseman, K. Barua, G. R. Beecher, D. Trainer, and J. Holden 1999. Isoflavones in retail and institutional soy foods. J. Agric. Food Chem. 47:2697-2704. Muthyala, R.S., Y.H. Ju, S. Sheng, L.D. Williams, D.R. Doerge, B.S. Katzenellenbogen, W. G. Helferich, and J. A. Katzenellenbogen. 2004. Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biologi-

with an emphasis on prostate cancer risk and prostate-specific antigen levels. J. AOAC Int. 89:1121-1134. Messina, M. J. and C. L. Loprinzi 2001. Soy for breast cancer survivors: a critical review of the literature. J. Nutr. 131(11 Suppl):3095S-3108S. Messina, M., and G. Redmond 2006. Effects of soy protein and soybean isoflavones on thyroid function in healthy adults and hypothyroid patients: a review of the relevant literature. Thyroid. 16:249-258. Messina, M., C. Nagata, and A. H. Wu 2006. Estimated Asian adult soy protein and isoflavone intakes. Nutr. Cancer 55:1-12. Messina, M. J. and C. E. Wood. 2008. Soy isoflavones, estrogen therapy, and breast cancer risk: analysis and commentary. Nutr. J. 7:17 doi:10.1186/14752891-7-17. Milerová, J., J. Cerovská, V. Zamrazil, R. Bílek, O. Lapcík, and R. Hampl 2006. Actual levels of soy phytoestrogens in children correlate with thyroid laboratory parameters. Clin. Chem. Lab. Med. 44:171-174. Miyanaga, N., H. Akaza, S. Hinotsu, T. Fujioka, S. Naito, M. Namiki, S. Takahashi, Y. Hirao, S. Horie, T. Tsukamoto, M. Mori, and H. Tsuji 2012. Prostate Cancer Chemoprevention Study: An investigative

cal activity through estrogen receptors alpha and beta. Bioorg. Med. Chem. 12:1559–1567. Nagata, C. 2010. Factors to consider in the association between soy isoflavone intake and breast cancer risk. J. Epidemiol. 20:83-89. Nagata, Y., T. Sonoda, M. Mori, N. Miyanaga, K. Okumura, K. Goto, S. Naito, K. Fujimoto, Y. Hirao, A. Takahashi, T. Tsukamoto, H. Akaza 2007. Dietary isoflavones may protect against prostate cancer in Japanese men. J. Nutr. 137:1974–1979. Nechuta, S. J., B. J. Caan, W. Y. Chen, W. Lu, Z. Chen, M. L. Kwan, S. W. Flatt, Y. Zheng, W. Zheng, J. Pier, and X. O. Shu 2012. Soy food intake after diagnosis of breast cancer and survival: an in-depth analysis of combined evidence from cohort studies of US and Chinese women. Am. J. Clin. Nutr. 96:123–132. Nestel, P. J., T. Yamashita, T. Sasahara, S. Pomeroy, A. Dart, P. Komesaroff , A. Owen, and M. Abbey 1997. Soy isoflavones improve systemic arterial compliance but not plasma lipids in menopausal and perimenopausal women. Arterioscler. Thromb. Vasc. Biol. 17:3392-3398. Nestel, P. J., S. Pomeroy, S. Kay, P. Komesaroff, J. Behrsing, J. D. Cameron, and L. West 1999. Isoflavones from red clover improve systemic arterial compliance but not plasma lipids in menopausal

randomized control study using purified isoflavones in men with rising prostate-specific antigen/ Cancer Sci. 103:125-130. Mortensen, A., S. E. Kulling, H. Schwartz, I. Rowland, C. E. Ruefer, G. Rimbach, A. Cassidy, P. Magee, J.

women. J. Clin. Endocrinol. Metab. 84:895-898. Nishio, K., Y. Niwa, H. Toyoshima, K. Tamakoshi, T. Kondo, H. Yatsuya, A. Yamamoto, S. Suzuki, S. Tokudome, Y. Lin, K. Wakai, N. Hamajima, and A. Tamakoshi 2007. Consumption of soy foods and

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the risk of breast cancer: findings from the Japan Collaborative Cohort (JACC) Study. Cancer Causes Control. 18:801–808. North, F. M., and K. Sharples 2001. Changes in the use of hormone replacement therapy in New Zealand from 1991-1997. N. Z. Med. J. 114:250-253. Pendleton, J. M., W. W. Tan, S. Anai, M. Chang, W. Hou, K. T. Shiverick and C. J. Rosser 2008. Phase II trial of isoflavone in prostate-specific antigen recurrent prostate cancer after previous local therapy. BMC Cancer 8:132 doi:10.1186/1471-2407-8-132. Poirier, L.A., D. R. Doerge, D. W. Gaylor et al. 1999. An FDA review of sulfamethazine toxicity. Regul. Toxicol. Pharmacol. 30:217-222.

ing glucosinolate and iodine intake via rapeseed meal diets on serum thyroid hormone level and total iodine in the thyroid in growing pigs. Endocrinol. Exp. 24:415–427. Schroder, C., A. Matthies, W. Engst, M. Blaut, and A. Braune 2013. Identification and expression of genes involved in the conversion of daidzein and genistein by the equol-forming bacterium Slackia isoflavoniconvertens. Appl. Environ. Microbiol. 79:3494–3502. Setchell, K. D. 1998. Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. Am. J. Clin. Nutr. 68:1333S– 1346S.

Rabiau, N., M. Kossaï, M. Braud, N. Chalabi, S. Satih, Y. J. Bignon, and D. J. Bernard-Gallon 2010. Genistein and daidzein act on a panel of genes implicated in cell cycle and angiogenesis by polymerase chain reaction arrays in human prostate cancer cell lines. Cancer Epidemiol. 34:200-206. Reynolds, K., A. Chin, K. A. Lees, A. Nguyen, D. Bujnowski, and J. He 2006. A meta-analysis of the effect of soy protein supplementation on serum lipids. Am. J. Cardiol. 98:633–640. Rivas, M., R. P. Garay, J. F. Escanero, P. Cia Jr., P. Cia, and J. O. Alda 2002. Soy milk lowers blood pressure in men and women with mild to moderate essential hypertension. J. Nutr. 132:1900-1902. Rock, C. L., C. Doyle, W. Demark-Wahnefried, J. Meyerhardt, K. S. Courneya, A. L. Schwartz, E. V. Bandera, K. K. Hamilton, B. Grant, M. McCullough, T. Byers, and T. Gansler 2012. Nutrition and physical activity guidelines for cancer survivors. CA Cancer J. Clin. 62:242-274. Rolfe, B.G. 1988. Flavones and isoflavones as inducing substances of legume nodulation. Biofactors. 1:3–10. Sacks, F. M., A. Lichtenstein, L. Van Horn, W. Harris, P. Kris-Etherton, and M. Winston 2006. Soy protein, isoflavones, and cardiovascular health: an Ameri-

Setchell, K. D., N. M. Brown, and E. Lydeking-Olsen 2002. The clinical importance of the metabolite equol-a clue to the effectiveness of soy and its isoflavones. J. Nutr. 132:3577-3584. Setchell, K.D.R. and A. Cassidy. 1999. Dietary isoflavones: Biological effects and relevance to human health. J. Nutr. 129:758-767. Setchell, K.D., C. Clerici, E.D. Lephart, S.J. Cole, C. Heenan, D. Castellani, B. E. Wolfe, L. NechemiasZimmer, N. M. Brown, T. D. Lund, R. J. Handa, and J. E. Heubi 2005. S-equol, a potent ligand for estrogen receptor beta, is the exclusive enantiomeric form of the soy isoflavone metabolite produced by human intestinal bacterial flora. Am. J. Clin. Nutr. 81:1072–1079. Severson, R. K., A. M. Nomura, J. A. Grove, and G. N. Stemmermann 1989. A prospective study of demographics, diet, and prostate cancer among men of Japanese ancestry in Hawaii. Cancer Res. 49:1857-1860. Shu, X. O., F. Jin, Q. Dai, W. Wen, J. D. Potter, L. H. Kushi, Z. Ruan, Y. T. Gao, and W. Zheng 2001. Soyfood intake during adolescence and subsequent risk of breast cancer among Chinese women. Cancer Epidemiol. Biomarkers Prev. 10:483–488. Sites, C. K., B. C. Cooper, M. J. Toth, A. Gastaldell,

can Heart Association Science Advisory for professionals from the Nutrition Committee. Circulation. 113:1034-1044. Schone, F., G. Jahreis, R. Lange, W. Seffner, B. Groppel, A. Hennig, and H. Ludke 1990. Effect of vary-

A. Arabshahi, and S. Barnes 2007. Effect of a daily supplement of soy protein on body composition and insulin secretion in postmenopausal women. Fertil. Steril. 88:1609–1617. Slavin, M., W. Kenworthy, and L. L. Yu. 2009. Anti-

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oxidant properties, phytochemical composition, and antiproliferative activity of Maryland-grown soybeans with colored seed coats. J. Agric. Food Chem. 57:11174-11185. Smit, E., F. J. Nieto, C. J. Crespo and P. Mitchell 1999. Estimates of animal and plant protein intake in us adults: results from the Third National Health and Nutrition Examination Survey, 1988–1991. J. Am. Diet. Assoc. 99:813-820. Synder, H.E. and T.W. Kwon. 1987a. Oriental soy food products in Soybean Utilization. Van Nostrand Reinhold: New York, pp. 218–241. Synder, H.E. and T.W. Kwon. 1987b. Processing of soybeans in Soybean Utilization. Van Nostrand Re-

ton, and M. Wolz 1992. Total morbidity and mortality from heart disease, cancer and stroke from 1950 to 1987 in 27 countries. National Institutes of Health Publication No. 92–3088, Bethesda, MD. Tice, J. A., B. Ettinger, K. Ensrud, R. Wallace, T. Blackwell, and S. R. Cummings 2003. Phytoestrogen supplements for the treatment of hot flashes: The isoflavone clover extract (ICE) study: A randomized controlled trial. JAMA 290:207-214. Tsuchida, K., S. Mizushima, M. Toba, and K. Soda 1999. Dietary soybeans intake and bone mineral density among 995 middle-aged women in Yokohama. J. Epidemiol. 9:14-19. Tsuda, M., T. Kitazaki, T. Ito, and T. Fujita 1986. The

inhold: New York, pp. 74–144. Squadrito, F., D. Altavilla, A. Crisafulli, A. Saitta, D. Cucinotta, N. Morabito, R. D’Anna, F. Corrado, P. Ruggeri, N. Frisina, and G. Squadrito 2003. Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study. Am. J. Med. 114:470-476. Squadrito, F., D. Altavilla, N. Morabito, R. D’Anna, F. Corrado, P. Ruggeri, G. M. Campo, G. Calapai, A. P. Caputi, and G. Squadrito 2002. The effect of the phytoestrogen genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium dependent vasodilation in postmenopausal women. Atherosclerosis 163:339-347. Steinberg, F. M., N. L. Guthrie, A. C. Villablanca, K. Kumar, and M. J. Murray. 2003. Soy protein with isoflavones has favorable effects on endothelial function that are independent of lipid and antioxidant effects in healthy postmenopausal women. Am. J. Clin. Nutr. 78:123–130. Taku, K., K. Umegaki, Y. Sato, Y. Taki, K. Endoh, and S. Watanabe. 2007. Soy isoflavones lower serum total and LDL cholesterol in humans: A meta-analysis of 11 randomized controlled trials. Am. J. Clin. Nutr. 85:1148–1156. Teas, J., L. E. Braverman, M. S. Kurzer, S. Pino, T. G.

effect of ipriflavone (TC-80) on bone resorption in tissue culture. J. Bone Miner. Res. 1:207-11. Tufano, A., P. Marzo, R. Enrini, L. Morricone, F. Caviezel, and B. Ambrosi 2004. Anthropometric, hormonal and biochemical differences in lean and obese women before and after menopause. J. Endocrinol. Invest. 27:648–653. Ursin, G., S. London, F.Z. Stanczyk, E. Gentzschein, A. Paganini-Hill, R.K. Ross and M. C. Pike 1999. Urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio and risk of breast cancer in postmenopausal women. J. Natl. Cancer Inst. 91:1067–1072. USDA. 2008. USDA database for the isoflavone content of selected foods. Available at: http://www. ars.usda.gov/SP2UserFiles/Place/12354500/Data/ isoflav/Isoflav_R2.pdf Accessed 28 April 2014. van Erp-Baart, M. A., H. A. Brants, M. Kiely, A. Mulligan, A. Turrini, C. Sermoneta, A. Kilkkinen, and L. M. Valsta 2003. Isoflavone intake in four different European countries: the VENUS approach. Br. J. Nutr. 89 (Suppl 1):S25-30. Villa, P., B. Costantini, R. Suriano, C. Perri, F. Macri, L. Ricciardi, S. Panunzi, and A. Lanzone 2009. The differential effect of the phytoestrogen genistein on cardiovascular risk factors in postmenopausal women: relationship with the metabolic status. J.

Hurley, and J. R. Hebert 2007. Seaweed and soy: companion foods in Asian cuisine and their effects on thyroid function in American women. J. Med. Food. 10:90–100. Thom, T. J., F. H. Epstein, J. J. Feldmen, P. E. Leaver-

Clin. Endocrinol. Metab. 94:552–558. Vincent, A. and L. A. Fitzpatrick 2000. Soy isoflavones: Are they useful in menopause? Mayo Clin. Proc. 75:1174-1184. Wakai, K., Y. Ohno, K. Genka, K. Ohmine, T. Kawamu-

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ra, A. Tamakoshi, Y. Lin, T. Nakayama, K. Aoki, and S. Fukuma 1999. Risk modification in lung cancer by a dietary intake of preserved foods and soyfoods: findings from a case-control study in Okinawa, Japan. Lung Cancer 25:147-159. Walker, H. A., T. S. Dean, T. A. Sanders, G. Jackson, J. M. Ritter, and P. J. Chowienczyk 2001. The phytoestrogen genistein produces acute nitric oxidedependent dilation of human forearm vasculature with similar potency to 17b- Estradiol. Circulation 103:258-262. Wang, H. J. and P. A. Murphy. 1996. Mass balance study of isoflavones during soybean processing. J. Agric. Food Chem. 44:2377–2383.

breast cancer in Asian-Americans. Cancer Epidemiol. Biomark. Prev. 5:901–906. Wu, J., J. Oka, J. Ezaki, T. Ohtomo, T. Ueno, S. Uchiyama, T. Toda, M. Uehara, and Y. Ishimi 2007. Possible role of equol status in the effects of isoflavone on bone and fat mass in postmenopausal Japanese women: a double-blind, randomized, controlled trial. Menopause 14: 866–874 Wu, J., J. Oka, M. Higuchi, I. Tabata, T. Toda, M. Fujioka, N. Fuku, T. Teramoto, T. Okuhira, T. Ueno, S. Uchiyama, K. Urata, K. Yamada, and Y. Ishimi 2006. Cooperative effects of isoﬂavones and exercise on bone and lipid metabolism in postmenopausal Japanese women: a randomized placebo-con-

Warri, A., N. M. Saarinen, S. Makela, and L. HilakiviClarke 2008. The role of early life genistein exposures in modifying breast cancer risk. Br. J. Cancer 98:1485-1493. Washburn, S., G. L. Burke, T. Morgan, and M. Anthony 1999. Effect of soy protein supplementation on serum lipoproteins, blood pressure, and menopausal symptoms in perimenopausal women. Menopause 6:7-13. Weggemans, R. M., and E. A. Trautwein 2003. Relation between soy-associated isoflavones and LDL and HDL cholesterol concentrations in humans: a meta-analysis. Eur. J. Clin. Nutr. 57:940-946. WHO. 2011a. Global status report on noncommunicable disaeses 2010. Geneva, World Health Organization. http://www.who.int/nmh/publications/ ncd_report2010/en/ Accessed 11 March 2014. WHO. 2011b. Global atlas on cardiovascular disease prevention and control. Geneva, World Health Organization. http://www.who.int/cardiovascular_diseases/ publications/atlas_cvd/en/ Accessed 11 March 2014. Wu, A. H., P. Wan, J. Hankin, C. C. Tseng, M. C. Yu, and M. C. Pike 2002. Adolescent and adult soy intake and risk of breast cancer in Asian-Americans. Carcinogenesis 23:1491-1496. Wu, A. H., M. C. Yu, C. C. Tseng, and M. C. Pike 2008.

trolled trial. Metabolism. 55:423–433. Xiao, C. W., M. R. L’Abbé, S. Gilani, G. Cooke, I. Curran, and S. A. Papademetriou 2004. Dietary soy protein isolate and isoflavones modulate hepatic thyroid hormone receptors in rats. J. Nutr. 134:743–749. Yan, L., and E. L. Spitznagel 2009. Soy consumption and prostate cancer risk in men: a revisit of a metaanalysis. Am. J. Clin. Nutr. 89:1155-1163. Yokoyama, S. and T. Suzuki 2008. Isolation and characterization of a novel equol-producing bacterium from human feces. Biosci. Biotechnol. Biochem. 72:2660–2666. Yokoyama, S., K. Oshima, I. Nomura, M. Hattori, and T. Suzuki 2011. Complete genomic sequence of the equol-producing bacterium Eggerthella sp. strain YY7918, isolated from adult human intestine. J. Bacteriol. 193:5570–5571. Yuan, J.M., Q.S. Wang, R.K. Ross, B.E. Henderson, and M.C. Yu 1995. Diet and breast cancer in Shanghai and Tianjin, China. Br. J. Cancer 71:1353–1358. Zhan, S., and S. C. Ho 2005. Meta-analysis of the effects of soy protein containing isoflavones on the lipid profile. Am. J. Clin. Nutr. 81:397–408. Zhang, X., X. O. Shu Y. T. Gao, G. Yang, Q. Li, H.

Epidemiology of soy exposures and breast cancer risk. Br. J. Cancer. 98:9–14 Wu, A. H., R. G. Ziegler, P. L. Horn-Ross, A. M. Nomura, D. W. West, L. N. Kolonel, J. F. Rosenthal, R. N. Hoover, and M. C. Pike 1996. Tofu and risk of

Li, F. Jin, and W. Zheng. 2003. Soy food consumption is associated with lower risk of coronary heart disease in Chinese women. J. Nutr. 133:2874–8. Zhang, Y. B., W. H. Chen, J. J. Guo, Z. H. Fu, C. Yi, M.

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Zhang, and X. L. NA 2013. Soy isoflavones supplementation could reduce body weight and improve glucose metabolism in non-Asian postmenopausal women-A meta- analysis. Nutr. 29:8-14. Zhou, J. R., L. Yu, Z. Mai, and G. L. Blackburn 2004. Combined inhibition of estrogen dependent human breast carcinoma by soy and tea bioactive components in mice. Int. J. Cancer 108:8-14. Zubik, L. and M. Meydani 2003. Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women Am. J. Clin. Nutr. 77:1459– 1465.

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VOLUME 4 ISSUE 1 Case Studies 8

Introduction Special Issue P. G. Crandall

A Personal Hygiene Behavioral Change Study at a Midwestern Cheese Production Plant J. A. Neal, C. A. O’Bryan and P. G. Crandall

Preventing Post-Processing Contamination in a Food Nugget Processing Line When Language Barriers Exist J. A. Neal, C. A. O’Bryan and P. G. Crandall

Behavioral Change Study at a Western Soup Production Plant C. A. O’Bryan, J. A. Neal, and P. G. Crandall

Salmonella in Cantaloupes: You Make Me Sick! B. A. Almanza

The Hurricane Sandy Dilemma B. A. Almanza

Intellect-u-ale: A Smart Approach to Quality Assurance in a Micro-Brewery A. J. Corsi, M. Goodman, and J. A. Neal

Introduction to Authors 61

Instructions for Authors

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INSTRUCTIONS TO AUTHORS MANUSCRIPT SUBMISSION

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Aerobic microbiology Aerobiology Anaerobic microbiology Analytical microbiology Animal microbiology Antibiotics Antimicrobials Bacteriophage Bioremediation Biotechnology Detection Environmental microbiology Feed microbiology Fermentation Food bacteriology Food control

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Variability, Replication, and Statistical Analysis To properly assess biological systems independent replication of experiments and quantification of variation among replicates is required by AFAB. Reviewers and/or editors may request additional statistical analysis depending on the nature of the data and it will be the responsibility of the authors to respond appropriately. Statistical methods commonly used in the bacteriology do not need to be described in detail, but an adequate description and/or appropriate references should be provided. The statistical model and experimental unit must be designated when appropriate. The experimental unit is the smallest unit to which an individual treatment is imposed. For bacterial growth studies, the average of replicate tubes per single study per treatment is the experimental unit; therefore, individual studies must be replicated. Repeated time analyses of the same sample usually do not constitute independent experimental units. Measurements on the same experimental unit over time are also not independent and must not be considered as independent experimental units. For analysis of time effects, assess as a rate of change over time. Standard deviation refers to the variability in the biological response being measured and is presented as standard deviation or standard error according to the definitions described in statistical references or textbooks.

Results Results represent the presentation of data in words and all data should be described in same fashion. No discussion of literature is included in the results section.

Discussion The discussion section involves comparing the current data outcomes with previously published work in this area without repeating the text in the results section. Critical and in-depth dialogue is encouraged.

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Results and Discussion Results and discussion can be under combined or separate headings.

Conclusions State conclusions (not a summary) briefly in one paragraph.

Acknowledgments Acknowledgments of individuals should include institution, city, and state; city and country if not U.S.; and City or Province if in Canada. Copies being reviewed shall have authors’ institutions omitted to retain anonymity.

References a) Citing References In Text Authors of cited papers in the text are to be presented as follows: Adams and Harry (1992) or Smith and Jones (1990, 1992). If more than two authors of one article, the first author’s name is followed by the abbreviation et al. in italics. If the sentence structure requires that the authors’ names be included in parentheses, the proper format is (Adams and Harry, 1982; Harry, 1988a,b; Harry et al., 1993). Citations to a group of references should be listed first alphabetically then chronologically. Work that has not been submitted or accepted for publication shall be listed in the text as: “G.C. Jay (institution, city, and state, personal communication).” The author’s own unpublished work should be listed in the text as “(J. Adams, unpublished data).” Personal communications and unsubmitted unpublished data must not be included in the References section. Two or more publications by the same authors in the same year must be made distinct with lowercase letters after the year (2010a,b). Likewise when multiple author citations designated by et al. in the text have the same first author, then even if the other authors are different these references in the text and the references section must be identified by a letter. For example

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“(James et al., 2010a,b)” in text, refers to “James, Smith, and Elliot. 2010a” and “James, West, and Adams. 2010b” in the reference section.

Book Chapter: Author(s) of the chapter. Year. Title of the chapter. In: author(s) or editor(s). Title of the book. Edition or volume, if relevant. Publisher name, Place of publication.

b) Citing References In Reference Section In the References section, references are listed in alphabetical order by authors’ last names, and then chronologically. List only those references cited in the text. Manuscripts submitted for publication, accepted for publication or in press can be given in the reference section followed by the designation: “(submitted)”, “(accepted)’, or “(In Press), respectively. If the DOI number of unpublished references is available, you must give the number. The year of publication follows the authors’ names. All authors’ names must be included in the citation in the Reference section. Journals must be abbreviated. First and last page numbers must be provided. Sample references are given below. Consult recent issues of AFAB for examples not included in the following section. Journal manuscript: Author(s). Year. Article title. Journal title [abbreviated]. Volume number:inclusive pages.

Examples: Chase, G., and L. Erlandsen. 1976. Evidence for a complex life cycle and endospore formation in the attached, filamentous, segmented bacterium from murine ileum. J. Bacteriol. 127:572-583. Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van Doesburg, and A. J. M. Stams. 2009. A typical one-carbon metabolism of an acetogenic and hydrogenogenic Moorella thermioacetica strain. Arch. Microbiol. 191:123-131.

Inclusive pages of chapter.

Examples: O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010. Assessing consumer concerns and perceptions of food safety risks and practices: Methodologies and outcomes. In: S. C. Ricke and F. T. Jones. Eds. Perspectives on Food Safety Issues of Food Animal Derived Foods. Univ. Arkansas Press, Fayetteville, AR. p 273-288. Dissertation and thesis: Author. Date of degree. Title. Type of publication, such as Ph.D. Diss or M.S. thesis. Institution, Place of institution. Total number of pages.

Maciorowski, K. G. 2000. Rapid detection of Salmonella spp. and indicators of fecal contamination in animal feed. Ph.D. Diss. Texas A&M University, College Station, TX. Donalson, L. M. 2005. The in vivo and in vitro effect of a fructooligosacharide prebiotic combined with alfalfa molt diets on egg production and Salmonella in laying hens. M.S. thesis. Texas A&M University, College Station, TX. Van Loo, E. 2009. Consumer perception of ready-toeat deli foods and organic meat. M.S. thesis. University of Arkansas, Fayetteville, AR. 202 p. Web sites, patents:

Book: Author(s) [or editor(s)]. Year. Title. Edition or volume (if relevant). Publisher name, Place of publication. Number of pages.

Examples: Hungate, R. E. 1966. The rumen and its microbes Academic Press, Inc., New York, NY. 533 p.

Examples: Davis, C. 2010.

Salmonella. Medicinenet.com.

http://www.medicinenet.com/salmonella /article. htm. Accessed July, 2010. Afab, F. 2010, Development of a novel process. U.S. Patent #_____

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Abstracts and Symposia Proceedings: Fischer, J. R. 2007. Building a prosperous future in which agriculture uses and produces energy efficiently and effectively. NABC report 19, Agricultural Biofuels: Tech., Sustainability, and Profitability. p.27 Musgrove, M. T., and M. E. Berrang. 2008. Presence of aerobic microorganisms, Enterobacteriaceae and Salmonella in the shell egg processing environment. IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10) Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Options and risks by using diagnostic gene chips. Program and abstracts book , The 8th Biennieal Congress of the Anaerobe Society of the Americas. p. 86 (Abstr.)

Data Presentation in Tables and Figures Figures and tables to be published in AFAB must be constructed in such a fashion that they are able to “stand alone” in the published manuscript. This

means that the reader should be able to look at the figure or table independently of the rest of the manuscript and be able to comprehend the experimental approach sufficiently to interpret the data. Consequently, all statistical analyses should be very carefully presented along with variation estimates and what constitutes an independent replication and the number of replicates used to calculate the averages presented in the table or figure. Each table and figure must be on a separate page in the submitted paper. In addition, you will need to submit all data for charts, tables and figures in native format when possible (e.g., Microsoft Excel, Powerpoint). Photographs should be submitted as high-resolution (600 dpi) .jpg or tif. files. All figures should be clearly presented with well defined axis and units of measurement. Symbols, lines, and bars must be made distinct as “stand alone” black and white presentations. Stippling, dashed lines etc. are encouraged for multiple comparison but shades of gray are discouraged. Color images, micrographs, pictures are recommended and there is no additional fee for their submission.

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