Issuu on Google+

International Journal of Agricultural Science and Research (IJASR) ISSN(P): 2250-0057; ISSN(E): 2321-0087 Vol. 4, Issue 2, Apr 2014, 105-112 Š TJPRC Pvt. Ltd.

STARTER CULTURE DEVELOPMENT OF SINDOL-A FERMENTED FISH PRODUCT OF ASSAM MANOJ KUMAR DAS Department of Biotechnology, Cotton College, Guwahati, Assam, India

ABSTRACT Fermented fish offers a great source of nutrition irrespective of season, time and place. Sindol, a fermented Moa fish (Amblypharyngodon mola) from Kamrup District of Assam was subjected to organoleptic, physicochemical and microbiological analysis for anticipation of starter culture. Raw Moa fish along with samples at different stages of indigenous fermentation were investigated. Among 9 different bacterial isolates, two of them are initially identified as Lactobacillus spp. These two isolates were predominant throughout the fermentation and have been chosen as probable starter culture. Laboratory fermented samples, inoculated with Lactobacillus spp., were compared with indigenous product in terms of organoleptic parameters at 3, 4, 5 and 6weeks of fermentation. The best result was obtained after 4 weeks of fermentation with Lactobacillus sp. II, which is confirmed as Lactobacillus plantarum based on conventional taxonomic techniques. This inoculum has made available the desired product within a shorter period of fermentation with characteristic taste, odour and texture and can be recommended as the starter culture of Sindol.

KEYWORDS: Fermented Fish, Sindol, Moa Fish, Amblypharyngodon mola, Starter Culture INTRODUCTION Fermented fish products are consumed almost everywhere in south-east Asia including India, generally as a condiment for rice dishes (Borgstrom, 1962). It is not just a dietary source of food but also a good source of nutrition. It has become very popular in the developed countries due to their high nutritive value and organoleptic characteristics (Sanjeev et al., 1990). Bacterial starters have been produced for a variety of fermented products to improve their sensory and other qualities. Spontaneous fermentation has been used for the production of fermented foods based on the microflora present in the raw material (Vogel et al., 2002). The direct addition of selected starter cultures to raw materials is a milestone in the production of fermented foods, which may help control the overall standardization of the fermentation process and quality of the end product. Lactic acid bacteria (LAB) used as starter culture may help to improve the quality and shelf life of the products. Several species belonging to the genera Leuconostoc, Weissella, Pediococcus, Lactococcus, Enterococcus and Streptococcus have been isolated from fermented fish, but, Lactobacillus strains are the most abundant. The characteristic smell of fermented fish is the result of enzymatic and microbiological activity in the fish muscle. It is suggested that the organic acids produced during the fermentation of fish in Asia are mainly lactic acid (Zakhia and Cuq, 1991).

www.tjprc.org

editor@tjprc.org


106

Manoj Kumar Das

'Sindol' is an indigenous fermented fish product of Assam, India. It is prepared from small sundried fish Amblypharyngodon mola, in the presence of salt for 2 to 3months or more at room temperature. It has become an important commodity amongst the people of North-east India, hence its development and production in shorter period is necessary to meet the demand of the growing population. Attention should also be paid on the microbiological aspects of this product from quality point of view and to safeguard the consumers' health. Therefore, the present study aims at anticipation of suitable starter culture for the production of Sindol.

MATERIALS AND METHODS Sample Collection Raw Moa fish (Amblypharyngodon mola) and its fermented product, Sindol were collected from the various markets in Kamrup (Rural) District of Assam, India in the month of January, 2012 in sterile gas jar to ensure safety during transportation. The samples were stored at 4째C until used. Indigenous Method of Sindol Production Raw Moa fish, generally collected from fresh water, are sun dried for two days after washing. Fishes are then smoked, grinded to a powdery form using stone roller and sprinkled with table salts. Mixture are then filled into already smoked earthen pots or glass bottles and are covered well with cloth to avoid air contact. Vessels are open after 50 to 60 days and marketed. This process is practiced by the local people, from the month of August to January every year. Experimental Design Raw Moa fish and mature indigenous product Sindol were subjected to organoleptic, physicochemical and microbiological analysis. Samples were tested at intervals of 3, 10, 20, 30, 40, 50 days and end product (60 days) of indigenous fermentation. Predominant microbial species, throughout fermentation, have been chosen as probable starter culture. Comparative evaluation were carried out for indigenous product with starter inoculated laboratory samples at 3rd, 4th, 5th and 6th weeks of fermentation. The best of laboratory fermented product (inoculated with starter) was further compared with indigenous product in terms of organoleptic and physicochemical parameters. Organoleptic Evaluation The raw and fermented fish samples were analyzed for its organoleptic qualities like texture, aroma and colour. Physicochemical Analysis The physicochemical qualities of the raw and fermented samples at different stages of its fermentation were tested for moisture content, pH and acidity. Moisture content was determined by gravitmetric method and expressed as percentage of moisture. pH was determined using a digital pH meter (NIG, Model No.333). The homogenized fish sample were titrated against 0.1 N NaOH using phenolphthalein as indicator. Microbial Analysis Microorganisms were isolated by serial dilution and pour plate technique. One gram of fish sample was homogenized with 100 ml. of sterile physiological saline. Decimal dilutions up to 10-7 were prepared. Total aerobic bacteria were enumerated on plate count agar (PCA, Himedia) (Lonner et al., 1986). MRS (Himedia) was used for total

Impact Factor (JCC): 4.3594

Index Copernicus Value (ICV): 3.0


Starter Culture Development of Sindol-A Fermented Fish Product of Assam

107

Lactic Acid Bacterial count (Man et al., 1960). The bacterial strains were maintained on Nutrient Agar (Himedia) and MRS agar. Total colony forming units of fungi were determined by serial dilution method (APHA, 1976) using Potato Dextrose Agar (Himedia). Acidified potato dextrose agar (Himedia), pH 3.7 was used for enumeration of yeasts (Okada et al., 1992). Fungal slants were made on Potato Dextrose Agar media and kept at 4°C. Coliform and E. Coli, were tested by Most Probable Number (MPN) method using Brilliant Green Lactose Broth (Himedia) and Eosine Methylene Blue agar (Himedia) respectively. Isolation and Identification of Lactobacilli Presumptive lactobacilli were selected from Nutrient agar and MRS agar and tested for cell morphology and Gram reaction. Isolates were purified by successive streaking on MRS agar before characterization. The agar plates were incubated for 24 h at 30°C for the isolation of lactobacilli. The Gram positive strains were evaluated for several physiological and biochemical characteristics. Strains were tested for production of acid from ribose, galactose, D-glucose, D-fructose, D-manose, ramnose, manitol, sorbitol, maltose, lactose and sucrose. Amylase activity was tested using Nutrient Agar media modified with starch and catalase activity was tested by adding 3% H2O2 directly on the plate. H2S formation is detected on SIM Agar (Himedia). The selected strain identified by conventional taxonomic techniques specific to bacteria (Pederson, 1936; Bergey’s Manual of Systematic Bacteriology, 1986; Banu, 2000; Tatiana Vassu et al., 2001). The pure bacterial cultures were inoculated in MRS broth, incubated for 24 h at 30°C, centrifuged at 3000 rpm for 15 min and the supernatant was decanted. The cell pellets were re-suspended in sterile MRS broth containing 15% (v/v) glycerol. The suspension was aseptically transferred into sterile cryotubes and stored at -20°C for future use in starter culture development. Formulation of Starter Culture and Evaluation of Laboratory Fermented Product Bacterial inoculums were prepared by transferring a loopful of individual pure culture in to 20ml of sterile MRS broth. Broths were centrifuged and the pellets (bacterial cells) were suspended in phosphate buffer saline (pH 4.3) after 36 hours of incubation at 320+10 C. Suspensions of two predominant bacterial strains were made to a concentration of 109 c fu ml-1. Sterile glass bottles containing 100g of pasteurised Moa fish with 1% salt, were separately inoculated with the suspension of the bacterial isolates selected and with their combination as well. Neck of the bottles were sealed properly with parafilm and allowed to ferment at 30 °C in BOD incubator. All these process were carried out under aseptic condition using laminar flow cabinet. Rate of fermentation is monitored by organoleptic characters such as aroma, texture and colour at 3rd, 4th 5th and 6th weeks of fermentation and compared with indigenous product. The best of laboratory fermented product, inoculated with starter culture, was compared with indigenous product for certain physicochemical parameters.

www.tjprc.org

editor@tjprc.org


108

Manoj Kumar Das

RESULTS AND DISSCUSSIONS Organoleptic Evaluation Findings of organoleptic characters are summarized in Table 1. Table 1: Organoleptic Evaluation of Raw and Fermented Fish at Different Intervals Sample Raw fish 3rd Day 10th Day th

20 Day 30th Day 40th Day 50th Day End product (60th Day)

Texture Soft Soft Soft with little stiffness Stiff Stiff Stiff Stiff Stiff

Aroma Fishy Fishy

Colour Light Brown Light brown

Fishy

Brown

Strong fishy smell Strong fishy smell Strong fishy smell Strong fishy smell

Brown Brown Dark Brown Dark Brown

Strong fishy smell

Dark Brown

Physicochemical Analysis Findings of physicochemical characters are summarized in the following Table 2. Table 2: Physicochemical Characteristics of Raw and Fermented Fish at Different Intervals Sample

Moisture Content (%)

pH

Raw fish 3rd Day 10th Day 20th Day 30th Day 40th Day 50th Day End product (60th Day)

68.9 65.0 56.5 48.8 39.0 35.0 28.3

7.64 6.6 6.34 5.9 5.6 5.9 6.2

Titrable Acidity 0.15 0.21 0.28 0.42 0.49 0.44 0.36

25.24

6.3

0.25

Microbial Analysis Altogether nine different types of bacterial isolates detected from the raw fishes and at different stages of fermentation. These bacteria had distinct morphology, Gram reaction and colony characteristics (results not shown). The preliminary investigations particularly, microscopic analysis (Gram-positive bacilli), negative catalase reaction and other biochemical characteristics, permitted the classification of the selected bacterial isolates as the member of Lactobacillus genus. As per the findings recorded in the Table 3, the isolate that had achieved best results of fermentation under laboratory condition, can be classified as the Lactobacillus plantarum with reference to Bergey’s Manual of Systematic Bacteriology, 1986 (Pederson,1936; Banu, 2000; Tatiana Vassu et al., 2001). Table 3: Characteristics of the Selected Strain Used for Starter Culture Development I. Taxonomic classification: – family – genus - species Impact Factor (JCC): 4.3594

Lactobacilaceae Lactobacillus L. plantarum Index Copernicus Value (ICV): 3.0


109

Starter Culture Development of Sindol-A Fermented Fish Product of Assam

Table 3: Contd., II. Morphological and culture characteristics -colour -shape -motility -Gram coloration -development on solidifiedmedium -development on liquid medium

cream-coloured - beige isolated little sticks (rods), straight with rounded ends non-motile positive smooth round colonies uniform turbidity, sediment formation

III. Biochemical characteristics Acid from: - ribose - galactose - D-glucose - D-fructose - D-manose - rhamnose - manitol - sorbitol - maltose - lactose - sucrose Enzyme production: - catalase - amylase H2 S formation

Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No No Yes

Two fungal isolates were detected from the raw fishes but are not investigated further due to their absence throughout the indigenous fermentation. None of the sample was tested positive for coliforms and E. coli during this study. Anticipation of Starter Culture Depending on microbial load, succession, dominance and Gram reaction, only two presumptive isolates, Lactobacillus sp. I and Lactobacillus sp. II, were selected for application as probable starter culture. Laboratory fermented product with these starter culture, when compared with indigenous product Sindol in terms of organoleptic parameters , the best result was obtained (Table 4) after 4 weeks of fermentation with Lactobacillus sp. II isolated. This isolate is characteristically quite similar and has been identified as Lactobacillus plantarum (Table 3). The laboratory developed product was further compared with mature Sindol (Table 4). Table 4: Comparison of Indigenous Sindol with Laboratory Fermented Product

www.tjprc.org

Parameters

Indigenous Product

Texture Aroma Color Bacterial load Moisture content (%) pH Titrable Acidity

Stiff Strong fishy smell Dark Brown 106 c fu g -1 25.24 6.3 0.25

Laboratory Fermented (Starter Inoculated) Moderately stiff Strong fishy smell Brown 108c fu g -1 23.22 6.54 0.22

editor@tjprc.org


110

Manoj Kumar Das

The total bacterial counts reached up to 106 and 108 c fu g

-1

for indigenous and laboratory samples respectively.

Increase in the viable count of microflora from 10 6 to 10 9 in 72 h of fermentation using starter culture was reported in African fermented fish (Aseidu and Sanni, 2002). This indicates the bacterial role in fermentation, which finally resulted in the production of flavours. Bacteria may contribute to development of flavour and odours in fermented fish products due to their proteolytic and lipolytic activities (Sand and Crisan, 1974). They also assist in the breakdown of fish tissue and development of flavour and aroma, which are essential for the quality of the final product (Beaumont, 2002). Comparatively more softening of fish muscle was observed in the samples with higher bacterial load. pH level of 6.54 was observed for starter fermented Sindol while a pH of 6.3 was recorded for the indigenous one. This finding was similar to that of 'Hentak' (a fermented fish product of Manipur) with a pH value 6.90 (Sarojnalini and Vishwanath, 1987). Higher pH allows bacteria to become dominant and also favours the anaerobic breakdown of proteins that releases amine compounds. Lactobacillus sp .I and Lactobacillus sp. II selected for application as probable starter culture were found to be dominant in Sindol fermentation. Best result was obtained with Lactobacillus plantarum. This isolate provided the desired product within a shorter period of fermentation with characteristic odour and texture. The importance of lactic bacteria as a whole and the Lactobacillus plantarum in particular, was already established as the potential model for fermentation studies (McDonald, 198; de Roissart and Luquet 1994; Banu, 2000).

CONCLUSIONS Fermented fish processing is an artisanal activity and the process differ from one country to another. The formulated starter culture for Moa fish fermentation does not bring about adverse changes in flavour and texture. The organoleptic analysis showed that the product obtained with starter culture remained comparable to indigenous one. The laboratory inoculum has accelerated the rate of Sindol fermentation with desired characteristic and most likely the starter culture of Sindol. The uses of starter cultures are recommended in order to prevent the growth of undesirable microbes and to improve the nutritional quality. Starter culture can make the product available all the time irrespective of season and place. Further study on genotype shall uphold this recommendation.

REFERENCES 1.

APHA (1976). Compendium of Methods for Microbiological examination of Foods, Speck, M. L. (Ed.), American Health Association, Washington.

2.

Asiedu, M. and Sanni, A. I. (2002). Chemical composition and microbiological changes during spontaneous and starter culture fermentation of enam ne-setaakye, a west African fermented fish-carbohydrated product. Eur. Food Res. Technol., 215(1): 8-12.

3.

Banu, C. (2000). Biotehnol. In industriaalimentar, Ed. Tehnic, Bucuresti.

4.

Beaumont, M. (2002). Flavouring composition prepared by fermentation with Bacillus spp. Int. J. Food Microbiol., 75: 189-196.

Impact Factor (JCC): 4.3594

Index Copernicus Value (ICV): 3.0


111

Starter Culture Development of Sindol-A Fermented Fish Product of Assam

5.

Borgstrom, G. (1962). Fish in world nutrition. In : Borgstrom, G. (Ed.), Fish as Food. Vol. 2, Academic Press, New York.

6.

De Roissart, H. and Luquet, F.M.(1994). Bacteries lactiques, Aspect fundamentaux et technologiques tome 1, Ed. Lorica, pp. 380-410.

7.

Holt, J. G. , Sneath, P.r H., Krieg, N. R. (1986). Bergey's Manual of Systematic Bacteriology (Vol. 2). Springer, New York.

8.

Lonner, C. T., Welander, N. M. and Dostalek, M. (1986). The microflora in a sour dough started spontaneously on typical swedish rye meal. Food Microbiol., 3: 3–12.

9.

McDonald, P. (1981). The biochemistry of silage, Chincester: John Wiley and Sons.

10. Okada, S. M., Ishikawa, T., Yoshida, T., Uchimura, N. O. and Kozaki, M. (1992). Identification and characteristics of lactic acid bacteria isolated from sour dough sponges. Biosci. Biotechnol. Biochem., 56: 572–575. 11. Pederson, C.S. (1936). A Study of the Species Lactobacillus plantarum (Orla-Jensen) Bergey et al. J. Bacteriol., 31(3):217-224. 12. Sand, A. and Crisan, E. V. (1974). Microflora of fermented Korean seafoods. J. Food Sci., 39: 1002-1005. 13. Sanjeev, K. S.and Dhanwant, K. S. (1990). Indian fermented foods; microbiological and biochemical aspects, Indian J. Microbiol., 30(2): 135-157. 14. Sarojnalini, C. and Vishwanath, W. (1987). Biochemical composition and fungal flora of fermented fish paste 'Hentak' of Manipur.Int. J. Acad. Ichthyol., 8(1): 9-12. 15. Vassu,T., Smrndache, D., Stoica, I., Ssrman, E., Fologea, D., Muat, F., Csutak, O., Nohit, A.M., Oana Iftime,O., Gherasim, R. (2001). Biochemical and genetic characterization of Lactobacillus plantarum cmgb – 1 strain used as probiotic, Roum. Biotechnol. Lett. Vol. 7 (1): 585-598. 16. Vogel, R.F., Ehrmann, M.A., Ganzle, M.G. (2002). Development and potential of starter lactobacilli resulting from explorationof the sour dough ecosystem. Antonie van Leewenhoek, 81(1-4): 631-638. 17. Zakhia, N. and J.L. Cuq, (1991). Aperçu sur la qualité du tilapia séché et commercialisé au Mali. In Proceedings of the FAO expert consultation on fish technology in Africa, Accra, Ghana, 22-25 October, 1991.

www.tjprc.org

editor@tjprc.org



12 agri starter manoj kumar das