Journal of Modern Agriculture January 2014, Volume 3, Issue 1, PP.1-8
Analysis and Control of Flora in Cream Vegetarian Salad Xiaolan Feng 1, Yunsheng Jiang2, YangYuan2 1. Yangzhou Commerce Higher Vocational School, Yangzhou 225127, China 2. School of Tourism and Culinary Science, Yangzhou University, Yangzhou 225127, China Email: email@example.com
Abstract A survey of microbial pollution and health quality change was conducted during the process of the production and refrigeration of Cream Vegetarian Salad food, to provide a basic reference for the catering industry to carry out HACCP management system for food like this and ensure the safety of food. Then a basic formula of Cream Vegetarian Salad was made according to the literature, further the total number of bacteria, pseudomonas, lactic acid bacteria, enterobacter, cocci and microzyme was tested in the main raw material and finished product detection according to the national standard method, Without changing the taste of the finished production, the raw materials were blanched, to measure the reduced bacteria rate, and determine the improved formula; the salad was stored at 4°C, then the number of bacteria was counted according to the time sequence and flora analysis was made to draw up the date of minimum durability. The total bacterial count of the Cream Vegetarian Salad by the basic formula is 2.3×104 CFU/g, among which coriander occupies 87%, sweetbell 7%, the rest is of small scale. Thus, the Cream Vegetarian Salad made in kitchen can be seriously polluted by microbial, mainly due to parsley, sweetbell red pepper and other raw materials, through the method of sterilization, storage in low temperature secondary pollution can be avoided, and the bacterial count can be controlled effectively, then the shelf life was extended and the risk of food poisoning was reduced, which did a lot of benefit to the food safety management of the catering industry. Keywords: Cream Vegetarian Salad; Total Number of Colonies; New Formula; Date of Minimum Durability; Food Safety
1 INTRODUCTION Salad is a cooking style of fruit and vegetable from Europe and America. There are many kinds of salad, including veggies, creams, fruits and other kinds of salad that have native characteristics.  Salad has bright color, beautiful appearance, fresh and relaxed.  With the exchange of Chinese and western food culture, salad has been seen in the guesthouses, the restaurants of the western hotel, western-style fast-food chains and dancing halls, coffee shops of our country.  The main materials of salad are vegetables and fruits. Though nutrient loss can be reduced during the process, the production of raw materials is very suitable for the growth of microorganism, because the nutrition of materials is rich. What’s more, salad is hardly to be heated thoroughly, easier to cause food accident.  How is it to set up a preventive monitoring system in the process of production and to improve the food security level? All above are greatly important. So far, most of the available researches are about the investigation and analysis of the quantity of pathogen, but they have not analyzed the cause and corresponding control measures. So it is not convenient to carry out HACCP management system. More attention should be paid to the microbial pollution during the salad production and the refrigeration, the health quality change and the relative control techniques in the paper.
2 MATERIALS AND METHODS 2.1 Materials Cucumber, tomato, carrot, onion, sweetbell, coriander, chili sauce, salt, monosodium glutamate and pepper, all are -1www.jma-journal.org
bottled or bagged products and bought from Auchan supermarket in Yangzhou.
2.2 Reagents and Culture Medium The reagents used include beef extract, peptone, yeast extract, glucose, lactose, D-mannitol, agar, bile salts, Fan red, as BR level; NaCl, NaOH, I2, KI, and anhydrous magnesium chloride, anhydrous potassium sulfate, magnesium sulfate, manganese sulfate, calcium carbonate, hydrogen phosphate, dipotassium and diammonium citrate, sodium acetate, ethanol, AR grade; Gram staining solution configured according to the literature. Nutrient agar: peptone 10 g, beef extract 3 g, NaCl, 5 g, 18 g agar, water 1000 mL, pH 7.2-7.4, at 121°C for 15 min, all of which is for the separation and the counting of the total bacterial. For Culture Collection users, agar is reduced to 8 g, and the rest are the same? PSA medium: peptone 20 g, anhydrous magnesium chloride, 1.4 g of anhydrous potassium persulfate 1 g Agar 13.6 g glycerol 10 ml distilled 1000 mL pH 6.9-7.1, 121°C 15 min 25°C 48 h, the culture condition is for the separation and counting of the Pesudomonas. MRS medium: 10 g of peptone, 10 g of beef extract, yeast extract 5 g, dipotassium hydrogen phosphate, 2 g, diammonium citrate 2 g, sodium acetate, 5 g, glucose 20 g, Tween 80 1 mL, magnesium sulfate 0.58 g, manganese sulfate 0.25 g agar 15 g, calcium carbonate 20 g, distilled water 1000 mL pH 6.2-6.4, 121°C 15 min culture conditions of 30°C 48 h for isolation and counting of the lactic acid bacteria. VRBGA medium: yeast extract 3 g, peptone 7 g, Bile 1.5 g NaCl 5 g lactose 10 g, neutral red 0.03 g, the crystal violet 0.002 g, agar 15 g, 1000 mL of distilled water, pH was adjusted to 7.3-7.5, 121 15 min culture conditions 30°C 48 h, for isolation and counting of the Enterobacteriaceae. MSA medium: beef extract 1 g, peptone 10 g, 10 g of D-mannitol, NaCl 75 g, agar 13 g, phenol red 0.025 g distilled water 1000 mL, pH 7.2-7.6, plus 1% of the phenol red solution 2.5 mL Mix, 121°C 15 min, culture conditions of 30°C 48 h, to determination aureus and Micrococcus.
2.3 Equipment XS-18 biological microscope, DT-200 Electronic Balance, pHS-3C pH meter, DFG30/HG101 electric blast drying oven, HG303 type electric drying incubator, Automatic electric pressure LDZX-40B2 vertical steam sterilizer, Adjustable million electric furnace, BCD-195WIV refrigerator, HH-8 number substantially constant temperature water bath Electric Appliance, SW-CJ-1F type clean bench, pH 5.5-9.0 Precision dipstick . Other equipment includes alcohol lamp, inoculation needles, surgical scissors, sterilized pipette, sterile tubes, sterilized dishes, flask, and magnifying glass.
2.4 Method 1) Protocol of a Basic Formula of Cream Vegetarian Salad A basic formula of Cream Vegetarian Salad was studied according to the literature shown in Table 1. TABLE 1 BASIC FORMULA OF CREAM VEGETARIAN SALAD
materials Usage amount(g)
monosodium glutamate 1.0
2) Preparation of Cream Vegetarian Salad Samples Wash carrots, sweetbell, tomato, coriander and cucumber were cut into thin slices; the onion was washed, then cut into mince. By adding of chili powder, salt, monosodium glutamate, pepper. The above was transferred into the sauce. Preparation is done.  3) Cream Vegetarian Salad Contamination Investigation and the Source Analysis The raw materials and the finished goods of respectively 25 g were sampled with sterile procedure. Make 1:10 -2www.jma-journal.org
incremental dilution, 1 mL pour plate; then two plates for each dilution, pour nutrient agar approximately at 45°C15 mL, and rotate the dish to mix the solution; solidify the medium and then invert the medium in the incubator at 37°C for 24 h, finally get it out and count.  The number of bacteria in the main raw material is taken as the variable, the usage as weight, and the source of finished bacteria number is measured by the weighted average method.  4) Control of the Number of Bacteria in Cream Vegetarian Salad By the way of blanching, anti bacteria treatment was adopted on material. Put the tomato, carrot, cucumber, onion, pepper and coriander into 1L90°C hot water, keep 5 min, to record the rate of bacteria reduction.  5) The Change in Health Quality of the Cream Vegetarian Salad during Refrigeration Make the unit of 25 g salad samples respectively by the basic recipe and improved formulation, then place the unit placed in a sterile petri dish, cool and preserve them at 4°C refrigerator, and get them out per 24 h, by time sequence the number of bacteria was measured and the growth curve was graphed. Do sensory test of the product at the same time to evaluate the quality of their health, by which the salad shelf life is built. 
3 RESULTS AND ANALYSIS 3.1 Contamination Investigation and the Source Analysis of Cream Vegetarian Salad The number of bacteria in raw materials and finished product is shown in Table 2. TABLE 2 DATE FOR THE NUMBER OF BACTERIA IN RAW MATERIALS AND FINISHED PRODUCT
Items cucumber tomato carrot onion sweetbell coriander cream finished product
Weight(g) 200 100 100 50 50 50 50 600
The total number of bacterial colonies (CFU/g) 1.4×103 1.5×103 1.3×103 1.7×103 1.8×104 2.1×105 7.1×102 2.3×104
As we can see from table 2 that the coriander produces more bacteria than others, which is 2.1×105CFU/g. Sweetbell produces1.8×104CFU/g. Cucumber, tomato, carrot and onion produce lower number, respectively 1.4×103 CFU/g, 1.5×103CFU/g, 1.3×103CFU/g and 1.7×103CFU/g. Cream produces the lowest number 7.1×102 CFU/g. Results showed that the higher number of bacteria in the coriander is due to their large number of spores. The sweetbell with relatively high number of bacteria may be related to its growth mode, because it grows the soil, and is irrigated by manure and sewage, making it difficult to clean. Bacteria in onion and carrot may be related to the freshness, because they were tested two days after the purchase. Tomato belongs to fruit-type vegetables with lesser bacteria due to the hanging growth. The cream in touch with hands, knife, and chopping block was exposed to the air. Consequently, it is also provided with a small amount of bacteria. The bacterial number which has different kinds of raw material is the variable, usage amount is weight. The total bacterial count of the Cream Vegetarian Salad by the basic formula is 2.3×104 CFU/g, among which coriander occupies 87%, sweetbell 7%, the rest are of small scale. Which shows that the level of the number of bacteria in the product is also related to the operation of the kitchen. Due to the abundance of the raw materials, on one hand, e the operation time was extended at room temperature and so that the bacteria multiply, on the other hand, contacting contaminated media such as knives, chopping board frequently, exposure to air for a long time, stirring seasoning also inevitably made aerosols which make the bacteria contact food repeatedly. So all of these make the salad finished product a dramatic rise in the actual number of bacteria. 
3.2 Control the Number of Bacteria Control the number of bacteria of Cream Vegetarian Salad -3www.jma-journal.org
TABLE 3 DATE FOR THE NUMBER OF BACTERIA AFTER BLANCHING
Items cucumber tomato carrot onion sweetbell coriander finished product
The total number of bacterial colonies(CFU/g) 1.4×103 1.5×103 1.2×103 1.7×103 1.8×104 2.1×105 2.3×104
The total number of bacterial colonies after blanching (CFU/g) 5.6×102 4.3×102 6.3×102 6.8×102 2.7×103 1.7×104 1.3×104
The rate of reducing Bacteria (%) 60 71 48 60 85 92 43
From table 3, the rate of reducing bacteria was 43% after blanching. The rate of reducing bacteria of all kinds’ materials was higher than 50%, especially in the coriander reduction rate of bacteria, which was the most significant, reaching 92%. Then followed by sweetbell which reached 85% tt reflects that blanching is a very effective means of reducing bacteria, playing an important role in controlling the bacteria number of salad vegetables.
3.3 Changes in the Quality of Health during the Refrigerated Process of Cream Vegetarian Salad 1) The Changes for the Number of Bacteria during the Refrigerated Process of Cream Vegetarian Salad
The number of bacteria （CFU/g）
Basic formula Improved formula 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0
FIG. 1 THE CHANGES FOR THE NUMBER OF BACTERIA DURING THE REFRIGERATED PROCESS OF SALAD
From figure 1, it could be seen that the number of bacteria showed a linear increase in the basic formula and improved formula during refrigeration process of Cream Vegetarian Salad. Within two days, the improved formula was an order of improvement, and two days later, the number of bacteria in basic formula improved fast. The formula was an order of improvement in 4 days. Then it is known that the date of minimum durability of basic formula is 2d, and the improved formula is 4d.
The number of Pseudomonas （CFU/g）
2) The Changes for the Number of Pseudomonas during the Refrigerated Process of Cream Vegetarian Salad Basic formula Improved formula
6.0 5.0 4.0 3.0 2.0 1.0 0.0 0
FIG. 2THE CHANGES FOR THE NUMBER OF PSEUDOMONAS DURING THE REFRIGERATED PROCESS OF SALAD
Figure 2 shows that the basic formula and improved formula of Pseudomonas are in a downward trend in 2 days, then the basic formula grows fast and keeps a high trend. The improved formula keeps agent trend. So blanching has -4www.jma-journal.org
certain inhibition to the Pseudomonas.
The number of enterobacter （CFU/d）
3) The Changes for the Number of Enterobacter during the refrigerated Process of Cream Vegetarian Salad Basic formula Improved formula
6.0 5.0 4.0 3.0 2.0 1.0 0.0 0
FIG. 3 THE CHANGES FOR THE NUMBER OF ENTEROBACTER DURING THE REFRIGERATED PROCESS OF SALAD
Figure 3 shows that the number of enterobacter of basic formula and improved formula salad are increasing in the preservation period, and the growth is fastest in 0-1d, indicating that the low temperature has a weak inhibition to the enterobacter. A day later, the enterobacter growth is relatively slow, but enterobacter number in the improved formula is still below one order of magnitude compared to the basic formula, indicating that white vinegar is with strong inhibition to enterobacter.
The numberof cocci（CFU/g0
4) The Changes for the Number of Cocci during the Refrigerated Process of Cream Vegetarian Salad Basic formula Improved formula
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0
FIG.4 THE CHANGES FOR THE NUMBER OF COCCI DURING THE REFRIGERATED PROCESS OF SALAD
Figure 4 shows that cocci number exhibites an upward trend in the basic formula and improved formula, however, the total number is fewer than other flora minimals, so the cocci does not play a dominant role in the corrupting process.
the number of lactic acid （CFU/g）
5) The Changes for the Number of Lactic Acid during the Refrigerated Process of Cream Vegetarian Salad Basic formula Improved formula
5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0
FIG. 5 THE CHANGES FOR THE NUMBER OF LACTIC ACID DURING THE REFRIGERATED PROCESS OF SALAD -5www.jma-journal.org
Figure 5 shows that the basic formula and improved formula of lactic acid bacteria are in a downward trend, which can be inferred that lactic acid bacteria growth is at a disadvantage in the competition process, furthermore, white vinegar has a strong inhibition of lactic acid bacteria; as well as that the lactic acid bacteria is not superior in product corruption.
the number of microzyme（CFU/g)
6) The Changes for the Number of Microzyme during the Refrigerated Process of Cream Vegetarian Salad Basic formula Improved formula
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0
FIG. 6THE CHANGES FOR THE NUMBER OF MICROZYME DURING THE REFRIGERATED PROCESS OF SALAD
Figure 6 shows that although the number of microzyme is in a certain proportion during the whole storage period, the absolute content and relative content are gradually declining; which may be possibly relative to the suitable growth temperature of microzyme, The most suitable temperature is 20-30°C, and it will be not conducive to the growth when the temperature is lower than 4°C.
4 DISCUSSION 4.1 The Sources of Bacterial Hazards of Cream Vegetarian Salad Since the Cream Vegetarian Salad production of raw materials is contaminated, without pasteurization processing, it is bound to be the remnants of various types of bacteria. In 1990s, due to the operational raw mix of radish outbreak of acute hemorrhagic colitis induced by E. coli O157H7, caused thousands of people sick, and dozens of death toll in Japan. In 2010, salmonellosis outbreak in United Stated due to the consumption of spinach salad, causing many deaths.  Dongmei reported that the salad had detected Listeria monocytogenes and Staphylococcus aureus in Guangdong. So salad is really a high-risk category of the restaurant industry operating items.  Analysis of the sources of the Cream Vegetarian Salad bacterial hazards showed that raw materials contaminated or flora confounded are related to the varieties of vegetables used to make salads, the planting and growing process, and the contact with soil, manure, and the use of sewage for irrigation, leading to pathogen contamination . Existing pollution-free agricultural standards cannot achieve cucumber, tomato without pathogen detection.  For the food and beverage industry, the premise cannot ensure that raw materials are sterile; operating salad food must also be in line with The Food Safety Law of the People's Republic of China which stipulates of non-toxic harmless requirements, and which requires directly imported food having no pathogens, while the raw materials cannot ensure to be sterile. This brings confusion to the food and beverage industry.  The present experimental results show that a considerable part of the Cream Vegetarian Salad bacterial contaminants is from the kitchen during the operation, some of the contaminants originally adhere to the vegetable surfaces, and diffuse in the washing, peeling, cutting, and flavoring, etc. During the shelving, it grows gradually and increases the risk.
4.2 The Control of Bacterial Hazards of Cream Vegetarian Salad The results of this experiment show that it is necessary to control the bacteria of raw material of Cream Vegetarian Salad. Blanching the raw materials works well. In addition, some commercially available packed and bottled -6www.jma-journal.org
condiments are added with preservatives, so not any bacteria could be detected. During the 28th Olympic Games in Beijing, decontamination treatment has been used to control the bacteria in raw food materials, improved to successfully ensure the safety of salad food.  It is a general accepted measure to use the condiments with bactericidal effect in China's food and beverage industry.However, the condiment dosage of the existing recipes is mostly intended from seasoning perspective but not in the perspective of bactericidal effect, so that its bactericidal effect is unknown. Thus, the expected effect of ensuring salad food safety canâ€™t be achieved. So it is necessary to establish new salad recipe whose priority is to ensure food security as a prerequisite by adjusting the condiment dosage. To control the bacteria hazards of Cream Vegetarian Salad, regular health work cannot be ignored, such as the health of persons, hand disinfection, container and utensil dedicated, cryopreservation of food and raw materials, and the establishment of shelf life, especially the development of awareness of aseptic, avoiding direct contact with hands during cutting and seasoning, improving the level of mechanization of cooking operation. All of these can contribute to the control of bacteria. 
4.3 The Establishment of Cream Vegetarian Salad food Safety Standard There is no salad foods standards in China's national food safety standards and industry standards, and local standards rarely can be found, which brings the blank of hygiene supervision and law enforcement of this type of food. Some companies assess their test results with reference to the sauce braised pork class standards, to evaluate the food and beverage industry products, which seems to be unreasonable.  It will have a positive effect on the management of the food and beverage industry and help to improve the level of supervision and law enforcement of government departments to formulate the limited value of microbiology indicators, such as the number of bacteria, to establish the judgment standard of salad freshness, improve the security processing technology specification of the Cream Vegetarian Salad, including storage conditions and shelf life, and building safety standards of salad food. Thus, it will protect the health interests of the consumers, and contribute to building a harmonious society.
Yuyan Xie, Donghao Zhang, Jun Hua. Natural Raw Food Therapy [M]. Shanghai:East China Normail University Press,1999,13.
Wenhua He. Western Cooking Techniques [M]. China Business Press, 1981, 60.
Haiming Zhu, Mandan Song, Weidong Lai, etc. Analysis of Staphylococcus aureus food poisoning [J]. China Tropical Medicine, 2006, 6 (3):523-524.
Yuqin Guo, Xiaolin Li. Health survey for salad processing [J]. China Sanitary Engineering, 2001, 10(4):153-154.
Libing Wang, Changfang Dai, Shaohong Gu, etc. HACCP on the control of sauce salad microbial [J], South China Journal of Preventive Medicine, 2004, 30(3):47-50.
Yunsheng Jiang. Cooking Microorganisms [M], China Light Industry Press, 2007, 328-329.
Baoding Zhu. How to make a salad [J]. Delicious Food, 2009, (4):62-63.
GB 4789. 2-2010. Microbiological examination of the national standard of the People's Republic of China Food Safety. Determination of the total number of colonies.
Yunsheng Jiang, Jian Chen. Fan on health research [J], China Cooking Research. 1997(3):26-30.
 Wenjuan Zhang, Yunsheng Jiang, Jie Dong, etc. The analysis and quality control for cold fresh split products flora [J]. Chinese Poultry, 2009, 31(10):15-18 and 22.  Xinzhang Huang. Some vegetables refrigerated conditions and storage period [J]. Northern Horticulture, 1991 (09):32.  Ligen Zou, Yun Zhao, Le Zhang, etc. The research for the change of vegetable nitrite in the pickled fermentation process [J]. Hangzhou Agricultural Science and Technology, 2006(4):10-12.  Yongchang Guo. Analysis of pasteurization and meat quality [J]. Meat Industry, 1996(9):30-31.  Yunsheng Jiang. Raw food cuisine of cucumber for health research [J]. Food Science, 1997, 18(11):49.  Dongmei He, Hanzhong Ni, Jiwen Yan, etc. The survey for food contaminated with Listeria monocytogenes in Guangdong (2003) [J]. South China Journal of Preventive Medicine, 2004, 30(5):48-49. -7www.jma-journal.org
 Xinqian Guan. Microbiological control of fresh-cut fruits and vegetables [J]. Xinjiang Chemical, 2004(3):51-53.  Zhijun Wang, Yunsheng Jiang. Catering food safety [M], China Light Industry Press, 2010, 8-9.  Meihua Fan, Jianhua Zhang, Xinhong Dou. Food security is not just for the Olympics [J].Chinese Poultry Tribune, 2008, 25(16):2-5.  Chaoying Zhang, Xiaoqing Lu, Hong Teng. The performance and effect of vinegar to kill bacteria [J].Medical Journal, 2007, 22(3): 196-198.  Yunsheng Jiang. Cooking health and security [M], China Light Industry Press, 2008, 252.  DB31195-2007.Shanghai local standard. Salad hygiene standards.  Mandan Song, Dongmei He, Bing Yang, etc. The analysis of salad foods microbial contamination in Guangdong [J], South China Journal of Preventive Medicine, 2006, 32(4):65-67.