Separation, Identification and Growing Characteristics ...

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Abstract - The microorganisms causing soymilk spoilage were isolated and identified with traditional biochemical, physiological and morphological methods, ...
Separation, Identification and Growing Characteristics of the Spoilage Microorganisms in Soymilk Xilin Xu

Hongwei Liu

Haiying Guo

College of light industry and food science South China University of Technology Guangzhou, Guangdong Province, China e-mail: [email protected]

College of light industry and food science South China University of Technology Guangzhou, Guangdong Province, China e-mail: [email protected]

College of light industry and food science South China University of Technology Guangzhou, Guangdong Province, China e-mail: [email protected]

liquid environment(pH,6~7) of this beverage, soymilk is of high susceptibility to spoilage microorganisms and is liable to deteriorate without food control intervention. Sources of spoilage microorganisms are believed to be raw ingredients or environmental contaminants(Pascall, Melvin, A., 2006), and the physical structure of soymilk influences the course and extent of spoilage greatly. In neutral or alkaline foods, bacteria are more dominant in spoilage and putrefaction. Temperature is also vital extrinsic factors in determining whether a soymilk would spoil(Bai, Yong, 1998). Thermal sterilization is the most effective method for sterilizing products, while optimum sterilization temperature and duration for different foods are different due to different properties of foods substrate, which requires in depth investigations, thus the nutrients and the odor of soymilk could be best maintained after thermal treatment (Kin-Chor Kwork, 1995). Generally, the shelf life of the unsterilized fresh soymilk sold on Chinese market is rather short, even just 1 day in summer. Correspondingly, storage and distribution of soymilk are inevitably cumbered due to its short shelf life. Hence, it is of great significance to study the spoilage microorganisms in the soymilk and implore an effective method to inhibit them. Until now, rare articles concerning the control of spoilage microorganisms in soymilk are reported. Our research aimed to find out the main spoilage microorganisms in the soymilk and studied their growth characteristics in depth in order to provide necessary theoretical and experimental basis for spoilage microorganisms control and shelf life extension.

Abstract - The microorganisms causing soymilk spoilage were isolated and identified with traditional biochemical, physiological and morphological methods, and they were Klebsiella oxytoca, Serratia marcescens, Klebsiella ozena and Serratia plymuthica. The spoilage course of soymilk was researched by supervising the microbial changes. The growing characteristics of spoilage microorganisms in soymilk under different sterilization conditions, storage temperatures and pH as well as different concentrations of soymilk was studied. The studied results will provide theoretical principle for controlling soymilk spoilage and prolonging the shelf life of products. Index Terms – Soymilk; Spoilage microorganisms; Separation; Identification; Characteristics of growth

I. INTRODUCTION Soymilk, a popular soy beverage in Asian countries, is recognized as the most inexpensive source of high nutritional quality protein and therefore is the predominant commercially available vegetable protein in the world. It is abundant in minerals, vitamins, soy cephalin and soy isoflavones, a subclass of flavnoids. It has been widely proved that soy isoflavones has the potential to prevent prostate and mammary cancer, cardiovascular diseases and osteosporosis (Setchell, K. D. R, 1998, Miguel Rivas, 2002, Anna H.Wu, 2002, Atmaca, 2008). Soymilk is extracted from integrate soy seeds, therefore it contains all the nutrients in the seeds. Salubrious and therapeutic components determines that soymilk is sort of prophylactic drink for people(Wang, H.; Murphy, P. A., 1994). Drinking soymilk helps relieve women perimenopause syndrome(Atmaca, 2008), and reduce diabolic blood pressure, attenuate the development of hypertension in spontaneously hypertensive people(Miguel Rivas, 2002). Additionally, the Soy lecithin rich in this beverage is essential component for membrane, encephalon and cerebral nerve, frequent intakes of soymilk improves memory conditions(Alicia A. Thorp, Natalie Sinn, Jonathan D. Buckley.et.al, 2009). Compared with cow milk, soymilk is free of cholesterol and low energy, soy-based diets have health benefits in reducing weight and blood lipids (Fang-Hsuean Liao, M.S., Ming-Jer Shieh, Ph.D., Suh-Ching Yang.et.al, 2007). As detrimental effects of abundant nutrients and neutral

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II. MATERIALS AND METHODS A. Microbiological test of fresh soymilk The soymilk used for experiment was made in laboratory with a soya-bean milk machine. The soybean was purchased from a local supermarket. No other nutrients and food additives were added. The media used for isolation, maintenance, growth, and inhibitive tests of the spoilage microorganisms were Eosin-Methylene Blue Agar(EMB), Nutrient Broth(NB), Potato Dextrose Agar(PDA), Rose Bengal Medium. These media were made up with the method described in GB 4789.28. The species of isolates were primarily identified with their macroscopic morphology on media. In order to supervise the microbiological counts of well accepted soymilk, we

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determined its original total amount count of bacteria and coliform bacteria as reference. The quantity determination of bacteria and corresponding morphological characteristic analysis were guided by China national food safety standard GB4789.2-2010(Food microbiological examination ˖ Aerobic plate count) and GB/T4789.3-2008 (Microbiological examination of foodhygiene-Detection of Coliform bacteria). Lactose peptone broth was prepared based on GB 4789.28, and the number of coliform bacteria in food systems can be expressed by MPN, which refers to coliform bacteria number in 100 ml liquid sample. B. Microorganisms isolation and spoilage test Fresh soymilk was cultured at 36 ± 1°C in an incubator, changes of soymilk like liquid stratification, coagulation, malodor, and pH were observed and recorded accurately every 2h during the cultivation. Isolation of spoilage microorganisms in the deteriorate soymilk was carried out with the pour-plate technology. Deteriorated soymilk was used as source for isolation of spoilage bacteria. 1.0 ml of the solution was diluted stepwise up to 10-5 with 0.1% sterile peptone water and 100ul of the dilution was spread on nutrient agar plates. After incubation at 37 °C for 24 hours, strains were isolated by shape and color of colony, then spread plate and Streak method were employed to purify the isolates separated from decomposed soymilk. Purified culture was stored at 4 °C for further studies. Spoilage tests with these isolates from spoiled soymilk were performed. We inoculated the autoclaved (120°C at 15 lb/in2 for 20 min) soymilk contained in flasks with these microorganisms. Situations of liquid stratification, coagulation, odor and total amount of bacterial changes during the soymilk cultivation at 36 ± 1°C in the incubator were observed and recorded. Then the microorganisms which possess spoilage capacity were selected for continuous study. C. Spoilage microorganisms identification Standard microbiological methods were used to fix the cells to slides for Gram staining and observation. Traditional macromorphology and micromorphology observations, physiological tests were used to identify subcultured bacterial isolates from soymilk. Physiological and chemical tests includes chromatogenous test, oxidase test, catalase test, nitrate reduction test, glucose ultilization were done under the instruction of taxonomic outline of the prokaryotes bergey’s manual of systematic bacteriology (second edition). Further identification to species was achieved with the GYZ-15E system(produced by Hangzhou Tianhe microbial reagents company). D. Growing characteristics of the spoilage microorganisms Different temperature and time for sterilization, storage temperature, pH were selected as the test conditions, the concentration of bacterial suspension in nutrient broth medium was 106 CFU/ml. After being treated with designed conditions, samples were cultured in reciprocating shaker bath at 37 °C for 24 h. Its OD was determined sequentially, the greater the OD was, indicating the greater concentration of microorganisms was in the soymilk. 1) Sterilization temperature experiment: Heat treatment is still the most available method for food sterilization, and

high temperature accompanied with high pressure endows foods with complete sterile state, therefore the shelf life of foods are greatly extended by this way. Autoclaved soymilk was inoculated with spoilage bacteria that previously isolated, then pasteurization was performed. Different pasteurization conditions, which carried out in water bath, were as follows (1) 62°C for 30min, (2) 70 °C for 15min, (3) 90°C for 5min, (4) 100°C for 2min. Treated soymilk was cultivated at 37 °C for 24h and its OD value (600nm) was measured. 2) Effects of storage temperature on the spoilage microorganisms mass: Temperature is the cardinal factor controlling the growth rate since other factors such as nutrient status and available water are non-limiting and no microbial interactions occur until maximum cell densities are reached. Therefore a knowledge of the effect of temperature on the rate of growth of the spoilage flora may be used to monitor the time-temperature history of expired shelf life of the product(Frits Schut, et.al, 1993). On the basis that growth temperature of bacteria ranging from 0~65°C, and taking into account the general conditions for soymilk storage (room or refrigerator), storage temperatures were selected as follows: (1) 0~4 °C, (2) 8~10 °C, (3) 18~20°C, (4) 28~30 °C (5) 36~37°C. Dealt soymilk was cultivated at 37 °C for 24h and OD value (600nm) was measured. 3) pH value effects on sterilization: Ideal pH range for bacteria growth is 4.00 ~ 9.00. pH value of the milk used was 4.00 ~ 7.00, which covers the whole pH value changes of deterioration. Therefore pH value of nutrient broth was selected as 4.00~8.00. Dealt soymilk was cultivated at 37 °C for 24h and OD value (600nm) was measured. 4) Effects of soymilk concentration on its sterilization: Soy protein, lipid and carbohydrate macromolecules may have some protective effects on microbes in the soymilk. In this experiment, the concentration of soymilk used were 100%, 75%, 50%, 25%, 0%. Freshly made soy milk was regarded as the 100%, and then it was diluted with distilled water, respectively into the concentration of 75%, 50%, 25%. These soymilk samples with different concentration were autoclaved at 121°C for 15min, then the isolated bacteria were inoculated into it and autoclaved for 1min. Dealt soymilk was cultivated at 37 °C for 24h and OD value (600nm) was measured. III. RESULTS A. Results of microbiological condition of fresh soymilk and spoilage test Total microbial account of both normal soy milk and deteriorated soymilk were determined according to China national food safety standard GB4789.2-2010(Food microbiological examination ˖ Aerobic plate count) and GB/T4789.3-2008(Microbiological examination of foodhygiene-Detection of Coliform bacteria). Corresponding results were shown in table 1. Test results showed that no coliform bacteria were detected in fresh soymilk, the amount of coliform bacteria in deteriorated soy milk was 2.0 MPN/100ml. Aerobic plate count of normal milk was 100 CFU/ml, this number as for spoiled soymilk was 5×106 CFU/ml. The pH of deteriorated

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soymilk significantly decreased from 6.23 to 4.08. Microorganisms isolated from the soymilk were numbered from No.1 to No.10, among which, microorganisms No.12 deteriorated the autoclaved soymilk in 12~24h, No.10 deteriorated sterilized milk in 12h, No9, No11 caused the deterioration in 18~20h, No.12 deteriorated soymilk in 24h. No spoilage effects of No.1~No.10 were found on soymilk. Aspergillus and yeast were not found, which indicated that the deterioration of the soymilk was mainly caused by bacteria. B. Microbiochemical identification of these four bacteria International Enterobacteriaceae Committee(IEC), taxonomic outline of the prokaryotes bergey’s manual of systematic bacteriology (2rd edition) and Enterobacteriaceae identification(W,H Ewing) all defined enterobacteriaceae as gram negative, aerobic or faculative anaerobic, oxidase test negative, and sporeless bacterium, can utilize glucose and produce gas or acid, able to reduce NaNO3 to NaNO2. Spoless, gram stain negative micromorphological features of these four bacteria were observed by microscope(oil 100×100), combined with results in the table 3, according to the definition of enterobacteriaceae described above, these four bacteria were primarily identified as enterobacteriaceae. GYZ-15E system used for further identification of the microorganisms is composed of 15 test items. 73 species of enterobacteria can be identified with this system. The identification results were showed in table 4. Bacteria No.9 is Klebsiella oxytoc, No.10 is Serratia marcescens, No.11 is Klebsiella ozena, and No.12 is Serratia plymuthica C. Results of growing characteristics performed on No.9, No.10, No.11, No.12 As response for pasteurization of different temperature, the OD value of soymilk changed slightly at 62°C for 30min, 70°C for 15min, 90°C for 5min and only the water bath pasteurization was at 100°C, OD value of four bacteria significantly reduced, which indicating that heating treatment at 100°C has a greater impact on the growth of bacteria, therefore autoclaving treatment at 100°C was chosen for further research(figure1). Water bath pasteurization at 100°C for10 min(Figure 2), OD value of bacterium decreased significantly, suggested the growth of bacteria was greatly impacted. Due to no spores formed by these four species of Enterobacteriaceae, sterilization conditions at 100°C have good effects. From figure 3, bacteria No.9,No.10,No.11,and No.12 cultured at 36~37°C had larger OD value, indicating the better growth of microorganisms at this temperature, at 0~4°C OD value of bacterial suspension was low, the growth of microorganisms was greatly inhibited at this temperature range. Therefore, 0~4°C storage conditions can be used as hurdles in the hurdle technology. Figure 4 shows the test results of different concentrations of soymilk, that soymilk of increasing concentration possesses increased microorganisms concentration was found in the soymilk. When its concentration decreased to 25% and 0% , heated at 121°C for 1min, bacteria can be almost all killed. From Table 3, poor microbial growth when pH value was of 4.00 was found. while pH was5.00,6.00,7.00,8.00 OD value of soymilk didn’t change significantly. Proper conclusion is

these four spoilage microorganisms inoculated in the sterile soymilk were largely inhibited at low temperature environment. IV. DISCUSSION In this article, we studied causes of spoilage of the most commonly consumed beverage in Asian countries, soymilk. The main spoilage microorganisms were isolated, they are Klebsiella oxytoc, Serratia marcescens, Klebsiella ozena, and Serratia plymuthica, among which Serratia marcescens could spoil soymilk most quickly, that is within 12 h at 37°C. The Serratia plymuthica, although deteriorated the soymilk with slowest speed, compared with Klebsiella oxytoc, Serratia marcescens, Klebsiella ozena, could also deteriorated soymilk within 24 h. Thus result helps explain the reality that shelf life of the soymilk sold on the market is less than one day in summer. There was no Aspergillus and Yeast were isolated from the spoiled soymilk, therefore, the enterobacteria is responsible for the spoilage of this beverage, and which might contribute to the neutral environment of the soymilk , a substrate more fit for the growth and propagation of bacteria. The growth of microorganisms in soymilk are greatly inhibited by low storage temperature and sterilization for 10 min would killed all of these four bacteria in the soymilk. Therefore, we can draw the conclusion safely that sterilization at 100°C for 10 min and conserving temperature at 4°C can be treated as vital control points in the soymilk production. Such conditions is easy to be achieved in factory production. While, although acidic liquid environment has great capacity of inhibitive effects on those spoilage microorganisms, it was far away from a ideal method to control spoilage when considering the odor changes when lactic acid, Hcl, acetic acid, or citric acid are added. ACKNOWLEDGMENT We appreciated the work of teacher Xiaoshan Lin(Bioengineering Academy) and engineer Ying Shi(Light industry and food Sciences academy), their help greatly contributed to the accomplishment of the integrate experiment. REFERENCES [1]

[2]

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Bjarne K. Jacobsen, Synnùve F. Knutsen, and Gary E. Fraser, “Does high soy milk intake reduce prostate cancer incidence? The Adventist Health Study (United States),” Cancer Causes and Control, vol.9, pp. 553-557, 1998. Setchell, K. D. R., “Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones,” Am. J. Clin. Nutr, vol. 68 (Suppl.), pp. 1333S-1346S, 1998 Miguel Rivas, Ricardo P. Garay, Jesu´ s F. Escanero, Pedro Cia, Jr., Pedro Cia and Jose´ O. Alda. “Soy Milk Lowers Blood Pressure in Men and Women with Mild to Moderate Essential Hypertension,” Journal of Nutrition, vol. 2, pp. 1900-1902,2002. Anna H.Wu, Peggy Wan, Jean Hankin, Chiu-chen Tseng, Mimi C.Yu and Malcolm C.Pike. “Adolescent and adult soy intake and risk of breast cancer in Asian-Americans,” Carcinogenesis, vol. 23(9), pp. 14911496, 2002. Atmaca, Aysegul MD, Kleerekoper, “Soy isoflavones in the management of postmenopausal osteoporosis,” The North American Menopause Society, vol. 15(4), pp. 748-757,2008. Wang, H., Murphy, P. A, “Isoflavone content in commercial soybean foods,” J. Agric. Food Chem, vol. 42, pp. 1666-1673, 1994.

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Alicia A. Thorp, Natalie Sinn, Jonathan D. Buckley, Alison M. Coates and Peter R. C. Howe, “Soya isoflavone supplementation enhances spatial working memory in men,” British Journal of Nutrition, vol. 102, pp. 1348-1354, 2009. [8] Fang-Hsuean Liao, M.S., Ming-Jer Shieh, Ph.D., Suh-Ching Yang, “Effectiveness of a soy-based compared with a traditional low-calorie diet on weight loss and lipid levels in overweight adults,” Nutrition, vol. 23, pp. 551–556, 2007. [9] Kwok, K. C., Liang, H. H., Niranjan, K, “Optimizing conditions for thermal processes of soymilk,” J. Agric. Food Chem, vol. 50, pp. 48344838, 2002. [10] Pascall, MelvinA., Ravishankar, Sadhana, Ghiron, Ken, “evaluation of magnetic resonance for detection of bacteria contamination in low-acid,

TABLE II TEST OF THE COLIFORM GROUP BETWEEN THE NORMAL AND SPOILAGE SOYMILK

TABLE ҇ THE EFFECT OF ON THE GROWTH OF MICROORGANISMS

Normal soymilk Total microbial account

1.0×10

2

Spoiled soymilk 5.0×10

6

Fresh

Blank 0

Results(MPN/100mL)

-

spoiled 2.0

Coliform bacteria +

Blank

TABLE III THE PHYSIOLOGICAL AND CHEMICAL TESTS FOR MICROORGANISMS IN THE NUTRIENT AGAR

Test Results

Test Items Chromatogenous test Glucose utilization test Oxidase test Catalase test Nitrate reduction test

No.9

No.10

No.11

No.12

+ + +

+ + +

+ + +

+ + +

Figure 1. The effect of the sterilization on growth of microorgani

Figure 3. The effect of the storage on growth of microoganisms

TABLE IV

pH 4.0 5.0 6.0 7.0 8.0

No9 0.10 0.72 0.77 0.66 0.66

Figure 2. The effect of the heating time on growth of microoganisms

Figure 4. Total bacteria number with the different concentration of soymilk

THE EFFECT OF TH PH ON THE GROWTH OF MICROORGANISMS

No10 0.18 0.75 0.80 0.74 0.71

No11 0.15 0.74 0.79 0.71 0.78

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No12 0.11 0.78 0.78 0.72 0.82

-