APPENDIX. Test carbohydrate 10.0 g. Bromocresol purple (2%) 2 ml. Mycological peptone 5.0g. Dipotassium hydrogen phosphate. Triammonium citrate 2.

Transcription

1 Appendix

2 Publications

3 APPENDIX CARBOHYDRATE FERMENTATION BROTH Peptone 10.0 g Sodium chloride 5.0 g Test carbohydrate 10.0 g Distilled water 1000 ml Bromocresol purple (2%) 2 ml MALT EXTRACT AGAR Malt extract 30 g Mycological peptone 5.0g Agar 20.0 g Distilled water 1000 ml ph 5.4 MRS AGAR Peptone 10.0 g Meat extract 10.0 g Glucose 20.0 g Yeast extract 5.0 g Tween 80 1 ml Dipotassium hydrogen phosphate 2.0 g Sodium acetate 5.0 g Triammonium citrate 2.0g Magnesium sulphate 0.2 g Manganese sulphate 0.05 g Distilled water 1000 ml

4 MRSc AGAR (Medium for Bifidobacteria) MRS agar with 0.2% Cysteine HCl. M17 AGAR Sodium glycerophosphate 19.0 g Soy peptone 5.0 g Casein peptone 2.5 g Beef extract 5.0 g Ascorbic acid 0.50 ml Lactose 5.0 g Magnesium sulphate 0.25 g Meat peptone 2.50 g Agar g ph 7.2± 0.2 NITRATE BROTH Peptone 5.0 g Beef extract 3.0 g Potassium nitrate 1.0 g Distilled water 1000 ml ph 7.0 MUELLER HINTON AGAR Beef infusion 300 ml Casein hydrolysate 17.5 g Starch 1.5 g Agar 15.0 g Distilled water 1000 ml ph 7.4 ±0.2

5 REAGENTS CRYSTAL VIOLET Crystal violet 10.0g Absolute alcohol 100 ml Distilled water 900 ml Dissolve the dye in the alcohol, filter and add the water. Crystal violet is used at the concentrations of 0.5 2%. GRAM S IODINE Iodine 1.0g Potassium iodide 2.0g Water 300 ml SAFRANIN Safranin saturated alcohol solution (2.5 g/ 100 ml of 95% alcohol) 10 ml Water 90 ml BROMOCRESOL PURPLE It is prepared as 0.2 % solution. For use 2.5 ml of this solution is added to 100 ml of medium to give the final concentration 0.005%. Bromocresol purple is yellow at ph 5.2 and violet purple at ph 6.8.

6 Available online at INTERNATIONAL JOURNAL OF CURRENT RESEARCH ISSN: xxxxxxxxxxx International Journal of Current Research xxxxxxxxxxxxxxxxxxxxxxxxxx RESEARCH ARTICLE PRELIMINARY SCREENING AND CHARACTERIZATION OF BIFIDOBACTERIUM FROM FAECAL SAMPLES P. Bhuvaneswari 1 and S. Ahmed John 2 1Department Of microbiology, A.V.C. College (Autonomous), Mannampandal, Mayiladuthurai 2 PG and Research Department of Plant Biology and Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirapalli ARTICLE INFO Article History: Received xxxxxxxxxxxxxx Received in revised form xxxxxxxxxxxxxx Accepted xxxxxxxxx Published online xxxxxxxx Key words: Bifid bacterium, Bifidus shunt, Acid bile resistance. INTRODUCTION ABSTRACT Human faecal samples were used as a source for Bifidobacterium, Gram positive bacteria found in normal microbiota of human beings. There are thirty faecal samples were collected from the new born infants (2 4 months). Bifidobacterium strains were isolated (MRSc medium), Out of sixty three isolates twenty six showed positivity for bifidus shunt and its species identified based on the biochemical profile. According to the bile and acid tolerance only seven strains were isolated, among this S2 (Bifidobacterium bifidum) showed more resistant capacity against both ph 2.5 and bile 0.5% of oxgall (at 4 hrs of treatment). Copy Right, IJCR, 2011, Academic Journals. All rights reserved MATERIALS AND METHODS Bifidobacteria play an important role in the microbial ecology of the human and animal gut. They have health promoting properties by maintaining an improved intestinal bacterial composition (Bezkorovainy, 2001), stimulation of the immune response, possible anticarcinogenic activity and protection against infections (Saavedra et al., 1994). The gastrointestinal microflora is influenced by diet, age, environmental conditions and by the host genotype. Tissier (1906) showed that Bifidobacterium species were the predominant microflora in breast fed infants and speculated that infant diarrhoea could be treated by giving large dose of bifidobacteria orally. Bifidobacterium is the predominant species of human colonic and faecal microbiota. It has been extensively introduced as probiotics in industry and pharmaceutical application (Guarner and Malagelada, 2003). Human faecal samples were used as a source of Bifidobacterium strains which are resistant to both acid and bile (Chung et al., 1999). The objective of this study was to isolate and identify the Bifidobacterium sp., from the infant faecal material. The effective strains were identified based on acid and bile tolerance test. Probiotics are defined as live microbial feed supplement that beneficially affects the host by improving its intestinal balance (Fuller, 1989). Most probiotic microorganisms are lactic acid bacteria such as Lactobacillus plantarum, Lactobacillus casei, Lactobacillus acidophilus and Streptococcus lactis (Sindhu and Khetarpaul, 2001). *Corresponding author: bhuvanak.micro@gmail.com Collection of Sample: Thirty fresh faecal samples were collected from the new born infants (aged 2 to 4 months). They were carefully transferred to the laboratory by using saline water containing sterilized screw capped bottles. Isolation of bacteria Isolation of strains: One gram of fresh feces was transferred in to the flask containing 9 ml of 0.2 % cysteinehcl and the suspension was homogenized for 2 minutes. Serial dilutions were made and using spread plate technique, the suspension was inoculated in MRSc agar, Bifidobacterium agar and TP agar. Then the plates were incubated at 37 C for 48 hours in the anaerobic chamber. Colony with distinct morphology was selected for further analysis. Morphological (Gram staining) and their metabolic properties were analyzed with selected colonies. F6PPK Test: According to Scardovi (1986) method, cells were grown in 5 ml of MRSc broth at 37 C for 48 hrs under anaerobic condition and the cells were harvested by centrifugation at 5000g for 10 minutes. The pellet was twice washed with 5 ml of 0.5 g/l phosphate cysteine buffer. After centrifugation, the pellet was collected in 1 ml buffer and disrupted by the addition of 0.4 ml of cetridium bromide (0.4 mg CTAB in 1 ml of distilled water).

7 Gram Staining Motility Catalase Oxidase Carbohydrate fermentation Arabinose Cellobiose Fructose Galactose Glucose Inulin Lactose Maltose Mannose Melibiose Raffinose Rhamnose Ribose Salicin Sorbitol Sucrose Trehalose Xylose Table 1 Characteristic feature of isolated strains Biochemical tests S1 S2 S3 S4 S5 S6 S7 ve, rod ve, rod ve, rod ve, rod ve, rod ve, rod S1, S3, S5, S6 and S7 Bifidobacterium bifidum,; S2 and S4 Bifidobacterium longum ve, rod ACID TOLERANCE TEST Acid tolerance capabilities were confirmed by viable count (Gilliland et al., 1984). MRSc broth for tolerance test and MRSc agar for Bifidobacterium enumeration were used. One ml of the isolate grown in the MRS broth for three generations having an optimal density of at 600 nm were inoculated in 9 ml of sterile MRSc broth whose ph was adjusted 2.5 with 0.5 N HCl. The inoculated broth was incubated at 37 hrs after inoculation. With different interval 1 hr, 2, 3, and 4 hrs, one ml of sample was taken and serially diluted with normal saline (0.84% sodium chloride) in order to neutralize the acidity of th inoculated onto MRSc agar plates. The agar plates were incubated at hrs. The colonies were counted using colony counter. The reduction in log cycle after exposure to low ph for 4 hrs as compared to control was considered as the criteria for acid tolerance (Wright and Klaenhammer, 1983). Percentage of survivability of the strain to acidic ph was calculated using the formula. % survivability = (log cfu 4 th hour/ log cfu 0 th hour) 100 BILE TOLERANCE TEST Carbohydrate fermentation test: The carbohydrates fermentation was determined on TPY containing bromocresol purple (0.04g/l) as a ph indicator and supplemented with 1% of the following carbohydrates: Arabinose, Cellobiose, Fructose, Galactose, Glucose, Inulin, Lactose, Maltose, Mannose, Melibiose, Raffinose, Rhamnose, Ribose, Salicin, Sorbitol, Sucrose, Trehalose and Xylose To ensure the anaerobic condition, each tube was supplemented with two drops of sterile liquid paraffin after inoculation. Bile salt tolerance capability of isolated strains was confirmed by viable count method. MRSc broth (ph) for the tolerance test and MRSc agar for the Bifidobacterium enumeration were used (Gilliland et al., 1984). From MRSc broth culture having an optical density of at 600 nm, 1 ml of the inoculums was inoculated in a 9 ml of sterile MRS broth enriched with oxgall (0.5 %) (w/v) and incubated at 37 (reflecting the time spent by the food in the small intestine). With different time interval 0, 1, 2, 3 and 4 hrs, 1 ml of culture was taken and serially diluted with sterile saline solution MRSc agar. The plates were incubated for hrs at 37 and the colonies were counted using colony counter. The reduction in the log values of survival after exposure to 0.5%

8 of bile salts for 4 hrs was compared with the values at 0 hr (as control) was considered as criteria for bile salt tolerance. The method was slightly modified as instead of 0.3% of sodium thioglycollate, 0.5 % of oxgall was used. Then the ph of the medium was maintained as alkali condition that reflects the ph of the small intestine. Percentage survivability of the strains to 0.5% oxgall was calculated using the formula. % survivability = (log cfu 4 th hour/ log cfu 0 th hour) 100 RESULTS AND DISCUSSION Based on the cultural characteristics (creamy white colony, puncti form and mm in diameter) morphology (Gram positive) the isolates were purified and presumed as lactic acid bacteria. Sixty three isolates were catalase, oxidase, and nitrate reduction negative. The presence of fructose 6 phosphate phosphoketolase enzyme was revealed by the appearance of reddish brown colour. This phenotypical characteristics and enzyme assay described by scardovi (1986) and Tamine et al., (1995) resulted the identification of Bifidobacterium. Among the sixty three isolates, twenty six isolates showed positive results for bifidus shunt. According the method of Vlkova et al., (2002), modified F 6PPK test was used for the identification of Bifidobacterium. Species level identification is accomplished based on the carbohydrate fermentation (Tab.1) (Miloud et al., 2005). Based on the acid and bile tolerance assay effective strain was identified (% of survivability). There are seven strains showed high % of survival rate that five isolates belongs to Bifidobacterium bifidum and two belongs to Bifidobacterium longum (Fig.1 and Fig, 2). Among the seven strains S2 (Bifidobacterium bifidum) showed maximum survivability against both acid and bile. REFERENCES Bezkorovainy, A., Probiotics determinants of survival and growth in the gut. Am. J. Clin. Nutr., 73 (suppl): Chung, H.S., Kim, Y.B., Chun, S.L. and Ji, G.E., Screening and selection of acid and bile resistant bifidobacteria. International journal of Food Microbiology. 47: Fuller, R., Probiotics in man and animals. Journal of Applied Bacteriology 66, Gilliland, S.E., Staley, T.E. and Bush, L.J., Importance of bile tolerance of Lactobacillus used as dietary adjunct. J. Dairy Sci., 67 (12): Guarner, F. And Malagelada, J.R., Gut flora in health and disease. Lancet. 361: Miloud hadadji, Rabha benama, Noureddine Saidu, djamal Eddine Henni and Mebrauk., Identification of cultivable Bifidobacterium species isolated from breast fed infant faeces in WestAlgeria. African Journal of Biotechnolgy. 4(5): Saavedra, J.M., Bauman, N.A., Oung, I., Perman. J.A., and Yolken, R.H., Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospitals for prevention of diarrhoea and shedding rotavirus. Lancet.344: Scardovi, V The genus Bifidobacterium. Bergeys manual of systematic bacteriology. J.G. holt ed. Willians and Wilkins Co., Baltimore.D. p Sindhu, S.C., and Khetarpaul, N., Probiotic fermentation of indigenous food mixture: effect on antinutrients and digestability of starch and protein. Journal of Food composition and Analysis 14: Tamine, A.Y., Marshall, V.M.E., and Robinson, E.K Microbiological and technological aspects of milks fermented by bifidobacteria. J. Dairy sci. 62: Tissier, H., Traitement des infections intestinales par la method de la flore bacterienne de l intestin. C.R.Soc. Biol. 60: Wright, C.T. and Klaenhammer, T.R., Survival of Lactobacillus bulgaricus during freezing and freeze drying after growth in the presence of calcium. J.Food Sci.48: nuctrans.html. Vlkova, E., Medokova, J., and Rada V., Comparison of four methods for identification of bifidobacteria to the genus level. Czech. J. Food sci. 20; *******

9 ISSN: xxxxxxxxxxx Available online at SPECIAL ISSUE International Journal of Current Research xxxxxxxxxxxxxxxxxxxxxxxxxx RESEARCH ARTICLE INTERNATIONAL JOURNAL OF CURRENT RESEARCH PREPARATION OF ACIDOPHILUS SOY MILK AND ITS THERAPEUTIC EFFECT AGAINST GASTROINTESTINAL PATHOGENS 1, *Bhuvaneswari, P. and 2 Ahmed John, S. 1Department of Microbiology, A.V.C.College (Autonomous), Mannampandal, Mayiladuthurai. 2PG and Research Department of Plant Biology and Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirapalli ARTICLE INFO Article History: Received xxxxxxxxxxxxxx Received in revised form xxxxxxxxxxxxxx Accepted xxxxxxxxx Published online xxxxxxxx Key words: Lactobacillus acidophilus, Soy milk, Antimicrobial activity ABSTRACT This study evaluates the therapeutic effect of acidophilus soy milk against intestinal infection causing pathogens. Lactobacillus acidophilus strains were isolated from curd and identified based on the colony morphology and biochemical profile. Using Lactobacillus acidophilus fermented soy milk was prepared. Its antimicrobial efficiency was identified against five pathogens such as Escherichia coli, Salmonella typhi, Shigella dysentriae, Vibrio cholera and Staphylococcus aureus. Among these five pathogens Vibrio cholerae was effectively inhibited by acidophilus soy milk. Copy Right, IJCR, 2011, Academic Journals. All rights reserved INTRODUCTION Soy protein is the best among the plant protein and is inferior to animal protein only in being deficient in sulphur containing amino acids. It can be used as substitute of cow milk by people with intolerance and for infants and children who are allergic to human or cow milk (Johnson et al., 1992). To make it more palatable, attempts are now being made to remove beany flavour of soy milk. Many approaches have been used like heat treatment or during initial processing to destroy lipoxidase, which causes the production of undesirable flavours during oxidation of lipids (Mital and Steinkraus, 1976). Fermentation technology, which can be used to improve the flavour (Wang et al., 1974). Lactic acid bacteria play an important role in food fermentation processes. Lactobacillus acidophilus, nonpathogenic and a member of the normal intestinal micro flora is widely used in fermented dairy products and is considerable industrial medical interest because it has been reported to aid in the reduction of the levels of harmful bacteria and yeast in the small intestine and to produce laccase, an enzyme which is important for the digestion of milk (Deraz et al., 2007). Many strains belonging to the L.acidophilus groups have been reported to produce antimicrobial compounds which show a great antagonistic activity against both Gram positive and Gram negative *Corresponding author: bhuvanak.micro@gmail.com bacteria (Alla et al., 2003). The present study was planned to isolate the Lactobacillus acidophilus from curd and to prepare soy based fermented product that can be used to evaluate the antibacterial potentialities against gastro intestinal pathogens. MATERIALS AND METHODS Isolation of strains: One ml/gm of curd was transferred to 100 ml of MRS broth for enrichment for 48 hrs and using streak plate method cultures were transferred to the plate containing media MRS (de Man Rogosa and Sharpe) agar medium. The plates were incubated at 37 Well isolated colonies were picked up for further analysis. Identification: The strain was identified based on the morphology and biochemical profile. Preparation of Soymilk: Hundred grams of soybeans were first washed and soaked overnight in distilled water. After decanting the water, the soaked soybeans were mixed with distilled water of 10 times their weight and the mixed in a blender for 3 minutes. The resultant slurry was filtered through a double layered cheese cloth to yield soy milk was pasteurized (Wang et al., 2002). Fermentation: The culture was inoculated (around 2%) in a respective containers and incubated at C for 3 4 h.

10 Estimation of ph and titratable acidity: The ph values were determined using a digital ph meter. Two drops of 1% phenolphthalein was added with 5 ml of fermented milk, mixed well and titrated with 0.1N NaOH until the appearance of pink colour. Total acidity was calculated using formula. Acidity (%) = volume of titrate (ml) Normality of NaOH /volume of sample Antibacterial activity: Antibacterial activity of acidophilus soymilk was tested by the well diffusion method (Bauer et al., 1966) using five pathogens such as Escherichia coli, Salmonella typhi, Shigella dysentriae, Vibrio cholera and Staphylococcus aureus. The pathogens were procured from IMTECH, Chandigarh. Bacterial strains were suspended in a peptone broth and incubated at 37 Then the cultures were spreaded over the Mueller Hinton agar respectively; the wells were made with the aid of flamed cork borer on the surface of the agar plates. Cell free extracts of fermented products obtained by centrifugation of the samples membrane filter (Padmanabha Reddy et al., 2006). Approximately 0.1 ml of cell free extract was transferred to the wells respectively, incubated at 37 C for h. RESULT AND DISCUSSION Based on the colony morphology (white, rounded, smooth colonies on MRS agar), cell wall type (Gram positive), non motile, catalase and oxidase negative isolates were purified and maintained as LAB. Table.1 showed the biochemical profile, which determines the isolates were Lactobacillus acidophilus ( John G Holt et al., 1982). Table 1. Characteristics of isolated strain Biochemical tests Gram Staining Motility Catalase Oxidase Carbohydrate fermentation Arabinose Cellobiose Fructose Esculin Galactose Glucose Inulin Lactose Maltose Mannose Melibiose Raffinose Rhamnose Ribose Salicin Sorbitol Sucrose Trehalose Xylose S1= Lactobacillus acidophilus S1 ve, rod By using the isolated strain, acidophilus soy milk was prepared. Soymilk preparation was adopted by the method of Wang et al., (2002). With different interval during fermentation were prescribed for the measurement of ph and titratable acidity. Fig. 1 showed that when incubation increased, ph of the fermented product was decreased, in contrast titratable acidity reach optimum level. Sarker and Banerjee (1996) revealed antibacterial activity of Lactobacillus against a variety of Gram positive and Gram negative bacteria. There are six gastrointestinal pathogens were selected in this study. Maximum zone of inhibition was noted in Vibrio cholera and followed by Shigella dysentrieae and then Escherichia coli. Minimum inhibition was noted in Staphylococcus aureus. This result supports the previous findings. REFERENCES Alla, A., Aroutcheva, Jose., Simoe, A., and Faro.s., Antimicrobial protein produced by vaginal Lactobacillus acidophilus that inhibits Gardenella vaginalis. Parthenon Publishing 9: Bauer, A.W., Kirby, W.M.M., Truck, H., and Sheries, J.C., Antibiotic susceptibility Testing by standardized single disc method. American J. Clin. Pathol. 45: De Man, J.C., Rogosa, M. and Sharpe, M.E., A medium for the cultivation of Lactobacilli. J. Appl. Bacteriol. 23: Deraz S., Karlsson, N.E., Khalil, A.A. and Mattiasson, B., Mode of action of acidocin D20079, a bacteriocin produced by the potential probiotic strain, Lactobacillus acidophilus DSM J. Ind. Microbiol. Biotechnol. 34: John, G. Holt., Noel, R. Krieg., Peter, H.A. Sneath., James, T. Staley., and Stanley, T. Williams., Bergey s manual of determinative bacteriology, 9 th Ed. Lippincott Williams &Wilkins, Baltimore.

11 Johnson, L.A., Myers, D.J., and Burden, D.J.,1992. Soy proteins history, prospects in food, feed. Inform. 3: Mital, B.K., and Steinkraus, K.H., Flavour acceptability of unfermented and lactic acid fermented soy milks. J. Milk Food Technol. 39: Padmanabha Reddy V, Christopher, M.D., and Sankara Reddy, T., Antimmicrobial activity of Lactobacillus acidophilus, J. Veterinary & Animal Sci. 2(4): Sarker, P. K. and Banerjee, S., Antibacterial activity of lactic acid bacteria isolated from Natural habitats. J. Food Sci. Technol. 33: Wang, H.L., Kraidej, L., and Hesseltin, C.W., Lactic acid fermentation of soy milk. J. Food Sci. Technol. 37: Wang, Y.J., Yu, R.C. and Chou, C.C., Growth and survival of bifidobacteria and lactic acid bacteria during the fermentation and storage of cultured soymilk drink. Food Microbiolo. 19: *******

12 INFLUENCE OF DIFFERENT TYPES OF TEMPEH AND ITS INHIBITORY EFFECTS ISSN : J. Ecotoxicol. Environ. Monit (6) Palani Paramount Publications Printed in India INFLUENCE OF DIFFERENT TYPES OF TEMPEH AND ITS INHIBITORY EFFECTS ON SELECTED BACTERIAL STRAINS P BHUVANESWARI 1 AND S AHMED JOHN 2 1 DEPARTMENT OF MICROBIOLOGY, A.V.C.COLLEGE, MANNAMPANDAL MAYILADUTHURAI , TAMIL NADU, INDIA. 2 PG AND RESEARCH DEPARTMENT OF PLANT BIOLOGY AND BIOTECHNOLOGY, JAMAL MOHAMED COLLEGE, TIRUCHIRAPALLI , TAMIL NADU, INDIA. E.mail : bhuvanak.micro@gmail.com ABSTRACT Tempeh is a traditional fermented food made from soaked and cooked soybean inoculated with Rhizopus oligosporous. In this study five types of substrates (soybean, soybeanbarley, soybeanrice, soybeanragi & soybeanwheat) were inoculated with R. oligosporous, incubated at 30 o C for 1822 h. After fermentation crude protein content was identified, cell free extract from the food products were used for the identification of antibacterial potentiality against pathogens such as Salmonella typhi, Shigella dysentriae, E.coli, Klebsiella sp., Bacillus cereus, Micrococcus sp., Staphylococcus aureus and Pseudomonas aeruginosa. Among five types of tempeh soy tempeh showed high protein content (6.63%) followed by Soy beanwheat (6.55%). Soy tempeh showed maximum zone of inhibition against Shigella dysentriae and Bacillus cereus where as Staphylococcus aureus and Salmonella typhi had more zone of inhibition in soywheat. Soyrice tempeh which showed maximum inhibitory effect against E.coli and Klebsiella sp., But both Soy and Soywheat inhibited Pseudomonas aeroginosa effectively. Among the five types, cell free extract collected from Soybarley showed moderate inhibitory activity. Key words : Tempeh, Fermentation, Soybean, Rhizopus oligosporous INTRODUCTION Tempeh is a solid substrate fermentation patty consisting of cooked acidified beans and grains. The fermentation is carried out by the mold Rhizopus oligosporous which is added as either a starter culture or a previous preparation of tempeh. Soy protein is the best among plant protein and is inferior to animal protein in being deficient in sulfur containing amino acids (Bai Y et al 1999). Apart from soy, tempeh can also be prepared using various other whole grains. Production of tempeh like fermented food using wheat, sorghum, oats, rice, barley, corn and rice is possible. The different grains influence the products appearance, flavour, texture Journal and aroma of Ecotoxicology and patty integrity & Environmental (Hachmeister Monitoring. & Fung Vol ). (2010)

13 560 P BHUVANESWARI AND S AHMED JOHN Tempeh contains antibacterial activity substances against Gram positive bacteria (Wang & Hesseltine 1972). The anti diabetic and anti hypertensive potential of aqueous extracts of soybean enriched for phenolic content of the bioprocessing of dietary fungus, Rhizopus oligosporous. The objective of the present study was undertaken to prepare the tempeh using soy and with combination of other substrates (barley, ragi, rice & wheat) by Rhizopus oligosporous (isolated from the soil) as an inoculum and to detect their crude protein content and antibacterial potentiality against pathogens. MATERIALS AND METHODS Preparation of substrate : Dried, dehulled yellow seeded soybeans (Glycine max), ragi, wheat, barley were soaked overnight in tap water using accelerated acidification (Nout et al 1987). Rice grains were soaked in water for 4 hours. Subsequently, the beans and grains were washed with tap water, cooked in fresh tap water (1:3 W/V) for 20 minutes, cooled and superficially dried at room temperature and then mashed. Five types of substrates were prepared as soybean, soybeanbarley(1:1), soybeanrice (1:1), soybeanragi (1:1) and soybeanwheat (1:1). Preparation of inoculum: Sporangiospore suspension was obtained by scraping off the sporangia from the slant cultures of Rhizoporous oligosporous grown a malt extract agar for 7 days at 30 C and suspending them in sterile distilled water with 0.85% NaCl and 0.1% peptone. The mash was inoculated with sporangiosporespore suspension (1% V/W), then the substrates were packed into hard plastic perforated boxes and incubated at 30 C for h. Fermented products were dried for 6 h at 60 C, ground using a 1mm screen and stored at 20 C. Preparation of cell free extract: Five mg of tempeh mixed with 5 ml of double distilled water and centrifuged at 1000 rpm for 10 minutes. Supernatant was collected for further analysis. Estimation of crude protein: Quantity of crude protein was estimated by determination of the total nitrogen following the kjeldahl method (AOAC 1970) with conversion factor of Well diffusion method: Mueller Hinton agar medium having plates were prepared; the wells were made with the aid of flamed cork borer on the surface of the agar plates. Bacterial pathogens such as Escherichia coli, Klebsiella, Salmonella typhi, Shigella dysentriae, Micrococcus sp., Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus were swabbed over the plates respectively. Approximately 0.1 ml of cell free extract was taken and delivered into the well and in another set, prepared food products that directly transferred the well respective manner for the detection of antibacterial potentiality (Bauer et al 1966). After inoculation the plates were incubated at 37 C for 24 h. RESULTS AND DISCUSSION Among the fermented soy foods, tempeh is widely accepted. According to the method formulated by Nout and Kiers (2005), tempeh preparation was done using different substrates including soy and with the combination of barley, ragi, rice and wheat. After fermentation has occurred, a dense cottony mycelium that bound the substrate to form a compact cake. Journal of Ecotoxicology & Environmental Monitoring. Vol. 20 (2010)

14 INFLUENCE OF DIFFERENT TYPES OF TEMPEH AND ITS INHIBITORY EFFECTS 561 The crude protein content was analysed through kjeldahl procedure. Fig.1 revealed that soy tempeh had more crude protein followed by soy wheat, soybarley, soyrice and then soyragi compared to other mixed tempeh products. Fig. 1 Crude Protein content of different types of Tempeh Table 1 Antibacterial activity of different types of Tempeh. S. No Test organisms Soy Soy barley Zone of inhibition (mm) Soy ragi Soy rice Soy Wheat Salmonella typhi Shigella dysentriae E.coli Klebsiella sp., Bacillus cereus Micrococcus sp., Staphylococcus aureus Pseudomonas aeruginosa Journal of Ecotoxicology & Environmental Monitoring. Vol. 20 (2010)

15 562 P BHUVANESWARI AND S AHMED JOHN Rhizopus oligosporous has been reported to produce antibacterial compounds as well as phenolic compounds that inhibit the growth of pathogenic bacteria. Kobayashi et al (1992) reported that antibacterial activity of R. oligosporous against three bacteria such as Staphylococcus aureus, Bacillus sp. and Streptococcus sp. The antagonistic property of tempeh (Table 1) was evaluated against eight pathogenic bacteria. Soy tempeh effectively inhibit the growth of Shigella dysentriae and Bacillus cereus likewise combination of Soy with rice showed maximum zone against E.coli and Klebsiella sp., Soywheat which showed maximum zone against Salmonella typhi, Staphylococcus aureus and Pseudomonas aeruginosa. These results support the previous findings of Kobayashi et al (1992)and Kiers et al (2002). REFERENCES AOAC 1970 Official methods of analysis 11th ed. Association of official analytical chemists, Washington D.C. Bai Y, Lester A and Wilson Glatz 1999 Quantity of commercial shelf stable soymilk Products. J. Food Prot. 61: Bauer A W, Kirby W M M, Truck H and Sheries J C 1966 Antibiotic susceptibility testing by standardized single disc method. American J. Clin. Pathol. 45: Hachmeister KA and Fung DYC 1993 Tempeh: a mold modified indigenous fermented food made from soy beans and / or cereal grains. Crit. Rev. Microbiol.19: Kobayashi S Y, Okazaki N and Koseki T 1992 Purification and characterization of an antibiotic substance produced from Rhizopus oligosporous IFO Biosci. Biotechnol. Biochem. 56: Kiers J L, Nout M J R, Rombouts M J A, Nabuurs and Van der Meulen 2002 Inhibition of adhesion of enterotoxigenic Escherichia coli K88 by soya bean tempeh. Lett. Appl. Microbiol. 35: Nout M J R and Kiers J L 2005 Tempeh fermentation, innovation and functionality: Update In to the third millennium. J. Applied Microbiol. 98: Wang H L and Hesseltine C W 1972 Wheat tempeh. Cereal Chem. 43: Journal of Ecotoxicology & Environmental Monitoring. Vol. 20 (2010)

16 COMPARATIVE ANALYSIS OF ANTIBACTERIAL POTENTIALITYOF COWMILKAND SOY MILK YOGHURT ISSN : J. Ecotoxicol. Environ. Monit (6) Palani Paramount Publications Printed in India COMPARATIVE ANALYSIS OF ANTIBACTERIAL POTENTIALITY OF COW MILK AND SOY MILK YOGHURT P BHUVANESWARI 1 AND S AHMED JOHN 2 1 DEPARTMENT OF MICROBIOLOGY, A.V.C.COLLEGE, MANNAMPANDAL MAYILADUTHURAI , TAMIL NADU, INDIA. 2 PG AND RESEARCH DEPARTMENT OF PLANT BIOLOGY AND BIOTECHNOLOGY, JAMAL MOHAMED COLLEGE, TIRUCHIRAPALLI , TAMIL NADU, INDIA E.mail : bhuvanak.micro@gmail.com ABSTRACT Lactobacillus lactis and Streptococcus thermophilus were isolated from the commercially available yoghurt that can be used as an inoculum for the preparation of cow milk and soy milk yoghurt. Antagonistic activity was detected against eight bacterial isolates such as Bacillus cereus, Proteus vulgaris, Streptococcus pyogenes, Staphylococcus aureus, Shigella dysentriae, Escherichia coli, Salmonella typhi and Pseudomonas areoginosa. Among the two fermented products, soy milk yoghurt showed maximum inhibitory effect against Streptococcus pyogenes, Pseudomonas aeroginosa and minimum zone against E.coli. Key words : Lactobacillus lactis, Streptococcus thermophilus, Yoghurt, Soy milk INTRODUCTION Yoghurt is a dairy product by bacterial fermentation of milk. It is semi solid in nature which originated in Turkey in which a mixed culture of Lactobacillus bulgaricus and Streptococcus thermophilus produce lactic acid during fermentation of lactose. The nutritive quality of soybean protein is at the principle of food sources available from the plant world. When compared to cow's milk, the high amounts of arginine and aspartate should be mentioned (Lee et al 1990). High protein soymilk has been shown to lower serum cholesterol when soybean protein is replacing protein from cow's milk completely (Sirtori et al 1999). Many consumers find the taste of plain soymilk unappealing (Liu & Lin, 2000). The distinct aroma of soymilk may be one of the factors for the low consumption. The taste of soymilk can be improved by decreasing the beany, grassy or soy flavour by lactic acid bacteria fermentation (Granata & Morr 1996; Liu & Lin 2000). The present work was undertaken to screen the probiotic organisms from the commercially available yoghurt, used as an inoculum for the preparation of Journal yoghurt of and Ecotoxicology detect its efficiency & Environmental against Monitoring. pathogens. Vol. 20 (2010)

17 590 P BHUVANESWARI AND S AHMED JOHN MATERIALS AND METHODS Probiotic organisms were isolated from Yoghurt sample, by appropriate dilutions with saline (0.85% NaCl), platted on MRS (De Man et al 1960) agar ( Protease peptone 10 g, Beef extract 10 g, yeast extract 5 g, dextrose 20 g, polysorbate 80 1 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulphate 0.1 g, manganese sulphate 0.05 g, disodium phosphate 2 g, agar 15 g Distilled water 1000ml, ph 6.5) and M17 agar (Peptone 5 g, yeast extract 5 g, lactose 5 g, ascorbic acid 0.5 g, disodium glycerophosphate 19 g, magnesium sulphate 0.5 g, distilled water 1000ml, ph 6.8) respectively and incubated at 37 o for 2 3 days. Well isolated colonies were picked up and transferred to the broth. Preparation of substrate: Three hundred ml of cow milk was collected and boiled (reduce the volume up to 2/3 of its original volume & then homogenized). Three hundred grams of clean, dry soy was taken and washed with water, then soaked for 6 16 h. Then soaked materials rinsed again with water and grounded. Using cheese cloth, filtrate was collected. Milk samples were pasteurized. Fermentation: The culture was inoculated (around 2%) in a respective containers and incubated at C for 3 4 h. Antibacterial activity: Antibacterial activity of cow milk (c) and soymilk(s) yoghurt were tested by the well diffusion method (Bauer et al 1966; John G Holt et al 1982) using eight bacterial strains which include Bacillus cereus, Proteus vulgaris, Streptococcus pyogenes, Staphylococcus aureus, Shigella dysentriae, Escherichia coli, Salmonella typhi and Pseudomonas areoginosa. Bacterial strains were spread over the Mueller Hinton agar; the wells were made with the aid of flamed cork borer on the surface of the agar plates. Cell free extracts of fermented products obtained by centrifugation of the et al 2006). Approximately 0.1 ml of cell free extract was transferred to the wells respectively, incubated at 37 C for h. RESULTS AND DISCUSSION Gram positive rods and cocci (in chains) were isolated and identified based on their morphological, cultural and biochemical characteristics (De Man et al 1960; Bergy). Table 1 depicts as the isolates are Lactobacillus lactis and Streptococcus thermophilus. The strains were maintained in MRS broth and M 17 broth, respectively. According to the method of Bourlioux and Pochart, yoghurt (cow milk & soy milk) were prepared. Rubin and Vaughan (1978) reported that the inhibitory effect of yoghurt against Salmonella typhimurium. In this study, antibacterial activity of fermented foods was analyzed against eight pathogens. From this in vitro analysis, soy yoghurt showed high zone of inhibition to the pathogens i.e. it effectively control the pathogenic bacteria (Fig.1). Sarker and Banerjee (1996) revealed antibacterial activity of Lactobacillus against a variety of Gram positive and Gram negative bacteria. Among the eight pathogens, soymilk yoghurt showed more antibacterial activity than cow milk yoghurt. The maximum zone of inhibition was observed Journal of Ecotoxicology & Environmental Monitoring. Vol. 20 (2010)

18 COMPARATIVE ANALYSIS OF ANTIBACTERIAL POTENTIALITYOF COWMILKAND SOY MILK YOGHURT 591 Fig.1 Antibacterial activity of cow and soymilk yoghurt Table 1 Biochemical properties of isolated bacterial strains from commercially available yoghurt Characteristics S1 S2 Gram staining Motility Catalase Oxidase Carbohydrate fermentation Arabinose Cellobiose Esculin Galactose Glucose Inulin Lactose Maltose Mannitol Mannose Melibiose Raffinose Rhamnose Ribose Salicin Sorbitol Sucrose Trehalose xylose Positive rods ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve Positive cocci ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve Identified organisms Lactobacillus lactis Streptococcus thermophilus Journal of Ecotoxicology & Environmental Monitoring. Vol. 20 (2010)

19 592 P BHUVANESWARI AND S AHMED JOHN in Streptococcus pyogenes and Pseudomonas aeroginosa, followed by Staphylococcus aureus, Salmonella typhi, Bacillus cereus, Proteus vulgaris, Shigella dysentriae and E.coli, whereas cow milk showed maximum inhibition against P. aeroginosa and minimum activity against Bacillus cereus. The results support the previous findings. It may be concluded from this study that soy milk is good substrate for the growth of L. lactis and S.thermophilus. Compared to cow milk yoghurt, soy milk yoghurt has good therapeutic value. REFERENCES Bauer A W, Kirby W M M, Truck H and Sheries J C 1966 Antibiotic susceptibility testing by standardized single disc method. American J. Clin. Pathol. 45: Bourlioux P and Pochart P 1988 Nutritional and health properties of yoghurt world. Nutr. Diet. 56: De Man J, Rogosa M and Sharpe M E 1960 A medium for the cultivation of Lactobacillus acidophilus. J. Appl. Bacteriol 23: Granata L A and Morr C V Improved acid, flavor and volatile compound production in a high protein and fiber soymilk yoghurt like product. J. Food Science 61: John G Holt, Noel R Krieg, Peter H A Sneath, James T Staley and Stanley T Williams 1982 Bergey's manual of determinative bacteriology, 9th ed. Lippincott Williams & Wilkins, Baltimore. Lee S Y, Morr C V and Seo A Comparison of milk based and soymilk based yoghurt. J. Food Science 55: Liu J R and Lin C W Production of kefir from soymilk with or without added glucose, lactose, or sucrose. J. Food Science 65: Padmanabha Reddy V, Christopher MD and Sankara Reddy T 2006 Antimmicrobial activity of Lactobacillus acidophilus, J. Veterinary Animal Sci. 2(4): Rubin H E and Vaughan F 1976 Elucidation of the inhibitory factors of yoghurt against Salmonella typhimurium J. Dairy Sci. 62(12): Sarker P K and Banerjee S 1996 Antibacterial activity of lactic acid bacteria isolated from natural habitats. J. Food Sci. Technol 33: Sirtori C R, Pazzucocconi F, Colom L, Battostom P, Bondiol A and Descheemaeler K Double blind study of the addition of high protein soya milk vs. cow's milk to the diet of the patients with severe hypercholesterolaeia and resistance to strains. British J. Nutrition 82: Journal of Ecotoxicology & Environmental Monitoring. Vol. 20 (2010)

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