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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access APPLICATION OF POLYMERASE CHAIN REACTION FOR RAPID EVALUATION OF HYGIENIC STATUS OF MILK(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2008) DEEPA MARY. J. J.; B. SunilRapid assessment of the bacterial load and detection of pathogens in milk is of public health significance. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogens. Therefore efficient extraction of DNA directly from milk is a major step. DNA could be efficiently extracted directly from milk by a prior sample preparation so as to remove the fat and milk proteins. The phenol chloroform method of DNA extraction was modified to reduce the time require for the procedure. The use of lysozyme helped the release of DNA from lysed gram positive Staphylococcus aureus. The extracted DNA was used as template in PCR. PCR was carried out with already published primers. PCR was modified with the use of PCR buffer containing PCR facilitators (BSA and Tween 20) to overcome PCR inhibition. The standardized procedure was used to assess the bacterial load and to detect Escherichia coli and S. aureus directly from milk. To assess the bacterial load dilutions of milk were made uptolO"'". DNA was extracted from each dilution with which PCR was carried out with primers specific for Pseudomonas. Aerobic Plate Count was also done for the same samples and compared with PCR. It could be concluded that the approximate APC of the milk sample by PCR is next lower dilution to the dilution giving the PCR amplification. The total time taken for the analysis was approximately five hours. Extraction of DNA and PCR was done with primers for detection of E. coli from the same milk samples and compared with culture. Percentage of samples positive both by culture and PCR was 50 and negative by both methods were 30. Twenty percentage of the samples were positive by PCR and negative by culture. Extraction of DNA and PGR was done with primers for detection of S. aureus from the same milk samples and compared with culture. Percentage of samples positive both by culture and PGR was 60 and negative by both methods were 20. Twenty percentage of the samples were negative by PGR and positive by culture. Hence, protocol developed for detection of 5. aureus needs further refinement to take care of false negative results by PGR, probably due to the low number of organisms present in milk.ThesisItem Open Access ASSESSMENT OF MICROBIAL QUALITY, ADULTERANTS AND PRESERVATIVES IN PASTEURIZED MILK(College of Veterinary and Animal Sciences, Mannuthy., 2008) BHAGYALAKSHMI P. S.; Dr. E. NanuIn the present study 200 pasteurized milk samples were collected from the retail shops of Thrissur and Palakkad districts. From Thrissur district, 20 samples each were collected from five brands viz., A, B, C, D and E, and an equal number of samples were obtained from the brands F, G, H, 1 and J of Palakkad district. All the samples were analyzed for the microbial quality by estimating various bacterial counts and yeast and mould count and also assessed the presence of certain pathogenic and spoilage bacteria. The milk samples were also tested to detect the presence of adulterants viz., starch and cane sugar and preservatives like carbonates and formaldehyde. The study revealed that the samples belonging to Thrissur district had an overall mean total viable count, coliform count, Escherichia coli count, psychrotrophic count, faecal streptococcal count and yeast and mould count of 5.08 ± 0.05, 2.89 ± 0.09, 0.53 ± 0.11, 5.30 ± 0.01, 3.40 ± 0.14 and 1.89 ± 0.08 logio cfu/ml, respectively. The corresponding count in the samples of Palakkad district was 5.24 ± 0.04, 3.01 ± 0.20, 0.78 ± 0.12, 4.99 ± 0.05, 3.20 ± 0.07 and 2.03 ± 0.09 logio cfu/ml. According to the total viable count limit prescribed by BIS (1992) 50 per cent samples from brand E were graded as satisfactory and the samples from all other brands were graded as unsatisfactory. The highest mean total viable count was seen in the samples of brand A (5.39 ± 0.04 logio cfu/ml). Of the samples collected from Thrissur district, the lowest count (4.51 ± 0.08 logic cfu/ml) was recorded from the samples of brand E. The samples collected from 1 brand of Palakkad district had the highest mean total viable count (5.35 ± 0.05 logic cfu/ml) and the lowest count (5.19 ± 0.09 logic cfu/ml) was observed in the samples of brand G. Ot the 100 samples collected from Thrissur district, the samples belonging to brand A had the highest mean colifonri count (3.40 ±0.17 logic cfu/ml). An equal number of samples collected from Palakkad district revealed that the highest mean count (4.39 ± 0.02 logio cfu/rnl) was observed in the samples belonging to brand I. According to the bacterial count limit prescribed by BIS (1992) 70 per cent of the samples from brand H were graded as satisfactory and the samples belonging to all other brands were graded as unsatisfactory. The overall mean coliform count of the samples belonging to various brands from Thrissur and Palakkad districts were at the level of two and three logio cfu/ml, respectively. The samples belonging to brand E of Thrissur and brand H of Palakkad had the lowest mean count. The samples collected from brand A of Thrissur district had the highest mean Escherichia coli count (0.92 ± 0.31 logio cfu/ml) and the lowest count (0.10 ±0.10 logic cfu/ml) was observed in the samples belonging to the brand E. Among the samples collected from Palakkad district, the highest mean count (1.54 ±0.11 logic cfu/ml) was observed in the samples of the brand I and the lowest count (0.43 ± 0.23 logic cfu/ml) was seen in the samples belonging to the brand H. The highest mean psychrotrophic count (5.39 ± 0.02 logic cfu/ml) was seen in the samples belonging to brand C of Thrissur district and the lowest count (5.29 ± 0.05 logic cfu/ml) was observed in the samples of the brand B. Among the samples from Palakkad district, the highest mean count (5.43 ± 0.01 logic cfu/ml) was seen in the samples of the brand 1 and the lowest count (4.63 ±0.10 logic cfu/ml) was observed in the samples of the brand ff. Of the samples collected from Thrissur district, the lowest mean faecal streptococcal count (2.95 ±0.17 logic cfu/ml) was seen in samples belonging to brand C and the highest count (3.78 ±0.16 logic cfu/ml) was observed in the samples of the brand B. Among the samples belonging to various brands of Palakkad district, the lowest mean count (2.88 ±0.12 logic cfu/ml) was seen in samples of the brand G and the highest count (3.51 ± 0.21 logic cfu/ml) was observed in the samples of the brand 1. Among the samples collected from the five brands of Thrissur district, the highest mean yeast and mould count (2.46 ±0.17 log lo cfu/ml) was seen in the samples of the brand A and the lowest count (1.35 ± 0.08 log lo cfu/ml) was observed in the samples of the brand E. Of the samples belonging to the five brands of Palakkad, the highest mean count was seen in the samples of the brand J (2.40 ± 0.24 logio cfu/ml) and the lowest count was observed in the samples of the brand F (1.66 ± 0.09 logio cfu/ml). A highly significant (P<0.01) difference was noticed among the mean total viable count, coliform count, faecal streptoccal count and yeast and mould count of various brands of pasteurized milk from Thrissur district. Similarly a highly significant (P<0.01) difference was noticed among the mean coliform count, Escherichia coli count and psychrotrophic count of the samples belong to the five brands of pasteurized milk from Palakkad district. Escherichia coli was isolated from 10 per cent of the samples belonging to Thrissur and the isolates consisted of serotype of 04 (1), rough strains (2) and untypable strains (7). The organism was isolated from 11 per cent of the samples collected from Palakkad. One of the isolates was serotyped as 04 and three isolates were serotyped as O60. One isolate fell in the class rough and six isolates were untypable. Two isolates each from Thrissur and Palakkad districts revealed Congo red binding charecteristics. Staphylococcus aiireus could not isolate from the samples obtained from Thrissur and Palakkad districts. Pseudomonas organism was isolated from four and six per cent of the samples from Thrissur and Palakkad. The isolates were identified as Pseudomonas aeniginosa, Pseudomonas flourescens, Pseudomonas cepacia and Pseudomonas putida. Bacillus cereus was isolated from three samples obtained from Thrissur district and two samples belonging to Palakkad district. None of the samples from Thrissur and Palakkad districts revealed the presence of the adulterants like starch and cane sugar and preservative like bicarbonates. But formaldehyde was detected from 19 per cent of the samples from Thrissur and 47 per cent of the samples from Palakkad.ThesisItem Open Access ASSESSMENT OF BACTERIAL QUALITY AND SHELF LIFE OF PASTEURIZED MILK(College of Veterinary and Animal Sciences, Mannuthy., 2007) ASHA. K.; Dr. E. NanuIn the present study raw and pasteurized milk samples were collected from two processing plants viz., DPi and DP: and pasteurized milk from retail shops. A total of 254 samples were analyzed for the bacterial quality by estimating various bacterial counts and also assessed the presence of certain bacteria of public health importance. The bacterial, physical and organoleptic qualities of pasteurized milk samples from two dairies stored under refrigeration (4 ± CC) were evaluated. Raw milk revealed an inferior bacterial quality with 50 per cent samples graded as fair (based on total viable count) and 85.7 per cent as poor quality (based on coliform count). The total viable count from both dairies was obtained at the level of 7 logio cfu/ml but coliform count was high in the samples obtained from DP| (3.34 ± 0.05 loglO cfu/ml). The psychrotrophic count and faecal streptococcal count in the samples belonging to both sources were at the level of 7 and 3 logio cfu/ml. respectively. Bacteria of public health significance like Escherichia coli. Staphylococcus aiireus and Pseudomonas was detected from a few samples. Pasteurization reduced the level of total viable count, coliform count, psychrotrophic count and faecal streptococcal count to a highly significant (P<0.01) level. Pasteurized milk under refrigeration (4 ± 1°C) showed an increase in total viable count and psychrotrophic count throughout the storage period with a difference of more than 3 log with that of fresh sample. However, coliform count. Escherichia coli count, and faecal streptococcal count of samples belonging to DP| initially showed increasing tendency up to si.\ days and thereafter the counts decreased. The increase in total viable count, coliform count, Escherichia coli count, psychrotrophic count and faecal streptococcal count between zero and 10''^ day from DP; was 4.8. 1.95. 2.08. 4.78 and 2.32 logm cfu/ml. respectively. The increase in the counts during storage may lead to the reduction in shelf life due to bacterial deterioration of milk. Isolates oi' Escherichia coli was obtained from [)P| on all days c.xcept eighth and lO"' day. A total of si.\ isolates were obtained from DIA- I'hc isolates belonged to 01 16 (3). 022. 046. 065 (2). 095 and the rest were rough varietv. Sluphvlococcus ctiircus was also isolated Irom two samples stoied on sixth da\' and three from the samples stored on zero, second and fourth da>. respectively (DPi). from DP:, three isolates were obtained from the samples stored on lO"' da\ and one from fresh samples. A total of 22.62 and 20.24 per cent Pseiuiomonus were isolated from DP| and DP:, respectively and the isolates were identified as I'seudomonus piUida. Psendomonus ucnipinosu and Pseudomonas Jloiirescens. Sensory and physico-chemical (COB test) analyses of refrigerated milk samples showed an overall reduction in the score of colour and appearance, llavour. odour and body as the storage period increased, fhe mean total scores Irom Di'i revealed that the samples were of excellent quality for up to .second day of storage, fhe sensory qualit\ of the samples stored on fourth day was good and then the quality of milk remained lair till eighth day and on 10''" day the quality became poor. In DP: samples had excellent quality for upto second day of storage, fhe sensory quality of the sample stored up to si.xth day was good and thereafter the quality ol milk remained as fair till the end of storage period. COB test of samples from DP| showed positive test on all samples stored on 10*'^ day. However, one sample stored on day six was COB test positive, fhe samples belonging to DP: showed that three samples stored on lO"' day and one sample stored on eighth day was COB positive. ■fhe bacterial prollle of the retail milk samples of the brands A. B. C. D. P and P was assessed and the samples belonging to the brand D had highest mean total viable count (5.94 ; 0.09 loglO efu/ml), psychrotrophic count (5.09 i 0.16 logm cfu/ml) and faecal streptococcal count (2.87 i 0.24 logi,, cfu/ml). Highest coliform count was seen in the samples of brand A (2.40 i 0.14 logiu ctu/ml) and Escherichia coli count (3.44 ± 0.72 logio cfu/ml) in samples of the brand C. Low counts especially total viable count (4.89 t 0.79 logio cfu/ml) and coliform count (1.19 i 0.42 logio cfu/ml) were seen in the samples of the brand F. Escherichia coli were detected from 20.8 per cent samples and the isolates consisted of the serotypes 046, 065, 095. 01 16, 0166 and 0171. Out of 15 isolates obtained six showed a positive congo red reaction indicating their property of invasiveness. Staphylococciis aureiis was isolated from only six samples (6.94 per cent). All retail milk samples were also tested for the isolation and identification of Pseiidomonas and the organism was isolated from 16 (22.22 per cent) samples. 1 he isolates were identified as Pseiidomonas pntida (7), Pseudomonas aeniginosa (6) and Pseiidomonas floiireseens (3). Polymerase Chain reaction was employed to identify and confirm the Eseherichia coli isolates obtained from the milk samples and a 366 bp product was obtained.ThesisItem Open Access EFFECT OF REFRIGERATION ON THE QUALITY OF BEEF FRANKFURTER AND CHICKEN PEPPERONI(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2004) AMBILI , V. S; Latha, C.The present study was conducted to assess the tnicrobial, physicochemical and organoleptic qualities of beef frankfurter and chicken pepperoni at chilled and frozen storage. Effect of storage on these products was studied by estimating various mierobial counts, assessing the presence of certain pathogenic and spoilage bacteria and studying the changes in pH and TEARS number and organoleptic qualities like color, odor and presence of sliminess. In chilled samples (4-7°C) the mean total viable count, faecal streptocoecal count, psychrotrophic count and yeast and mold count were found to increase significantly as storage period progressed. In both the products, eoliforms reached a detectable level by second day of chilled storage and thereafter the count increased. E. coli were detected only from samples of chilled beef frankfurter from fourth day onwards and the count remained at 10 cfu/g level. Aeromonas hydrophila, A. sobria and A. caviae were the three species of Aeromonas isolated from the chilled samples of both the products. Hemolytic and hemagglutination assays of these isolates were also carried out which is indicative of enteropathogenic effects. Escherichia coli were isolated from beef frankfurter samples. Among the isolates 84.62 per cent belonged to the serotype 02 ( Enterohaemorrhagic E. coli). Salomonellae could not be detected from any of the samples. A number of samples revealed the presence of Pseudomonas aeruginosa. Important species of lactobacilli isolated were Lactohacillus brevis, Lactobacillus curvatus, Lactobacillus fermentum and Lactobacillus sake. The mean pH and TBARS values were increased during chilled storage, indicating the progress of development of acidity and rancidity. Color and odor scores gradually increased from fourth day ouwards and slight discoloration was not.=d on tne products. Surface slime was developed on fourth day m be f fra k urt samples and from sixth day onwards in chicken pepperon, samples. Thus, shelf life was found to be four days fee beef frankfurter and less than s,x days or chicken pepperoni stored at 4-7° C. When samples were stored at -20-C. It was observed that mean total viable counts of fresh and frozen beef frankfurter samples did not differ signi y Frozen samples of chicken pepperoni had the total viable count stgmfican y (P<0.05) lower than that of fresh samples. Aero.,oms hydrophila, A. sobrla and A. caviae were isolated front frozen samples of both the products. Many of the isolates were hemolytie and hemagglutinating. Ps.udo„.o.as aen,gi..os. were also detected. £ coU , salmonellae. and lactobacilli were not isolated from any of the frozen samples. Frozen sausage samples had lower mean pH values when compared to fresh samples and mean TBARS values were found to decrease gradually during the frozen storage. Color and odor scores remained the same during frozen storage and slime formation was not observed in frozen samples. Study revealed that frozen samples of both products had a shelf-life of 90 days. In order to identify various critical points of bacterial contamination, samples of air, water, rinse samples from equipment, hand washing of personnel in the processing line and packaging material were examined for their hygienic quality. The mean total viable count and yeast and mold count of air samples were found to increase after processing. Among the water samples, the high microbial count was recorded for hand washings, reflecting unsanitary working practices. Among the equipment, sausage filler tvas found to contribute maximum to the total microblal load of the product. Among the raw ingredients, samples of beef used for preparation of beef frankfurter and samples of pork used for chicken pepperoni were fourtd to possess high bacterial load. Coliforms were present at 2 log efu/g level in all the ingredients. Faecal streptococci were detected in ail the ingredrents except beef. E. coli were present only in samples of spices. study reflects the importance of quality assurance during every step of preparation of ready-to-cook meat products to avoid the early spoilage and to safe guard consumer health. Presence of pathogenic organisms in these produets is o great public health significance as improper cooking can cause outbreaks of food borne diseases.