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Canadian Journal on Scientific and Industrial Research Vol. 2, No. 2, February 2011

Bacterial quality of raw and packed milk Muhammad Rizwan, Arshid Pervez, Jamil Khan* Department of Environmental Sciences/Chemistry, COMSATS IIT Abbottabad Pakistan Abstract Present study has been carried out to evaluate the hygienic quality of milk at milking, transportation, storage stages and to identify the predominant bacteria in milk. The bacterial load in milk depends upon the hygienic environment. The results provided evidence that contamination of raw milk is originated during milking, transportation and storage. The hygienic packaging of milk would result in decline of milk contamination. It is highly recommended to drink milk after proper pasteurization. Keywords: Bacteria, Hygienic quality, Raw and packed milk

INTRODUCTION Bacteria multiply rapidly in milk due to its rich nutritional composition. Boycheva et al., 2002, have been studied bacterial quality of milk shows the presence of different types of bacteria in milk like Escherichia species, Listeria species, Staphylococcus species and Clostridium species (Boycheva et al., 2002). The isolation of pathogenic and coliform bacteria from the milk indicates that milk may be contaminated from udder of animals, utensils used for milking or the water used (Bonfoh et al., 2003). Microbial load in fresh milk is although very low i.e. less than 10-3 CFU/L but this level may increase up to 100 fold if this milk is stored for many days at normal temperature. In the past as well as recent time E. coli 0157:H7 emerged as a serious threat to the dairy industry as there are many outbreaks reported in developed countries ranging from mild diarrhea to potentially fatal hemolytic uremic syndrome (HUS), hemorrhagic colitis and thrombotic thrombocytopaenic purpura (Wells et al., 1991). Salmonellosis which is a disease of cattle and calves is caused by different species of Salmonella especially Salmonella enterica subspecies enterica serovars Typhimurium and Dublin (Veling et al., 2001). Environment plays important role in the spread of Salmonellosis e.g. contamination of animals may occur through fodder water wildlife or pasture. Worldwide Salmonella prevail between 2.6 and 25.3% from bulk tank milk, retail meat and fecal samples of cull dairy cow (Jayarao and Henning, 2001). Escherichia coli and coliform bacteria can enter milk and milk products very easily and their presence in the milk is an indication of contamination of milk. The presence of E. coli is the indicator of fecal contamination as well as it indicates the presence of toxigenic or enteropathogenic bacteria which are major public health hazard. In past as well as recent time E. coli 0157:H7 is becoming a serious threat in the developed countries and is causing wide range of diseases from mild diarrhoea to potentially fatal hemorrhagic colitis, HUS and Thrombotic thrombocytopaenic purpura. Shigella specie is a major cause of food borne gastrointestinal illness (Aslam et al., 2003). It is mostly found in the intestinal tract of humans and other primates. Thus, the source of Shigella to food is waters polluted with fecal material and infected food handlers. Due to debate on raw and pasteurised milk it was

*

Corresponding Author: Jamil Khan, Department of Chemistry, COMSATS IIT Abbottabad, Pakistan Email: [email protected] 86

Canadian Journal on Scientific and Industrial Research Vol. 2, No. 2, February 2011

possible to identify the pathogenic bacteria in milk. There are some organizations which are in favour of pasteurizing milk before consumption like Centers for Disease Control (CDC), the Food and Drug Administration (FDA) and some other organizations. On the other hand Weston A. Price Foundation thinks that the complete benefits of milk are destroyed due to pasteurizations so they think milk should be produced hygienically (FDA 2007). The major objective of this study was to evaluate the hygienic quality of milk collected from different places of Abbottabad city at milking, transportation and storage stages and to identify the predominant bacteria in milk. This study will help in identifying the various bacteria present in raw milk.

BACTERIAL QUALITY OF RAW AND PACKED MILK Dairy sector In order to improve dairy industry a private sector has been established which is supported by government. There are several improvements to be made in the dairy sector by improving available research facilities capacity building and training of farmers, training of veterinaries, improvement in milk chillers to improve cold chain, improvement of pasteurized milk, development of model commercial dairy farms, improvement of breed facilitation of credit financing to dairy farmers and the linkage of rural areas farmer with market (Magnusson et al., 2007).

Isolation of E. coli Using PCR Assay The most probable number method for the analysis of bacterial quality and safety of raw milk sources in Taif region (Western Saudi Arabia) for the natural contamination of fecal coliform and Escherichia coli. API 20E was used to identify E. coli isolates inorder to identify species and screened for markers of STEC (Stx1, Stx2), ETEC (ST, LT), (EaeA) and ExPEC (CNF1, CNF2, SfaS, PapA, CdtB, IutA, FyuA, TraT) using PCR assays. Thirty three raw milk samples contain an E. coli strain which shows milk is contaminated by fecal coliform. In four of strains NTEC was detected. TraT (17 strains), IutA (11 strains) and FyuA (8 strains) were the most frequent virulence markers. The results show E coli in raw milk which is a potential public health threat (Abdullah et al., 2009).

Campylobacter enteritis in raw milk In Netherland there were two outbreaks of Campylobacter enteritis in raw milk during the year 2005. Campylobacter jejuni was isolated from the stool sample of 11 children out of 34 who visited the dairy farm and suffers from diarrhoea. The epidemiology of the diarrhoea was associated with the drinking of raw milk at dairy farm. Out of 10 fecal samples collected from dairy cattels 3 were C. jejuni positive. There was similarity in restriction patterns and flagellin typing in the isolates of C. jejuni from human and animal source and it was confirmed using pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction­ restriction fragment length polymorphism (PCR­RFLP) respectively. Raw milk was considered as the source of pathogenic bacteria like C. jejuni after bacteriological and 87

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epidemiological results but it was not isolated from either bulk milk tank neither filters used during milk processing. The consumption of bulk tank milk at the dairy farm in 2007 resulted in enteritis outbreak. Sixteen people out off nineteen who drink raw milk become ill. There was no illness in people who did not drink raw milk. There was close relation between tasting raw milk and development of illness (risk difference=0.84, p=0.0011). The isolate of C. jejuni from one bulk tank milk sample was having similar APCR­RFLP genotype to those which were isolated from stool samples of patients. The bacteriological and epidemiological findings confirm raw milk as the source of enteritis infection in humans. Distribution and sale of untreated milk will result in these kinds of outbreaks in feature as well if government will no take important decisions. Serving of raw milk at farms should be avoided at dairy farms (Heuvelink et al., 2009).

Detection of HAV antigen in Raw Milk In Mashhad, Iran ELISA technique was used for the detection of HAV antigen in raw cow milk. Different HAV antigens dilutions were added to UHT milk inorder to determine the most suitable recovery method for the detection of HAV antigen in raw milk, different HAV antigens dilutions (0.00, 1.0, 10-3, 106 and 109 ml/l) were added to UHT milk. The results showed that acidic coagulation of milk than its filtration with paper filters and membrane filters results in maximum optical density and was considered the best method for the recovery of HAV antigen. HAV antigen was determined in raw milk during two seasons i.e. spring and summer. The results shows HAV antigen in raw milk was 13.3% and 34.48% during spring and summer respectively (Mortazavi et al., 2008).

Bacterialand Chemical Composition of Buffalo Milk Bacterialand chemical composition of one hundred and twelve buffalo milk samples from four locations in China was analyzed. The fat, total solids, crude protein, and ash contents of milk from multi crossbreed buffalo were higher than those of river buffalo's, but lower than those of crossbreed F1 and F2 buffalo's. In all of the samples bacterialtotal count for different microrganisms was as fallows mesophilic aerobic bacteria of 5.59 log cfu/ml, bacterial endospores 2.31, lactic acid bacteria 4.62, fungi 1.79, coliforms 2.42, Escherichia coli 1.53, and Staphylococcus aureus 1.68. Listeria species were below detection level. The bacterialquality of milk was not good and it needs to improve hygienic standards. The bacterial count of milk was not satisfactory however presence of indicator and pathogenic bacteria like E. coli, coliforms, and S. aureus in milk might lead to hazard for public health (Han et al., 2007).

Analysis of Raw Milk Quality Using Headspace The quality of raw cow milk was done using headspace analysis of volatile components. In this study deterioration of milk quality due bacteria, chemicals and enzymes was studied. In the raw milk seven volatile components were present naturally without any quality defect. The application of heating and homogenization of raw milk resulted in increase of volatile compounds up to ten folds. Headspace analysis was unable to detect 88

Canadian Journal on Scientific and Industrial Research Vol. 2, No. 2, February 2011

Pseudomonas growth in the early stage. This process was good method for the determination of raw milk quality as it could detect many quality defects at a time (Hettinga et al., 2008). Effect of Refrigeration on Milk Quality There is minute alternation in properties of good quality milk from a single cow like bacterial growth and less curd production if the milk is refrigerated. There are more chances of deterioration of milk quality in bulk milk tanks as compared with milk from single cow i.e. there will be high bacterial count and high loss of curd yield and it was obvious in the farm bulk milk tanks. The comparative study between milk from two sources i.e. high quality milk of the cow having no infection and milk of infected cows from farms and dairy silos it was concluded that there might be loss in curd production due to the infection of udder (Leitner et al., 2008).

Microbiology of Raw and Bottled Commercially Pasteurised Milk Raw and bottled commercial pasteurised milk for mesophilic, psychrotrophic, proteolytic and lipolytic bacteria was analyzed. Milk was taken from two processing plants in Gaborone, Botswana. Proteolytic and psychrotroph counts in both types of milk ranged between 101 and 105 CFU/ml. Corynebacterium pseudodiphtheriticum (44%) and Bacillus brevis (72%) predominated in were the two dominant bacteria in raw and commercial pasteurised milk respectively. Chymotrypsin and Trypsin were found in isolates of Bacillus circulans, Micrococcus lentus and Pseudomonas cichorii. Due to post process contamination and ineffective pasteurisation the quality and shelf life of pasteurised milk was deteroriated. Due to post process contamination the bacterialquality of commercially pasteurised milk produced by the two dairy plants in Gaborone was poor (Aaku et al., 2004).

Microbiology of Raw Milk from Milk Collection Centers The study was conducted on 360 dairy farms in Peninsular Malaysia for bacterialsafety of raw milk. The collection of milk samples was done randomly from 40 Milk Collection Centers (MCC) from four regions, namely, Southern (Johor/Melaka), Central (Selangor/Negeri Sembilan), Northern (Perak/Kedah) and Eastern (Kelantan/Terengganu). Total Plate Count (TPC) method was used to count the bacteria like Staphylococcus aureus, coliform and E. coli as well as pathogenic bacteria like Listeria monocytogenes, E. coli 015:H7 and Salmonella species. In price incentive programme TPC less than 106 CFU/ml is considered as milk collection centers (MCC) standard. Out of the total 930 milk samples tested for bacterial contamination 90% of the samples were contaminated by coliform bacteria and 65% by E. coli the mean count was between 103 and 104. In 60% of the samples tested Staphylococuss aureus was present and the mean count was 12×103/ml. E. coli 0157:H7 was found in 312 (33.5%) samples. Out of all 930 milk samples analyzed three serotypes of Salmonella were identified these were S. muenchen, S. anatum and S. agona. There were 47 strains of listeria found in milk with overall percentage of 4.4% with L. innocua (2.1%) Quality of raw milk samples and the products made from them was greatly affected by the presence of high bacterial count. Drinking of raw milk in rural areas could be health concern due to the presence of E. coli 0157:H7, Listeria species and Salmonella species in the raw milk (Fook et al., 2004). 89

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Isolation of E. coli O157:H7 from Raw Milk and Pickled Cheeses It was isolated from the faeces of healthy bovine. There is a great risk of spreading of these pathogenic bacteria if the milk would not be pasteurized before using it in production of milk products. In the above research 150 samples were tested for the presence of E. coli O157 there were 100 bovine's raw milk samples and 50 white pickled cheeses. In addition to this physical and chemical properties of milk were also analyzed. There is potential threat of E. coli O157 infection if unpasteurized milk would be used in white pickled cheese production (Oksuz et al., 2004).

Effect of Homogenization on Milk Proteins There is alternation in casein micelles and whey protein by reduction in fat droplet size during the homogenization step of marketed milk processing. Homogenization and heat treatment sequences tell the structural changes which would happen in milk proteins. When we consider human health homogenized milk is easily digested as compared to the milk which is untreated. In animals homogenization of milk may produce milk allergy but there is no effect of homogenization in lactose intolerant adults and children allergic to milk. The debate is going on that the homogenization of milk increases the biological activity of some useful casein peptides and milk fat globule milk proteins. The consumption of early milk cow may result in type 1 susceptible children if milk is not homogenized but the effect of unhemogenized milk in other population is still not been studied (Michalski and Januethere, 2006).

Availability of Pathogenic free Food The bacterialfree food availability is only possible if pathogenic bacteria in food samples will be monitored using standard methods. In order to find the accuracy of standard analytical procedures for the isolation of pathogenic bacteria like Listeria monocytogenes and Salmonella from milk samples experiment was performed. In the experiment different concentrations of these pathogenic bacteria along with different concentration of mesophilic aerobes are added to raw milk which was to be processed. After addition these bacteria were isolated from the milk by using standard methods. The concentration of Listeria monocytogenes and Salmonella enteritidis was 0.2­5.2 log CFU/mL in treatments with 1.8­ 8.2 log CFU/mL and 0.9­3.9 log CFU/mL in treatments with 3.0­8.2 log CFU/mL respectively. According to the results it was very difficult in order to isolate pathogenic bacteria from the treated milk as mesophilic aerobes were numerous as compared to the pathogenic bacteria (Nero et al., 2007).

Isolation of Staphylococcus aureus from Dairy Processing Plant In dairy processing plant of Goia´s State, Brazil during February 2004 to march 2005 in which total 140 samples out of which there were 92 food handlers, 24 raw milk and 24 90

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Minas Frescal cheese samples all these samples were examined for presence of Staphylococcus aureus. According to the findings it was confirmed that S. aureus contamination of cheese was raw milk (Millogo et al., 2010). Bifidobacter in Raw Milk In order to isolate biifidobacter in raw milk of three farms fifty eight samples were collected. The bifidobacteria which were isolated from raw milk were compared with the dung of same cows from which raw milk was collected. In 95% of dung samples and 885 of raw milk samples Bifidobacterium pseudolongum subsp. Globosum was isolated (Beerens et al., 2000).

Rapid Method for Estimation of BacterialActivity On the basis of amino peptidase activity of Gram-negative psychrotrophic bacteria a quick method for the estimation of dominant bacteria in refrigerated raw milk was developed. Amino peptidase activity was measured using direct and indirect test. Bacterial estimination using amino peptidase activity method and by conventially used method shows a good correlation (r=0.93­0.95). The sensitivity of the indirect assay was 2 ×104 CFU/mL (Manzano et al., 2005).

Isolation of Enterococci and Lactobacilli Bacteria from Raw Milk Among the intestinal microflora of humans and animals Enterococci and lactobacilli dominates. From one form only 362 lactobacilli and 56 putative enterococci were isolated from bovine faeces (n=26), cows' teats, raw milk, the milking machine and the milking environment. Pulsed-Field Gel Electrophoresis was used for finding the clonal relationships of each group. Majority of enteroccal isolates in bovine faeces were Aerococcus viridans. Out of all the isolates seven of them belong to a potential novel Aerococcus spesies while only one isolate was found to be potential second novel Aerococcus spesies. Majority of Lactobacilli in bovine faeces were Lactobacillus mucosae and Lactobacillus brevis with few Lactobacillus plantarum.. There were only one E. casseliflavus and one L. parabuchneri/kefir out of 76 and 247 isolates of enterococci and lactobacilli respectively among all milk isolates. It was found that equipments used for milking were the main source of these bacteria (Kagkli et al., 2007).

High Nutritive Value of Milk Microbial contamination of raw milk is likely to occur if it will be kept at room temperature. As the time passes on the milk will become sour as well. This souring of milk is due to the activity of Lactic acid bacteria. In order to strengthen or maintain milk lactic acid bacteria were added to fresh milk. The addition of these bacteria to milk results in controlled acidification as well as good flavour. In different parts of the world many types of fermented milk and its products were manufactured historically. Different products can be made from milk which depends mainly on milk used, pre treatment of milk and climatic conditions of 91

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fermentation and on technology used for treatment. Although in fermented milk mostly lactic acid bacteria are present but in some cases some other bacteria may also be present in milk like yeast and moulds. In order to optimize the manufacturing process, starter culture for dairy products was developed. The starter culture either consists of multiple strains starter cultures of specific pure culture or mixed strains of starter culture which comprises of undefined mixture of different bacteria. The quality of milk products is positively affected by using starter culture but it may also result in limiting diversity of fermented dairy products (Wouters et al., 2002).

Detection of E. coli using Fluorescents Bacteriophage Assay The fluorescent bacteriophage assay (FBA) was used in order to detect E. coli O157:H7 in raw milk and ground beef. In two steps FBA assay works i.e. combines immunomagnetic separation, in order to separate the target organism from mixed culture, with a highly specific fluorescently stained bacteriophage to label the E. coli O157:H7 cells. When FBA was combined with flow cytometry it was able to detect 2.2 CFU/g of artificially contaminated ground beef after six hours of enrichment. After ten hours of enrichment raw milk was contaminated artificially having 10 CFU/ml of bacteria. The results show that for the preliminary detection of E. coli O157:H7 in food through FBA is potentially useful as a quick technique (Goodridge et al., 1999).

CONCLUSION The general hygiene at milking affects the numbers of bacteria in the milk. The hygienic packaging of milk would result in decline of milk contamination at the selling spot. The bacterial load in milk from the udder to the shops depends upon the hygienic environment. The overall hygienic condition was not satisfactory as the farmers are not having the habit of washing there hands with soap prior to milking. The animals in the farm were in rooms with no proper ventilation which resulted in opportunity for the growth of anaerobic bacteria. The decrease in time between the farm and the market, addition of sodium hypochlorite to water for its quality improvement and by controlling sub clinical mastitis at the end of lactation stage will improve milk hygiene. The improvement in the shelf life of the milk and acknowledgment incentive will definitely facilitate in order to boost the stakeholder's compliance. The tetra pack milk was bacteria free and safe for consumption. After this study following are some of the recommendations: · Training and guidance programmes should be started in order to develop awareness among farmers emphasizing the need for hygienic practice at farm level. Transportation and storage of raw milk should be at low temperature to avoid bacterial growth in raw milk. Farm environment should be improved in order to decrease the breeding ground for pathogenic bacteria. Utensils should be properly washed prior to transferring milk to them. 92

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·

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Canadian Journal on Scientific and Industrial Research Vol. 2, No. 2, February 2011

· ·

It is highly recommended to drink milk after proper pasteurization. Sale and distribution of raw milk should be banned

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Jayarao, B.M., Henning. D.R., Prevalence of food borne pathogens in bulk tank milk, J Dairy Sci. vol. 84, pp2157-2162, 2001. Kagkli, D.M., Vancanneyt, M., Hill, C., Vandamme, P., Cogan, T.M., Enterococcus and Lactobacillus contamination of raw milk in a farm dairy environment, Internat J Food Microbiol. vol. 114, pp243-251, 2007. Leitner, G., Silanikove, N., Jacobi, S., Weisblit, L., Bernstein, S., Merin, U., The influence of storage on the farm and in dairy silos on milk quality for cheese production, Internat Dairy J. vol. 18, pp109-113, 2008. Magnusson, M., Christiansson, A., Svensson, B., Bacillus cereus Spores During Housing of Dairy Cows: Factors Affecting Contamination of Raw Milk, J Dairy Sci. vol. 90, pp27452754, 2007. Manzano, S., Ordo, J.A., Hoz, L., Ferna, M., A rapid method for the estimation of the bacterialquality of refrigerated raw milk based on the aminopeptidase activity of gramnegative bacteria, Internat Dairy J. vol. 15, pp79-84, 2005. Michalski, M.C., Januel, C., Does homogenization affect the human health properties of cow's milk, Trends in Food Sci. and Technol. Vol. 17, pp423-437, 2006. Mortazavi, A., Najafi, M.B., Habibi, Y.M., Barouei, J., Application of commercial immuno assay (ELISA) technique for determination of hepatitis A antigen (HAV) in raw milk, Food Control. vol. 19, pp551-556, 2008. Millogo, V., Svennersten, S.K., Ouedraogo, G.A., Agenas, S., Raw milk hygiene at farms, processing units and local markets in Burkina Faso, Food Control. vol. 21, pp1070-1074, 2010. Nero, L.A., Mattos, M.R., Beloti, V., Barros, M.A.F., Pontes, N.D., Nogueira, P.J.P.A., Hazards in non-pasteurized milk on retail sale in Brazil: prevalence of Salmonella species, Listeria monocytogenes and chemical residues, Brazilian J Microbiol. vol. 35, pp211-215. 2004. Oksuz, O., Arici, M., Kurultay, S., Gumus, T., Incidence of Escherichia coli O157 in raw milk and white pickled cheese manufactured from raw milk in Turkey, Food Control. vol. 15, pp453-456, 2004. Veling, J., Van, Z.F.G., Van, Z.V.B.A.M., Shcukken, Y.H., Barkema, H.W., Evaluation of two enzyme-linked immunosorent assays for detecting Salmonella enterica subsp. enterica serovar Dublin antibodies in bulk milk. Clinical. Diagnostic, Lab. Immunology. Vol. 8, pp1049-1055, 2001. Wells, J.G., Shipman, L.D., Gren, K.D., Sowers, E.G., Green, J.H., Cameron, D.N., Downers, P.P., Martin, M.L., Griffin, P.M., Ostroff, S.M., Potter, M.E., Tauxe, R.V., Wachsmuth, I.K., Isolation of Escherichia coli serotypes 0157: H7 and other shiga-like toxin-producing E. coli from dairy cattle. J. Clinical Microbiol. vol. 29, pp985-988, 1999. Wouters, J.T.M., Ayad, E.H.E., Hugenholtz, J., Smit, G., Microbes from raw milk for fermented dairy products, Internat Dairy J. vol. 12, pp91-109, 2002. 94

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