|Year : 2012 | Volume
| Issue : 1 | Page : 88-94
Microbiological profile of milk: Impact of household practices
Amit Agarwal1, Vandana Awasthi2, Ajit Dua3, Sanjeev Ganguly4, Vivek Garg5, Satwinder S Marwaha6
1 Project Fellow, Punjab Biotechnology Incubator, Mohali, Punjab, India
2 Scientist Biology, Punjab Biotechnology Incubator, Mohali, Punjab, India
3 Senior Scientist, Punjab Biotechnology Incubator, Mohali, Punjab, India
4 Head, Medical and Scientific Affairs, Nestlé India Limited, Mohali, Punjab, India
5 Manager Medico Marketing, Nestlé India Limited, Mohali, Punjab, India
6 Chief Executive Officer, Punjab Biotechnology Incubator, Mohali, Punjab, India
|Date of Web Publication||6-Jun-2012|
Satwinder S Marwaha
Chief Executive Officer, Punjab Biotechnology Incubator, SCO: 7&8 (Top Floor), Phase-V, SAS Nagar (Mohali) 160059, Punjab
Source of Support: The study was conducted with fi nancial support from Nestlé India Ltd., Conflict of Interest: Sanjeev Ganguly and Vivek Garg are employees of Nestlé India Ltd. There are no other confl icts of interest.
| Abstract|| |
Background: Milk is susceptible to contamination by many microorganisms including microbial pathogens responsible for causing diseases. Various processes including pasteurization, boiling or storage under refrigerated conditions are undertaken to minimize the microbial contamination of milk. Objective: This study was undertaken with an objective to evaluate the effect of household practices on the microbiological profile of milk. Materials and Methods: Milk samples of pasteurized, ultra heat treated (UHT) as well as unpasteurized milk (Vendor's milk) were collected. The effect of different storage practices and treatments on the microbiological profile (standard plate count (SPC), coliform, E. coli, Salmonella, Shigella, Staphylococcus aureus, yeast and moulds, anaerobic spore count, and Listeria monocytogenes) of milk was studied using National/ International Standard Test Methods. Results: Average SPC in vendor's milk was found very high as compared to pasteurized milk. Coliform, yeast and moulds, E. coli, and Staphylococcus aureus were detected in the samples of vendor's as well as pasteurized milk. Boiling the milk reduces SPC and kills the other microorganisms. Storage of boiled milk under room temperature or refrigerated condition resulted in a similar increase in SPC at the end of 24 h, but storage of un-boiled milk even under refrigerated conditions increased SPC manifold after 24 h. Conclusion: The pasteurization process and hygienic conditions at the milk processing units along with cold chain of milk from suppliers to end users needs improvement. Currently, even pasteurized milk does not match the microbiological standards. It is recommended that milk should be boiled before consumption and refrigerated for storage to improve its shelf life/keeping quality.
Keywords: Boiling, Bovine milk, Microbiological profile, Milk pathogens, Milk storage, Pasteurized milk, UHT milk
|How to cite this article:|
Agarwal A, Awasthi V, Dua A, Ganguly S, Garg V, Marwaha SS. Microbiological profile of milk: Impact of household practices. Indian J Public Health 2012;56:88-94
|How to cite this URL:|
Agarwal A, Awasthi V, Dua A, Ganguly S, Garg V, Marwaha SS. Microbiological profile of milk: Impact of household practices. Indian J Public Health [serial online] 2012 [cited 2018 Oct 18];56:88-94. Available from: http://www.ijph.in/text.asp?2012/56/1/88/96984
| Introduction|| |
Milk is an important part of the Indian diet and is a source of important nutrients. Being a rich source of nutrients, milk is susceptible to contamination by many microorganisms including pathogenic microbes, which can cause the food-borne illness and are a threat to consumer's health. However, milk has no protection from external contamination and can be contaminated easily when it is separated from the source animals like cows/buffaloes.  Moreover, the milk acts as a good growth medium for the further multiplication of these pathogens.
These food-borne pathogens found in milk may cause various symptoms ranging such as nausea, vomiting, fever, loose stools and in severe cases, death. Various outbreaks of food-borne illness associated with the consumption of dairy products have been documented previously. 
There are country-specific legal standard for the upper limit of microflora in pasteurized milk. In India, the Prevention of Food Adulteration Act, 1954 (PFA) was followed till September 2011.  The Food Safety and Standard Act has been enacted w.e.f. 5th August, 2011.  PFA specifies the maximum upper limit of microflora in pasteurized milk as 30,000 cfu/g. Further this act specifies the limits for coliform as absent/0.1 g for E. coli, Staphylococcus aureus, yeast and moulds, anaerobic spore former, Listeria monocytogenes as absent per gram for each and for Shigella and Salmonella as absent per 25 g of pasteurized milk. 
Varied microflora in raw milk may arise from several sources, such as the exterior surfaces of the milch animals and the surfaces of milk handling equipment such as milking machines, containers, etc.  The health status of animals, the nature of their feed (forage, ensilage, etc.), and the storage conditions of raw milk are also important factors that determine the composition of their microbial flora. 
Milk from healthy cows/buffaloes contains relatively few bacteria (10  -10  cfu/ml); however, the bacterial load may increase up to 100 fold or more once it is stored for some time at normal temperature.  The average standard plate count (SPC) for raw milk depends upon temperature as well as handling conditions.
There are various ways to protect the milk from microbial spoilage. Pasteurization, a thermal process (75 °C for 15 s), is carried out with an objective to remove pathogenic microorganisms, minimize health hazards, and improve product shelf life. However, due to processing defects, the presence of pathogenic microorganisms in the pasteurized milk has been reported in the past. ,, At the household level, the practices such as boiling of milk and storage under refrigerated conditions are used to achieve these objectives.
In this study, the microbiological quality of unpasteurized (vendor's milk, VM), Pasteurized milk and ultra heat treated (UHT) milk was assessed along with the effect of various household practices such as boiling and storage at room temperature and under refrigerated conditions on the microbiological quality of milk. This study is intended to spread awareness regarding the microorganisms present in the milk, associated health hazards and simple household measures to improve the microbial quality of milk.
| Materials and Methods|| |
The study was conducted during the period September 2010 to November 2010, at Punjab Biotechnology Incubator, Mohali (Punjab). Different brands (in 500 ml pouch packing) of pasteurized milk samples having different fat content, i.e. standardized milk (SM) with fat: 4.5%, double toned milk (DTM) with fat: 1.5% and full cream milk (FCM) with fat: 6.0%, were collected from the local market for this study. Samples of unpasteurized milk (raw milk) with fat content varying from 4.1% to 6.7% were collected from local vendors. Three (03) samples of vendor's milk (VM), twelve (12) samples of pasteurized milk of different brands (6 samples of SM, 4 samples of DTM and 2 samples of FCM) and two (02) samples of UHT milk were analyzed. The samples were tested for microbial parameters: SPC, coliform, E. coli, Salmonella, Shigella, Staphylococcus aureus, yeast and moulds, anaerobic spore count and Listeria monocytogenes for recording the pretreatment values.
Boiling and Storage Methodology
To assess the impact of boiling on microbiological quality of milk, the milk samples were boiled as per simulated household conditions, i.e. boiled at minimum heat by setting the knob of the gas burner at minimum indicator under covered conditions. The effect of boiling was assessed by the post-boiling analysis of all the milk samples for the microbiological parameters. Lethality was calculated as the difference between the log of colony count of un-boiled (N0 ) and boiled (N1 ) milk samples (log 10 N0 − log 10 N1 ). 
The effect of storage conditions on the microbiological profile of milk samples was studied after boiling. In one set, the sample was boiled, and a part was kept at room temperature (25-28 °C) for 24 h with intermittent boiling at 12 h and in the other set boiled milk was stored at refrigerated conditions (2-8 °C) for 24 h without any intermittent boiling. The samples were analyzed at 0 h without boiling and 6, 12, and 24 h after boiling.
The effect of storage of un-boiled pasteurized milk under refrigerated conditions was also investigated. Pasteurized milk pouch was opened. A part it was taken out and analyzed as such and the remaining milk in pouch was stored in the refrigerator. The sample was analyzed after 6 and 24 h of storage.
Standard National, i.e. Indian Standard-IS methods (SPC per g,  coliform per 0.1 g,  E. coli per 1 g,  Salmonella per 25 g,  Shigella per 25 g,  Staphylococcus aureus per g,  yeast and moulds per g,  and Listeria monocytogenes per g  ) and International test methods (presence/absence of anaerobic spores per g  ) were used for the analysis of milk samples during the course of present investigation.
| Results|| |
Microbiological Profile of Different Types of Milk
Standard Plate Count: Average SPC in vendor's milk was found very high as compared to pasteurized milk [Figure 1]. The average SPC even in all the pasteurized milk samples was found to exceed the limit specified (i.e. 30,000 cfu/g) as per the Prevention of Food Adulteration Act, 1954 (PFA). 
|Figure 1: Average standard plate count (SPC) in different types of vendor's milk (VM), pasteurized milk (standardized milk (SM), double toned milk (DM), full cream milk (FCM)) and ultra heat treated (UHT) milk samples|
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Other Microflora: Pathogenic organisms (E. coli, Salmonella, Shigella, Staphylococcus aureus), coliform, and yeast and moulds were analyzed for the presence/absence in milk samples as per the requirement of the Prevention of Food Adulteration Act, 1954 (PFA). 
Coliforms as well as yeast and moulds were present in all the samples of vendor's as well as pasteurized milk (SM, DTM, FCM). E. coli was present in two samples each of vendor's milk, standardized milk and double toned milk. Staphylococcus aureus was present in one sample each of the vendor's milk and the standardized milk, double toned milk and full cream milk. Anaerobic spore formers were also present in one sample of double toned milk. Salmonella, Shigella, and Listeria monocytogenes were not present in any of the milk samples.
However, no microorganism from the assessed parameters was present in the UHT milk samples investigated in this study.
Effect of Boiling on Microflora of Milk
Standard Plate Count: A considerable reduction in SPC was observed in vendor's and pasteurized milk samples analyzed after boiling. The SPC observed after boiling was under permissible limits [Figure 2]. The lethality values ranged from 4.1 to 6.4 log 10 cfu/g.
|Figure 2: Effect of boiling on standard plate count (SPC) of pasteurized standardized milk|
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Other Microflora: E. coli, coliforms, Staphylococcus aureus, anaerobic spore formers and yeast and moulds present before were not present after boiling in any sample.
Effect of Storage Temperature on Microflora of Milk
Storage of Boiled Milk:
Standard Plate Count: SPC of boiled milk increased during storage at room temperature conditions. It reduced after boiling at 12 h, while under refrigerated conditions; SPC remained almost unchanged up to 12 h. Under storage of SM at room temperature and refrigerated conditions, the SPC increased in both groups and was similar at the end of 24 h [Table 1].
|Table 1: Effect of storage at room and refrigerated temperature conditions for 24 h on SPC of pasteurized standardized milk|
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Other Microflora: The coliforms, E. coli, Salmonella, Shigella, anaerobic spores, Listeria monocytogenes and yeasts and moulds in all milk samples were absent after the boiling process and remained negative during storage under refrigerated conditions at 24 h. However, coliforms and E. coli were present in few samples of boiled milk (DTM and FCM) stored at room temperature for 24 h, whereas yeast and moulds were present in all types of milk samples at 24 h [Table 2].
|Table 2: Effect of storage at room temperature (25 ± 2°C) for 24 h on the microflora of boiled milk|
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Storage of Un-boiled Milk
Standard Plate Count: In pasteurized milk samples stored under refrigerated conditions for 24 h without boiling, there was reduction in SPC up to 6 h but at the end of 24 h, a manifold increase in SPC was observed [Figure 3].
|Figure 3: Effect of storage under refrigeration without initial boiling on standard plate count (SPC) of pasteurized standardized milk|
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Other Microflora: There was no change observed in the status of other microorganisms tested as compared to the baseline data.
| Discussion|| |
The microbiological quality and safety of milk is of utmost importance in the interest of public health. This study demonstrates a considerably high SPC in vendor's milk as compared to the pasteurized milk samples. Interestingly, none of these milk samples met the specified legal standards of the country.  The earlier studies have also reported a total bacterial count (TBC) of 13 × 10  cfu/ml in raw milk, which is considerably higher than 71 × 10  cfu/ml in pasteurized milk samples tested.  The higher number of microflora in raw milk might be due to contamination from the animal and unhygienic milking procedures or equipment leading to entry of pathogens from dairy utensils and milk contact surfaces (exterior of the infected udder and adjacent areas). , Contamination of mastitis milk with fresh clean milk and storage conditions might also affect the microbiological quality of the milk. , The cleaning of milking utensils with detergents and good quality water helps remove the milk remains including microorganisms. 
Coliform and yeast and moulds have been observed in all the samples of vendor's and pasteurized milk. E. coli was present in two samples each of vendor's milk, standardized milk and double toned milk. Staphylococcus aureus was present in one sample each of the vendor's milk and the standardized milk, double toned milk and full cream milk. The presence of coliform in most of raw milk samples, E. coli in 80% of the samples and Staphylococcus aureus in 62% of the milk samples has been reported earlier. 
In a similar study from Malaysia, 930 raw milk samples were tested where ~90% samples were contaminated with coliforms, 65% with E. coli, and 61% were found contaminated with Staphylococcus aureus.  In a study from eastern India, 46.23% of the cows were found to be infected with Staphylococcus aureus. 
Another study from Guwahati reported the contamination of Staphylococcus aureus in all the milk samples (n = 20) tested, with a mean bacterial count of 4.60 10  per ml. E. coli was also present in 12 samples with a mean bacterial count of 4.12 × 10  per ml. The microorganisms detected were found to be resistant to one or multiple commonly used antibiotics. 
The presence of coliforms (an indicator of fecal contamination) has been related to an unsanitary environment.  Detection of coliforms and pathogens in milk indicates a possible contamination of milk either from udder, milk utensils, or water used in milching as well as postmilching practices. 
E. coli is a good indicator of fecal pollution and its presence in milk products indicates the presence of enteropathogenic microorganisms which constitute a serious public health hazard.  Several outbreaks of E. coli 0157 have been reported in developed countries ranging from mild diarrhea to potentially fatal hemolytic uremic syndrome (HUS), hemorrhagic colitis, and thrombotic thrombocytopenic purpura.  Enteropathogenic E. coli can also cause severe diarrhea and vomiting in infants, and young children.
Handling of the infected cattle is one of the important reasons for Staphylococcus contamination of the bovine milk  as S. aureus is a major causative agent of clinical and subclinical mastitis in dairy cattle.  In humans, S. aureus can cause minor skin infections (pimples, boils and abscesses) and life-threatening diseases such as pneumonia and septicemia.
Spoilage of dairy products by moulds is frequent and a matter of concern for human health. Yeasts and moulds in milk might act as allergen and an irritant to human health. ,,
Anaerobic spore formers were also present in one sample of double toned milk. Some bacterial species are capable of forming heat-resistant spores that survive high temperature short time (HTST) pasteurization  and some strains are able to germinate and grow at refrigerated temperature. , Salmonella, Shigella, and Listeria monocytogenes were not present in any of the milk samples tested. This is important as all Salmonellae are of public health concern which may cause mild self-limiting gastroenteritis, septicemia or life-threatening typhoid fever.  L. monocytogenes may cause flu-like symptoms, diarrhea, meningitis and abortions. Vulnerable population like pregnant women, newborns, elderly, and immune-compromised people are at greater risk to develop serious disease if infected with virulent strain of L. monocytogenes. ,
During this study, UHT milk was found to be free from all the microorganism tested, which is in line with earlier reports. 
In this study, a considerable decrease in SPC was observed after boiling of milk. Earlier studies have also reported a significant decrease in bacterial counts associated with boiling of milk. , We also observed that boiling kills coliforms, E. coli, Staphylococcus aureus, and yeast and moulds present in milk samples. It has been reported that the count of mesophilic bacteria in milk are reduced significantly after boiling. 
The effect of storage conditions on microflora of milk observed during this study are in line with the earlier studies. Refrigeration of the pasteurized milk after boiling helps in keeping the bacterial count low.  The shelf life of pasteurized milk was found 6 days, when milk is stored at 7 °C. However, milk has a shelf life of only 20-24 h, when stored at 25 °C.  Storage temperature below 7 C has been reported to extend the shelf life of pasteurized milk. 
The storage of pasteurized milk under refrigerated conditions without boiling initially decreases SPC for short periods, but increases rapidly after 6 h. Hence, it is advisable to boil even the pasteurized milk before keeping under refrigerated conditions in order to improve its microbial quality.
| Conclusion|| |
This study provides important information regarding the presence of pathogenic microorganisms in the vendor's as well as pasteurized milk. It is a serious public health concern, especially for the vulnerable population such as infants, growing children, pregnant women, and elderly people. Microbial types and their extent in commercially available milk are associated with hygienic conditions at the cattle farm and the practices followed from milching to its delivery to the public.
The current study clearly underscores the attention required to improve the pre- and postpasteurization processes including hygienic conditions at milk processing units. Efforts should be also be made to maintain the cold chain of milk from suppliers to end users. Milk vendors should be educated adequately to implement the hygienic milching practices. UHT milk has a better microbial quality as compared to other types of milk available. It is recommended that simple household steps like good personal hygiene, use of clean utensils, practice of boiling the milk before consumption and refrigeration for storage should be undertaken to improve the microbiological as well as keeping quality of the vendor's and pasteurized milk.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]
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