Chapter III.

Microbiological analysis of milk, milking

equipments

and

processing environment

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milk

3.1 Introduction There is an increasing focus on milk quality and hygiene in the dairy industry. Producing high quality milk requires effective udder health programs at a herd level (Bhutto et al., 2010). The safety of milk is an important attribute for consumers of milk and dairy products. Milk and products derived from milk of dairy cows can harbor a variety of microorganisms and can be important sources of foodborne pathogens. The presence of foodborne pathogens in milk is due to direct contact with contaminated sources in the dairy farm environment and due to excretion from the udder of an infected animal (Oliver et al., 2005). Entry of foodborne pathogens via contaminated raw milk into dairy food processing plants can lead to persistence of these pathogens in biofilms, and subsequent contamination of processed milk products and exposure of consumers to pathogenic bacteria (Latorre et al., 2010). Inadequate or faulty pasteurization will not destroy all foodborne pathogens. Furthermore, pathogens can survive and thrive in postpasteurization processing environments, thus leading to recontamination of dairy products. These pathways pose a risk to the consumer from direct exposure to foodborne pathogens present in unpasteurized dairy products. The safety of dairy products with respect to food-borne diseases is of great concern around the world. This is especially true in developing countries where production of milk and various milk products takes place under unsanitary conditions and poor production practices (Mogessie, 1990). A major factor determining milk quality is its microbial load. It indicates the hygiene practiced during milking, like cleanliness of the milking utensils, condition of storage, manner of transport as well as the cleanliness of 73

the udder of the individual animal (Spreer 1998; Gandiya 2001). Milk from a healthy udder contains few bacteria but it picks up many bacteria from the time it leaves the teat of the cow until it is used for further processing. These microorganisms are indicators of both the manner of handling milk from milking till consumption and the quality of the milk. Milk produced under hygienic conditions from healthy animals should not contain more than 5 x 105 bacterial/ml (O’Connor 1994). The detection of coliform bacteria and pathogens in milk indicates a possible contamination of bacteria either from the udder, milk utensils or water supply used (Bonfoh et al., 2003). Fresh milk drawn from a healthy cow normally contains a low microbial load (less than 1000 ml-1), but the loads may increase up to 100 fold or more once it is stored for sometimes at normal temperatures (Richter et al., 1992). However, keeping milk in clean containers at refrigerated temperatures immediately after milking process may delay the increase of initial microbial load and prevent the multiplication of microorganisms in milk between milking at the farm and transportation to the processing plant (Adesiyun, 1994; Bonfoh et al., 2003). Contamination of mastitis milk with fresh clean milk may be one of the reasons for the high microbial load of bulk milk (Jeffery and Wilson, 1987). The current research includes all the independent factors that are able to affect the food safety level of the end product of the whole dairy chain, i.e., the consumed fluid pasteurized milk. Transportation between the stages is also considered, i.e., transport of raw milk to the processing factory, and delivery of pasteurized milk to the sale unit (retailer/catering establishment).

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Agriculture is the base of Indian economy. Livestock production including dairy plays a multipurpose role in the agriculture systems of India. Dairy plays a dynamic role in India’s agro-based economy. Today, India ranks the first in the world in terms of milk production. In Goa, there is ample scope for income generation through livestock production. The territory has about 100,000 cattle and 45,000 buffaloes. The assessment of microbial load at various stages of manufacture or processing may serve as a useful tool for quality assessment and improvement which will result in longer shelf life which is a desirable market requirement. Keeping fresh milk at an elevated temperature together with unhygienic practices in the milking process may result in microbiologically inferior quality. Apparently, these are common practices for small-scale farmers who produce fresh milk and sell it to local consumers or milk collection centers (Chye et al., 2004). Thus, this study was carried out to investigate the microbiological quality and safety of locally produced raw milk and to identify the relevant sources of contamination and critical point in the chain of locally produced raw bovine milk.

3.2 Materials and Methods 3.2.1 Samples All the samples were collected aseptically and processed immediately as per the standard protocols. A total of 933 samples comprising of milk from dairy animals collected at different levels of collection and processing (udder, milking utensils, dairy cooperative society (DCS), receiving dock and bulk coolers) and swabs from cans and milk processing line within Goa region were 75

collected during 2006–2009. For collection of the samples, the udder was washed with antiseptic solution, wiped dry with clean cloth (Fig 3.1) and then disinfected with cotton ball dampened with 70% alcohol, the foremilk was discarded and 20 ml of pooled milk was collected (5 ml from each quarter) Details of samples collected are summarized in Table 3.1.

Fig 3.1. Cleaning of udder before milking. Table 3.1. Details of samples collected from different sources for analysis of microbiological parameters. Source of samples

No. of samples collected

Udder

147

Milking utensils

147

DCS

147

Receiving Dock

267

Market

120

Swabs

60

Environmental samples

30

Bulk coolers

15

Total

933

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The samples were collected after the cleaning and sanitation of the plant as per guidelines of Bureau of Indian Standards, IS 7005:1973 Code of hygienic conditions for production, processing, transportation and distribution of milk. Samplings were done at quarterly interval (January, April, July and October). Prior to milking, the contamination of the surface of the material and the containers was determined by flushing all containers with 100 ml of sterile water. A total 60 swab from cans and milk processing line and 15 milk samples from bulk milk coolers were also collected. All the samples were collected in sterile screw cap tubes. Samples were collected early in the morning at udder, milking utensils, and dairy cooperative society levels. For sampling at udder level, milking animals were randomly selected at randomly selected farms. After milk was transferred in the can from the same cow; from this can second milk sample was collected. When this can reached at DCS, third sample was collected. At DCS the milk got transferred in another big can (Fig 3.2), which came to receiving dock at processing unit, where fourth sample was collected. All the samples were kept in the icebox, transported to the laboratory under chilled conditions and processed for microbiological analysis. The time between milking and transportation to the processing unit was also assessed. At each visit, farm management and general hygiene were evaluated with emphasis on milking procedures, cleaning of containers and materials used.

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Fig 3.2. Filtering of milk after receiving at dairy cooperative society.

3.2.2 Milk analysis 3.2.2.1 California Mastitis Test California mastitis test (CMT) was carried out according to the method described by Schalm and Noorlander (1957), at cow side by mixing an equal volume of milk CMT reagent (3 gm of Sodium lauryl sulphate and 300 mg of bromocresol purple added in 100ml distilled water). Each quarter milk samples from the cow was collected in cups of the CMT paddle. Equal amount of the CMT reagent was added to quarter milk samples. As the CMT paddle was rotated gently, any colour changes or formation of a viscous gel were interpreted: in brief, scores were given within the range 0–4, with 0 for no reaction, 1 for a trace, 2 a weak positive, 3 a distinct positive and 4 a strong positive.

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3.2.2.2 Methylene Blue Reduction Test The methylene blue reduction test (MBRT) was performed according to the IDF (1990). One ml of methylene blue was added to 10 ml of each of the raw milk samples, shaken. The test tubes were then incubated at 370C in hot water bath for 30 min and the change in color was carefully observed. In case the methylene blue decolorized during the incubation period, the MBRT was recorded to be 30 min. After the initial 30 min reading, the subsequent readings were taken at hourly intervals.

3.2.2.3 Total Plate Counts Total microbial count was carried out as described in IS: 5402-2002. For enumeration of bacteria, the samples were serially diluted in peptone water (Himedia, Mumbai) and appropriate dilutions were plated on plate count agar using the spread plate method. The plates were incubated at 370C for 24 h for aerobic mesophilic counts. The enumerations were done as per ICMSF (1978).

3.2.2.4 Coliform count For enumeration of coliforms procedure described in IS: 5401(part 1)2002 was used. The market milk samples were serially diluted in peptone water (Himedia, Mumbai) and appropriate dilutions were plated on MacConkey’s agar using the spread plate method. The plates were incubated at 370C for 24 h for coliform counts.

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3.2.2.5 Swabs A total of 60 swab samples were also collected from cans and milk processing line. The swab samples were collected in sterile saline and then transported to the laboratory for further analysis. Both total plate and coliform counts of the swabs were determined.

3.2.2.6 Airborne bacterial counts A total of 30 samples for bacterial count in air were collected by exposing nutrient agar plates inside the sheds for 10 min. The lid of the petri plate was covered and incubated for 24 h in an incubator at a temperature of 370C to study the bacterial count and airborne emission to the immediate environment.

3.2.2.7 Data analysis The data was analysed using paired t-test using statistical package WASP.2 (www.icargoa.res.in).

3.3 Results and Discussion The farmers used mainly steel containers (59%) and aluminum containers for milking the animals. The milk passed through at least four to five containers, two funnels and two sieves before reaching the container, which is processed at the processing unit. The containers were cleaned thoroughly. Soap is used sometimes; washing of milker’s hands and cow’s udders was a common practice. Milk samples were collected for bacteriology, and the CMT was performed cow-side. In this study, subclinical mastitis 80

(SCM) was found in 23.8% of the animals (at least one positive quarter per cow) by CMT. Subclinical mastitis was found more important in India (varying from 10-50% in cows and 5-20% in buffaloes) than clinical mastitis (1-10%) (Joshi and Gokhale, 2006). In another study, of the 507 milk samples collected, 454 (89.5%) were California mastitis test (CMT)-positive (Adesiyun, 1994). The California mastitis test (CMT), first described and used in 1957 (Schalm and Noorlander, 1957), has been accepted as a quick, simple test to predict somatic cell count (SCC) from individual quarters or composite milk (Sanford et al., 2006). The CMT is an inexpensive, fast and cow-side test that can be performed by any individual with minimal training. With increasing SCC or total leukocyte count in milk, the CMT score also increases (Schalm and Noorlander, 1957; Dohoo and Meek, 1982). At the cow-level, the sensitivity and specificity of the CMT (using the four quarter results interpreted in parallel) for identifying all pathogens were estimated at 70 and 48%, respectively (Sanford et al., 2006). During an evaluation of CMT for diagnosing precalving intramammary infection (IMI) on a total of 428 dairy heifers from 23 dairy herds Holstein heifers,

at the quarter level, the

sensitivity and specificity of CMT were 68.9% and 68.4%, respectively to identify all IMI. However, at the heifer level sensitivity and specificity of CMT for major pathogens were 91.0% (81.5-96.6) and 27.5% (22.8-32.6), respectively (Roy et al., 2009). Bacteriological culture is often accepted as the gold standard for the identification of IMI. SCC can be useful for detecting IMI and is cheaper than cultures (Sargeant et al., 2001). CMT scores at drying off might be good indicators of IMI and a significant association between the frequency of

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isolation of major pathogens and the CMT score in milk samples obtained one week before and those at drying off has been reported (Bhutto et al., 2010). CMT could be used reliably to identify subclinical mastitis in lactating cows, and it might be useful in identifying such affected quarters that require antibiotic treatment and early drying off (Barkema et al., 1997). The California Mastitis Test has previously been adapted for use in an inline, cow-side sensor and relies on the fact that the viscosity of the gel formed during the test is proportional to the somatic cell concentration (Verbeek et al., 2008). The CMT has been reported to play a useful role in dairy herd monitoring programs as a screening test to detect fresh cows with IMI caused by major pathogens (Sargeant et al., 2001). In this study, the average methylene blue reduction time decreased from the farm to the processing unit. The reduction time was significantly correlated (P < 0:001) with the critical control point of milk collection in the chain i.e., between udder and dairy cooperative society, and udder and receiving dock. This denotes the exponential increase in contamination from the udder to the processing point. Estimation of microbial load in raw milk is crucial in relation to its spoilage and keeping quality. Several techniques are currently available for determining the total viable cell count as well as microbial load including the laboratory methods for determining total viable cell count include direct microscopic count (DMC), most probable numbers (MPN), and standard plate count (SPC) (Ahmed and Jindal, 2006). However, the most frequently used methods for indirect estimation of the microbial load in raw milk in the dairies and milk collection centers are based on dye reduction. Among dye reduction methods, methylene blue reduction time

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(MBRT) is widely used at the milk collection centers (Ahmed and Jindal, 2006). In the present study, the total viable counts varied from