Chapter III.

Microbiological analysis of milk, milking equipments and milk

processing environment

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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 (OConnor 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 Indias 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 20062009. 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 Udder Milking utensils DCS Receiving Dock Market Swabs Environmental samples Bulk coolers Total No. of samples collected 147 147 147 267 120 60 30 15 933

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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 04, 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 MacConkeys 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 milkers hands and cows 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 <103 to 4.75x105 CFU/ml at udder level, 5x103 to 4.95x105 CFU/ml at milking utensils, 1.98x104 to 5.94x106 CFU/ml at dairy cooperative society and 9.4x104 to 6.93x106 CFU/ml at receiving dock. The average counts were 2.9x105, 3.88x105, 1.6x106 and 2.8x106 CFU/ml at udder, milking utensils, collection centres and receiving dock (processing point) levels, respectively. The data is presented in Table 3.2 and Fig. 3.3. The total counts at udder level and for samples from milking utensils differed significantly (P<0.005) from that of receiving dock level. Similar findings were reported by Godefay and Molla (2000) in Ethiopia while studying the bacteriological quality of raw cow's milk taken at different sampling points from four dairy farms and a milk collection centre. A high increase in the mean total aerobic plate count was observed in milk samples taken from the bucket (1.1 x 105 CFU/ml), storage container before cooling (4 x 106 CFU/ml) and upon arrival at the processing plant (1.9 x 108 CFU/ml). Table 3.2. Average total plate counts in cfu/ml of milk samples at different levels (season wise) Level Udder Milk Uten. DCS Dock Jan 2.2x105 5x105 3.8x106 2.6x106 April 6.6x105 5.4x105 7.5x105 4.7x106 Jul 2.7x104 3.2x105 8.9x105 2x106 Oct 2.4x105 1.7x105 1x106 1.8x106

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Lack of knowledge about clean milk production, use of unclean milking equipment and lack of potable water for cleaning purposes were some of the factors which contributed to the poor hygienic quality of raw milk in the study farms (Godefay and Molla, 2000). The total aerobic plate count per ml of preprocessed raw milk was found to be high ranging from 5.8 x 105 to 5.7 x 108 in Trinidad (Adesiyun, 1994). The average total viable counts of can rinse were 3.11x106. Fresh milk drawn from a healthy cow normally have a low microbial load, but the loads may increase up to 100 fold or more once it is stored for some time at normal temperatures (Richter et al., 1992). 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). Highest microbial load occurred during summer season, while the lowest counts occurred during winter season. The total counts of the samples collected during October at udder level were significantly different from the counts of the samples collected during January (Table 3.3; Fig 3.4). Also the total counts of the samples collected during January significantly (P<.005) differed from the counts of the samples collected from milking utensils during April. The seasonal value indicated that temperature of the environment also matters in the microbial quality of milk. Comparatively high value during summer indicated increased microbial growth. It was observed that the load of the

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Table 3.3. Average total plate counts at different levels of collection. Levels Udder Milk Uten. DCS Dock Market 5.94x106 6.93x106 5.9x105 1.98x104 9.4x104 <100 1.6x106 2.8x106 2.2x105 4.75x105 4.95x 104 Total plate count in cfu/ml Maximum Minimum <103 5.0x 103 Average 2.9x 105 3.88x105

Fig 3.3. Average total plate counts at different levels of collection. microorganism was high in raw milk in all seasons. The high microbial load indicated unhygienic practices prevailing at the production level. Proceeding time and normal environmental condition allows favorable growth of

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microorganism increasing microbial load. In some cases, from DCS to dock microbial load increased by 1-3 log unit per ml. Enumeration of mesophilic aerobes (MA) is the main quality and hygiene parameter for raw and pasteurized milk. High levels of these microorganisms indicate poor conditions in production, storage, and processing of milk, and also the presence of pathogens (Freitas et al., 2009). In this study, the coliform counts of the market milk ranged between < 10 to 5.6x104 with an average of 2.4x103. The average total counts of swabs were 0.73x104 for aluminum cans, 0.23x104 for steel cans, and 6.33x104 at processing lines (Table 3.4). The average total counts of milk samples from bulk coolers were 4.5x105. The detection of coliform bacteria and pathogens in milk is an indication of possible contamination of bacteria either from the udder, milk utensils or water supply used (Olson and Mocquot, 1980; Bonfoh et al., 2003). While determining the total coliform counts in 250 samples of kraals and indigenous milk products in the coastal savannah zone of Ghana, coliforms exceeded 10 CFU/ml in 66.0% (Addo et al., 2011). Coliforms were detected in 62.3% of 131 bulk tank milk samples in eastern South Dakota and western Minnesota (Jayarao and Wang., 1999). In a study in Zimbambwe, the coliform counts ranged from <10 to 6.0x103 CFU/ml; 10% of milk samples on delivery had more than 103 CFU/ml coliform counts (Gran et al., 2002). Investigation of various factors associated with bulk milk coliform count (CC) in dairy farms (n = 10) indicated geometric mean in-line milk CC (ILCC) to be 37 CFU/ml which varied by farm, ranging from 5 to 1,198 CFU/ml (Pantoja et al., 2011). Rate of fall-offs, rate of cluster washes, outdoor

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and indoor temperature, indoor humidity, sampling duration, and parity group were unconditionally associated with ILCC. The nature of the associations between liner CC, rate of cluster washes, rate of milking units fall-offs, and ILCC indicated that managing and monitoring such events had the potential for Table 3.4. Analysis of swab samples and bulk coolers. Source Total plate count in cfu/ml Maximum Aluminium can Steel Can Processing line Bulk coolers 6.8x104 1x104 26.2x104 7.6x105 Minimum <10 <10 <10 5.8x104 Average 0.73x104 0.23x104 6.33x104 4.5x105 0 0 3 0 0 0 0 0 E. coli Listeria spp.

improving bacteriological quality of farm bulk milk (Pantoja et al., 2011). At three smallholder dairies in Zimbabwe 83% utensils used for milking had >300 cfu per 20 cm2 (Gran et al., 2002). In order to reduce contamination of the milk, utensils used for milking should be rinsed, cleaned using detergent and disinfected immediately after use (Dodd & Phipps, 1994; FAO and WHO, 1997b; IDF, 1990). The use of detergents and good quality water for cleaning the equipment could be expected to remove milk remains, including microorganisms, and thereby affect the microbiological quality of the milk. Use of disinfection, either chemical or hot water, would mostly reduce the numbers of microorganisms (Gran et al., 2002). The farmers' personal hygiene and their hygiene practice in milk handling could be expected to influence the number of microorganisms in raw milk. 87

The mean value of total bacterial count in the barn air was 1.73 x 103 with minimum of 1.5 x 102 and maximum of 2.1 x 104. Earlier study

(Matkovic et al., 2006) reported the mean value of total bacterial count in the barn air ranging from 2.82 104 CFU/m3 at noon to 7.76 104 CFU/m3 in the evening in Croatia. Total airborne bacterial count has been reported to be directly influenced by air temperature, relative humidity and air flow velocity, and also could be attributed to daily animal and human activities in the barn (Matkovic et al., 2006) whereas, the outdoor air bacterial emission depends on the source of contamination, position of air outlet on the barn roof or wall, ground configuration, air flow, air temperature, humidity and sunlight (Matkovic et al., 2006). The presence of bacteria in barn air is a natural phenomenon, their primary source being the animals themselves, then the fodder and humans. Bacteria are only one of the many groups of air pollutants. Bacterial count may also depend on the construction and technical characteristics of the housing, number of animals kept in the housing, temperature and humidity in the housing, and feeding, grooming, milking, and other activities (Lange et al., 1997; Seedorf et al., 1998). Bacterial count in the air of a dairy barn may provide appropriate data on the hygienic condition at the farm from where milk starts on its way to the consumer. In addition, for the assessment of the effect of dairy barns on the local environment, bacterial count in the barn air and monitoring of its emission from the barn to the adjacent environment are important parameters (Matkovic et al., 2006). Seasonal changes in airborne fungi, bacteria and in the incidence of S. aureus resistant to antibiotics at a dairy cattle concentrated

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animal feeding operation has been reported in the southwest United States (Alvarado et al., 2009). High spore counts can occur at the dairy farm and feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk (Scheldeman et al., 2005). Good hygienic measures could probably reduce the contamination level of raw milk, thereby minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. In view of the current concerns of effects of climate change, it may be interesting to know the type of dominant species of pathogens prevalent in the barn environment and their association with infections. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems. In order to produce milk product conforming to high quality standard, it is important that milk should be collected, transported and cooled immediately under strict hygienic conditions. Ideally all the milk leading to the dairies should be bulk cooled. Possible cooling at DCS will decrease the load of microorganisms. Quality management on dairy farms becomes more and more important regarding the different areas of animal health, animal welfare and food safety. Monitoring animals, farm conditions and farm records can be extended with risk identification and risk management. The hazard analysis critical control point system is useful as an on farm strategy to control the product as well as the production process on the areas of animal health, animal welfare and food safety.

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An integrated approach to controlling food safety throughout the entire food chain (farm to table) has become an important issue in guaranteeing a greater food safety level for consumer products (Valeeva et al., 2004). In line with these ideas about food safety, a number of countries have developed and introduced new regulations to assure food safety at different stages of the food production chain. Most of these regulations stipulate that improving food safety should focus not only on assuring safe food production within a single stage but also on assuring other links relating to this stage. The health of the dairy herd, milking and pre-storage conditions are also basic determinants of quality (Aumaitre, 1999). Another source of contamination by microorganisms is unclean teats. However, in the present study the bacteriological counts in milk due to unclean udders was low but intense manipulation of small quantities of milk using several containers increased the count of microflora in milk. The use of unclean milking and transport equipment contributed also to the poor hygienic quality of the milk. These observations are in line with findings in Ethiopia (Godefay and Molla, 2000). The initial microbiological quality of milk varies considerably and depends for the most part on the cleanliness of containers. The number of containers used in the milk chain was the main source of contamination. High ambient temperatures coupled with general lack of refrigeration and poor standard of hygiene means that milk, which often contains a large number of bacteria, acidifies on its way to the processing unit. The production of high-quality milk and safe milk should be of great importance to the economy of the farmer and the sustainability of the dairy industry in this country.

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Microbial contamination of raw milk may occur from 3 main sources: from within the udder (mastitis associated organisms), from environmental organism transfer via dirty udder and teat surfaces, and from improperly cleaned and sanitized milking equipment. Additionally, improper cooling and prolonged storage of milk can also influence bacterial count by increasing the rate of bacterial growth during storage of milk (Elmoslemany et al., 2009). It was observed that the period between time of collection of milk and its transportation to the processing unit was critical for change in microbial count. On an average, it required 4.5 h between milking and arrival at processing unit. The milk produced at farmers field was of the best quality except on few occasions. However, further handling of the milk adds to the microbial contamination. Presence of mastitis increases the microbial count of the raw milk. As far as possible the time duration between milking and arrival of milk at processing unit need to be decreased or reduced. Chilling plants may be

established at far off places for initial cooling of milk so that the bacterial multiplication is minimal. Clean milk production starts at the farm therefore animals, shed, utensils and the milking personnel all contribute to the quality of milk. A backward linkage of quality of milk and status of animal health together with the milking surroundings need to be established. This will help in taking corrective actions and breaking the unhealthy link. High microbial counts and the occurrence of pathogens are likely to affect the keeping quality and safety of raw milk as well as products derived from it. Therefore, it is recommended that training and guidance should be given to farms owners and their workers responsible for milking. Meanwhile, information on health hazards associated

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with contaminated raw milk should be extended to the public, so that consumption of untreated raw milk could be avoided.

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