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 post-

pasteurization 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 micro-

organisms 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

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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

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(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 Jan April Jul Oct

Udder 2.2x105 6.6x105 2.7x104 2.4x105

Milk Uten. 5x105 5.4x105 3.2x105 1.7x105

DCS 3.8x106 7.5x105 8.9x105 1x106

Dock 2.6x106 4.7x106 2x106 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 pre-

processed 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 Total plate count in cfu/ml

Maximum Minimum Average

Udder 4.75x105 <103 2.9x 105

Milk

Uten.

4.95x 104 5.0x 103 3.88x105

DCS 5.94x106 1.98x104 1.6x106

Dock 6.93x106 9.4x104 2.8x106

Market 5.9x105 <100 2.2x105

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 10³ 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 E. coli Listeria spp.

Maximum Minimum Average

Aluminium can

6.8x104 <10 0.73x104 0 0

Steel Can 1x104 <10 0.23x104 0 0

Processing line

26.2x104 <10 6.33x104 3 0

Bulk coolers

7.6x105 5.8x104 4.5x105 0 0

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.

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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

88

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.

89

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

91

with contaminated raw milk should be extended to the public, so that

consumption of untreated raw milk could be avoided.

92