DAIRY BACTERIOLOGY AND MICROBIAL ANALYSIS OF MILK
USING SYRINGE FILTER
*ARULRAJ P, ASARUDHEEN I, JASMIN T,
SAHNAS N K, VEDHA SATHEESH,
VENKATANARAYANAN R
Department of Pharmaceutical Biotechnology,
RVS College of Pharmaceutical Sciences,
Coimbatore.
*Corresponding author
Arulraj P
Associate Professor,
Department of Pharmaceutical Biotechnology,
RVS College of Pharmaceutical Sciences,
Sulur, Coimbatore - 641 402.
Web: www.bpharmpharmd.webly.com,
E mail: arulraj.p@rvsgroup.com
Mobile: (+91)9842234215
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ABSTRACT
Objective: To find the effect of syringe filter for the sterilization of different milk samples. Raw
cow milk, commercial Pasteurized milk and goat milk was taken as the samples for the analysis.
Method: The sterility testing was done for the three samples with direct inoculation method
using pour plate method and spread plate method. The filtration sterilization was performed by
using single use syringe filter. Result: Highlighted spread plate method as it gives maximum
number of colonies. 437 number of colonies were observed in the goat milk, 184, 326 number of
colonies were found in commercial pasteurized milk and raw cow milk respectively.
Conclusion: We decided to find the effectiveness of the sterilization of the above milk samples
by using syringe filter (minisart single use syringe filter Non-pyrogenic 0.2 micrometer). After
sterilization the number of colonies were nil in both commercial pasteurized milk and goat milk
but 21 number of colonies were still observed in raw cow milk. Hence concluded that the use of
syringe filter shall be for commercial and goat milk but not effective for raw cow milk.
Key words: Milk analysis, test for sterility, direct inoculation method, Syringe filter
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1. INTRODUCTION
Milk is an important and popular source of nutrition that is widely consumed around the world.
Milk for human consumption can be obtained from a number of domesticated animals including
sheep, goat buffalo and cow, whose milk is by far the most consumed. Fresh cow milk contains
approximately 3.5%protein, 80% casein, 15% whey protein, as well as vitamins, and lipids, all of
which provide necessary ingredients for growth. The number of bacteriods present in colon was
huge as much in milk.
[1-4]
It is well documented the composition of milk changes between
breeds, feed and of course species.
[5-7]
. Because they make up a significant proportion of the
content, milk proteins have been a focus of research in order to better understand its makeup,
quality, and health-related properties.
A typical dairy case at a major grocery store today contains numerous choices for the customer.
There is milk labeled with different levels of fat content, and where retail raw milk sales are
allowed, the consumer may choose between conventional, organic, and raw milk products, as
well as homogenized or non-homogenized. In addition to fluid milk, other dairy products include
butter, cheese, cream, ice cream, colostrum, yogurt, kefir, and other fermented dairy products. [812]
Below are some basic definitions of raw and pasteurized milk.
“Raw” or “unpasteurized” refers to a dairy product that has received no heat treatment to destroy
pathogens or spoilage organisms. WAPF promotes a more refined definition for raw milk,
termed “real milk,” that also includes organic, non-homogenized, “grass fed,” and produced from
certain breeds of cattle as criteria. [13-15]
1.1 Sources and Causes of High Bacteria Counts in Raw Milk:
Milk is synthesized in specialized cells of the mammary gland and is virtually sterile when
secreted into the alveoli of the udder. Beyond this stage of milk production, microbial
contamination can generally occur from three main sources from within the udder, from the
exterior of the udder and from the surface of milk handling and storage equipment. The health
and hygiene of the cow, the environment in which the cow is housed and milked, and the
procedures used in cleaning and sanitizing the milking and storage equipment are all key factors
in influencing the level of microbial contamination of raw milk. [16-18] equally important are the
temperature and length of time of storage, which allow microbial contaminants to multiply and
increase in numbers. All these factors will influence the total bacteria count and the types of
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bacteria present in bulk raw milk.
1.2 Milk Storage Temperature and Time:
Refrigeration storage, while preventing the growth of non-psychrotrophic bacteria, will select for
psychrotrophic microorganisms that enter the milk from soiled cows, dirty equipment and the
environment. Minimizing the level of milk contamination from these sources will help prevent
psychrotrophs from growing to significant levels in the bulk tank during the storage period on
the farm or at the dairy plant. [19-21] In general these organisms are mostly not thermoduric and
will not survive pasteurization. The longer raw milk is held before processing (potential up to 5
days; 2 days on the farm, 3 at the plant), the greater the chance that psychrotrophs will increase
in numbers. Holding milk near the legal limit of 7.2°C (45°F) allows much quicker growth than
milk held below 4.4°C (40°F). Although milk produced under ideal conditions may have an
initial psychrotroph population of less than 10% of the total bulk tank count, psychrotrophic
bacteria can become the dominant microflora after 2-3 days at 4.4°C (40°F). [22]
1.3 Syringe filter technique
This syringe filter is well suited for maximum sample recovery such as tissue culture media
preparation, sterile or non sterile filtration and clarification of biological fluids, protein and
enzyme filtrations, hybridization buffers, and other aqueous solutions. Its mainly recommended
to filter probe solutions. Syringe filters are designed to sterilize and remove particulates from
proteinaceous samples. Due to the extremely low holdup volume the negligible protein-binding
characteristics, these syringe filter units are ideal for applications requiring maximum sample
recovery from a standard or presterilized syringe filter.
2. MATERIALS AND METHODS
2.1 Materials: Raw cow Milk, Pasteurized Milk, Goat Milk collected from nearby areas at Sulur,
Coimbatore. Nutrient agar medium, sterile disc paper, cork borer, Syringe filters, digital Colony
Counter singhla make, chemicals are supplied from S.D Fine chemicals Mumbai.
2.2 Methods: Nutrient media was prepared and sterilized by autocloaving at 1210C, 15
lb/Sq.inch for15 minutes. The Glasswares required for the work was sterilized by using hot air
oven at 1800C for 45 minutes. The media and other materials after sterilization were been taken
to the aseptic area under laminar air flow.
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The milk samples were tested for its sterility by direct inoculation technique with pour plate
method and spread plate method. Excluded streak late method as it gives isolated colonies. Upon
completing the direct inoculation technique, the labeled, inoculated petriplates were kept under
incubation condition at 370C for 18-24 hrs. The obtained results were recorded.
The effectiveness of the use of syringe filter for the sterilization of above milk samples were
performed with minisart single use syringe filter, Non-pyrogenic, 0.2 micrometer. Test for the
sterility was performed after sterilizing with syringe filter. The results obtained were recorded by
digital colony counter.
3. RESULTS AND DISCUSSION
The effectiveness of the sterilization of milk samples before and after use of minisart single use
filter, Non-pyrogenic, 0.2 micrometer was compared and analysed. Before sterilization,
maximum 437 number of colonies were obtained in goat milk. Also 326 and 184 number of
colonies was obtained in raw milk and commercial pasteurized milk respectively (table.1). Even
after sterilization, we found around 21 number of colonies in the raw milk where as other two
milk samples have became sterile.
Table.1 Number of colonies observed by direct count method
S.
No
1
2
NUMBER OF COLONIES
SAMPLE
Before
After
Sterilization
Sterilization
spread plate
326
21
pour plate
318
18
spread plate
184
-
pour plate
178
-
spread plate
437
-
pour plate
425
-
Raw Cow milk
Commercial
Pasteurized milk
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METHODS
Goat milk
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3.1 Before sterilization
Figure 1 - Cow raw milk
Figure 2 - Pasteurized commercial milk
Figure 3 - Goat milk
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3.2 After sterilization
Figure 4 - Raw cow milk
Figure 5 - pasteurized commercial milk
Figure 6 - goat milk
Hence the present investigation suggests use the disposable syringe filter is applicable only for
commercial pasteurized milk and goat milk and also not to use syringe filter for raw cow milk as
the effectiveness is not much in restricting all kind of microbes present in the raw milk samples.
4. CONCLUSION
Coxiella burnetii, E-coli, Mycobacterium bovis or tuberculosis, Mycobacterium para
tuberculosis, Salmonella species, Yersini enterocolitica, lacto bacillus, enterobacterogens were
found to be present in all the milk samples. The above bacterial species were identified and
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confirmed by biochemical reactions. The current investigation suggests, that the use of minisart
single use syringe filter, Non-pyrogenic, 0.2 micrometer for the sterilization was found to be
effective with commercial pasteurized milk and goat milk sample. The number of colonies
obtained by direct inoculation technique of test for sterility has become nil after sterilization. At
the same time we are not ready to suggest the same syringe filter for the purpose of sterilizing
raw milk where it fails to restrict many number of microbes which may cause serious hazards to
the human being
5. ACNOWLEDGEMENT
The researchers would like to thank Mr. K Senthil Ganesh, Managing trustee, RVS Educational
trust, Dr. R Venkatanarayanan, Principal, RVS College of Pharmaceutical Sciences,
Mr. C S Kandasamy, Mr. Sam Johnson, research and collaboration wing, RVS College of
Pharmaceutical Sciences, Ms. Bhuvaneswari, skilled microbiology technician for their great
support and guidance for completing this research work successfully.
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