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A Microb ial Study o f

Locally ProducedPasteu rised Milk

Presented by:

Yujna Devi Banjeet

ID: 1115365

Course: BSc

Microbiology (Year 3)

Date: 09/06/2014
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OUTLINE

Introduction

Aims of the study

Methodology Results

Discussion

References
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Page 3

INTRODUCTION

Milk is:

A highly nutritious food.

An ideal medium for microbial growth.

Easily contaminated by pathogenic andnon-pathogenic microorganisms.


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Page 4

INTRODUCTION

Pathogenic microorganisms in milk:

Salmonella spp

Listeria monocytogenes

Staphylococcus aureus

Pathogenic strains of Escherichia coli


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Page 5

INTRODUCTION

Non-pathogenic microorganisms:

Non-pathogenic strains of Escherichia coli

Enterobacteriacea such as Klebsiella spp

and Citrobacter spp

Psychrotrophs such as Pseudomonas

spp.


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Page 6

INTRODUCTION

Milk is pasteurised:

Safer for consumption

Eliminate pathogenic and non-pathogenicmicroorganisms

Increase the shelf life of milk

Pasteurised milk can get contaminatedPost pasteurisation contamination


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AIMS OF THE STUDY

To test the presence of the potential non-

pathogenic, spoilage microorganisms.

To test if the microbial load increases as

the expiry date approaches.

To test if the presence of these

microorganisms affected the components

in milk


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METHODOLOGY

Samples:

3 different brands used (Brand V, SC and

M)

A total of 45 samples were used

5 samples of each brand were analysed

weeklySample collection:

Samples were collected from 3 different

outlets


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METHODOLOGY

Experimental design:

All the microbiological tests were carried

out in duplicates

A tenfold dilution was carried out up to a

concentration of 10-4.

Figure 1: The steps involved in serial dilution
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METHODOLOGY

1. Total Viable Count (Pour plate method and Milk Plate Count agar)

2. Coliform and Staphylococcal count (Spread plate method; MacConkey and

Baird Parker agar.)

3. Further detection of coliforms (Brilliant Green Bile Lactose Broth and Eosin

Methylene Blue agar.)

4. Physico-chemical and biochemical tests of milk such as peroxidase test.

5. Identification (Gram stain and a set of biochemical tests such as IMViC

tests and Catalase test)

6. Confirmatory molecular analyses (DNA extraction and Polymerase Chain

Reaction with a set of specific primers)

7. Samples giving no positive PCR result (amplified with16S primer and

sequenced)


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METHODOLOGY

The primers used for each microorganism

are shown in Table 1.

Table 1: The primers used for each microorganismMicroorganisms Primers Sequences

Escherichia coli TEcol553

Tecol 754

5-TGGGAGCGAAAATCGTG-3

5-CAGTACAGGTAGACTTCTG-

3

Klebsiella

pneumoniae

KP(F)

KP(R)

5-CAACGGTGTGGTTACTGACG-3

5-TCTACGAAGTGGCCGTTTTC-3

Staphylococcus

aureus

GSEBR-1

GSEBR-2

5-GTATGGTGGTGTAACTGAGC-3

5-

CCAAATAGTGACGAGTTAGG-3


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RESULTS

Total Viable Count:

Brand V: 1.51x1024.74x103CFUs/ml

Brand SC: 06.82x102 CFUs/ml

Brand M: 1X1023.69X103CFUs/ml


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RESULTS

Coliform count:

Brand V: 1x1023.75x105CFUs/ml

Brand SC: 02x104CFUs/ml

Brand M: 5.55x1034.15x104 CFUs/ml


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RESULTS

Staphyloccus count: Colonies were formed on plates inoculated

with the mother solution (MS) only.

Brilliant Green Bile lactose Broth:

All three brands gave similar results:

MS: Cloudy + Gas formation

10-1, 10-2: Cloudy only

Figure 2: Test-tubes of BGBLB

showing turbidity


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RESULTS

Morphological characteristics:

MacConkey agar:

Small, circular, raised, pink and non-mucoid (Escherichia coli)

Large, dome-shaped, pink

mucoid (Klebsiella spp)Few yellow round (Shigella)

Figure 3:Pink and yellow colonies formed

on MacConkey agar


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RESULTS

Morphological characteristics:

Baird Parker agar:

Black, shiny and convex colonies with anopaque zone

Eosin Methylene Blue agar:

Pink mucoid (Klebsiella spp)Green with a metallic sheen

(Escherichia coli)Figure 4:The colonies formed on EMB

E.coli (left) and Klebsiella (right).


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RESULTS

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6

logc

fu

Replicate number

Brand V

4 days beforeexpiration

1 day beforeexpiration


00.5

1

1.5

2

2.5

0 2 4 6

lo

gc

fu

Replicate number

Brand SC

1 day beforeexpiration

3 days before

expiration


0

0.5

11.5

2

2.5

3

0 2 4 6

logcfu

Replication number

Brand M

3 days before

expiration6 days beforeexpiration


The graphical representations below give an indication of the changes in

the log CFU of the TVC of the three different brands.

Figures 5,6&7: The changes in the log cfu of the different replicates of brand V, SC

and M respectively


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RESULTS

Statistical results:

Kruskal Wallis test:

Very small difference in the TVC of the threebrands over the three weeks.

Tukeystest (5% significance level):Significant difference in the coliform count

between V and the two others.


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RESULTS

Fat content:

V:3.4%, SC: 3.8%, M:

2.4%

Titratable acidity:V: 0.167, SC: 0.171, M:

0.142

pH:

V: 6.91, SC:6.76, M:6.72

Peroxidase test:

All gave negative results

0

0.5

1

1.5

2

2.5

3

3.5

4

Week 1

Week 2

Week 3

Figure 8: The difference in the average fat content of

the three brands

Physicochemical and biochemical results:


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RESULTS

Biochemical tests results: IMViC results for coliforms

The results are depicted in Table 2.

Biochemical test Results

Indole 44.4% (+), 55.6% (-)

Methyl Red 55.6% (+), 44.4% (-)

Voges Proskauer 88.9% (-)Citrate 55.6% (+), 44.4% (-)

Catalase results for Staphy lococcus

spp:

They all gave positiveresults.

Table 2: The percentage of samples which gave positive or negative results for

each biochemical test.


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RESULTS

Table 3: The samples present in the

labelled lanes and the band sizes

Lane Sample Size of

band

Size of

the

amplicon

H SC1R1 400 bp 212 bp

I SC2R2 400 bp 212 bp

N M1R1 500 bp 212 bp

S M3R2 500 bp 212 bp

PCR results for suspected E.coli :

Figure 9: The gel electrophoresis for thePCR result of the suspected E.coli


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RESULTS PCR results for suspected Klebsiel la

spp:

Presence of bands

in all the lanes.

Size of amplicon:

108 bp

Sizes of bands:

200 bp-500 pb


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RESULTS

PCR results for suspected

Staphylococcus spp:

No bands with specific primers (GSEBR-1

&GSEBR-2)

Sequencing results:

The microorganism identified as being

either an Enterobacter spp or

Lactobacillus plantarum


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DISCUSSIONPhysico-chemical changes observed:

Fat con ten t:

Brand SC and V had a normal fat content.

Brand M had lowest fat content- milk was diluted

with water.

Titratab le acid i ty and pH:

They were within the required range for the three

brands.

Biochemical test of milk:

Peroxidase test:

All negative results; Milk was heated properly.


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DISCUSSION

PCR identification of suspected E.col i :

Primers successfully used to identify

environmental E.coli.

Primers bind to tufgene; amplicon of size

212 bp.

Bands were of different sizes; E.coli

contain more than 1 tuf gene.


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DISCUSSION

PCR identification of suspected

Klebsiel la spp:

The primers used to identify Klebsiella

pneumoniae.

Primers amplify the rpoBwith a product

size of 108 bp.

The bands were of different sizes; the

strains were not K.pneumoniae.


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DISCUSSION

Microbiological analysis of milk samples:

Brand V the most contaminated one.

The number of microorganisms do not increase

as expiry date approaches.

The predominating coliform isolated was

Klebsiella spp.

Presence of microorganisms:Use of unclean equipment

Unethical practices

Unhygienic handling


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CONCLUSION

Regular verification of processing plants.

Microbiological analysis of raw material

and end-products.

Tests on environmental samples.

Operators have to follow the principles of

the HACCP system.


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REFERENCES Alam, K., Dwipayan, S., Hossain, T. 2010. Chemical and Microbiological Quality

Assessment of Raw and Processed Liquid Market Milks of Bangladesh. Journal ofDairy Sciences. 4(4), 28-34.

Albanell, E., Caja, G., Casals, R., Rovai, M., Salama, A., Such, X. 2003.

Determination of Fat, Protein, Casein, Total Solids and Somatic Cell Count in Goats

Milk by Near-Infrared Reflectance Spectroscopy. Journal of AOAC International. (86),

746-753.

Mirkena, A. 2010. Microbiological Safety of Pasteurized and Raw Milk from MilkProcessing Plants in and around Addis Ababa. Thesis (MSc). Addis Ababa

University. [online]. Available at:

http://etd.aau.edu.et/dspace/bitstream/123456789/3306/1/ABERRA%20ASSEFA%20

MIRKEMA.pdf [Accessed 20th December 2013].

Park, Y.K., Koo, H.C., Kim, S.H., Hwang, S.Y., Jung, W.K., Kim, J.M., Shin, S., Kim,

R.T. and Park, Y.H. 2007. The Analysis of Milk Components and Pathogenic BacteriaIsolated from Bovine Raw Milk in Korea. Journal of Dairy Science. [Website:

http://www.journalofdairyscience.org/article/S0022-0302(07)72013-1/fulltext]

[Accessed 18th August 2013]

Murphy, S. 1996. Sources and causes of high bacterial count in raw milk. Cornell

University. New York. Pp 32-34.
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