1. Comparison of different phenol- chloroform based ...shodhganga.inflibnet.ac.in/bitstream/10603/7221/4/04_chapter 3.pdf · In this part of the study, four different phenol chloroform

Results and Discussion

163

1. Comparison of different phenol- chloroform based methods for DNA

isolation from E.coli, S.aureus and S.typhi

In this part of the study, four different phenol chloroform based methods were

evaluated for DNA extraction from the three bacterial strains namely E. coli, S.

typhi and S. aureus.

Extraction of DNA was carried out by using 4 different phenol-chloroform based

methods. DNA was extracted and the concentration of DNA was checked by UV-

spectrophotometer.

Table 1, Table 2 and Table 3 shows the O.D. of extracted DNA from E. coli, S.

aureus and S. typhi respectively. Figure 1 shows the comparison O.D. of E. coli

DNA samples obtained by four different methods. It can be seen that method 4

gives the 260/280 ratio near to 1.8 for E. coli DNA, indicating that highest DNA

was recovered from E. coli bacterial cells using this method. Thus, out of the four

studied methods, method 4 is most efficient for E. coli DNA extraction.


164

Table 1. O.D. of extracted DNA by different methods from E. coli

Method O.D. at 260

nm

O.D. at 280

nm

260/280 ratio

Method 1 0.77 0.40 1.91

Method 2 0.68 0.404 1.68

Method 3 0.75 0.436 1.72

Method 4 0.83 0.453 1.83

Results are mean of three observations


165

0

0.5

1

1.5

2

2.5

Method 1 Method 2 Method 3 Method 4

Different methods for DNA extractions

O.D

.

O.D. at260 nm

O.D. at280 nm

260/280ratio

Figure 1. O.D. of E. coli DNA samples obtained by four different phenol-

chloroform based DNA isolation methods


166

Table 2 and Figure 2 show the comparison O.D. of S. aureus DNA samples

obtained by four different methods. It can be seen that method 2 gives the

260/280 ratio near to 1.8 for the S. aureus DNA, indicating that out of four studied

methods, method 2 is most efficient for S. aureus DNA extraction.

Table 3 and Figure 3 show the comparison O.D. of S. typhi DNA samples

obtained by four different methods. It can be seen that method 4 gives the

260/280 ratio near to 1.8 for the S. typhi DNA, indicating that out of four studied

methods, method 4 is the most efficient for S. typhi DNA extraction.


167

Table 2. O.D. of extracted DNA by different methods from S. aureus

Method O.D. at 260

nm

O.D. at 280

nm

260/280 ratio

Method 1 0.73 0.426 1.71

Method 2 0.78 0.421 1.85

Method 3 0.65 0.329 1.97

Method 4 0.63 0.370 1.70


168

0

0.5

1

1.5

2

2.5


different methods for DNA extraction

O.D

.

O.D. at 260 nm

O.D. at 280 nm

260/280 ratio

Figure 2. O.D. of S. aureus DNA samples obtained by four different phenol-



169

Table 3. O.D. of extracted DNA by different methods from S. typhi

Method O.D. at 260 nm O.D. at 280 nm 260/280 ratio

Method 1 0.72 0.444 1.62

Method 2 0.69 0.367 1.88

Method 3 0.62 0.352 1.76

Method 4 0.78 0.428 1.82


170

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2


Different methods for DNA extraction

O.D

.

O.D. at 260 nm

O.D. at 280 nm

260/280 ratio

Figure 3. O.D. of S. typhi DNA samples obtained by four different phenol-



171

Extraction of genomic DNA in a reasonably intact and pure state forms the first

step in studies attempting to understand the molecular aspects of bacterial

pathogenesis, physiology, and epidemiology. Chemical methods for extraction of

genomic DNA rely primarily on the use of lysozyme in conjunction with other

lipolytic and proteolytic enzymes. However, many bacteria, such as

Staphylococcus aureus, are resistant to lysozyme (Lachica et al., 1971).

Increased recovery of genomic DNA, in particular from gram-positive bacteria

was obtained when the cells were pretreated with 70% ethanol (Kalia et al.,

1999). It is probable that 70 % ethanol exposure induces changes in the bacterial

cell wall and membrane, thereby increasing cellular porosity that could

accentuate subsequent bacterial lysis resulting in greater recovery of genomic

DNA.

When DNA is isolated from organisms, frequently there remains protein present

in the DNA solution; protein is tightly bound to DNA and complete removal of

protein is not always possible. To determine the concentration and purity of the

DNA solution, the absorbance of UV light is measured in a spectrophotometer.

Both protein and DNA absorb UV light, but they have different absorbance

curves. The peak of light absorption is at 260 nm for DNA and at 280 nm for

protein. When a spectrum of absorbance with varying wave length is run, both

curves slightly overlap in the area between, and including, 260 and 280 nm.

Thus, when a solution contains both protein and DNA, absorbance at 260 nm is

mainly due to the DNA present, but a little bit by the protein. At 280 nm, it is due

to the presence of protein. By dividing the two absorbance values, one can

calculate the purity of the DNA solution. If the solution relatively free of protein,

then one can take the absorbance at 260 nm as a measure for concentration of

DNA (Peng et al., 2007).


172

Of the 4 phenol chloroform based methods, method 4 was the most efficient

method for DNA extraction from E. coli. The same method also showed optimum

results when used for DNA isolation from S. typhi. However, for DNA extraction

from S. aureus, method 2 showed the best results.


173

2. Molecular and bacteriological examination of milk from

different milch animals with special reference to coliforms

The present study was planned to assess the quality of milk from different milch

animals to detect the presence of coliforms. For this, microbiological as well as

molecular methods (PCR) were used.

A total of twenty samples of milk were analysed. Raw milk of cow (5 samples),

buffalo (5 samples) and goat (5 samples) were collected from local vendors and

farmers of Vallabh Vidyanagar and Anand in the month of April, 2006. Amul

brand pasteurized milk (5 samples) was purchased from retailed outlets.

Cow milk samples were labeled as C1 to C5, buffalo milk samples from B1 to B5,

goat milk samples from G1 to G5 and pasteurized milk samples from P1 to P5.

The study was carried out in two stages. Milk samples from different milch

animals and pasteurized milk samples were analyzed during the first stage by

agar plate and sugar fermentation methods. In the second stage, confirmation of

bacteria was carried out by Polymerase Chain reaction.

Microbial analysis of milk samples

Total Plate count of milk samples

The results of total plate count are presented in Table No. 1. Total plate count in

milk samples ranged from 3.26 x 103 to 3.44 x 105 cfu/ml. Total plate count in

cow milk samples ranged from 0.83 x 104 to 3.44 x 105 cfu/ml and of buffalo milk

sample ranged from 1.07 x 104 to 9.31 x 104 cfu/ml. Among all milk samples,

goat milk showed highest plate count results ranging from 3.26 x 103 to 6.55 x

105. Plate count results in pasteurized milk samples ranged from 0.54 x 104 to

8.71 x 104 cfu/ml. Among all milk samples, pasteurized milk samples showed the

least number of cfu/ml.


174

VRBA count of milk samples

The results of VRBA count are presented in Table No. 2. VRBA count in milk

samples ranged from 1.53 x 101 to 8.73 x 102 cfu/ml.

VRBA count in cow milk samples ranged from 4.17 x 102 to 6.81 x 102 cfu/ml and

in buffalo milk samples, it ranged from 2.28 x 102 to 5.21 x 102 cfu/ml. Among all

milk samples, goat milk samples showed VRBA results ranging from 4.87 x 102

to 8.73 x 102 cfu/ml. VRBA results in pasteurized milk samples ranged from 1.53

x 101 to 1.31 x 102 cfu/ml.

Yeast and mold count of milk samples

The results of Yeast and mold count are presented in Table No. 3. Yeast and

mold count in all milk samples ranged from 1.02 x 101 to 2.23 x 102 cfu/ml. Yeast

and mold count in cow milk samples ranged from 1.04 x 102 to 2.23 x 102 cfu/ml

and in buffalo milk samples, it ranged from 1.41 x 102 to 2.12 x 102 cfu/ml. Goat

milk showed Yeast and mold count results ranging from 1.49 x 102 to 2.17 x 102

cfu/ml. Only one pasteurized milk sample showed the presence of Yeast and

molds, which was 1.02 x 101 cfu/ml. Highest count of yeast and mold was

observed in cow milk samples, while the least count of yeast and mold was

observed in pasteurized milk samples.

BGLB results of milk samples

BGLB (Brilliant Green Lactose Bile broth) results are presented in Table 4. Out

of a total of 20 milk samples, 9 samples showed positive results in BGLB broth.

LST results of milk samples

LST (Lauryl Sulphae broth) results are presented in Table 5. Out of total 20 milk

samples, 9 samples showed positive result in LST broth confirming the presence

of coliform group of bacteria. The same 9 samples were also positive for BGLB

test.


175

Table 1. Total Plate count of milk samples

Sample

No.

Total Plate

count

(cfu/ml)

Total Plate

Count

(log cfu/ml)

C1 4.51 x 104 4.451

C2 5.74 x 104 4.574

C3 3.44 x 105 5.344

C4 0.83 x 104 4.083

C5 6.58 x 104 4.658

B1 3.94 x 104 4.394

B2 4.12 x 104 4.412

B3 7.83 x 104 4.783

B4 1.07 x 104 4.107

B5 9.31 x 104 4.931

G1 2.74 x 105 5.274

G2 2.01 x 104 4.201

G3 6.55 x 105 5.655

G4 7.72 x 104 4.772

G5 3.26 x 103 3.326

P1 8.71 x 104 4.871

P2 0.54 x 104 4.054

P3 4.76 x 104 4.476

P4 2.64 x 104 4.264

P5 8.15 x 104 4.815

cfu = colony forming unit; C1-C5: Cow milk

B1-B5: Buffalo milk; G1-G5: Goat milk

P1-P5: Pasteurized milk


176

Table 2. VRBA count of milk samples

Sample

No.

VRBA

count

(cfu/ml)

VRBA

Count

(log cfu/ml)

C1 6.81 x 102 2.681

C2 4.81 x 102 2.481

C3 5.71 x 102 2.571

C4 6.70 x 102 2.670

C5 4.17 x 102 2.417

B1 2.28 x 102 2.228

B2 4.87 x 102 2.487

B3 5.21 x 102 2.521

B4 5.10 x 102 2.510

B5 2.82 x 102 2.282

G1 5.71 x 102 2.571

G2 8.73 x 102 2.873

G3 7.7 x 102 2.770

G4 5.76 x 102 2.576

G5 4.87 x 102 2.487

P1 2.21 x 101 1.221

P2 1.31 x 102 2.131

P3 1.71 x 101 1.171

P4 1.53 x 101 1.153

P5 1.68 x 101 1.168





177

Table 3. Yeast and mould count of milk samples

Sample

No.

Yeast and

mould

count

(cfu/ml)

Yeast and

mould

Count

(log cfu/ml)

C1 1.37 x 102 2.137

C2 2.23 x 102 2.223

C3 -- --

C4 1.04 x 102 2.104

C5 -- --

B1 1.41 x 102 2.141

B2 -- --

B3 -- --

B4 2.12 x 102 2.212

B5 -- --

G1 1.49 x 102 2.149

G2 -- --

G3 2.08 x 102 2.208

G4 2.17 x 102 2.217

G5 -- --

P1 -- --

P2 -- --

P3 -- --

P4 1.02 x 101 1.102

P5 -- --





178

Table 4. Gas production by milk samples in

Brilliant Green Lactose Bile Broth (BGLB)

Sample

No.

BGLB

reaction

C1 --

C2 --

C3 + ve

C4 --

C5 + ve

B1 + ve

B2 + ve

B3 --

B4 + ve

B5 + ve

G1 --

G2 + ve

G3 --

G4 + ve

G5 + ve

P1 --

P2 --

P3 --

P4 --

P5 --

-- = absent; + ve = Present

C1-C5: Cow milk; B1-B5: Buffalo milk

G1-G5: Goat milk; P1-P5: Pasteurized milk


179

Table 5. Gas production by milk samples in

LST (Lauryl tryptose broth)

Sample

No.

LST

reaction

C1 --

C2 --

C3 + ve

C4 --

C5 + ve

B1 + ve

B2 + ve

B3 --

B4 + ve

B5 + ve

G1 --

G2 + ve

G3 --

G4 + ve

G5 + ve

P1 --

P2 --

P3 --

P4 --

P5 --

-- = absent; + ve = Present

C1-C5: Cow milk; B1-B5: Buffalo milk

G1-G5: Goat milk; P1-P5: Pasteurized milk


180

ImViC test

Results from ImViC tests are presented in Table 6. Out of the total 20 milk

samples tested, 9 samples showed positive result for indole production. None of

the samples showed positive result in VP broth. 9 samples showed positive

results in Methyl Red test. None of the samples were found to be positive for

citrate production.

Indole positive bacteria such as Escherichia coli produce tryptophanase, an

enzyme that cleaves tryptophan, producing indole and other products. When

Kovac's reagent (p-dimethylaminobenzaldehyde) is added to a broth with indole

in it, a dark pink color is developed (Bacteriological Analytical Manual, 1998). The

methyl red (MR) and Voges-Proskauer (VP) tests were read from a single

inoculated tube of MR-VP broth. After 24-48 hours of incubation the MR-VP broth

was split into two tubes. One tube was used for the MR test; the other was used

for the VP test. MR-VP media contains glucose and peptone. All enteric bacteria

oxidize glucose for energy; however the end products vary depending on

bacterial enzymes. Both the MR and VP tests were used to determine what end

products result when the test organism degrades glucose. E. coli is one of the

bacteria that produce acids, causing the pH to drop below 4.4. When the pH

indicator methyl red is added to this acidic broth it will be cherry red, a positive

MR test (B.A.M., 1998).

In the present study, the pasteurized milk samples showed the presence of

bacteria. Gruetzmacher and Bradley (1999) stated that factors that limit the shelf

life of refrigerated pasteurized milk and the microbial quality of raw milk are time

and temperature of pasteurization, presence and activity of post pasteurization

contaminants, types and activity of pasteurization resistant microorganisms and

the storage temperature of milk after pasteurization.


181

Table 6. ImViC test of milk samples

Sample

No.

Indole

production

MR-VP Methyl Red Citrate

production

C1 - ve - ve - ve - ve


C3 + ve - ve + ve - ve


C5 + ve - ve + ve - ve

B1 + ve - ve + ve - ve


B3 - ve - ve - ve - ve



G1 - ve - ve - ve - ve

G2 + ve - ve + ve - ve

G3 - ve - ve - ve - ve



P1 - ve - ve - ve - ve





+ ve = Positive, - ve = Negative


182

PCR Analysis

PCR analysis of milk samples is expressed in Figure 1. Samples C3, C5, B1, B2,

B4, B5, G2, G4 and G5 showed amplification using primers specific for E. coli.

Approximately 150 bp fragment was amplified by the primers. Amplification was

observed in two samples of cow milk, four samples of buffalo milk and three

samples of goat milk. Pasteurized milk samples did not show any amplification.

This could be due to the pasteurization time and temperature employed at which

majority of the heat sensitive microorganisms get destroyed.


183

Figure 1. Samples from different milch animals showing amplification by E. coli

specific primers

M1- 100 bp marker; M2- 10 bp marker; C- control;


184

Pathogenic bacteria in milk have been a major public health concern since the

early days of the dairy industry. Many diseases are transmissible via milk

products. Traditionally raw or unpasteurised milk has been a major vehicle for

transmission of pathogens (Vasavada, 1988). The health of dairy herd, milking

conditions etc. are basic determinants of milk quality. Another source of

contamination by microorganisms is unclean teats. The use of unclean milking

and transport equipment also contribute to the poor hygienic quality (Bonfoh et

al., 2003). In the present study, the samples of raw milk examined contained

coliform group of microorganisms. This indicates that the analyzed milk samples

can prove to be a potential risk for public health when consumed or when used in

the production of dairy products such as cheese, butter, cream and ice cream

without being pasteurized or when subjected to insufficient heat processing.

From the results of the present study, it was found that majority of the samples

were contaminated by coliform bacteria. Coliform bacteria were found in milk

samples of different origin of milk. In operational conditions, mainly a failure to

observe hygienic rules of milking process contributes to the impairment of

microbial quality of milk (Jayarao et al., 2004). Tando et al. (2000) investigated

more than reported that 35.2 % of food handlers were asymptomatic carriers of

staphylococcus aureus, and that 90.4 % of raw milk samples among more than

3200 investigated dairy products by them.

Oksuz et al. (2004) reported E. coli 0157:H7 at the rate of 1% in 100 samples of

raw milk. Soomro et al. (2002) isolated E. coli in 57% of the 100 raw milk

samples. Coliforms and S. aureus are good indicators of the standard of hygiene

and handling. According to Harrigan and McCance (1976), coliform bacterial

count should be less than 200 cfu/g in food. The existence of the Coliforms has

been considered as leading to the fact that the product was subject to process

under inefficient hygienic conditions (Harrigan and McCance, 1976; Altug and

Bayrak, 2003).


185

A high level of coliform of the fresh farm milk can indicate the evidence of

unhygienic conditions of the product (Altug and Bayrak, 2003). Collins et al.

(1995) reported that E. coli and coliform bacilli which belong to the family of

Enterobacteriaceae may indicate evidence of contamination or pollution

especially of fecal nature. Enterobacteriaceae include other organisms, like

important pathogens such as salmonella and various non-lactose fermentors that

may be present in human and animal faeces. The bacterial count of milk is used

to measure its sanitary quality and most grading of milk is on the basis of some

method for estimating numbers (Collins et al., 1995).

Post pasteurization contamination has received most of the attention and is

considered to be the factor, which limits shelf life in the majority of cases (Waes,

1982). Waes (1982) found out that Pasteurized milk, which was collected from

the local shops, showed different values for standard bacterial counts. The higher

count of coliform bacteria in the milk might be due to improper handling, poor

cleaning and storage of equipments etc. as stated by Hayes et al. (2001).

The total viable count of fresh milk samples in the present study showed a mean

value of 4.572 log cfu/ml. Milk can be contaminated with different kinds of

microorganisms due to direct or indirect contact with any source of external

contamination during the steps of milking, collection, packing and transport.

Direct physical contact of milk with unclean surfaces such as those of milking

utensils, udders and teats, and the hands of milkers besides environmental

factors such as the design and cleanliness of buildings and installations, the

adequacy of the water supply, the manner in which the manure and other wastes

are disposed of, and the amount of dust in the immediate surroundings are

important in so far as they may contribute to the microbial contamination of

surfaces with which milk comes into contact (Hayes et al., 2001).

During milking operation, however, milk may be exposed to contamination from

the animal, especially the exterior of the udder and adjacent areas. Bacteria

found in manure, soil, and water may enter from this source. Such contamination

can be reduced by clipping the cow, and washing the udder with water or a

germicidal solution before milking. Contamination of cow with manure, soil, and


186

water may also be reduced by paving and draining barnyards, keeping cows from

stagnant pools, and cleaning manure from the barns or milking parlors.

Pasteurization kills pathogens that may enter the milk and improves the keeping

quality of milk (Hayes et al., 2001).

PCR methods are mostly used for the detection of microorganisms in different

types of food materials. These methods often allow better specificity compared to

traditional biochemical identification methods. In the present study, colonies

growing on the Nutrient agar plate were given pre-enrichment. This pre-

enrichment step was performed to achieve appropriate sensitivity. Another critical

component of the PCR assay is the inclusion of an internal positive control that

indicates PCR failures, e.g., through carry over of PCR inhibitors. Differentiation

of bacterial foodborne pathogens beyond the species level also provides exciting

opportunities to better understand the biology of bacterial strains and subtypes,

including differences in their ability to cause human foodborne disease (Maurer,

2006).

Samples analyzed in the present study can contribute a potential risk for public

health in cases when it is consumed or used in the production of dairy products

such as cheese, butter, cream and ice cream without being pasteurized or being

subjected to insufficient heat processing. Moreover, PCR is less labor intensive

and more rapid than bacterial culturing followed by conventional methods of

bacterial identification (Maurer, 2006).

The obtained results indicate that strict hygienic measures should be applied

during production, processing and distribution of milk and it’s products to avoid

contamination. Periodical inspection must be done by specialists on the dairy

farms to minimize milk contamination with different types of microorganisms.

Efficient cleaning and sanitization of farm dairy utensils must be done to improve

the quality of raw milk and consequently the related dairy products. Milk and milk

products should be kept under refrigeration at all times and the practice of

storage at room temperature should be discouraged.


187

3. Microbiological and molecular detection of E. coli, S. typhi

and S. aureus from milk samples

This part of study was carried out to detect the presence of three organisms

namely E.Coli, S. aureus and S. typhi in different milk samples available in the

local market.

10 different raw milk samples were collected from the local areas of V.V. Nagar

under aseptic conditions, samples were serially diluted and after pre trials, the

aliquots from 10-4 dilution was plated on nutrient agar, VRBA and EMB, for total

plate count, coliform count and E. coli count, respectively. From nutrient agar

loopful of colony was preserved in either glycerol or sterile D/W. Prior to analysis,

the culture was transferred to enrichment broth (Luria broth) for 18 hours for

activation.

For PCR analysis, preserved colonies were transferred to sterile distilled water,

vortexed for 5 to 10 sec and boiled at 95°C for 10 min and immediately

transferred to ice for 5 min, centrifuged and supernatant was directly taken for

PCR reaction.

Bacterial counts and PCR results are discussed in this section.

Microbiological analysis of milk samples

Collected milk samples were analyzed for total plate count, EMB count, VRBA

and yeast and mold count.

Total plate count

The total plate count results of milk samples are presented in Table No. 1. Total

plate count in all samples ranged from 2.31 × 104 to 5.12 × 106 cfu/ml. Sample

No. 5 showed highest total plate count while sample No. 1 showed lowest plate

count.

EMB count

The EMB (Eosin Methylene Blue) plate count results of milk samples are

presented in Table No. 2. EMB count in all samples ranged from 2.13 × 101 to


188

3.14 × 102 cfu/ml. Sample No. 1 showed highest plate count while sample No. 7

showed lowest plate count on EMB plates.

VRBA count results

The VRBA (Violet Red Bile Agar) plate count results of milk samples are

presented in Table No. 3. VRBA count in all samples ranged from 1.61 × 101 to

7.24 × 101 cfu/ml. Sample No. 2 showed highest plate count while sample No. 10

showed lowest plate count results in VRBA plates.

Yeast and Mold count results

The yeast and mold count results of milk samples are presented in Table No. 4.

Yeast and mold count in all samples ranged from 2.21 × 101 to 1.29 × 102 cfu/ml.

Sample No. 4 showed highest count while sample No. 8 showed lowest count on

Potato Dextrose Agar plates.


189

Table 1. Total plate count of milk samples

Sample No. CFU/ml log CFU/ml

1 2.31 × 104 4.23

2 7.92 × 104 4.79

3 1.21 × 106 6.121

4 4.15 × 104 4.415

5 5.12 × 106 6.512

6 8.27 × 105 5.827

7 5.83 × 105 5.583

8 1.79 × 106 6.179

9 8.14 × 105 5.814

10 3.86 × 105 5.386


190

Table 2. EMB plate count of milk samples

Sample No.

CFU/ml

EMB plate count

(log CFU/ml)

1 3.14 × 102 2.314

2 1.12 × 102 2.112

3 7.61 × 101 1.761

4 8.27 × 101 1.827

5 8.55 × 101 1.855

6 1.83 × 102 2.183

7 2.13 × 101 1.213

8 3.37 × 101 1.337

9 3.20 × 101 1.320

10 2.72 × 101 1.272


191

Table 3. VRBA plate count of milk samples

Sample No.

CFU/ml

VRBA plate

count (log

CFU/ml)

1 5.21 × 101 1.521

2 7.24 × 101 1.724

3 2.85 × 101 1.285

4 3.32 × 101 1.332

5 2.91 × 101 1.291

6 1.97 × 101 1.197

7 1.77 × 101 1.177

8 2.28 × 101 1.228

9 2.39 × 101 1.239

10 1.61 × 101 1.161


192

Table 4. Yeast and mold count of milk samples

Sample No. Yeast and mold

count (CFU/ml)

Yeast and

mold count

(log

CFU/ml)

1 8.24 × 101 1.824

2 6.12 × 101 1.612

3 7.31 × 101 1.731

4 1.29 × 102 2.129

5 4.96 × 101 1.496

6 5.18 × 101 1.518

7 7.91 × 101 1.791

8 2.21 × 101 1.221

9 3.83 × 101 1.383

10 6.38 × 101 1.638


193

PCR results of milk samples

Figures 1, 2 and 3 show the PCR results of milk samples. Figure 1 shows PCR

results for E. coli. It can be seen from the figure that sample numbers 2,4,5,6, 8,

9 and 10 were found to be positive for the presence of E. coli. Figure 2 shows the

PCR results for S. aureus. Sample number 2,4,5,6 and 10 were found positive

for the presence of S. aureus. Figure 3 shows the PCR results for S. typhi. It can

be seen from the figure that none of the samples were found to be positive for

salmonella.


194

Figure 1. Detection of E. coli from milk samples by PCR

C: Control; M: Marker, 1-10: milk samples


195

Figure 2. Detection of S. aureus from milk samples by PCR



196

Figure 3. Detection of S. typhi from milk samples by PCR



197

The quality of milk is determined by aspects of composition and hygiene. Due to

it’s complex biochemical composition and high water activity, milk serves as an

excellent culture medium for the growth and multiplication of many kinds of

microorganisms. Therefore in the processing of milk, some of the

microorganisms may produce undesirable effects and some micro-organisms

produce food infections which can increase the likelihood of infection of the

consumer‘s food. The contamination of milk and milk products is largely due to

the human factor and unhygienic conditions. Usually milk is contaminated with

different kinds of microorganisms at milk collecting places. Milk is a major part of

human food and plays a prominent role in the Indian diet. Approximately 50

percent of the milk produced is consumed as fresh or boiled, one sixth as yoghurt

or curd and the remaining is utilized for manufacturing of indigenous varieties of

milk products such as Ice cream, Butter, Khoa, Paneer, Rabri, Kheer, Burfi and

Gulabjaman (Anjum et al., 1989). The manufacture of these products is based on

traditional methods without any regard to the quality of raw material used and/ or

the hygienic quality of the products. Under such conditions many microorganisms

can find access to the milk products. Among all micro-organisms, Escherichia

coli is the frequently contaminating organism and is a reliable indicator of fecal

pollution generally in insanitary conditions of water, food, milk and other dairy

products (Diliello,1982). Martin et al., (1986) reported two cases of hemolytic

uraemic syndrome which provide evidence that raw milk may be a vehicle of

transmission of E.coli O157: H7, both affected persons consumed raw milk.

Recovery of E. coli from food is an indicative of possible presence of

enteropathogenic and/or toxigenic micro-organism which could constitute a

public health hazard. Enteropathogenic E. coli (EEC) can cause severe diarrhoea

and vomiting in infants and young children (Anon, 1975). In 1971 USA faced an

outbreak of food poisoning in which 387 persons were suffered with

Enteropathogenic E. coli due to the consumption of imported French cheese

(Marrier, 1973).


198

In the present study, the microbiological and molecular analysis of the milk

samples revealed that the highest number of samples was contaminated with E.

coli as compared to S. aureus. However, S. typhi was found to be absent in all

the milk samples.

According to Mosupye and Van Holy (1999), the method of production, handling,

transportation and marketing of milk is entirely dependent upon the traditional

system. Such a system could pose a favourable environment for bacterial

contamination. The unclean hands of workers, poor quality of milk, unhygienic

conditions of manufacturing unit, inferior quality of material used and water

supplied for washing the utensils could be sources of accelerating the bacterial

contamination of milk products and the post manufacturing contamination

(Grewal and Tiwari, 1990; Kulshrestha, 1990).

Although E. coli is a frequently occurring organism in milk and its products, the

incidence of the species of E. coli itself in milk and milk products as a possible

cause of food borne disease is insignificant because E. coli normally is a

ubiquitous organism (Hahn, 1996). However, the occurrence of pathogenic

strains of E. coli in milk products can be unhygienic, which could be hazardous

for consumers.


199

4. A comparison of methods for the detection of Escherichia

coli O157:H7 from artificially-contaminated dairy products

using PCR

The present study was planned to evaluate various DNA extraction methods for

detection of E. coli O157:H7 from artificially contaminated dairy products (liquid

skim milk, skim milk powder, cheese) with the help of PCR. The methods

evaluated were (i) Solvent method and (ii) Concentration method

Comparison of PCR detection limits for the solvent method and the

bacterial concentration method

Solvent method and concentration method were evaluated for their efficacy of E.

coli DNA extraction from liquid skim milk, skimmed milk powder and cheese. The

extracted DNA was amplified by PCR using E. coli specific primers. The

amplified PCR products were run on agarose gel electrophoresis to evaluate the

efficacy of the methods used for DNA extraction from food materials.

Results are expressed in Table 1. When both the methods were applied to liquid

skim milk, the solvent-based method provided higher PCR detection limits

compared to the bacterial concentration technique. In this case, the final

detection limits were 103 cfu/ ml using the solvent technique and 105 cfu /ml for

the concentration method (Table 1 and Figure 1). It can be seen that PCR

amplicon was not obtained on the inoculum level of 101 and 102 cfu/ml in the

solvent method, whereas in bacterial concentration method PCR amplicons were

not obtained in the inoculum level upto 105 cfu/ml. This indicates the better

detection limits of the solvent method compared to the concentration method.


200

Table 1. Detection of PCR products in artificially contaminated dairy

products with two DNA extraction methods

Food

Material

Method Inoculum level of E. coli O157:H7

101 102 103 104 105 106 107

Liquid

Skim milk

Solvent - - + + + + +

concentration - - - - - + +

Skim milk

powder

Solvent - - - - - + +

Concentration - - - - + + +

cheese solvent - - - + + + +

concentration - - - - + + +

+ : presence of amplicon; - : absence of amplicon


201

Fig. 1. PCR amplification of Escherichia coli O157:H7 DNA isolated from

liquid skim milk using the (a) solvent-based method (b) the bacterial

concentration method

(a)

(b)

The initial inoculum levels are given above each gel lane. They are in 10x mode (where x = 1,2,3,4,5,6,7).

The PCR product in this and all other foods is a 1.5 kb segment of slt-II from E. coli O157:H7.

- and + indicate negative (no template) and positive controls, respectively.


202


powder skim milk using the (a) solvent-based method (b) the bacterial

concentration method

(a)

(b)





203


cheese using the (a) solvent-based method (b) the bacterial concentration

method

(a)

(b)





204

Similarly, when applied to cheese, the solvent-based extraction method allowed

a higher improvement in PCR detection limits with visible amplicons obtained at

contamination levels of 104 cfu/ ml while the bacterial concentration method

could detect only upto 105 contamination levels (Table 1). Both sample

preparation methods performed equally well for PCR amplification of DNA

extracted from cheese sample. Visible amplification bands were obtained from

samples with initial E. coli O157:H7 levels of 104 cfu/ ml or higher (Table 1 and

Figure 3).

Likewise, for skim milk powder, PCR detection limits were ≥ 106 cfu /ml for both

the solvent and concentration methods. Surprisingly, attempts at using the

solvent method on skim milk powder resulted in no detectable PCR amplicon,

even when skim milk powder was seeded with 105 cfu/ ml. Whereas, inoculum

level of 105 cfu/ ml was detectable by bacterial concentration method.

Using the concentration technique for skim milk powder, PCR amplicons from E.

coli O157:H7 could be visually detected at initial contamination levels of 105 cfu

ml-1 and above (Table 1 and Figure 2).

Overall, it can be concluded that the Solvent method showed better results than

the bacterial concentration method for detection of PCR amplicons from

artificially contaminated dairy products.

The detection limits of PCR-based pathogen screening methods for foods are

directly dependent on the efficiency of the nucleic acid extraction method

employed. Direct DNA extraction from a variety of foods has been applied

recently, with varying degrees of detection sensitivity (Dickinson et al. 1995;

Drake et al. 1996 and Abolmaaty et al. 1998). For example, the direct DNA

extraction and PCR detection method described by Dickinson et al. (1995)

reported detection limits between 103 and 104 cfu/ml. L. monocytogenes or

Aerococcus viridans in Camembert cheese. Lantz et al. (1994) achieved a


205

detection sensitivity of 104 cfu/ml when detecting L. monocytogenes from soft

cheese using an aqueous two-phase system.

A number of recent studies for detecting pathogens in food have made use of

bacterial concentration strategies, such as immunomagnetic separation (IMS) to

sequester cells for subsequent detection by PCR (Okrend et al., 1991; Jinneman

et al., 1995; Gooding and Choudary 1997; Tomoyasu 1998; Onoue et al., 1999).

Lucore et al. (2000) described a novel method for concentrating Salmonella

enteritidis and L. monocytogenes from food samples using absorption by metal

hydroxides prior to RNA extraction and subsequent detection by RT-PCR. Cell

recovery efficiencies of 65-96 % were obtained from non-fat dry milk

artificially contaminated with these two pathogens. RT-PCR detection limits were

in the order of 101-102 cfu /25 ml of non-fat dry milk. As the target template used

in the Lucore et al. study was 16S rRNA, detection limits were high, partly

because of the high initial copy number of this macromolecule in viable cells.

The present findings indicate that in almost all cases, the solvent method

performed equally well or better than the bacterial concentration method. For the

milk and cheese sample used in this study, the quantity of DNA obtained using

the solvent extraction method was considerably higher than that obtained with

the concentration method, perhaps because bacterial concentration prior to

nucleic acid extraction may limit the amount of food-related DNA that is co-

extracted. The results for skim milk powder were not in accordance with these

findings. It was hypothesized by McKillip et al., (2000) that the lactose

component of skim milk powder either decreased the efficiency of adsorption to

metal hydroxides and/or the extraction of DNA.

Other investigators (Rossen et al., 1992) have attempted to improve PCR

detection limits by procedural changes and PCR additives and were largely

unsuccessful. Many of these strategies are commonly employed to reduce the

effect of carry-over inhibitors on the PCR reaction, and/or function to maximize

primer-template annealing, template availability and/or Taq polymerase activity.


206

Of these approaches, few had any positive impact on DNA yield, and none

increased PCR sensitivity. Gel-based detection of PCR products is undoubtedly

less sensitive than real-time fluorescent-based detection systems (McKillip and

Drake 2000).

The advantages and disadvantages of either the solvent extraction or the

concentration method vary depending on time constraints, PCR detection limits

desired, level of contamination and specific project objectives. The bacterial

concentration method involves fewer steps than the solvent method and does not

require the extensive use of organic solvents. It has the added advantage of

allowing an assessment of bacterial recovery/viability by plate counts

immediately following metal hydroxide adsorption. The solvent extraction method

offers no such opportunity for bacterial recovery prior to cell lysis. In contrast, the

solvent method takes less time to complete than concentration, and provides

better end-point detection limits for some foods. While some minor procedural

modifications were needed when adapting both methods to different dairy

commodities, it is expected that either method could readily be modified for a

variety of food products. It is crucial; however, that such procedural modifications

evaluate the efficiency of DNA extractions, as this appears to be an extremely

important and frequently overlooked variable impacting the overall detection

limits of PCR-based detection strategies (Mckillip et al., 2000).

The study indicates that the Solvent method showed better results than the

bacterial concentration method for detection of PCR amplicons from artificially

contaminated dairy products. The detection limits of PCR-based pathogen

screening methods for foods are directly dependent on the efficiency of the

nucleic acid extraction method employed.


207

5. Assessment of viability of probiotic bacteria and competitive

growth of Lactobacillus acidophilus in yoghurt during

refrigerated storage

In the present study, survival of lactic acid bacteria was evaluated using

molecular methods like PCR during refrigerated storage of three experimental

yoghurt samples. Competitive growth of Lactobacillus acidophilus was also

monitored in the presence of other lactic acid bacteria during refrigerated storage

of yoghurt for a period of 30 days.

Microbial Analysis of Yoghurt samples

Microbial analysis of yoghurt samples was carried out by analyzing for total

Lactobacillus count, L. acidophilus count, Yeast and Mold count and coliform

count. Monitoring the viability of 7 probiotic strains in yoghurt over 30 days has

indicated trends that are related to the different species of organisms tested. In

control yoghurt, count of total lactobacilli increased during first 15 days of storage

and then it decreased with increased storage time (during 15 to 30 days) at 4˚C

(Table 1 and Figure 1), although the increase and decrease was nonsignificant

(p>0.05). Similar trend was also observed in experimental yoghurt B and yoghurt

C. In experimental yoghurt A, total lactic acid bacteria decreased significantly

(p


208

during 30 days of storage at 4˚C. In this sample, L. acidophilus was inoculated

with all other lactic acid bacteria. In Exp. B and Exp. C yoghurt samples, L.

acidophilus count was also found to be decreasing during last 15 days of 30 days

of storage but it was nonsignificant (p>0.05).

Yeast and Mold are one of the most common groups of microbes responsible for

spoilage of fermented dairy products (Pitt & Hocking, 1997). These microbes

have the ability to reduce the shelf life of dairy products even after refrigerated

storage. There was no significant (p>0.05) growth of yeast and mould during the

storage of yoghurt for 30 days at 4˚C (Figure 3). Coliform count increased during

15 to 30 days, however it was also nonsignificant (p>0.05) (Figure 3).


209

Table 1. Total lactic acid bacteria count in yoghurt samples during storage

period of 30 days at 4˚C

Yoghurt Samples

Day 0

(CFU/ml)

Day 15

(CFU/ml)

Day 30

(CFU/ml)

Control 6.72 x 106 7.41 x 106 6.93 x 106

Experimental A 4.13 x 107 3.72 x 106 1.25 x 106

Experimental B 5.02 x 108 6.29 x 108 2.51 x 108

Experimental C 7.38 x 108 8.42 x 108 3.17 x 108



210

1

2

3

4

5

6

7

8

9

Control Experimental A Experimental B Experimental C

Yoghurt Samples

log

10

(CF

U/m

l)

0 day

15 day

30 day

Fig. 1. Total lactic acid bacteria count in yoghurt samples during storage period of

30 days at 4˚C


211

Table 2. Lactobacillus acidophilus count in yoghurt samples during

storage period of 30 days at 4˚C

Yoghurt Samples 0 Day (CFU/ml) 15 Day (CFU/ml) 30 Day (CFU/ml)

Experimental I 4.17 x 103 3.72 x 102 8.24 x 101

Experimental II 5.7 x 103 6.28 x 103 2.17 x 103

Experimental III 7.4 x 103 8.49 x 103 3.15 x 103



212

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 day 15 day 30 day

Days

log

10

(CF

U/m

l)

Experimental A

Experimental B

Experimental C

Fig. 2. Lactobacillus acidophilus count in yoghurt samples during storage period of

30 days at 4˚C


213

0.8

0.9

1

1.1

1.2

1.3

1.4

Day 0 day 30

Days

log

10(C

FU

/ml)

Yeast andMold

Coliform

Fig. 3. Yeast and mold and coliform count of yoghurt samples during storage period

of 30 days at 4˚C


214

PCR Analysis

Enzymatic nucleic acid amplification methods such as the polymerase chain

reaction (PCR) offer several advantages for the rapid and reliable detection of

microorganisms. DNA extraction and PCR analysis was performed from the

yoghurt samples to confirm the survival of each individual bacteria during the

storage period of 30 days at 4˚C. The results are presented in Table 3.

All the bacterial strains were found to be present i.e. amplification was

observed in all the strains by their respective primers in all the yoghurt samples

at the initial level i.e. on 0 day and at final level i.e. on day 30. In spite of the low

count of L. acidophilus in yoghurt samples (Exp. A, Exp. B and Exp. C), there

was sufficient DNA which was amplified by PCR cycles. It indicates that L.

acidophilus survives in the presence of other lactic acid bacteria, but it’s viability

gets reduced.

Studies indicate that, in the development of rapid detection methods,

fermented dairy products have been particularly challenging because they are

compositionally complex and contain food-associated components and high

level of background microflora that often interfere with detection assay, resulting

in less than optimal detection limits. In general, different authors have found

differences of 10 fold or more in detection limits when results from seeded dairy

matrices are compared to results from pure cultures, even after the incorporation

of procedural modifications such as increased Mg++ concentration to improve

amplification efficiency and prior DNA purification (Wernars, Heuvelman,

Chakraborty, & Notermans, 1991; Fluit, Torensma, Visser, Aarsman, Keller, &

Verhoef, 1993 and Wilson, 1997).


215

Table 3. Polymerase chain reaction amplification results of different bacterial

species in yoghurt samples during storage period of 30 days at 4˚C

Bacterial

species

Yoghurt samples


0

day

30

day

0 day 30 day 0 day 30 day 0 day 30 day

Lactobacillus

delbrueckii

subsp.

bulgaricus

+ + + + + + + +

Lactobacillus

plantarum

-- -- + + + + + +

Lactobacillus

acidophilus

-- -- + + + + + +

Lactobacillus

casei

-- -- + + + + + +

Streptococcus

thermophilus

+ + + + + + + +

Lactobacillus

fermentum

-- -- + + + + + +

Lactobacillus

paracasei

-- -- + + + + + +

+: Presence of amplification; --: not applicable


216

Proteolytic activity of Yoghurt samples during storage period

All the three experimental yoghurt samples showed lower proteolytic activity than

the control yoghurt. No significant change (p>0.05) in the proteolytic activity

(zone of clearance on the milk agar plate) of the yoghurt samples was observed

during 30 days of storage (Figure 4). Among all yoghurt samples, control sample

showed highest proteolytic activity, which was on day 30. Among the

experimental yoghurt samples, experimental sample C showed highest

proteolytic activity, which was on Day 0. L. acidophilus along with the other

thermophilus rods is adjudged to be more proteolytic and releases more amounts

of free amino acids (Alm, 1982). However, Amar & Lammerding (1980) on testing

the L. acidophilus strain found it to be relatively less proteolytic compared with

other lactic acid bacteria.

The proteolytic activity of yoghurt is mainly due to the action of Lactobacillus

bulgaricus (Tamine & Robinson, 1999 and Thomas & Mills, 1981). Lactobacillus

bulgaricus, a lactic acid bacterium with complex growth requirements, is

extensively used in the manufacture of cheese and yogurt (Law & Kolstad, 1983).

The pool of free amino acids and peptides present in milk is not enough to

ensure optimal bacterial growth (Mills & Thomas, 1981). The main source of

nitrogen for this species in milk is provided by the hydrolysis of caseins by the

action of L. bulgaricus proteases (Tamine & Robinson, 1999; Thomas & Mills,

1981). Expression of proteolytic activity is important in relation to symbiotic

growth with Streptococcus thermophilus during the production of yoghurt (Rasic

& Kurman, 1981). Results concerning the proteolytic activity of L. bulgaricus have

been obtained in rich media such as MRS broth (Argyle, Mathison, & Chandan,

1976), in which bacteria utilized free amino acids present in the broth. In contrast,

the proteolytic activity in milk has not been extensively studied. A recent study by

Laloi, Atlan, Blanc, Gilbert, & Portalier, 1991 regarding proteases of cells grown

in milk and in MRS broth showed identical patterns of hydrolytic products of α-

and β-caseins.


217

10.6

10.8

11

11.2

11.4

11.6

11.8

12

12.2

12.4


Yoghurt samples

Zo

ne o

f cle

ara

nce i

n m

m

Day 0

Day 30

Fig. 4. Proteolytic activity (Zone of clearance on milk agar plate) of yoghurt samples

during storage period of 30 days at 4˚C


218

Sensory Evaluation

Sensory evaluation was carried out from all yoghurt samples and it is presented

in Table 4. The results indicated that overall acceptability of experimental yoghurt

A obtained a score of 7.97 on a scale of 10 points which indicates very good

acceptability. Control yoghurt showed an overall acceptability score of 7.78. The

average flavour score was higher for experimental yoghurt B, although the

differences were non-significant (p>0.05). Overall acceptability was higher in the

experimental yoghurt A. Gupta, Mital & Garg (1997) reported no significant

differences in the textural characteristics of both the control and acidophilus

yoghurt. In the present study, organoleptic evaluation revealed that all the three

yoghurt samples were almost identical with respect to colour, flavour, texture and

overall acceptability with a score ranging from 7.72 to 7.97 on a 10 point scale.

According to Gilliland & Speck, 1975; the acidic nature of L. acidophilus

enables it to withstand storage in an acidic environment for a reasonable time

without loss in viability. The situation is different when L. acidophilus is mixed

with a medium, such as yoghurt, containing the metabolic products of other

microorganisms. In spite of reports that indicate that L. acidophilus can be added

to yoghurt successfully, no supporting data have been found for these

presumptions. By the use of a medium which can enumerate differentially L.

acidophilus in a mixture with yoghurt culture, we have shown that L. acidophilus

indeed is damaged markedly with respect to viability during storage with the

products contained in yoghurt. The microorganism in yoghurt responsible for the

antagonism for L. acidophilus was L. bulgaricus. The microorganism produces

some substance(s), other than acid during it’s growth which is the antagonistic

agent(s) (Servin & Coconnier, 2003). Gilliland & Speck, 1975 reported that

hydrogen peroxide seems to be the main agent responsible for the loss in

viability of L. acidophilus when mixed in yoghurt. Studies have shown that L.

bulgaricus produces hydrogen peroxide in milk at 5˚C (Gilliland & Speck, 1975;

Premi & Bottazii, 1972 and Ito, Sato, Kudo, Sato, Nakajima, & Toba, 2003). They

also reported that sufficient peroxide was produced to inhibit the growth of

psychrotrophic bacteria.


219

Table 4. Sensory evaluation of yoghurt samples on 0 day

Sensory Score ( out of 10 )

Product Colour Flavour Texture Overall

acceptability

Control

7.97 ±

1.02

7.17 ±

1.36

8.22 ±

1.26

7.78 ± 0.96

Experimental

Experimental

Aa

8.25 ±

0.94

7.33 ±

0.91

8.33 ±

1.28

7.97 ± 0.91

Experimental

Bb

7.80 ±

1.13

7.36 ±

1.03

8.03 ±

1.31

7.73 ± 0.99

Experimental

Cc

7.87 ±

1.13

7.22 ±

1.03

8.08 ±

1.31

7.72 ± 0.82

Mean of 10 judges ± S.D.

a: Addition of L. acidophilus along with other bacterial cultures

b: addition of L. acidophilus 2 hours after the inoculation of other bacterial cultures

c: Addition of L. acidophilus 2 hours before the inoculation of other bacterial cultures


220

PCR amplification was observed in the 0 day samples and 30 day samples.

Hence, all lactic acid bacteria were viable at the end of the storage study period

indicating survival of lactic acid bacteria during synbiotic growth in yoghurt. All

the prepared yoghurt samples had good overall acceptability. No significant

growth was observed in yeast and coliform count during storage. No significant

change in the proteolytic activity of the yoghurt samples was observed upon

storage. The study of the proteolytic activity of L. bulgaricus in milk can enhance

the knowledge base required for selection of starter cultures. The present study

indicates that yoghurt product is a suitable career for a variety of probiotic

bacteria but survival of L. acidophilus will need to be improved to provide

optimum health benefits to consumers. Further research can be carried out to

determine genetic relationship of antagonistic effect of microorganisms during

synbiotic growth in a model system.


221

6. Effect of nisin on growth and survival of selected food

pathogens

The present study was planned to evaluate the effect of the bacteriocin nisin on

selected food pathogens namely S. aureus, L. monocytogenes and S. typhi

during yoghurt fermentation and storage. Effect of nisin was also evaluated on

the growth of yoghurt starter cultures namely L. bulgaricus and S. thermophilus.

Parameters studied were:

1. Effect of Nisin on yoghurt fermentation

2. Effect of Nisin on S. aureus, S. typhi and L. monocytogenes

3. Effect of Nisin on S. aureus, S. typhi and L. monocytogenes in Yogurt

1. Effect of Nisin on yoghurt fermentation

As yogurt starter cultures i.e. L. bulgaricus and S. thermophilus is reported to be

sensitive to nisin (Kumar and Prasad, 1994; Kuma and Prasad, 1994b;

Vandenbergh, 1993), this preliminary experiment was carried out to determine

the concentration of nisin which can be used without affecting significantly normal

yogurt processing and acid production.

Nisin was added to the cups containing yoghurt to a final concentration of 10, 50

and 100 IU/ml. A sample not containing nisin was used as control. The

containers were incubated at 43˚C for 24 hours and then transferred to the

refrigerator (4 -7°C).

The pH and acidity were measured every 4 hours upto 24 hours. The change in

pH and acidity of control sample is shown in Table 1. Table 2 shows change in

pH and acidity of yoghurt sample having nisin concentration 10 IU/ml. Table 3

shows change in pH and acidity of yoghurt sample having nisin concentration 50

IU/ml, whereas Table 4 shows change in pH and acidity of yoghurt sample

having nisin concentration 100 IU/ml.


222

Table 1. pH of the prepared yoghurt during fermentation

Fermentation

Hours

pH of yoghurt samples

C Sample A Sample B Sample C

0 6.5 6.6 6.5 6.5

4 5.6 5.4 5.9 5.6

8 4.7 4.9 4.9 5.1

12 4.2 4.5 4.5 4.9

16 3.7 3.9 4.3 4.7

20 3.5 3.6 4.2 4.3

24 3.2 3.4 4.2 4.2

Sample A: Nisin Concentration. 10 IU/ml

Sample B: Nisin Concentration 50 IU/ml

Sample C: Nisin Concentration 100 IU/ml

C: Control


223

Table 2. Acidity of the prepared yoghurt during fermentation

Fermentation

Hours

Acidity of yoghurt samples (gm/Lit)

C Sample A Sample B Sample C

0 2.2 2.2 2.1 2.1

4 2.2 3.9 3.4 3.7

8 6.6 7.1 6.3 5.8

12 7.0 7.2 7.0 6.0

16 7.6 7.6 7.6 6.0

20 7.9 7.8 7.7 6.1

24 8.1 8.0 7.9 6.1

Sample A: Nisin Concentration. 10 IU/ml

Sample B: Nisin Concentration 50 IU/ml

Sample C: Nisin Concentration 100 IU/ml

C: Control


224

Table 1 and Table 2 show the decrease in pH and the increase in acidity during

fermentation of yogurt containing different concentrations of nisin. In the control

yoghurt sample, pH dropped and acidity increased as the time of fermentation

increased.

In the samples having 10 IU/ml and 50 IU/ml, the pH dropped and the acidity

increased in the same way as in the control. Milk coagulation in all samples was

normal; it occurred between 6 to 7 hours and the curd was firm and without

syneresis. It can be seen from Table 1 and Table 2 that a nisin concentration of

50 IU/ml or less had no noticeable effect on yoghurt fermentation.

However, in the samples containing 100 IU/ml of nisin, fermentation was greatly

retarded and the curd had an abnormal, viscous body. Yoghurt sample having

nisin concentration of 100 IU/ml showed 4.2 and 6.1 gm/Lit pH and titrable acidity

respectively at the end of 24 hours fermentation.

pH and titrable acidity in control sample were 3.2 and 8.1 gm/lit respectively, at

the end of 24 hours fermentation. This indicates that nisin has an inhibitory effect

on L. bulgaricus and S. thermophilus (yoghurt starter cultures). This result was

also observed by several other scientists (Benkerroum et al., 2003, Kumar and

Prasad, 1994).

Benkerroum et al. (2003) and Kumar and Prasad (1994) tested several strains of

lactobacilli for their sensitivity to nisin and found that the MIC ranged between 35

and 100 IU/ml for Lb. delbueckii subsp. bulgaricus strains at optimal growth

conditions and when an inoculum of 10 ml/Lit was used. These values doubled

for an inoculum of 20 ml/l (Benkerroum et al., 2003; Kumar and Prasad, 1994).

As matter of fact, a moderate delay in yogurt acidification may be suitable in

yogurt technology since in the conventional process the product usually develops

too much acidity towards the end of the storage and wheys-off. According to

Bayoumi (1991), nisin addition to the level of 50 IU/ml prevents such a defect


225

resulting in a 7 day increase of the shelf-life without affecting sensory

characteristics.

2. Effect of Nisin on S. aureus, S. typhi and L. monocytogenes

(A) Varying pH with constant Nisin concentration

Table 3 (Figure 1, Figure 2, Figure 3, Figure 4) shows the effect of nisin on S.

aureus growth at different pH values. It may be seen that numbers of S. aureus

increased at pH 6.8 in the controls (without nisin), while they decreased steadily

in test samples containing 50 IU/ml of nisin. Similar results were obtained at pH

5.5. At pH 4.5, a decrease in S. aureus counts was observed in both test and

control samples; however, the pathogen was eliminated from the test samples

within 48 hours while, in the control, few cells were still viable at 48 hours. An

almost similar trend was observed at pH 5.0.

Table 4 (Figure 5, Figure 6, Figure 7, Figure 8) shows the effect of nisin in

yoghurt on S. typhi at different pH values. At pH 6.8, S. typhi count in test

samples decreased steadily at the end of 48 hours, while it increased in control

samples. Similar trend was observed in pH 5.5 and 5.0. At pH 4.5, S. typhi was

eliminated from the test sample after 24 hours, while it took 48 hours to eliminate

S. typhi from control samples

Growth of L. monocytogenes in listeria selective agar plates at different pH

values in the absence or presence of 50 IU/ml of nisin is shown in Table 5

(Figure 9, Figure 10, Figure 11 and Figure 12). It may be seen that numbers of L.

monocytogenes increased at pH 6.8 in the control (without nisin), while they

decreased steadily in test samples containing 50 IU/ml of nisin. Similar results

were obtained at pH 5.5. At pH 4.5, a decrease in Listeria counts was observed

in both test and control samples; however, the pathogen was eliminated from the

test samples within 24 hours while, in the control, few cells were still viable even

at 48 hours. The same behavior was observed at pH 5.0 (Table 1). Similar

results were previously reported in TSB but with a higher nisin concentration

(Benkerroum et al., 2003). These data suggest that 50 IU/ml of Nisin is effective

in the control of L. monocytogenes in milk and dairy products. A concentration of


226

10 to 500 IU/g has been recommended in food preservation in general (Eapen et

al., 1983).

The use of nisin, the bacteriocin produced by Lactococcus lactis subsp. lactis, is

successfully used nowadays as an antibacterial agent in various food products.

Nisin affects several Gram-positive bacteria such as Listeria spp.,

Staphylococcus spp. but does not inhibit the majority of Gram-negative bacteria

(Abee et al., 1994; Martinis et al., 1997). Nisin has shown to be efficient in

inactivating Gram-negative bacteria when used together with chelating agents

(EDTA), causing an aberration in cell membrane lipopolisaccharide component

(Stevens et al., 1992).

EDTA was added in the tubes having S. aureus and S. typhi to enhance the

inhibitory activity of nisin. Nisin when used in combination with the chelating

agent EDTA; inhibits a wide variety of Salmonella and Staphylococcal species

(Grisi and Gorlach-Lira, 2005). Stevens et al. (1991), reported that inhibition of

Salmonella species by a combination of nisin and EDTA, is a time-dependent

phenomenon and the method of application (simultaneous versus sequential) is

critical to achieving the desired effect. Increasing the nisin concentration above

50 ug/ml will, most likely, increase the magnitude-of inactivation. Furthermore,

the observed inactivation by nisin can be extended to other gram-negative

bacteria. The observed population reductions by nisin are facilitated by the

chelation of magnesium ions, present in the outer membrane, by EDTA. The

removal of magnesium ions from the lipopolysaccharide layer of the outer

membrane results in the loss of lipopolysaccharide and an increase in cell

permeability (Nikaido et al, 1987).

This increase in outer membrane permeability to nisin is proposed to facilitate

inactivation of the cell via bactericidal action at the cytoplasmic membrane.

Applications involving simultaneous treatment with nisin and an outer membrane

modifying-chelating agent such as EDTA may be of value in controlling food-

borne Salmonella species as well as other gram-negative pathogens in foods.

Leonides et al (2008) reported that nisin is an efficient alternative to antibiotics for

the treatment of staphylococcal mastitis.


227

The use of the Generally Recognized As Safe (GRAS) lactic acid bacteria (LAB),

or the antimicrobial compounds they produce (bacteriocins), is a promising

ongoing development in food preservation (smid and Gorris, 2007). Bacteriocins

are antimicrobial peptides with activity mainly against Gram-positive bacteria has

gained great attention in recent years. Although the efficacy of bacteriocins can

be limited in food systems if applied alone, several bacteriocins have shown

additive or synergistic effects when used in combination with other antimicrobial

agents or processes such as chelating agents, heat, modified atmosphere

packaging and high hydrostatic pressure (HHP).


228

Table 3. Effect of different pH and Nisin (50 IU/ml) on the growth of S. aureus (log

CFU/ml) at 37˚C

pH

6.8 5.5 5.0 4.5

Hours + nisin - Nisin + Nisin - Nisin + Nisin - Nisin + Nisin - Nisin

0 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00

4 6.18 6.07 6.23 6.09 5.89 6.26 5.24 5.44

8 5.89 7.81 6.93 7.20 5.72 5.84 4.47 4.81

24 5.02 8.64 4.62 7.61 4.29 5.36 2.70 3.42

48 3.28 9.23 3.38 8.27 2.28 3.80 0.00 1.33

Nisin was used in combination with 20 mM EDTA

Media used: Baird-Parker Agar media


229

Table 4. Effect of different pH and Nisin (50 IU/ml) on growth of S. typhi (log

CFU/ml) at 37˚C

pH

6.8 5.5 5.0 4.5


0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0

4 6.32 6.74 6.26 6.38 5.78 6.26 5.37 5.72

8 6.18 7.61 6.79 7.22 5.20 5.49 4.12 4.96

24 5.29 8.47 4.92 8.04 4.06 4.20 0 2.69

48 3.77 9.58 3.95 9.09 2.22 3.48 0 0

Nisin was used in combination with 20 mM EDTA

Media used: Salmonella differential agar plates


230

Table 5. Effect of different pH and Nisin (50 IU/ml) on growth of L. monocytogenes

(log CFU/ml) at 37˚C

pH

6.8 5.5 5.0 4.5


0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0

4 6.64 7.15 6.33 6.24 5.7 6.3 4.6 5.3

8 7.63 8.86 7.22 7.61 5.8 5.92 4.4 4.8

24 4.21 9.76 3.31 7.38 3.2 5.80 0 2.92

48 1.72 9.71 1.4 8.52 0 2.3 0 0.65

Media used: Listeria selective agar plates


231

0

1

2

3

4

5

6

7

8

9

10

0 4 8 24 48

Time (in hours)

Lo

g C

FU

/ml

Nisin

Control

Figure 1. Growth of S. aureus in Nisin added (50 IU/ml) at pH 6.8


232

0

1

2

3

4

5

6

7

8

9

0 4 8 24 48

Time (in hours)

Lo

g C

FU

/ml

Nisin

Control



233

0

1

2

3

4

5

6

7

0 4 8 24 48

Time (in hours)

Lo

g C

FU

/ml

Nisin

Control

Figure 3. Growth of S. aureus in Nisin added (50 IU/ml) at pH 5


234

0

1

2

3

4

5

6

7

0 4 8 24 48

time (in hours)

Lo

g C

FU

/ml

Nisin

Control



235

0

2

4

6

8

10

12

0 4 8 24 48

Time (in hours)

Lo

g C

FU

/ml

Nisin

Control

Figure 5. Growth of S. typhi in Nisin added (50 IU/ml) at pH 6.8


236

0

1

2

3

4

5

6

7

8

9

10

0 4 8 24 48

Time (in hours)

Lo

g C

FU

/ml

Nisin

Control



237

0

1

2

3

4

5

6

7

0 4 8 24 48

Time (in hours)

Lo

g C

FU

/ml

Nisin

Control

Figure 7. Growth of S. typhi in Nisin added (50 IU/ml) at pH 5


238

0

1

2

3

4

5

6

7

0 4 8 24 48

Time (in hours)

Lo

g C

FU

/ml

Nisin

Control



239

0

2

4

6

8

10

12

0 4 8 24 48

Time (in Hours)

Lo

g c

fu/m

l

Nisin

Control

Figure 9. Growth of L. monocytogenes in nisin added (50 IU/ml) at pH 6.8


240

0

1

2

3

4

5

6

7

8

9

0 4 8 24 48

Time (in Hours)

Lo

g c

fu/m

l

Nisin

Control

Figure 10. Growth of L. monocytogenes in Nisin added (50 IU/ml) at pH 5.5


241

0

1

2

3

4

5

6

7

0 4 8 24 48

Time (in hours)

Lo

g c

fu/m

l

Nisin

Control

Figure 11. Growth of L. monocytogenes in Nisin added (50 IU/ml) at pH 5


242

0

1

2

3

4

5

6

7

0 4 8 24 48

Time (in hours)

Lo

g c

fu/m

l

Nisin

Control

Figure 12. Growth of L. monocytogenes in Nisin added (50 IU/ml) at pH 4.5


243

B. Effect of varying pH and varying Nisin concentration

Effect of varying pH and varying Nisin concentration are presented in Table 6,

Table 7 and Table 8 for S. typhi, S. aureus and L. monocytogenes, respectively.

Considering the fact that in practice there is a tendency to minimize the amount

of an additive in food preservation, this experiment was carried out in TSB using

different combinations of pH and nisin concentrations. At pH 6.8, no

concentration was inhibitory to L. monocytogenes; no significant difference (P

>0.05) between O.D. reached after 48 hours in samples containing up to 200

IU/ml and the control, was observed (Table 5). At pH 6.0, 5.5 and 5.0; however,

50, 100 and 200 IU/ml were all effective. A concentration of 10 IU/ml had no

significant (P> 0.05) effect on L. monocytogenes at pH 6.8 and 5.5 but was

significantly (p


244

concentration or to the polymerization of the molecule (Kumar and Prasad,

1994). In fact, nisin activity was completely and irreversibly lost after 4 days of

storage at pH 7.0 and ambient temperature. Results show that nisin action at a

given pH depends on the concentration and the medium used. In effect, 50 IU/ml

resulted in more than 4 log units reduction in Listeria counts in listeria selective

agar plates at pH 6.8 after 48 hours.


245

Table 6. Effect of varying pH and varying Nisin concentration on

growth of S. typhi in luria broth and incubated at 37˚C

pH Nisin (IU/ml)

O.D. at different time intervals

0 hr 2 hr 4 hr 8 hr 24 hr 48 hr

6.8 0 0 0.132 0.184 0.238 0.653 0.935

10 0 0.141 0.195 0.319 0.77 0.872

50 0 0.223 0.238 0.398 0.822 0.848

100 0 0.217 0.235 0.317 0.790 0.824

200 0 0.196 0.240 0.298 0.793 0.811

6 0 0 0.115 0.232 0.241 0.911 0.932

10 0 0.122 0.127 0.272 0.902 0.917

50 0 0.228 0.214 0.298 0.353 0.427

100 0 0.218 0.218 0.311 0.341 0.382

200 0 0.168 0.179 0.305 0.320 0.344

5.5 0 0 0.089 0.177 0.328 0.732 0.777

10 0 0.169 0.183 0.281 0.584 0.642

50 0 0.211 0.231 0.251 0.279 0.297

100 0 0.219 0.239 0.263 0.288 0.305

200 0 0.230 0.235 0.242 0.261 0.289

5 0 0 0.113 0.175 0.202 0.306 0.378

10 0 0.26 0.182 0.212 0.276 0.308

50 0 0.209 0.263 0.281 0.288 0.302

100 0 0.069 0.122 0.202 0.233 0.242

200 0 0.077 0.106 0.169 0.183 0.192


246


growth of S. aureus grown in luria broth and incubated at 37˚C

pH Nisin (IU/ml)


0 hr 2 hr 4 hr 8 hr 24 hr 48 hr

6.8 0 0 0.139 0.181 0.248 0.642 0.922

10 0 0.149 0.178 0.332 0.717 0.861

50 0 0.238 0.242 0.376 0.822 0.859

100 0 0.229 0.246 0.321 0.790 0.838

200 0 0.185 0.233 0.283 0.782 0.837

6 0 0 0.123 0.224 0.241 0.911 0.932

10 0 0.134 0.122 0.262 0.902 0.917

50 0 0.227 0.227 0.246 0.353 0.427

100 0 0.229 0.189 0.324 0.341 0.382

200 0 0.179 0.150 0.341 0.322 0.351

5.5 0 0 0.098 0.177 0.328 0.732 0.807

10 0 0.144 0.201 0.226 0.521 0.589

50 0 0.213 0.219 0.242 0.257 0.270

100 0 0.231 0.217 0.244 0.263 0.331

200 0 0.234 0.239 0.242 0.261 0.289

5 0 0 0.128 0.178 0.232 0.328 0.407

10 0 0.211 0.172 0.236 0.263 0.337

50 0 0.228 0.253 0.274 0.262 0.323

100 0 0.066 0.128 0.216 0.253 0.232

200 0 0.065 0.118 0.152 0.171 0.198


247


growth of L. monocytogenes in TSB incubated at 37˚C

pH Nisin (IU/ml)


0 hr 2 hr 4 hr 8 hr 24 hr 48 hr

6.8 0 0 0.72 0.197 0.316 0.766 0.917

10 0 0.214 0.239 0.382 0.753 0.878

50 0 0.168 0.228 0.351 0.717 0.778

100 0 0.188 0.205 0.279 0.639 0.741

200 0 0.62 0.188 0.251 0.651 0.722

6 0 0 0.131 0.166 0.27 0.783 0.933

10 0 0.43 0.169 0.203 0.742 0.849

50 0 0.188 0.212 0.417 0.481 0.509

100 0 0.174 0.188 0.356 0.408 0.431

200 0 0.161 0.84 0.321 0.361 0.393

5.5 0 0 0.057 0.17 0.203 0.763 0.781

10 0 0.049 0.101 0.213 0.641 0.664

50 0 0.136 0.212 0.267 0.328 0.34

100 0 0.118 0.233 0.241 0.281 0.315

200 0 0.114 0.227 0.281 0.319 0.342

5 0 0 0.042 0.063 0.127 0.228 0.276

10 0 0.055 0.058 0.089 0.131 0.165

50 0 0.117 0.157 0.171 0.149 0.117

100 0 0.125 0.141 0.147 0.158 0.128

200 0 0.061 0.112 0.123 0.118 0.083


248

Nisin, due to its legal status (European parlament of council, 1995) and

commercial availability is the bacteriocin that has been more widely studied,

although other bacteriocins such as sakacin K (produced by Lactobacillus sakei

CTC494) and enterocins A and B (produced by Enterococ-cus faecium CTC492)

have also been shown to reduce the counts of artificially inoculated L.

monocytogenes, Salmonella or S. aureus in meat products (Garriga et al., 2002;

Aymerich et al., 2005; Chung et al., 2005; Yuste et al., 1998; Ananou et al.,

2005) and to provide extra protection when breaks in the cold chain occurred

(Marcos et al., 2008).

3. Effect of Nisin on S. aureus, S. typhi and L. monocytogenes in Yogurt

Table 9 shows the growth of L. monocytogenes, S. aureus and S. typhi in yogurt

in the presence and absence of nisin during storage at 7˚C. Although a

significant decrease in Listeria counts was observed in yogurt without nisin, the

pathogen survived manufacture and 10 days of storage at 7˚C. However, in

yogurt containing 10 IU/ml of nisin, no Listeria was found after 24 hours. Table 5

shows the behaviour of S. aureus and S. typhi in yoghurt in the presence and

absence of nisin during storage at 7˚C. S. aureus and S. typhi were not able to

survive for 24 hours in the presence of Nisin in yoghurt. However in control

samples, they were able to survive until 10 days.


249

Table 9. Effect of nisin (10 IU/ml) on the growth of Listeria

monocytogenes, S. aureus and S. typhi in yogurt

L. monocytogenes S. aureus S. typhi

Days Log

CFU/ml

(sample)

Log

CFU/ml

(Control)

Log

CFU/ml

(sample)

Log

CFU/ml

(Control)

Log

CFU/ml

(sample)

Log

CFU/ml

(Control)

0 6.0 6.0 6.0 6.0 6.0 6.0

1 0 4.2 0 4.7 0 5.3

2 0 3.3 0 3.2 0 4.4

3 0 3.1 0 2.6 0 3.5

10 0 2.4 0 1.3 0 2.1

15 0 0 0 0 0 0

Nisin (10 IU/ml) was used in combination with 20 mM EDTA in samples inoculated with

S. aureus and S. typhi


250

Survival of pathogens in fermented dairy products, in spite of the antagonistic

effect of lactic acid bacteria used as starter cultures is well documented

(Benkerroum et al., 2000; De Buyser et al., 2001; Donnely, 1990). In yogurt and

in other dairy products fermented with the same starter [e.g. Lb. bulgaricus and

Str. thermophilus (ST:LB::1:1)] such as some cultured milks and Feta cheese, L.

monocytogenes survives manufacture and storage (Ribeiro and Carminati, 1996;

Rocourt, 1996). Papageorgiou and Marth (1989) showed that L. monocytogenes

could survive the manufacture and more than 90 days of storage at 4°C in Feta

cheese. This bacterium survived in cultured milk fermented with STLB and in

yoghurt for 1 to 12 weeks and 1 to 12 days, respectively (Piccinin and Shelef,

1995).

The same authors showed that survival of L. monocytogenes in yogurt depended

on the size of Listeria and starter culture inocula, the final pH reached the

temperature and duration of the fermentation, and Listeria strain. Shaack and

Marth (1988) showed that L. monocytogenes survives only between 9 to 15

hours during the actual fermentation process of yogurt. However, in a typical

yogurt fermentation of 4 to 6 hours, Listeria was able to grow during fermentation

and then survive during storage at 4 °C. Lammerding and Doyle (1989) also

could recover L. monocytogenes from yoghurt after 7 days of storage at 4˚C,

although the initial inoculum was relatively low (about 32 x 102

CFU/ml).


251

7. Bacteriocin production by free and immobilized Lactococcus

lactis

In the present study, bacteriocin production was carried out using Lactococcus

lactis. Free bacterial cells and immobilized bacterial cells were evaluated for

bacteriocin production. Bacteriocin production was carried out from free bacterial

cells using two different media: Media 1 (MRS broth) and Media 2 (MRS broth +

sucrose). The supernatants of the two different media and the immobilized

organism were assessed for bacteriocin activity.

Bacteriocin activity of all the three culture filtrates was compared using agar

diffusion assay. Firstly standard nisin concentrations over a range (10-50 IU/ml)

were used for assessing activity against each sensitive bacterial strain like M.

luteus, S. aureus and B. subtilis. Diameter of inhibition zones were measure

against each organism and a standard curve were generated.

Micrococcus luteus which is used as the indicator strain did show increase in

zone of inhibition with increase in the number of transfers. Maximum zone of

inhibition were obtained by supernatant of MRS + sucrose while only MRS did

show lower levels of inhibition. The results are presented in Figure 1.


252

0

0.5

1

1.5

2

2.5

3

3.5

1 2 3 4 5

No. of Transfers

Zo

ne o

f in

hib

itio

n (

cm

)

MRS

MRS + Sucrose

Immobilized

Fig. 1. Zone of inhibition of three different media at different transfers against

M. Luteus


253

In the case of the immobilized supernatant, good inhibition was achieved upto

two transfers but it decreased gradually in later transfers. S. aurues which is

used for assessing antimicrobial activity showed a good zone of inhibition using

different supernatants. In the case of MRS supernatant, increased inhibition with

an increase in the no. of transfers was observed. In the case of MRS + sucrose

supernatant, maximum zone of inhibition compared to other samples was

observed but it decreased in the 5th transfer. Immobilized culture filtrate showed

good inhibition zones in the 1st two transfers then remained stationary from the

third transfer. Results are presented in Figure 2. B. subtilis which was used for

assessing antimicrobial activity did not show good zone of inhibition compared to

S. aurues & M. luteus. B. subtilis further did not show any inhibition by

supernatant of MRS and MRS + sucrose supernatant in the 1st transfer.

In the 2nd transfer, both MRS and MRS + sucrose supernatants showed a slight

zone of inhibition. The immobilized filtrate showed a very large zone of

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