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22 International Journal of Research in Pure and Applied Microbiology 2011; 1(2): 22-31

Original Article

Isolation and Characterization of Microorganisms Responsible for Different Types of

Food Spoilages

Ashok Kumar1*

, Varun Bhushan1, Shikha Verma

1, Gaurav Srivastav

2 and Sushil Kumar

3

1 Department of Biotechnology, Himachal Institute of Life Sciences Rampurghat Road, Paonta Sahib -173025 Himachal Pradesh INDIA 2Research Scholar Biotechnology, Uttarakhand Technical University, Suddhowala, Dehradun-248002 Uttarakhand INDIA

3Department of Microbiology, Himachal Institute of Life Sciences Rampurghat Road, Paonta Sahib -173025 Himachal Pradesh INDIA

*Corresponding author: [email protected] Cont: +919450501471

Received 13 September 2011; accepted 20 September 2011 Abstract

Bacteria are the main and an important cause of food spoilage. They thrive where food and water are present and the

temperature is suitable. To resist harm, some bacteria can form spores tough reproductive cells that are able to survive under

adverse conditions, that can resist damage by heat as in cooking, by cold as in freezing and by chemicals such as disinfectants.

Bacteria need four hours to adapt to the new environment. 30 spoiled food samples were collected form Paonta sahib and seven

bacterial isolates viz. Bacillus, Klebsiella, Pseudomonas, E.coli, Lactobacillus, Staphylococcus, and Micrococcus were isolated and characterized on the basis of morphology and biochemical reactions. It was found that the Bacillus, Klebsiella and

Pseudomonas were the dominating species in the spoilage of every categories of food material. In fruits, vegetables and meat

Klebsiella was the most potent spoiling bacteria. In meat, milk and in fatty products Pseudomonas was found in most of the

spoilages. Although Klebsiella, Pseudomonas and Bacillus were dominant in spoilages E.coli, Staphylococcus and

Micrococcus was also recovered from considerable categories of the spoiled sample. The bacteria especially gram negative,

was key responsible for food spoilage.

© 2011 Universal Research Publications. All rights reserved

Key words: Food spoilage, Bacteria, Gram negative, Klebsiella, milk, Lactobacillus.

Introduction

There is potential for a wide range of vegetables and fruits

products to become contaminated with microorganisms. The

range of microorganisms associated with outbreaks linked to

fresh produce encompasses bacteria, viruses and parasites.

The products of most concern are sprouted seeds and

unpasteurized juices. Most of the reported outbreaks have

been associated with bacterial contamination, particularly

members of the Enterobacteriaceae. Of these, Salmonella

and Escherichia coli in sprouted seeds and fruit juices are of

particular concern. Outbreaks linked to protozoa e.g.

Cryptosporidium, Cyclospora, Giardia etc have been

associated more with fruits than with vegetables. Protozoa and viruses are most often associated with contaminated

water or food handlers. Fruits and vegetables normally carry

a non-pathogenic epiphytic microflora (Ray, 2004).

There is rapid progress in the field of chemical detection

technology; little of this technology appears to have found application in estimation of the remaining shelf life of foods

and early detection of spoilage. Predictive microbiology aims

to summaries the probable behavior of specific spoilage

organisms and the progression of spoilage processes in foods.

The quantitative knowledge generated in the field of

predictive microbiology provides a sound basis for the

rational development of devices with which to monitor loss

of product shelf life during storage, distribution and retail

sale. To predict remaining shelf life accurately it is necessary,

however, to consider the microbial ecology of the food

system (McMeekin et al., 1996).

A Bacillus strain was isolated from spoiled apple juice. This

strain was acidophilic with a growth range between pH 2.5

and 5.5. Lipid analysis demonstrated the occurrence of omega

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cyclohexane fatty acids and hopanoids. As these cell

constituents have among bacilli been found only in Bacillus

acidocaldarius strains, isolated microorganism seems to be

related to this species. The organism could be a threat to fruit

juices during storage at elevated temperatures, greater than or

equal to 26˚C because its spores were able to survive pasteurization conditions (Cerny et al., 1984). Species most

commonly implicated in fruit and fruit product disintegration

are Byssochlamy sfulva, Byssochlamys nivea, Neosartorya

fischeri, Talaromyces flavus and Eupenicillium brefeldianum.

They can survive heat treatments used for fruit processing

and can grow and spoil the products during storage at room

temperature, which results in great economic losses. Besides

spoilage, the heat-resistant molds produce a number of toxic

secondary metabolites, such as byssotoxin A; byssochlamic

acid; the carcinogen, patulin, the tremorgenic substances,

fumitremorgin A and C, and verruculogen; fischerin, which

caused fatal peritonitis in mice; and eupenifeldin, a compound possessing cytotoxicity as well as in vivo

antitumor activity (Tournas., 1994).

Strain A. acidoterrestris was identified as the causative agent

in spoilage of commercially pasteurized apple juice.

Alicyclobacillus sp. is soil borne bacteria, and do not strictly

require thermophilic and acidic environments.

Alicyclobacillus sp. possesses several distinct characteristics;

the major one is their ability to survive commercial

pasteurization processes and produce off-flavours in fruit

juices. Guaiacol and halophenols were identified as the offensive smelling agent in many Alicyclobacillus sp. related

spoilage. The Alicyclobacillus sp was identified as potential

spoilage bacteria in fruit juices (Chang et al., 2004).

Spoilage of milk products by Pseudomonas fragi is

characterized by the production of a strawberry like odour.

Ethyl esters of butyric, hexanoic, and 3-methylbutanoic acid

were shown to be the major contributors to the odour on the

basis of their relative concentration in the headspace

(Cormier et al., 1991). Proteolysis during storage of ultra high

temperature skim and whole milks processed by either direct or indirect systems has been studied. All the proteolysis

indices determined increased activities of both native milk

proteinase and proteinases of bacterial origin were observed

in skim ultra high temperature milks. The different behavior

of ultra high temperature skim and whole milks on storage

would have to be taken into account in establishing the

process conditions (Lopez et al., 1993). Proteolysis of milk

proteins is attributed to both native proteases and the

proteases produced by psychrotrophic bacteria during storage

of fresh raw milk. These proteases cause beneficial or

detrimental changes, depending on the specific milk product.

Plasmin, the major native protease in milk, is important for cheese ripening. A microbial protease from a psychrotrophic

microorganism can indirectly increase plasmin levels in the

casein curd. This relationship between the plasmin system

and microbial proteases in milk provides a means to control

levels of plasmin to benefit the quality of dairy products

(Nielsen, 2002).

The reason for the reported difference in spoilage behaviour

of skim and whole pasteurized milks was investigated. The rates of growth of psychrotrophic bacteria were not

significantly different in the two milks and the bacterial

types, all Pseudomonads, present at spoilage were also

similar. However, when representative spoilage organisms

were cultured into freshly pasteurized skim and whole milks,

skim milks exhibited predominantly bitter flavours while

whole milk showed mostly sour flavours. The different

spoilage behaviors can be largely explained by greater

proteolysis in skim milk than in whole milk, caused by higher

production of protease and greater susceptibility of the

protein to protease attack (Deeth et al., 2002).

Material and Methods:

1. Sample collection The samples were collected from the local market of Paonta

Sahib (Fig. 1). The sample collection was performed

according to standard method given by APHA 1998. The

sample names were coded for simplicity on the basis of their

types. The codes with their name are listed in table 1.

Fig. 1 Location Paonta Sahib in HP

2. Bacteriological Analysis: Bacteriological analysis was done by selective media method

by Presscott, 2002 and Sherman, 2005.

3. Morphological Characterization

The isolated microbes were characterized on the basis of

simple staining and gram staining (Holt et al., 1994;

Sherman, 2005).

4. Biochemical Characterization

The isolates were characterized by biochemical tests viz.

IMViC reactions i.e. indole test, Methyl Red test, Voges

Proskauer test and Citrate utilization test, Nitrate Reduction


Table 1: Sample Type and their codes

S No Sample Type Sample Code

1 Apple S1

2 Mango S2

3 Orange S3

4 Sugar cane S4

5 Pomegranate S5

6 Papaya S6

7 Litchi S7

8 Lady finger S8

9 Pumpkin S9

10 Guard S10

11 Bingil S11

12 Capsicum S12

13 Karella S13

14 Cucumber S14

15 Tomato S15

16 Egg S16

17 Fish S17

18 Pork S18

19 Hegot S19

10 Chicken S20

21 Milk S21

22 Cheese S22

23 Curd S23

24 Ice-cream S24

25 Ghee S25

26 Butter S26

27 Milk cream S27

28 Kneeled wheat S28

29 Bread S29

30 Honey S30

test, Lactose, Sucrose, Dextrose fermentation Reaction test by

standard method given by Sherman, 2005 and Holt et al.,

1994.

Results and Discussion

Bacteria can cause fruits and vegetables to get mushy or

slimy, or meat to develop a bad odor. There are different

spoilage bacteria which grow well at room temperature. The

large number of microorganisms and their waste products

cause the objectionable changes in odor, taste and texture. In

the present study different samples of spoiled food were

collected from the market of Paonta sahib.

Morphological Characterization

The seventy three isolates were recovered from the thirty samples of the various categories of food. The recovered

isolate were characterized on the basis of colour, shape,

texture, margin, arrangement and staining characteristics

(Table 2).

Seventy three isolates were characterized on the basis of

colony morphology and the staining characteristics. It was

observed that thirty-nine isolates were gram (-ve) rods, twenty-five isolates was gram (+ve) rods and nine isolates

was gram (+ve) cocci (Fig 2).

Biochemical Characterization The seventy three isolates were characterized on the basis of

biochemical tests (Table 3). The tests performed to

characterize the isolates were Indole, MR, VP, citrate

utilization, nitrate reduction, fructose, sucrose and dextrose

fermentation (Sherman, 2009).

In the present study, the thirty spoiled samples of various

categories of food such as fruits, vegetables, meat, milk and milk products, ghee, butter, milk cream, kneeled wheat, bread

and honey were collected from the local market of Paonta

sahib. The seventy three isolates were characterized on the

basis of biochemical identification (Sherman, 2009). The

result obtained from the data shows that the bacteria found in

spoiled samples was Bacillus, Klebsiella, Pseudomonas,

E.coli, Lactobacillus, Staphylococcus, Micrococcus and there

prevalence was 18%, 15%, 14%, 10%, 7%, 5%, 4%

respectively (Fig. 3). Klebsiella pneumonia is a potent

enteroinvasive food borne pathogen and causes serious illness

(Sabota et al., 1998).

In order to understand the prevalence of bacteria for different

categories of food individual study of different categories was

carried out. Sixteen isolates was recovered from the seven

samples of fruits. The bacteria found to spoil the fruits were

identified as Klebsiella, Bacillus, E.coli, Staphylococcus,

Pseudomonas, Lactobacillus and there prevalence was

31.25%, 25%, 18.75%, 12.5%, 6.25% and 6.25% respectively

(Fig. 4). A Bacillus strain was isolated from spoiled apple

juice. This strain was acidophilic with a growth range

between pH 2.5 and 5.5. This organism could be a threat to fruit juices during storage at temperatures greater than or

equal to 26°C because its spores were able to survive

pasteurization condition (Cerny et al., 1984). Lactobacillus

fermentum and Lactobacillus plantarum are the heat resistant

lactic acid bacteria obtained from spoiled acidified fruits

(Shearer et al., 2002).

Among the vegetable eight samples was collected and

twenty-one isolates were recovered from them. The bacteria

found to spoil the vegetables were identified as Klebsiella,

Bacillus, E.coli, Staphylococcus, Pseudomonas and there

prevalence in vegetable sample was found to be 33.33%, 23.80%, 14.28%, 14.28%, 14.28% respectively (Fig. 5).

Pseudomona and Klebsiella are responsible for spoilage even

in frozen vegetables (Manani et al., 2006).


Table 2: Morphological Characterization of Recovered Isolates

S.

No.

Sample

code

Isolate

code

Colony Morphology Simple

staining

Gram Staining Suspects

1 S1 S 1I1 Abundant, opaque, white waxy

growth

Rods Gram(+ve),

rods

Bacillus

2 S1 S1I2 Slimy, White, Somewhat

translucent, raised growth

Rods Gram(-ve), rods Klebsiella, E.coli,




4 S2 S2I2 White, moist and glistening growth Rods Gram(-ve), rods E.coli, Klebsiella

5 S3 S3I1 Small ,creamy, whitish, convex

colonies

Rods Gram(+ve),

rods

Lactobacillus

6 S3 S3I2 Slimy, White, Somewhat translucent, raised growth


7 S4 S4I1 Abundant, opaque, white waxy

growth

Rods Gram(+ve),

rods

Bacillus

8 S4 S4I2 Abundant, Opaque, golden growth Cocci Gram(+ve)

cocci

Staphylo-coccus,

Micrococcus




Rods Gram(-ve), rods E.coli, Klebsiella


growth

Rods Gram(+ve),

rods

Bacillus


cocci

Staphylo-coccus,

Micrococcus

13 S6 S6I3 Abundant, thin, white growth with

medium turning green

Rods Gram(-ve), rods Pseudomonas






growth

Rods Gram(+ve),

rods

Bacillus

17 S8 S8I1 Slimy, White, Somewhat translucent, raised growth







growth

Rods Gram(+ve),

rods

Bacillus









cocci

Staphylo-coccus,

Micrococcus



growth

Rods Gram(+ve),

rods

Bacillus




growth

Rods Gram(+ve),

rods

Bacillus







growth

Rods Gram(+ve),

rods

Bacillus


cocci

Staphylo-coccus,

Micrococcus



growth

Rods Gram(+ve),

rods

Bacillus


cocci

Staphylo-coccus,

Micrococcus








growth

Rods Gram(+ve),

rods

Bacillus




41 S17 S17I2 Soft, smooth, yellow growth Cocci Gram(+ve)

cocci

Micrococcus,

Staphylo-coccus,

42 S17 S17I3 Abundant, opaque, white waxy growth

Rods Gram(+ve), rods

Bacillus














growth

Rods Gram(+ve),

rods

Bacillus






colonies

Rods Gram(+ve),

rods

Lactobacillus

53 S21 S21I3 Soft, smooth, yellow growth Cocci Gram(+ve)

cocci

Micrococcus

Staphylo-coccus,


growth

Rods Gram(+ve),

rods

Bacillus

55 S22 S22I1 Small ,creamy, whitish, convex colonies


Lactobacillus


colonies

Rods Gram(+ve),

rods

Lactobacillus






colonies

Rods Gram(+ve),

rods

Lactobacillus


growth

Rods Gram(+ve),

rods

Bacillus




61 S25 S25I2 Soft, smooth, yellow growth cocci Gram(+ve)

cocci

Micrococcus,

Staphylo-coccus




63 S26 S26I2 Soft, smooth, yellow growth cocci Gram(+ve)

cocci

Micrococcus,

Staphylococcus

64 S26 S26I3 White, moist and glistening growth Rods Gram(-ve), rods E.coli ,Klebsiella





colonies

Rods Gram(+ve),

rods

Lactobacillus



Bacillus


growth

Rods Gram(+ve),

rods

Bacillus




Bacillus


colonies

Rods Gram(+ve),

rods

Lactobacillus


growth

Rods Gram(+ve),

rods

Bacillus


Among the meat products thirteen isolates were recovered

from the five samples. The bacteria found to spoil the

vegetables were identified as Pseudomonas, Klebsiella,

Bacillus, E. coli, Micrococcus and there prevalence in

vegetable sample was found to be 38.46%, 23.07%, 23.07%,

7.69%, 7.69% respectively (Fig. 6). A study of the bacteria

associated with spoilage of fresh meat was carried out. The

flora causing spoilage of meat includes Bacillus subtilis,

Escherichia coli, Klebsiella pneumoniae, Micrococcus

varians, Pseudomonas aeruginosa and Staphylococcus

aureus. Pseudomonas aeruginosa was the most dominant of

the isolated species. It was able to utilize glucose as its

primary carbon source and grew faster than the other meat

spoilage organisms (Olajuyigbe et al., 2006).

Among the milk and milk products nine isolate were recovered from the four samples. The bacteria found to spoil

the milk were identified as Lactobacillus, Pseudomonas,

Bacillus, Micrococcus and there prevalence in milk samples

was found to be 44.44%, 22.22%, 22.22%, 11.11%

respectively (Fig. 7). Mesophilic Lactobacillus sp. is the

dominant organisms in mature Cheddar cheese (Jordan et al.,

2002). Species of the genus Lactobacillus are widespread in

nature and can be found on plants or material of plant origin,

in manure and man-made habitats such as sewage, in

fermenting or spoiling food and also in association with the

intestinal tracts and mucous membranes of man and many


Table 3: Biochemical Characterization of the Recovered Isolate

Isolate code Indole MR VP CU NR Lactose Sucrose Dextrose Identified bacteria

S 1I1 - - - - + - A A Bacillus sp.

S1I2 - + - + + AG AG AG Klebsiella sp.

S2I1 - - - + + AG AG AG Klebsiella sp.

S2I2 + + - - + AG A AG E.coli

S3I1 - - - - + + + + Lactobacillus sp.


S4I1 - - - - + - A A Bacillus sp.

S4I2 - + + - + A A A Staphylococcus sp.

S5I1 + + - - + AG A AG E.coli sp.

S5I2 - - + + + AG AG AG Klebsiella sp.

S6I1 - - + - + - A A Bacillus sp.


S6I3 - - - + + - - - Pseudomonas sp.


S7I2 - + + + + AG AG AG Klebsiella sp.


S8I1 - - + + + AG AG AG Klebsiella sp.







S11I2 - + - - + A A A Staphylococcus sp.

S12I1 + + - - + AG A- AG E.coli sp.





S13I3 - - + + - - - Pseudomonas sp.



S14I3 - + - - + A A A Staphylococcus sp.








S17I2 - - - - - - - - Micrococcus sp.


S17I4 + + - - + AG A- AG E.coli sp.
































A= acid, G= gas, AG= acid, gas


animals (Chenoll et al., 2005). Lactobacilli from breast milk

could contribute to an anti-infective protection in neonates

and would be excellent candidates for the development of

infant probiotic products (Olivares et al., 2006). Degradation

of milk components through various enzymatic activities

associated with the contamination of dairy products by

Pseudomonas sp can reduce the shelf life of processed milk

(Cormier et al., 1991; Belgin et al., 2006).

On the basis of this observation it was estimated that the

bacteria causing spoilage in food product were Klebsiella,

E.coli, Bacillus, Lactobacillus, Pseudomonas, Micrococcus

and Staphylococcus.

Conclusion

From the present study it was concluded that seven

bacterial isolates viz. Bacillus, Klebsiella, Pseudomonas,

E.coli, Lactobacillus, Staphylococcus, Micrococcus was

isolated from the spoiled food samples. It was found that the

Bacillus, Klebsiella and Pseudomonas were the dominating species in the spoilage of every categories of food material.

In fruits, vegetables and meat Klebsiella was the most potent

spoiling bacteria. In meat, milk and in fatty products

Pseudomonas was found in most of the spoilages. Although

Klebsiella, Pseudomonas and Bacillus were dominant in

spoilages E.coli, Staphylococcus and Micrococcus was also

recovered from considerable categories of the spoiled sample.

Lactobacillus was recovered from the acidified spoilages as

well as from the milk spoilages. The strains of Lactobacillus

in fresh milk act as probiotic but in spoilages it is responsible

for the acid production and the sour taste and ranicity.

Acknowledgements:

Authors are thankful to Dr. Gaurav Gupta, Director Himachal

Institute of Life Sciences, Paonta Sahib (HP) INDIA for

providing the lab facility. The authors are also grateful to

Mrs. K.P. Rathoure for her technical support.

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Source of support: Nil; Conflict of interest: None declared

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