ejbps, 2015, Volume 2, Issue 4, 1261-1270.
Vaishnana et al.
Research Article
SJIF Impact Factor 2.062
2349-8870
European
Journal of of
Biomedical
and PharmaceuticalISSN
Sciences
European
Journal
Biomedical
Volume: 2
AND
Issue: 4
1261-1270
Pharmaceutical sciences
Year: 2015
http://www.ejbps.com
ISOLATION, CHARACTERIZATION AND DECARBOXYLASE
ACTIVITYOF LACTIC ACID BACTERIA ISOLATED FROM DAIRY
PRODUCTS
Sayani Chatterjee and Archana Vaishnana*
1
Department of Biotechnology, Dolphin (PG) Institute of Biomedical and Natural Sciences,
Deheradun.
Article Received on 08/07/2015
*Correspondence for
Article Revised on 29/07/2015
Article Accepted on 20/08/2015
ABSTRACT
Author
The relationship between certain food and health benefits has been
Dr. Archana Vaishnana
investigated for many years. In recent years, there has been a lot of
Department of
active research in the field of probiotics, due to the growing
Biotechnology, Dolphin
commercial interest in the probiotic food. Strains of Lactobacillus were
(PG) Institute of Biomedical
and Natural Sciences,
Deheradun.
introduced into dairy products because of the potential advantage of
consuming active LAB adapted to the intestine and to produce mildly
acidified yoghurts. In the study isolated bacteria were observed by
light microscope. It is clear that the bacteria were gram positive, rod shaped bacilli and
spherical shaped coccus occurring singly or in chains. The gram staining results indicated that
the isolated bacteria could be identified as lactobacilli. In performing catalase test, the
isolated bacterium is catalase negative. It is well known that Lactobacillus is catalase
negative. Isolated LAB strains were tested for different amino acid decarboxylase activities.
BA production was not detected in the Decarboxylase tests carried out by the isolated LAB.
In performing catalase test, no bubble was observed indicating that the isolated bacterium is
catalase negative and could not mediate the decomposition of H2O2 to produce O2. It is well
known that Lactobacillus is catalase negative.
Decarboxylation systems consist of a
decarboxylase and a precursor. Their combined action results in amino acid intake,
decarboxylation, and release of the corresponding BA. BA production was not detected in the
Decarboxylase tests carried out by the isolated LAB.
KEY WORDS: Lactic Acid Bacteria, Biogenic amines, Catalase, Oxidase, Decarboxylase
activity and Dairy products.
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INRODUCTION
Probiotic microorganisms are often incorporated in food in the form of yoghurt and yoghurt
type fermented food. Recently, there are probiotic ice cream, cheese, infant formulas,
breakfast cereals, sausages, luncheon meats, chocolate and puddings. Nondairy food also has
been manufactured with the addition of the same types of microorganisms. In fact, there are
also medical probiotics in the form of capsules and tablets.[4] Lactobacilli represent a
significant part of our intestinal microflora, and their friendship with the general state of
human health is under serious investigation0.[18] The genus Lactobacillus is one of the major
groups of lactic acid bacteria used in food fermentation and is thus of great economical
importance. Strains of L. acidophilus were introduced into dairy products because of the
potential advantage of consuming active LAB adapted to the intestine and to produce mildly
acidified yoghurts.[19]
In recent years, extensive work has been carried out on bacteriocin producing strains of lactic
acid bacteria (LAB) for their potential use as biopreservatives in food industries. The
preservation of foods by natural and microbiological methods may be a satisfactory approach
to solve economic losses due to microbial spoilage of raw materials and food products, as
well as to reduce the incidence of food borne illnesses.[8] Lactic acid bacteria (LAB) are a
heterogeneous group of Gram- positive, catalase negative rods and cocci usually non motile,
non spore forming. These microorganisms are aerotolerant, microaerophilic or facultative
anaerobic. They are mesophilic with optimal temperature of growth between 30 ºC and 40ºC,
but some strains able to growth at the temperature lower than 5ºC or higher than 45ºC. They
are protected from oxygen byproducts (e. g H2O2) because they have peroxidases. Lactic acid
was their major end product of these microorganisms.[2]
Some species of Lactic Acid bacteria (LAB) can produce Biogenic Amines (BAs). The
ability of microorganisms to decarboxylate amino acids is highly variable, often being strain
specific, and therefore the detection of bacteria possessing amino acid decarboxylase activity
is important to estimate the likelihood that foods contain BA and to prevent their
accumulation in food products. Moreover, improved knowledge of the factors involved in the
synthesis and accumulation of BA should lead to a reduction in their incidence in foods.
Their presence is undesirable because it can result in toxicological effect to consumers such
as hypertension, headache, diarrhea, rash, and localized inflammation when ingested in
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excessive amounts.[10,20] The aim of this study was to isolate and characterization of Lactic
Acid Bacteria from dairy products and its decarboxylase activity.
MATERIALS AND METHODS
Isolation and screening of microorganisms from naturally occurring processes have always
been the most powerful means for obtaining useful cultures for scientific and commercial
purposes. This is certainly true for lactic acid bacteria (LAB), which play an important role in
a large number of various traditional food fermentations. Among these traditional processes,
cheese and fermented milk are known to be essentially fermented by LAB, although often a
functional secondary flora develops. Some properties of LAB such as flavor and texture
formation are especially important to the food and feed industries because of their
applicability for a large variety of products. The dairy industry uses well-defined single strain
and multiple strain starter cultures to obtain dairy products of high and constant quality.
Therefore, a continuous need exists for the isolation of new strains with superior natural
qualities.
(i) Collection of samples
Dairy samples (milk, curd and cheese) were collected from nearby local markets in
Dehradun. Samples of each dairy product were collected aseptically, transferred to sterile
plastic bags and were directly transported to the laboratory under cold conditions. They were
stored at 4 °C and analyzed within 24 hours. 1gm. of Cheese sample was crushed in a mortar
pestle and dissolved in Phosphate Buffer Saline (PBS).
(ii) Preparation of Media
MRS (de Man, Rogosa and Sharpe) agar medium was prepared by dissolving 11.03gm of
MRS agar powder in 200ml of distilled water. MRS broth medium was prepared by
dissolving 5.5g of MRS broth powder in 100ml of distilled water.100ml of Nutrient broth and
Nutrient Agar medium was prepared. The media were autoclaved before use and the agar
medium was poured in sterile petriplates.
(iii)Isolation of Lactic Acid Bacteria
In 3 petriplates, 150 μl of milk, cheese and curd samples was spreaded on MRS agar medium.
In another 3 petriplates the dairy samples were streaked on the agar medium. The plates were
incubated for 24-48 hours at 40 °C. The colonies that showed different morphological
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characteristics were then identified by using various biochemical tests as described by Collins
and Lyne (1980).
(iv) Purification, Maintenance and Preservation of culture
Isolates were purified by subculturing on MRS agar plates and pure cultures were maintained.
A loop full of inoculum was transferred in MRS and Nutrient Broth and was incubated in an
Incubator Shaker at 40 °C for 24-48 hours. Turbidity in broth was observed after 36 hours.
Glycerol stocks were prepared by adding 5ml of autoclaved glycerol to 5ml of overnight
grown culture in Nutrient broth. Glycerol stocks were maintained in cryovials and preserved
at -20°C.
(v) Identification and characterization of isolated colonies
All strains were initially tested for Gram reaction and catalase pro-duction.[11,24] Cell morphology and colony characteristics on MRS were also examined and separation into phenotypic
groups was under-taken. Only the Gram positive, catalase negative isolates were further
identified.
(vi) Morphological characterization
Cell‘s morphology (Gram‘s reaction, cell shape and arrangement) of isolates were studied.
The gram stain reactions of the isolates were determined by light microscopy after gram
staining. Lactic acid bacterial cultures were known to be gram positive so, it means that they
give blue-purple color by gram staining.
(vii) Biological characterization
Fresh liquid cultures of isolates were used for catalase test by dropping 3 % hydrogen
peroxide solution onto 1ml of overnight cultures. The isolates, which did not give gas
bubbles, were known as catalase negative. The oxidase test is the another useful test for the
diagnosing the isolated strains. In performing oxidase test, disc colour did not change to
purple from white after addition of the culture. It is well known oxidase negative
reaction.[3, 21]
Testing Decarboxylase activity
The ability of the test strains to decarboxylate histidine, Arginine and Lysine, was detected as
suggested by.[12] Decarboxylase broth was prepared by adding gm of Decarboxylase broth
powder in 300 ml of distilled water. It was then equally divided in three conical flasks.
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Amino acids Histidine was added to one flask, Arginine to second flask and Lysine to third
flask. After dissolving the amino acids the broth was transferred to labeled test tubes and
autoclaved. Loop full of inoculum of pure cultures was transferred to each test tube and a
layer of mineral oil was laid above the broth to provide anaerobic condition. The test tubes
were incubated for 24-48 hours at 40°C.
RESULT
In the study isolated bacteria were observed by light microscope. It is clear that the bacteria
were gram positive, rod shaped bacilli and spherical shaped coccus occurring singly or in
chains. The gram staining results indicated that the isolated bacteria could be identified as
lactobacilli. In performing catalase test, the isolated bacterium is catalase negative. It is well
known that Lactobacillus is catalase negative. Isolated LAB strains were tested for different
amino acid decarboxylase activities. BA production was not detected in the Decarboxylase
tests carried out by the isolated LAB.
1. Identification of isolated Lactic Acid Bacteria (LAB)
Six isolates were chosen based on their growth appearance on MRS agar media. Gram
staining revealed that all isolates were Gram-positive. Isolates were then grouped based on
their, cell arrangements, Gram reaction, catalase production, and oxidase production
(Table1).
Table 1: Identification of recovered isolates based on Gram staining, Catalase and
Oxidase tests.
Isolates
A
B
C
D
E
F
Gram
character
+ VE
+ VE
+VE
+VE
+VE
+VE
Morphology
Catalase
Oxidase
Bacillus
Bacillus
Coccus
Coccus
Coccus
Coccus
-ve
-ve
-ve
-ve
-ve
-ve
+ve
-ve
-ve
-ve
-ve
+ve
Identified
organism
Weissella
Lactobacillus
Streptococcus
Pediococcus
Lactococcus
Leuconostoc
Based on the shape, form and cell arrangement observed under the microscope, the
microorganisms were then eventually grouped according to cell shape, as cocci and rods.
Two (2) of six (6) isolates were found to be the rod-shaped strains with short and rounded
ends, pairs or single cells and these could presumptively determined as Bacillus. Isolate A
was determined to be Weissella and isolate was found to be Lactobacillus. The rest of the
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isolates (4) were cocci, single and chain, therefore they presumptively referred to as coccus.
Isolate C, D, E and F were determined to be Streptococcus, Pediococcus, Lactococcus and
Leuconostoc respectively.
The catalase test is one of the most useful diagnostic tests for the recognition of
bacteria due to their simplicity. In performing catalase test, no bubble was observed
indicating that the isolated bacterium is catalase negative and could not mediate the
decomposition of H2O2 to produce O2. It is well known that Lactobacillus is catalase
negative.
All the strains tested for oxidase test. Maximum strains showed negative reaction i.e. disc
colour did not change to purple from white after addition of the culture. Two (2) of six (6)
isolates were found to be positive for oxidase test. The rest of the isolates (4) were oxidase
negative as most of the probiotics were oxidase negative in nature.
2. Determination of biogenic amines (BA) production through the Decarboxylase Activity
Lactic acid bacteria play a pivotal role in many food fermentations and sometimes represent a
health threat due to the ability of some strains to produce BA that accumulate in foods and
cause trouble following ingestion. These strains carry specific enzymatic systems catalyzing
the uptake of amino acid precursors (e.g. ornithine and lysine), the decarboxylation inside the
cell, and the release of the resulting biogenic amines (e.g., putrescine and cadaverine).
Decarboxylation systems consist of a decarboxylase and a precursor. Their combined action
results in amino acid intake, decarboxylation, and release of the corresponding BA (Table
2).
Table 2: Decarboxylase test results
Organism
Weissella
Lactobacillus
Streptococcus
Pediococcus
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Amino acids
Histidine
Arginine
Lysine
Histidine
Arginine
Lysine
Histidine
Arginine
Lysine
Histidine
Arginine
Lysine
Result
-VE
-VE
-VE
-VE
-VE
-VE
-VE
-VE
-VE
-VE
-VE
-VE
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Lactococcus
Leuconostoc
European Journal of Biomedical and Pharmaceutical Sciences
Histidine
Arginine
Lysine
Histidine
Arginine
Lysine
-VE
-VE
-VE
-VE
-VE
-VE
BA production was not detected in the Decarboxylase tests carried out by the isolated LAB.
DISCUSSION
The relationship between certain food and health benefits has been investigated for many
years. In recent years, there has been a lot of active research in the field of probiotics, due to
the growing commercial interest in the probiotic food. LAB are regarded as a major group of
probiotic bacteria.[1] Strains of Lactobacillus were introduced into dairy products because of
the potential advantage of consuming active LAB adapted to the intestine and to produce
mildly acidified yoghurts.
Lactic acid bacteria (LAB), a broad group of Gram positive, nonspore forming rods and cocci
have a role as commensal on mucosal surfaces and skin and inhabit the digestive tract of
many animal species including humans.[22] A large number of species of lactic acid
bacteria has been detected in the digestive tract but their prevalence and distribution varied
with the animal species.[24] In general, lactic acid bacteria are the organisms which first
colonize the digestive system of animals. Many lactic acid bacteria possess probiotic
property and are thus widely used in probiotic preparations. Lactic acid bacteria are
common inhabitants of many vegetables and fruits and thus form a part of fermented food
products prepared from these fruits and vegetables.
Based on the shape, form and cell arrangement observed under the microscope, the
microorganisms were then eventually grouped according to cell shape, as cocci and rods.
Two (2) of six (6) isolates were found to be the rod-shaped strains with short and rounded
ends, pairs or single cells and these could presumptively determined as Bacillus. Isolate A
was determined to be Weissella and isolate B was found to be Lactobacillus. The rest of the
isolates (4) were cocci, single and chain, therefore they presumptively referred to as coccus.
Isolate C, D, E and F were determined to be Streptococcus, Pediococcus, Lactococcus and
Leuconostoc respectively.[13]
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In performing catalase test, no bubble was observed indicating that the isolated bacterium is
catalase negative and could not mediate the decomposition of H 2O2 to produce O2. It is
well known that Lactobacillus is catalase negative.[15,17] Maximum strains showed oxidase
negative reaction i.e. disc colour did not change to purple from white after addition of the
culture. The rest of the isolates (4) were oxidase negative. As most of the probiotics were
oxidase negative in nature.[16] Lactobacillus is a gram positive, catalase negative and oxidase
negative bacterium.[7] Amino acid decarboxylation is also considered important criteria for
safety and commonly associated with food fermentation which may results in formation of
biogenic amines (BA). BA can trigger human health problems including palpitation,
hypertension, vomiting and headache.[14] Therefore, a strain with potentiality showing amino
acid decarboxylating activity should not be considered as safe for human welfare. In our
study, all the 10 strain showed negative response regarding biogenic amine production.[23]
Decarboxylation systems consist of a decarboxylase and a precursor. Their combined action
results in amino acid intake, decarboxylation, and release of the corresponding Biogenic
amine (BA). BA production was not detected in the Decarboxylase tests carried out by the
isolated LAB.[5] The results of this research work in addition to results obtained in other
studies carried out on enzymatic characterization of LAB isolated from Genestoso cheese
constitute an essential tool to select LAB strains with interesting characteristics from a
technological point of view.[9]
CONCLUSION
This study had established that wide variety of LAB are present in dairy products (cheese,
curd and milk) and lactobacilli are considered to be one of the most important potential
probiotics. Lactic acid bacterial cultures are designed to meet food safety, shelf life,
technological effectiveness and economic feasibility criteria. Specially selected strains may
also provide probiotic health benefits, and, if properly modified, then may be endorsed with
nutraceutical traits. Many isolates displayed varying promising capabilities but not all the
desirable properties were present in a single isolate. Very interestingly, the experimental
results indicated that some of the selected isolates can be further used to check their probiotic
potential in detail as they were resistance to Gastro-intestinal passage and may be work
effectively in human gut by modulating resident microflora.
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Therefore, isolation and screening of lactic acid bacteria from potential locally fermented
drinks are the basic sources for the discovery of new potential LAB for controlling and
treatment of infectious disease to improve the health quality of human beings.
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