Beruflich Dokumente
Kultur Dokumente
ABSTRACT
Lactic Acid Bacteria (LAB) commonly used as starter cultures in food technology are known to
manufacture antimicrobial products having great potential. The aim of this study is isolation of lactic
acid bacterial strain which has been potential for lactic acid production, to identify, characterize it and
to optimize culture conditions for the process. For this study, we have isolated different strains of
Lactic Acid Bacteria (LAB) from dairy sludge samples which were collected from 3 different dairy
plant of Gujarat, (India). Therefore, characterization of isolated strains through morphological,
physiological, biochemical and carbohydrate fermentation test were done. Lactic Acid Bacteria (LAB)
are a group of Gram-positive, non- spore forming, cocci or rod shaped, catalase-negative and fastidious
organisms, considered as Generally Recognized As Safe (GRAS) organism. For isolation, samples
were serially diluted and plated on MRS agar (DE MAN, ROGOSA and SHARPE agar. HI Media)
Plate. Well-isolated colonies with typical characteristics were picked from each plate and were further
sub cultured until pure isolates were obtained and transferred to MRS broth (HI Media) for further
experiments. Identification of lactic acid bacteria belongs to family Lactobacillaceae was done first
at genus level in the Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus.
Lactobacillus is rod shape and Streptococcus, Leuconostoc, Pediococcus are cocci shape. Most of
them are used in dairy starter culture, present in raw milk, milk-loving bacteria. Isolated colony was
identified by gram-staining, gram +ve and rod shape strain showed confirmation of lactobacillus .
L.bulgaricus and Streptococcus thermophilus were found as the most dominant species in common
sludge unit. While L.acidophillus was found as dominant species along with the L.bulgaricus and
Streptococcus thermophilus in the sludge of pro biotic acidophilus butter milk manufacturing unit
and L.casei, L.helveticus, L.brevis, L.lactis were present as the dominant species in the cheese
manufacturing unit along with the L.bulgaricus and Streptococcus thermophilus. Selected microbial
isolates obtained in this study will be used for production of lactic acid acts as monomer for the
synthesis of biodegradable polymer on my future study.
Key Words :LacticAcidBacteria(LAB),Isolation,Dairysludge,MRSagarplate,
MRS Broth, Bacterial strain
INTRODUCTION
To overcome the problem of environmental
pollution through outcome of industries, waste and
pollutant from different industries, unused packing
material and one is the possible solution of these
problems is to minimize the use or to enhance the
reutilization of waste material. But simplest way
to minimize the rate of pollution is by
*Author for correspondence
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Family
Spirillaceae
Pseudomonadaceae
Neisseriaceae
Enterobacteriaceae
Vibrionaceae
Rickettsiaceae
Microccaceae
Streptococcaceae
Bacillaceae
Lactobacillaceae
Propionibacteriaceae
Corynebacteriaceae
Actinomycetaceae
Mycobacteriaceae
Nocardiaceae
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Genus
Campylobacter
Pseudomonas
Brucella
Acinetobacter
Moraxella-like organisms
Escherichia
Enterobacter
Salmonalla,Yersinia
Aeromonas
Chromobacterium
Flavobacterium
Vibrio
Coxiella
Micrococcus
Staphylococcus
Steptococcus
Leuconostoc
Bacillus
Clostridium
Lactobacillus
Listeria
Propionibacterium
Corynebacterium
Arthrobacter
Microbacterium
Actinomyces
Mycobacterium
Nocardia
ve
ve
Rods Lactoballius
ho mo fe r me nta tiv e
Coc c i
Coc c i
le uc o no sto r s
Tetrad
Growth @ 15C
+Ve
pediococcus
Roads
Lactobacillu Roads
he te r o te me nta tiv e
Ve
Ve
thermobacterium
+Ve
tr epto bacter ium
Gr owth@
45 C
Ve
+Ve
Growth@10C
+Ve
Lactococcus
+Ve
Enterococcus
Ve
Lactococcus
Catalase test
Growth at different temperatures
A drop of 3% hydrogen peroxide was placed on
50l of overnight cultures were transferred into
a clean microscopic slide. With a nicrome wire
the tubes which contain 5 ml temperature test
loop pick up cells from the center of a well isolated
media. After inoculation, they were incubated for
colony of the test culture and transfer them into
7 days at 10 C, 40 C or 45 C. Cells growth at
the drop of hydrogen peroxide. Both were mixed
any of these temperatures was detected by the
and observed for gas bubble production. The
change in the color of the cultures, from purple to
strains showing gram-positive and catalase
yellow.
negative isolates were identified at species level.
Physiological and biochemical identification Growth at different NaCl concentrations
Each isolate was activated in 5 ml MRS broth for 50l of overnight cultures were transferred into
24 h at 30 C before use. Therefore, overnight the tubes which contain 5 ml NaCl test media.
cultures were used during all the identification Isolates were tested for growth at 2%, 4% or
procedures. Physiological and biochemical 6.5% NaCl concentrations. They were incubated
identifications were performed according to the for 7 days at 30C. The change of the color from
methods and criteria of Sharpe and Fryer; Garvie, purple to yellow taken as the evidence for cell
growth.
Devriese et. al., Teuber.
For the identification of rod shaped isolates, Arginine hydrolysis test
following tests were applied
The arginine hydrolysis, arginine MRS broth was
used instead of Reddy broth. 50l overnight
1. Gas poduction from glucose
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Fig. 3 : Streptococcu
bacteria colony
thremophilus
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Fig. 4 : Lactobacillus
bulgaricus
Table 3 : The percentage distribution of different genus of LAB in dairy sludge sample
and differential characteristics of lactic acid bacteria based on morphology
Lactic acid bacteria (%)
Sample
Pediococcus
Lactobacillus
40
10
45
15
50
35
50
30
10
55
35
10
45
25
10
55
Cocci
Cocci
Cocci
cocci in tetrads
Rods
10C
45C
6.5% NaCl
pH 4.4
pH 9.6
L, DL
D, L, DL
CO2fromglucose
Growth
Thermo bacterium
Strepto bacterium
Beta bacterium
Motility
Growth at 5c
Growth at 15c
Growth at 45c
Nitrate reduction
Homofermentation
Homofermentation
Heterofermentation
Fermentation
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G + ve
G + ve
G +ve
G+ ve
G + ve
G +ve
G +ve
G +ve
G +ve
G +ve
G +ve
Rods
Rods
Rods
Rods
Rods
Rods
Cocci
Rods
Cocci
Cocci
Cocci
L. acidophilus
L. casei
L. lactis
L. helviticus
L. brevis
thermophillus
Lactobacillus spp.
Streptococcus spp.
Leuconostoc spp.
Pediococcus spp.
reaction
stain
Gram
L. bulgaricus
Species
Cell shape
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-
activity
Catalase
glucose
from
Acid
lactose
from
Acid
glucose
from
Co2
Cocci
Single or in chain.
arrangement
Cellular
25-32C
15-35C
30-37C
30-50C
40-50C
30C
400-42C
40-43C
30C
35-38C
40C
temperature
Optimum
Table 4 : Morphological and simple physiological characterization of LAB isolated from the dairy sludge sample
L.casei
L.lactis
L.helveticus
L.brevis
S. thermophilus
L.acidophilus
L.bulgaricus
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-
Morpho 5C
logy
Lactic acid
S/N
15C
Growth at
Mann
itol
Sorbi
tol
Treha
lose
Sugar fermentation
Mal
tose
Lact
ose
Gala
ctose
CONCLUSION
Sludge samples were collected from 3 different
dairy plants. Identification of lactic acid bacteria
belongs to family Lactobacillaceae was done
first at genus level in the Lactobacillus,
Lactococcus, Streptococcus, Leuconostoc,
Pediococcus. Lactobacillus is rod shape and
Streptococcus, Leuconostoc, Pediococcus are
cocci shape. Most of them are used in dairy starter
culture, present in raw milk, milk-loving bacteria.
Isolated colony was identified by gram-staining,
gram +ve and rod shape strain showed
confirmation of Lactobacillus . L.bulgaricus and
Streptococcus thermophilus were found as the
most dominant species in common sludge unit.
While L.acidophillus was found as dominant
species along with the L.bulgaricus and
Streptococcus thermophilus in the sludge of pro
biotic acidophilus butter milk manufacturing unit
and L.casei, L.helveticus, L.brevis, L.lactis were
present as the dominant species in the cheese
manufacturing unit along with the L.bulgaricus
and Streptococcus thermophilus.
RECOMMENDATION
This species can be used in further study for
production of lactic acid from natural waste
resources.
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