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J. Environ. Res. Develop.Journal of Environmental Research And Development Vol. 7 No. 1A, July-September 2012

234

*Author for correspondence

ISOLATION, IDENTIFICATION AND CHARACTERIZATION

OF LACTIC ACID BACTERIA FROM

DAIRY SLUDGE SAMPLE

Choksi Nikita and Desai Hemangi*

TIFAC-CORE In Environmental Engineering, Sarvajanik College of Engineering and Technology

Surat, Gujarat (INDIA)

Received May 02, 2012 Accepted September 10, 2012

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 Lactobacil lus, 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 : Lactic Acid Bacteria (LAB), Isolation, Dairy sludge, MRS agar plate,

MRS Broth, Bacterial strain

INTRODUCTIONTo 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

manufacturing article from biodegradable material.Biodegradable material are those which can be

degrade in the presence of environmental

condition like soil, moisture, microorganism, light,

heat etc. and end products of this is not harmful

to environment.

Dairy industry produces huge volumes of wastes,

both solid and liquids. This waste poses escalating

disposal and pollution (High BOD) problems and



235


represents a loss of valuable biomass and

nutrients. However, despite their pollution and

hazard aspects, in many cases, dairy processing

wastes have a good potential of converting into

useful products of higher value as by-product, or

even as raw material for other industries. Organic

acids are examples of such valuable by-productof the fermentation of high carbohydrate

containing industrial substrates. They therefore

could be utilized cheaply as substrate for

microorganisms producing intermediate volume

high value organic acids like lactic acid. Lactic

acid is under increasing demand in food,

pharmaceutical and chemical industries and for

production of polylactic acid polymers, which

possess outstanding biomedical applications.

Wastes generated from dairy plants may be

regarded as a viable option for meeting this

growing demand for lactic acid and lactic acidhas received attention for use if a wide range of

applications mostly as it acts as a monomer for

the production of biodegradable poly (lactic acid)

or polylactide (PLA). PLA can be produced

chemically and biotechnologically but

biotechnologica l routes are mostly favored

because of environmental concerns and limited

nature of petrochemical feedbacks. Worldwide

efforts have been made for the production of lactic acid and PLA with good yield and low costmanagement.1,2

A long history record of lactic acid used infermentation, preservation of food stuffs is alsoavailable. Lactic acid was firstly discovered byScheela in, 1780 in sour milk and named byLavoisier is acid lactique in 1789 which becamethe origin of present terminology lactic acid.3 Lacticacid has been approved by the US FDA as GRAS

(Generally Recognized As Safe) for the

consumption of it as a food additive, cosmetic,

pharmaceutical, medical implantation4 It can be

produced by chemically and biotechnologically

both.5,6 But due to several serious problems, its

biotechnology route is more favorable because

racemic DL-lactic acid is produced by chemical

synthesis from petro chemical sources whereas

an optically pure L(+) or D(-) lactic acid can be

obtained by microbial fermentation of renewable

sources and higher physical properties of

polymerized Poly Lactic Acid (PLA) is crucially

dependent on the optically pure L(+) or D(-) lactic

acid which is suitable for commercial products.7,8

For the pilot scale production of lactic acid though

biotechnological route, there have been various

requirement for high productivity i.e. cheap raw

material , lactic acid producing microorganism,

fermentation approach, type of bioreactor andfinally purification of optically pure lactic acid for

production of high crystalline lactic acid.

So that production of PLA can be divided into

two step i.e. production of lactic acid and its

polymerization to PLA. Here, initially required

lactic acid producing bacteria which help to lactic

acid production with easily available raw material.

Lactic acid bacteria are a group of related bacteria

that produce lactic acid as a result of carbohydrate

fermentation. The concept of the group name

Lactic Acid Bacteria’ was created for bacteriacausing fermentation and coagulation of milk and

defines as those which produce lactic acid from

lactose. The family name Lactobacteriaceae

was applied by Orla-Jensen to a physiological

group of bacteria producing lactic acid alone or

acetic and lactic acids, alcohol and carbon dioxide.

Today, lactic acid bacteria are regarded as

synonymous by and large with the family

Lactobacteriaceae.

Lactic acid bacteria are widely distributed in the

nature. They could be isolated from soils, waters, plants, silages, waste products and also from the

intestinal tract of animals and humans (Axelsson).

Lactic Acid Bacteria (LAB) are characterized

as Gram - positive, usually non-motile, non -

sporulating bacteria that produce lactic acid as a

major or sole product of fermentative metabolism.

Kandler and Weiss have classified Lactobacillus

isolates from temperate regions according to their

morphology, physiology and molecular characters9

Schleifer classified LAB based on the molecular

characteristics10 LAB from food and their current

taxonomical status have been described by many11,12

These microbes are broadly used by us in the

production of fermented food products, such as

yogurt (Streptococcus spp. and Lactobacillus

spp.), cheese ( Lactococcus spp.), Sauerkraut

( Leuconost oc spp.) and sausage. These

organisms are heterotrophic and usually have

complex nutritional necessities because they lack




236

many biosynthetic capabilities. Most species have

multiple requirements of amino acids and vitamins.

Because of this, lactic acid bacteria are generally

abundant only in communities where these

requirements can be provided. They are often

associated with animal oral cavities and intestines;

plant leaves as well as decaying plants or animalmatter, compost, etc.

The classification of lactic acid bacteria into

different genera is largely based on morphology,

mode of glucose fermentation, growth at different

temperatures and configuration of the lactic acid

pr oduced ability to grow at high sa lt

concentrations and acid or alkaline tolerance.

Lactic acid bacteria are among the best studied

microorganisms for human health advantageous

effects and fermentation. Significant novel

developments have been made in the research of lactic acid bacteria in the area of multidrug

resistance, bacteriocins, osmoregulation, autolysins

and bacteriophages. Advancement has also been

made in the production of food grade genetically

modified lactic acid bacteria.

In the previous studies, LAB could be isolated

from many kinds of sources such as milk products,

sugar cane plants13, fresh water fishes14, but

studies on isolation of LAB from dairy sludge

sample remain scare. Therefore, dairy isolates of

lactic acid bacteria capable of degrading dairy

waste as well as working with other waste raw

material and converting them into lactic acid are

considered to be the key to the development of a

workable microbial fermentation based value

addition process for dairy wastes containing lactic

acid bacteria.

MATERIAL AND METHODS

The present study used sludge samples collected

from 3 different well known dairy plants in Gujarat

at India. Sludge sample collected inside ice box

and transferred to the laboratory for

microbiological analysis MRS agar and broth were

used for enumeration and culture of LAB.15 The

sludge samples serially diluted and pour plated on

MRS plates. The MRS plates overlaid with MRS

agar and incubated at 37 °C for 48–72 h. This is

optimal temperature for bacterial growth but

different lactobacillus strains isolated at particular

temperature set up here for colony isolation and

incubated at 300C, 370C , 400C, 420C and 500C

for 48-72 hours. Well - isolated colonies with

typical characteristics with entire margins were

picked from each plate and transferred to MRS

broth. Lactobacilli isolated on MRS agar should

be further confirmed biochemically.

Identification of lactic acid bacteria at thegenus level

For identification of lactic acid bacteria, overnight

cultures of each isolate in MRS broth were used.

All isolates were initially tested for gram reaction,

catalase enzyme and production of acid from

glucose in Hugh and Leifson medium by oxidation

or fermentation rection (Harrigan and

MacCance). Only gram positive bacteria with

catalase negative reactions were observed

(Schillinger and Lucke, Garvie and Weiss.) and

the representative isolates were purified bysuccessive streaking on to the same agar

substrate. For the gram-positive, catalase negative

rods, growth at various temperature 100C, 150C

and 450C ,tolerance of different salt levels (2,4

and 6.5% w/v NaCl), hetero- and homo-

fermentative activity (using MRS broth) with

inverted Durham tubes in MRS broth were

determined. Isolates from dairy sludge were then

selected based on the above tests for further

identification. The bacteria were characterized by

microscopic and by conventional biochemical and

physiological test. The culture were examined for

colony and cell morphology, motility, gram stain

and production of acid from glucose (Harrigan

and MacCance. In addition to the oxidation and

fermentation test according to Hugh and Leifson.

These preliminary test make it possible to classify

the isolates in genus on the basis of characteristic

and tests of identification mentioned by Harrigan

and MacCane, Hammes et al., Holzapfel and

Schillinger and Dicks et al.

Identification of lactic acid bacteria to the

species level

After their microscopic examination , gram +ve

and catalase –ve lactobacillus were tested for

their sugar fermentation pattern, production of

ammonia from arginine in addition to their ability

of growth at 150C and 450 C. according to

Harrigan and Maccance.16



237


RESULTS AND DISCUSSION

Isolation and identification

Colonies of lactic acid bacteria were observed

on the surface of MRS plates. More than one

colony was observed in most of the cases.

Cultural and morphological characteristics wereexamined with the help of microscope. Different

types of microorganisms were observed, majority

of them belonged to Gram+ve rods and cocci

shaped bacteria. The purification of isolates was

done by transferring Gram+ve rods and cocci

shaped bacteria to the plates of selective media

MRS respectively. These isolates were further

sub cultured until pure isolates were obtained.

Numbers of lactic acid bacterial cultures were

isolated from dairy sludge samples. After initial

identification, most of them were determined as

representative of the family Lactobacillaceae

and genus Lactobacillus and rest were referred

to genus Lact ococcu s, Streptococ cus,

Pedi ococcus, Leuconostoc and some of

Bi fidobacteria, Staphylococcus etc.17,18 A

summary of micro-organisms associated withmilk and milk products is given in Table 1.

This genus should be possible in sludge sample is

depend on many factors like dairy industries

region, raw milk sample, starter culture used for

production, production unit working such as crude

manufacturing, paneer manufacturing, different

types of cheese manufacturing, probiotic butter

milk, seasonal changes, temperature, weather

effects, drainage system etc. So, all time we could

not get same genus as well as same species

Section (According to 9th Family Genus

edition of Bergey’s Manual)

Bacteria Spirillaceae Campylobacter

Pseudomonadaceae Pseudomonas

Brucella

Neisseriaceae Acinetobacter

Moraxella-like organisms

Enterobacteriaceae Escherichia

Enterobacter

Salmonalla,Yersinia

Vibrionaceae AeromonasChromobacterium

Flavobacterium

Vibrio

Rickettsiaceae Coxiella

Microccaceae Micrococcus

Staphylococcus

Streptococcaceae Steptococcus

Leuconostoc

Bacillaceae Bacillus

Clostridium

Lactobacillaceae Lactobacillus

Listeria Propionibacteriaceae Propionibacterium

Corynebacteriaceae Corynebacterium

Arthrobacter

Microbacterium

Actinomycetaceae Actinomyces

Mycobacteriaceae Mycobacterium

Nocardiaceae Nocardia

Table 1 : Micro-organisms associated with milk and milk products




238

therefore, probability changes based on condition.

Here, we are interested to isolate lactic acid

bacteria which are potential for lactic acid

production in nutrient agar more of this were

isolated with it fungal and mould also grown and

contamination problem occurs. So MRS agar is

selected for study. Colonies were identifiedaccording to their morphological, cultural,

physiological and biochemical characteristics 10,11.

This all genus grown on agar plate is used in dairy

starter culture and some of it was present in raw

milk also. Further, genus lactobacillus was

studied on subgenus and species level by

biochemical test as well as some part icular

identification characteristics.

Although LAB are comprised of 11 genera,

only 6 of them are dairy associated

These are Lactococcus, Enterococcus, Streptococcus,

Leuconostoc, Pediococcus, and Lac tobacillus .

(Axelsson, Garvie) Identification test for the

same is shown in Fig. 1.

Pediococcus is a genus of gram-positive lacticacid bacteria, placed within the family of

Lactobacillaceae. They usually occur in pairs

or tetrads and they are purely homofermentative.

Pediococci are used as probiotics, and are

commonly added as beneficial microbes in the

creation of cheeses and yogurts. Recently, lactose

positive pediococci have been used instead of

Streptococcus thermophilus. Lactococcus is a

s

Gram Positive, catalase -negative

Gas from glucose

–v e

Cocci Rods Lac toball ius

homofermentative

Tetrad

Growth @15°C

+Ve pedioc occ us

–Ve

Growth@

45°C

–Ve +Ve

Growth@10°C

+Ve

Lac tococc us

–Ve

Lac toc occ us

+Ve

Enterococcus

Growth 6.5 NoCI

–Ve

thermobacterium

+Ve

treptobacterium

+ve

–v e

Cocci

leuconostorsRoads

Lac tobaci llu Roads

heterotementative

Fig. 1 : Route for identification of lactic acid bacteria at genus levelgenus of Gram-positive lactic acid bacteria that

were formerly included in the genus Streptococcus

Group N1. They are catalase-negative, non-motile

cocci that are found singly, in pairs, or in chains

and they are homofermentors. These organisms

are commonly used in the dairy industry in the

manufacture of fermented dairy products like

cheeses. They can be used in single-strain starter

cultures, or in mixed-strain cultures with other

lactic acid bacteria such as Lactobacillus and

Streptococcus. The bacteria also play a role in

the flavor of the final product.

Steptococcus is a genus of gram- positive lactic

acid bacteria. They are catalase-negative-cocci

usually found in chains or pairs. Some of species

are also great value as they used by dairy industry



239


for the manufacturing of fermented dairy

products.

Leuconostoc is a genus of gram-positive bacteria,

placed within the family of Leuconostocaceae.

They are generally avoid cocci in pairs or often

forming chains. Leuconostoc spp. are catalase-

negative (which distinguishes them fromStaphylococci). All species within this genus are

heterofermentative, i.e., glucose is fermented with

production of D(-) lactic acid, ethanol and CO2

and are able to produce dextran from sucrose.

They are generally slime-forming and used in

cheese manufacturing.

Lactobacillius are gram-positive rods, typically

non-motile, non-sporulating and non-acid fast,

lactobacilli are aerobic and facultatively

anaerobic, catalase negative and grow best at PH

6.0. the carbohydrates and polyalcohols are

changed by homofermentation to lactic acid or by heterofermentation to lactic acid and acetic

acid, alcohols and carbon dioxide. The genus was

subdivided by Orla-jensen into three groups

(Thermo bacterium, Strepto bacterium and beta

bacterium).

Identification test of Lactobacillus bacteria

All isolates were microscopically examined for

gram stain reaction, cell morphology and cellular

arrangement (Gerhardt et al., and Sneath et al.

Catalase test

A drop of 3% hydrogen peroxide was placed on

a clean microscopic slide. With a nicrome wire

loop pick up cells from the center of a well isolated

colony of the test culture and transfer them into

the drop of hydrogen peroxide. Both were mixed

and observed for gas bubble production. The

strains showing gram-positive and catalase

negative isolates were identified at species level.

Physiological and biochemical identification

Each isolate was activated in 5 ml MRS broth for

24 h at 30 ºC before use. Therefore, overnightcultures were used during all the identification

procedures. Physiological and biochemical

identifications were performed according to the

methods and criteria of Sharpe and Fryer; Garvie,

Devriese et. al., Teuber.

For the identification of rod shaped isolates,

following tests were applied

1. Gas poduction from glucose

2. Growth at different temperatures (15ºC, 45ºC)

3. Growth at 6.5% NaCl concentration

4. Arginine hydrolysis5. Carbohydrate fermentation

Gas production from Glucose

In order to further define ho mofermentative

isolates, CO2 production from glucose test was performed. For this purpose, MRS broths and

inverted Durham tubes were used. 50µl of

overnight cultures were transferred into the 8 ml

test media. After incubation for 5 days at 30 ºC,

gas accumulation in Durham tubes was taken as

the evidence for CO2 production from glucose.

Voges-Proskauer (V-P) Test

Inoculate the medium Glucose Phosphate Broth

(GPB) with culture and incubate the medium at

370 C for 24-48 hours. After incubation, add 0.6

ml of a- naphthol and 0.2 ml of KOH solution per ml of broth.(Reagents should be added in this

order only because a-naphthol exerts catalytic

effect only if added before the KOH). Shake well

after addition of each reagent and slope the tube

to increase the aeration. Development of pink-

red color within 15 minutes or more, it means V-

P positive for particular species so, we could

concluded this lactobacillus strain either

thermobacterium or streptobacterium and V-P

negative identified betabacterium.

Growth at different temperatures

50µl of overnight cultures were transferred into

the tubes which contain 5 ml temperature test

media. After inoculation, they were incubated for

7 days at 10 ºC, 40 ºC or 45 ºC. Cells growth at

any of these temperatures was detected by the

change in the color of the cultures, from purple to

yellow.

Growth at different NaCl concentrations

50µl of overnight cultures were transferred into

the tubes which contain 5 ml NaCl test media.

Isolates were tested for growth at 2%, 4% or

6.5% NaCl concentrations. They were incubated

for 7 days at 30ºC. The change of the color from

purple to yellow taken as the evidence for cell

growth.

Arginine hydrolysis test

The arginine hydrolysis, arginine MRS broth was

used instead of Reddy broth. 50µl overnight




240

cultures were inoculated into 5 ml arginine test

media, and were then incubated for 5 days at 30ºC.

After the incubation, ammonia production was

detected by using Nessler reagent. For this

purpose, 100 µl of culture broth were pipetted into

each well of the microtitre plates and 100 µl of

Nessler reagent were added. Immediate orangecolor formation was taken as the indication for

ammonia production. No color change indicated

that the strain could not hydrolyse arginine.

Identification of Cocci

The characteristic used for the identification of

cocci shaped LAB in this study were presented.

Except for the arginine test, all the other tests

were the same as those for bacilli shaped LAB.

Arginine hydrolysis and gas production from

citrate

In order to perform this test, 8 ml of Reddy broth

and inverted Durham tubes were used. Fifty µl of

overnight cultures were inoculated into the Reddy

broth and were then incubated for 5 days at 30ºC.

Arginine hydrolysis

The cultures which utilize arginine, change the

color of the broth first to yellow due to the lactic

acid production and to violet because of the

ammonia production (Cardinal et al). On the other

hand, the cultures which do not utilize arginine

assume a deep-yellow color by producing lactic

acid only.Gas production from citrate

The breakdown of the citrate results in production

of carbon dioxide. Gas accumulation in inverted

Durham tubes indicated citrate utilization.

Sugar fermentation tests

Carbohydrates differentiation discs (from Hi-

media) was used for sugar fermentation test.

Andrade peptone water, liquid media are

dispensed in 5 ml amount in test tube with

inverted Durham’s tube for testing fermentation

and sterilized. A single carbohydrate disc is added

to each tube aseptically and inoculated with test

organisms.

Incubation is carried out at 36±1.0º C for 18-48

hours and results are recorded at 18-24 hours and

again at 48 hours. The results should be frequently

observed since reversal of fermentation can take

place. In case of liquid medium gas produced

during fermentation is collected in the inverted

Durham’s tube while acid production changes color

of the medium. In case of fermentation, the color

of sugar was changed from red to yellow,

reflecting the test as positive.

Percentage distribution of different genus of lactic

acid bacteria were collected from 3 different dairy

industries A, B, C. Dairy sludgeA1(common

sludge unit), Dairy sludgeA2 (acidophilus

but termilk production unit ). dairy sludgeB1(common sludge unit), dairy sludgeB2(cheese

manufacturing unit),dairy sludgeC1(common

sludge unit), dairy sludgeC2(cheese manufacturing

unit). Differential characteristics of lactic acid

bacteria based on morphology and physiology

determined from various microbial and analytical

confirmation test is shown in rod shape

Lactobacillus is divided into three groups.

Summary of Differential characters of

lactobacillus is shown in Table 2. Rod shape

lactobacillus is divided into three groups.

Summary of Differential characters of lactobacillus is shown in Table 3.

The best available microscopic results of the

experiments are shown in Fig. 2 to Fig.4.

Morphological and simple physiological

Characterization of LAB which were isolated from

the dairy sludge sample based on microscopic

characterization and analytical test is shown in

Table 5.

Results of sugar fermentation test and growth

stability at various temperature based on chemical

test is shown inTable 2.

Fig. 2 : Lactic acid

bacteria colony

Fig. 3 : Streptococcu

thremophilus

Fig. 4 : Lactobacillus

bulgaricus



241


Sample Lactic acid bacteria (%)

Streptococcus Leuconostoc Lactococcus Pediococcus Lactobacillus

Dairy SludgeA1 40 5 10 - 45(common Sludge unit)

Dairy SludgeA2 (acidophilus 15 - 5 - 50

buttermilk production unit)

Dairy Sludge B1 35 5 5 5 50

(common sludge unit)

Dairy Sludge B2 30 5 10 - 55

(cheese manufacturing unit)

Dairy Sludge C1 35 10 5 5 45

(common sludge unit)

Dairy Sludge C2 25 5 10 5 55

(Cheese manufacturing unit)

Characteristics

Morphology Cocci Cocci Cocci cocci in tetrads Rods

CO2 from glucose* - - - - ±

Growth

10°C - + + ± ±

45°C ± - - ± ±

6.5% NaCl - - - ± ±

pH 4.4 - ± ± + ±

pH 9.6 - - - - -

Lactic acid configuration L L L L, DL D, L, DL

Table 3 : The percentage distribution of different genus of LAB in dairy sludge sample

and differential characteristics of lactic acid bacteria based on morphology

+Positive; -negative; varies between pecies

*test for homo- or hetero fermentation of glucose : -homofermentation, + heterofermentation

Test Thermo bacterium Strepto bacterium Beta bacterium

Motility – – –

Growth at 5ºc – – –

Growth at 15ºc – + V

Growth at 45ºc + + ?

Gas from glucose – – +

Voges- Proskaucer reaction + + –

Nitrate reduction – – –

Fermentation Homofermentation Homofermentation Heterofermentation

Table 3 : Differential characters of the three group of Lactobacillus

+ = Positive reaction; – = Negative reaction; V= Variable reaction




242

T a b l e 4 : M

o r p h o l o g i c a l a n d s i m p l e p h y s i o l o g i c a l c h a r a c t e r i z a t i o n o f L A B

i s o l a t e d f r o m

t h e d a i r y s l u d g e s a m p l e

S p e c i e s

C e l l s h a p e

G r a m

C a t a l a s e

A c i d

A c i d

C o 2

C e l l u l a r

O p t i m u m

s t a i n

a c t i v i t y

f r o m

f r o m

f r o m

a r r a n g e m e n t

t e m p e r a t u r e

r e a c t i o n

g l u c o s e

l a c t o s e

g l u

c o s e

L .

b u l g a r i c u s

R o d s

G + v e

-

+

+

-

s i n g l e , p a i r s a n d s h o r t c h a i n

s

4 0 ° C

L . a c i d o p h i l u s

R o d s

G + v e

-

+

+

-


s

3 5 - 3 8 ° C

L . c a s e i

R o d s

G + v e

-

-

+

-

S h o r t o r l o n g c h a i n r o d s .

3 0 ° C

L .

l a c t i s

R o d s

G + v e

-

-

+

-

L o n g , s i n g l e o r i n p a i r s

4 0 ° - 4 3 ° C

L .

h e l v i t i c u s

R o d s

G + v e

-

+

+

-


s

4 0 0 - 4 2 ° C

L .

b r e v i s

R o d s

G + v e

-

+

V

+

S i n g l e o r i n c h a i n .

3 0 ° C

t h e r m o p h i l l u s

C o c c i

G + v e

-

+

+

+

P a i r o r l o n g c h a i n C o c c i

4 0 º - 5 0 ° C

L a c t o b a c i l l u s s p p .

R o d s

G + v e

-

±

±

±

S h o r t o r l o n g c h a i n r o d s

3 0 º - 5 0 ° C

S t r e p t o c o c c u s

s p p .

C o c c i

G + v e

-

±

±

±

S p h e r i c a l a n d o v o i d s h a p e

3 0 º - 3 7 ° C

C o c c i

L e u c o n o s t o c s p p .

C o c c i

G + v e

-

±

±

±

P a i r o r s h o r t c h a i n c o c c i

1 5 º - 3 5 ° C

P e d i o c o c c u s s p p .

C o c c i

G + v e

-

±

±

±

T e t r a d s i n p a i r o r s h o r t c h a i n

2 5 ° - 3 2 ° C

C o c c i



243


T a b l e 5

: R e s u l t s o f s u g a r f e r m e n t a t i o n t e s t

G r o w t h a t

S u g a r f e r m e n t a t i o n

S / N

L a c t i c a c i d

M o r

p h o

5 ° C

1 5 ° C

4 5 ° C

A c i d a n d

N H 3

A r a b i

M a n n

M e i z i M

e l i b

R a f f i

S o r b i

T r e h a

M a l

L a c t

G a l a

l o g y

g a s f r o m

f r o m

n o s e

i t o l

t o s e i

o s e

n o s e

t o l

l o s e

t o s e

o s e

c t o s e

g l u c o s e

a r g i n i n e

1

L . b u l g a r i c u s

R

-

-

+

-

-

-

-

-

-

-

-

-

v

+

+

2

L . a c i d o p h i l u s

R

-

-

+

-

-

-

+

-

v

+

-

+

+

+

+

3

L . c a s e i

R

-

+

V

-

-

-

+

+

-

-

+

+

+

+

+

4

L . l a c t i s

R

-

-

+

-

-

-

+

-

-

+

-

+

+

+

+

5

L . h e l v e t i c u s

R

-

-

+

-

-

-

-

-

-

-

-

-

+

+

+

6

L . b r e v i s

R

-

+

-

+

+

+

-

-

+

v

-

-

v

v

v

7

S .

t h e r m o p h i l u s

C

-

+

V

-

+

+

-

v

v

v

v

v

+

+

+




244

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 Lact obac il lus ,

Lactococcus, Streptococcus, Leuconos toc,

Pediococcus. Lactobacillus is rod shape andStreptococcus, 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. bu lgar ic us 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 lact ic acid from natural waste

resources.

REFERENCES

1. Young-Jung Wee., Jin-Nam Kim and Hwa-Won Ryu., Biotechnological Production of Lactic Acid, Food Technol. Biotechnol., 44(2), 163–172, (2006).

2. Vijayakumar J., Aravindan R., and ViruthagiriT., Recent Trends in the production,

purification and application of lactic acid,Chem. Biochem. Eng.Q., 22(2), 245-264,(2008).

3. Benninga H., A history of lactic acid making, Kluwer Academic Publishes , Dordrecht, Netherlands, 1-61,(1990).

4. Datta R., Tsai S.P., Bonsignore P. and MoonS.H.et.al., Techological and economic

potential of poly (lactic acid) and lactic acidderivatives, FEMS Microbi ol . Rev. ,16(1),221-231, (1995).

5. Litch J.H., Microbiological production of lacticacid, adv. Appl. Microbiol. ,42(1),45-95,(1996).

6. Lunt J., Large scale production, propertiesand commercial application of polyacticacid polymers, Poly. Degrad Stabil ,59(2),145-152, (1998).

7. Soder G. and Stolt M., Properties of lacticacid based poltmers and their correlationwith composition, Prog. Poly. Sci.,

27(2),1123-1163,(2002).8. Ashe B. and Paul B. S. Isolation and

characterization of lactic acid bacteria fromdairy effluents, J. Environ. Res. Develop.,4(4), 983-991, (2010).

9. Kandler O. Weiss N., P. H. A. Sneath, N.S. Mair, M. E. Sharpe and J. G. Holt, InBergey’s Manual of SystematicBacteriology, (Eds), Baltimore: Williamsand Wilkins, 2(1), 1209 – 1234, (1986).

10. Schleifer K. H., lactic acid bacteriagenetics, metabolism and application

FEMS . Microb io l. Re v., 46 (1), 201-203,(1987).

11. Gonzalez C. J., Encinas J. P., M. L. Gracia-Lopez and A. Otero A., Lactobacillus

Food. Microbiol ., 17(1),383-391,(2000).12. Salminen S., A. von Wright, Lactic acid

bacteria microbiology and functionalaspects NewYork : Marcel Dekker Inc,2nd Edn.,180-193, ( 1998).

13. Liliana S.C., Lactic acid production by astrain of Lactococcus lactis subs lactisisolated from sugar cane plants,

Electron.J.Biotech, 9(1),152-156, (2006).14. Gonzales C. J., Encinas J.P., Gracia-Lopez

M.L. and Otero A., Characterization andidentification of lactic acid bacteria fromfreshwater fishes, Food Microb io l .,17(1),383-391,(2000).

15. De Man., Rogosa J. C., M. E. Sharpe, Amedium for the cultivation of lactobacilli,

J. Appl . Bacteriol . , 23 (1), 130-135.(1960).

16. Harr igan W.F. and McCance M.E. ,Laboratory methods in food and dairymicrobiology, Acad . Press , 1st Ed.,London, 25-29.(1976).

17. Yadav J.S., Grover S. and Batish V.K.,A

comprehensive dairy microbiology,MetopolitianBook Co Pvt. Ltd, New Delhi

164,(1993)18. Mane V.N. and Gandhi M.B.,Studies on

proteolytic thermoduric psychotrophicv ba cter ia in milk and fermented milk products, J. Environ. Res. Develop., 5(2),384-392, (2011).

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