British Journal of Dairy Sciences 2(1): 5-10, 2011
ISSN: 2044-2440
© Maxwell Scientific Organization, 2011
Received: December 25, 2010
Accepted: February 02, 2011
Published: April 25, 2011
Screening and Characterization of Lactic Acid Bacteria from Fermented Milk
B. Lavanya, S. Sowmiya, S. Balaji and B. Muthuvelan
School of Bio sciences and Technology (SBST), VIT University,
Vellore-632 014, India
Abstract: In this study, different strains were isolated from the fermented milk in Vellore and were subjected
to preliminary screening and 45 isolates were obtained and it was characterized and examined for the presence
of probiotic properties like cholesterol assimilation, exopolysaccride production and antibiotic resistance. The
cholesterol assimilation ranged from 28-83%, which is significantly highest and observed for the first time, and
exopolysaccride varied from 16-89%. Further, resistance to 8 commonly used antibiotics $-lactans (penicillin,
ampicilin), gram positive spectrum (vanomycin), broad spectrum (rifampin, trimethoprim) and aminogycosides
(kanamycin, streptomycin, and bacitracin) was assessed by disk diffusion method. Among the selected 45
strains, 20, 20, 60, 70, 90 and 100% were found to be exhibiting a significant degree of resistance to kanamycin,
trimetroprim, rifampicin, kanamycin, amphicilin and penicillin respectively. However, all strains were resistant
to penicillin and 90% were resistant to ampicillin. Usually all Lactobacillus and Bifidobacterium strains were
susceptible to $-lactum antibiotics but our isolates showed resistance which is contrary and new information
to the previous investigation. Based on the above characters 7 isolates were considered to be best for probiotic
applications. The strains thus obtained are under investigation for further studies.
Key words: Antibiotics resistance, cholesterol assimilation, exopolysaccharide production, probiotics
become unbalanced or greatly reduced in numbers due to
the administration of various antimicrobial agents
(Sanders and Huis in’t, 1999).
Further more many strains of dairy LAB manufacture
extra cellular polysaccharides (EPS). In nature, bacterial
EPS fulfills a variety of diverse functions including cell
protection, adhesion of bacteria to solid surfaces, and
participate in cell-cell interactions. Incorporation of EPS
or EPS-producing (EPS+) cultures in dairy foods can
provide viscosifying, stabilizing, and water-binding
functions. EPS also contributes to the mouth-feel, texture,
and taste perception of fermented dairy products. EPS
may even play a role in the probiotic activity of certain
LAB. Milk fermented with EPS+ dairy LAB generally
develops a ropy or viscous texture, and EPS+ strains of
LAB are widely used in yogurt manufacture to enhance
viscosity and reduce syneresis. Besides yogurt, other
dairy products where LAB EPS has been shown to
affect product quality include sour cream and
traditional fermented milks in Nordic countries
(Nagorska et al., 2008). More recently, researchers have
shown EPS+LAB can enhance the functional properties
of cheese. Because LAB EPS have excellent waterbinding properties and moisture retention is vital to
functionality in low fat cheese, the use of an EPS+ starter
has been shown to improve the moisture and melt
properties
of
low
fat
Mozzarella cheese
(Mansour et al., 2003; Parvez et al., 2006). With the
INTRODUCTION
Lactic Acid Bacteria (LAB) are typically involved in
a large number of spontaneous food fermentations such as
cheese, yoghurt, butter and kimchi. Furthermore, they are
closely associated with the human environment. LAB
associated with fermented foods include species of the
genera Carnobacterium, Enterococcus, Lactobacillus,
Lactococcus, Leuconostoc, Oenococcus, Pediococcus,
Streptococcus, Tetragenococcus, Vagococcus and
Weissella (Searcy and Bergquist, 1960; Stiles and
Holzapfel, 1997). There has been much recent interest in
the use of various strains of LAB as probiotics, since
because these bacteria, mainly lactobacilli and
bifidobacteria, may have several therapeutic functions
(Berg, 1996; Oberg et al., 1998). One of the beneficial
health effects related to probiotics is their ability to reduce
serum cholesterol levels. Lactobacillus acidophilus
actively takes up cholesterol from growth media would
function in vivo to exert a hypocholesterolemic effect.
In general, the probiotic strains should also have
desirable antibiotic resistance and sensitivity patterns, be
antagonist toward potentially pathogenic microorganisms
and have metabolic activities beneficial to the well being
of the host. In this context, many Lactic Acid Bacteria
(LAB) are resistant to antibiotics. On the other hand,
intrinsically antibiotic-resistant probiotic strains may
benefit patients whose normal intestinal microbiota has
Corresponding Author: B. Muthuvelan, School of Bio sciences and Technology, VIT University, Vellore-632 014, Tamil Nadu,
India. Tel: 91-416-2243091 Ext. 2556, Fax: 91-416-2243092
5
Br. J. Dairy Sci., 2(1): 5-10, 2011
Gram positive, catalase
negative
Gas from glucose
-ve
+ve
-ve
Cocci
Arginine
Rods Lactobacillus
homofermentative
Cocci
leuconostos
Tetrad
Rods
Growth @ 15°C
Lactobacillus
heterofermentative
-ve
+Ve
pediococcus
-Ve
thermobacterium
+Ve
streptobacterium
Growth @ 45°C
-ve
+Ve
Growth @ 6.5NaCl
Growth @ 10°C
+Ve
lactococcus
-Ve
streptococcus
+Ve Enterococcus
Fig. 1: Flow sheet for characterization of lactic acid bacteria (Salminen et al., 2002)
above addressed beneficial information on the LAB
organism, the present studies have been carried out with
objective to screen lactic acid bacteria from locally
available fermented milk for the possible application as
potent probiotics based on cholesterol reduction,
antibiotic resistance and exopolysaccride reduction.
at 4ºC for use as working culture. All the procedures were
adapted as proposed in earlier reports (Stiles et al., 1997).
Characterization of isolated cultures: The cultures were
further characterized based on differentiation scheme for
(Fig. 1) genera level identification of lactic acid bacteria
(Ramos et al., 2001).
MATERIALS AND METHODS
Screening of probiotic characters: Cholesterol reduction
assay: Cholesterol reduction measurement was done by
the method described by Searcy and Bergusst (1960).
LAB isolates were grown in MRS broth supplemented
with 0.3% bile salt (sodium thioglycollate, SRL). Further,
ten mg of cholesterol dissolved in 500 :L of ethanol was
added to 100 mL of MRS broth with bile salt. The
cultures were grown for 24 h at 37ºC. Cells were
harvested by centrifugation at 8000 rpm for 10 min at
4ºC. Spent broth was collected and used for cholesterol
assay. The uninoculated broth was considered as control.
To the 1 mL of spent broth, 3 mL of 95% ethanol
followed by 2 mL of 50% potassium hydroxide (KOH,
NICE chemicals) were added and the contents were
mixed well after addition of each component. The tubes
were heated for 10 min at 60ºC in a water bath. After
cooling, 5 mL of hexane was dispensed to all tubes and
Isolation of cultures: All the strains were isolated from
fermented milk samples collected from various sources
and places, during the period January 2010 to August
2010 at VIT University, Vellore, Tamil Nadu, India. The
samples were diluted serially from 10G1 to 10G9 and the
dilutions 10G4 to 10G9 were plated onto Man, Rogosa and
Sharpe media (MRS) agar. The individual colonies with
different morphology were picked using tooth pick and
grown in MRS broth. Further it was plated to check for
purity. These cultures were subjected to preliminary
screening of LAB with Gram staining and catalase
reaction (Ulrich and Friedrich, 1987). Glycerol stocks of
the screened isolates were prepared by mixing 1 mL of
40% glycerol with 1 mL of the culture broth and stored
at -20ºC. A set of MRS stabs were also made and stored
6
Br. J. Dairy Sci., 2(1): 5-10, 2011
chemicals) stock of 1 mg/mL was prepared and 20, 40,
60, 80, 100 :L of this solution was taken in individual test
tubes and the volume was made up to 100 :L with
distilled water. 50 :L of sample was taken and volume
was made to 100 :L with distilled water. Similarly a
blank with 100 :L of water was taken. 50 :L of phenol
solution was added to each test tube followed by addition
of 2 mL of concentrated sulphuric acid in a stream. The
tubes were allowed to stand in room temperature for 10
min. The absorbance was read at 490 nm. The
productivity was found using the following formula:
vortexed for 5 min at 20 sec interval. Then 3 mL of water
was added and mixed thoroughly. Tubes were allowed to
stand for 15 min at 30ºC to permit phase separation. After
that, 2.5 mL of hexane layer was transferred to a fresh test
tube and allowed to dry completely. 1.5 mL of ferric
chloride reagent was added to each test tube and allowed
to stand for 10 min. Then, one ml of concentrated
sulphuric acid (NICE chemicals) was added along the
sides of the tube. The mixture was vortexed and allowed
to stand for 45 min at 30ºC. The optical density was
measured at 540 nm in UV spectrophotometer (UV
ultrospec1100 pro). The concentration of cholesterol was
determined using cholesterol standard graph. The
percentage assimilation was calculated using the formula:
Productivity (%) =
Conc. of carbohydrate in sample- Conc. of cholesterol in control
Conc. of carbohydrate in control
Assimilation (%) =
Conc. of cholesterol in control- Conc. of cholesterol in sample
Conc. of cholesterol in control
RESULTS
In the present study the isolated strains were
identified based on differential scheme for genera level
characterization of lactic acid bacteria. The identification
was based on thermal stability (15, 10 and 45ºC), gas
production and production of ammonia from arginine.
Among those six strains were found to be Lactobacillusheterofermentative (no 15, 14, 31, 47, 56 and 57), two
strains were Leuconostoc, (no 48 and 19), fifteen strains
were Lactobacillus - homofermentative, three strains was
Streptococcus (no 28, 41and 55), fifteen strains was
Enterococcus (no 13, 20, 21, 24, 33, 38, 45, 46, 49, 50,
51, 52, 53, 58 and 32) and five strains was found to be
Pediococcus (no 22, 59, 16, 29 and 54). Table 1 shows
the results of the characterization. The numbers was given
from 13 onwards for the isolated strains. And they were
compared with the 12 standard strains.
Some of the organism are capable of reducing the
cholesterol levels naturally and shows anticholesterol
activity. Table 2 shows the results of cholesterol reduction
by isolates from fermented milk in the presence of 0.3%
bile salt. The assimilation ranged from 28-83%. The
following strains had assimilation greater than 70% (13,
14, 16, 18, 22, 29, 31, 59, 57, 47, 44 and 43).
Most of the lactic acid bacteria are resistant to many
antibiotics. Table 3 shows the result of the resistance
characteristics. Strain 43 was resistant to 7 antibiotics out
of 8, strains 10, 9, 7, 47 were found to be resistant to 6-8
atibiotics, strains 22, 18, 12, 8, 6, 2, 4, 27 and 42 were
resistant to 5-8 antibiotics. Almost all the strains tested
were resistant to penicillin and 10% were susceptible to
ampicillin, ($ lactum antibiotics). Around 20% of the
strains were resistant to kanamycin and streptomycin
(aminoglycosides), 70% were resistant to rifampicin, 20%
to trimethoprim and only 6% were resistant to bacitracin.
In the screening for EPS production the range of
production varied from 16-89% and the strains 43, 29, 16,
10 had the productivity above 80%. Almost 30% of the
LAB will show EPS producing characteristics.
Antibiotic susceptibility test: Disk diffusion method
proposed by Bauer et al. (1966), was followed for
antibiotic susceptibility test. Each strain was inoculated
into the MRS broth, which was incubated at 37ºC for 12
h. Plates were made with muller hinton agar (Hi media)
and allowed to solidify. By spread plate technique the
cultures were inoculated in the plates using sterile swab.
The antibiotic discs of kanamycin, penicillin,
vancomycin, ampicillin, streptomycin, bacitracin,
trimethoprim, rifampicin (Hi media) were placed in the
plates. Agar plates with antibiotic disks were then
incubated for 24 h. The diameters of the inhibition zones
were measured using a ruler under a colony counter
apparatus (Servewell instruments, Banglore). The results
were expressed as sensitive (S), intermediate (I) and
resistant (R) as per the recommended standards.
Screening of exopolysaccride producers: Screening of
strains for exopolysaccride was done by protocol given by
Vijayendra et al. (2008). The cultures were streaked onto
MRS agar plates and incubated for 24 h at 37ºC and the
strains, which produced slimy colonies, were recorded as
capable of producing exopolysaccrides. The selected
producers were inoculated to 10 mL of MRS broth and
incubated overnight. Then it was centrifuged at 10000
rpm for 20 min and the supernatant was transferred to
fresh tube and twice the volume of ice cold isopropanol
(NICE chemicals) was added and exopolysaccride was
allowed to precipitate overnight. It was centrifuged at
12000 rpm for 30 min. The pellet was reprecipitated with
isopropanol for decolourisation. Then the pellet was
dissolved in 1 mL of sterile distilled water.
The amount of EPS was found by phenol sulphuric
acid method (Dubois et al., 1956). In hot sulphuric acid
glucose is dehydrated to hydroxymethylfurfural and this
forms a green coloured product with phenol and it has
absorption maximum of 490 nm. A glucose (S.D fine
7
Br. J. Dairy Sci., 2(1): 5-10, 2011
Table 1: Identification of Genus for the strains isolated from fermented milk
Genus
Lactobacillus - Heterofermentative
Leuconostoc
Lactobacillus - Homofermentative
Streptococcus
Enterococcus
Pediococcus
Isolates
15, 14, 31, 47, 56, 57
48, 19
17, 18, 25, 26, 27, 30, 34, 35, 36, 37, 39, 40, 42, 43, 44
28, 41, 55
13, 20, 21, 24, 33, 38, 45, 46, 49, 50, 51, 52, 53, 58, 32
22, 59, 16, 29, 54
Table 2: Cholesterol assimilation by the isolates
Cholesterol reduction (%)
Less than 50 %
50-70 %
Greater than 70 %
Isolate
1, 13, 4, 16, 17, 22, 26, 27, 32, 33, 34,
3, 6, 7, 9, 15, 25, 26, 32, 33, 41, 42
13, 14, 16, 18, 22, 29, 31, 59, 57, 47, 44, 43.
Table 3: Antibiotic resistance by isolates from fermented milk
Resistant to antibiotics
----------------------------------------------------------------------------------------------------------------------Category
No. of samples Sample No. Va
P
Ri
Tr
Ba
Ka
Am
St
> 7 ab
1
43
R
R
R
R
S
R
R
R
Resistant to 6 Ab
4
47
S
R
R
R
S
R
R
R
7
R
R
R
S
S
R
R
R
9
R
R
R
R
S
S
R
R
10
S
R
R
R
R
S
R
R
Resistant to 5 Ab
9
4
R
R
R
R
S
I
R
S
2
R
R
R
R
S
S
R
S
6
R
R
R
R
I
S
R
S
8
R
R
R
R
I
I
R
S
12
R
R
R
S
S
R
R
I
18
R
R
R
R
S
I
R
I
22
R
R
R
R
I
S
R
S
27
S
R
R
S
R
R
R
I
42
S
R
R
S
S
R
R
S
Va: Vancomycin; P: Penicillin; Ri: Rifampicin; Tr: Trimethoprim; Ba: Bacitracin; Ka: Kanamycin; Am: Ampicillin; St: Streptomycin; R: Resistant;
I: Intermediate; S: Susceptible
Table 4: Probiotic characters for selected strains
Isolates
Genera
Cholesterol assimilation
L08
L. pentosum
77.55
L16
L. reuteri
83.33
L18
L. fermentum
78.57
L43
L. casei
81.97
L47
L. brevis
75.17
Exopolysaccride productivity
63.44
95.20
64.60
90.81
75.87
DISCUSSION
Resistant character
VRTA
RTA
VRTA
VRTKAS
RTKAS
Bifidobacterium HJB -4 had the best hypocholesterolemic
effects (57, 64.4 and 58.8%, respectively) in MRS broth
with soluble cholesterol containing 0.3% oxgall. There are
reports that lactic acid bacteria can reduce the serum
cholesterol level up to 50% in the presence of bile salt in
48 h (Guslandi et al., 2003). In the present study the
isolates 16, 43 and standard strain L. Brevis has the ability
to reduce the cholesterol level upto 80% in 24 h and may
be the important finding. It has been reported that the
ability of the organism to reduce the cholesterol level was
due to assimilation of cholesterol within bacterial cell and
increased excretion of bile salts due to deconjugation by
the bile salt hydrolase (Salminen et al., 2002).
All most all the strains tested were resistant to
penicillin and 10% were susceptible to ampicillin,
($-lactum antibiotics). This is contradictory to the result
predicted by Zhou et al. (2000a) they have reported that,
the Lactobacillus and Bifidobacterium strains were
susceptible to $-lactum antibiotics (Penicillin, ampicillin)
One of the main criteria needed to be fulfilled by a
probiotic organism is, it should be non-pathogenic
(Dubois et al., 1956; Ljungh and Wadstrom, 2006). From
the 47 isolates 33 were chosen for further studies. Due to
the clinical infection by Enterococcus like urinary tract
infection, bacteremia, bacterial endocarditis and
meningitis, these strains were eliminated from further
studies. From a medical standpoint, the most important
feature of this genus is their high level of endemic
antibiotic resistance to $-lactum antibiotics and
aminoglycosides.
Elevated level of certain blood lipids are a greater
risk for cardiovascular disease. A few research
reports describe the use of L. acidophillus to decrease
the serum cholesterol levels in human and animals
(Lee et al., 1992). As per studies by Hyeong-Jun et al.
(2004) Streptococcus HJS-1, Lactobacillus HJL-37 and
8
Br. J. Dairy Sci., 2(1): 5-10, 2011
this may be due to the difference in source of isolation.
Among antibiotic resistances, vancomycin resistance is of
major concern because vancomycin is one of the last
antibiotics broadly efficacious against clinical infections
caused by multidrug resistant pathogens. Some LAB
however, including strains of L. casei, L. plantarum and
Leuconostoc spp., L. bulgaricus, L. fermentum etc were
found to be resistant to vancomycin. Such resistance is
usually intrinsic, that is, chromosomally encoded and
nontransmissible (Zhou et al., 2000b).
In this study, it has been observed that, many of the
strains manufacture EPS. In nature, bacterial EPS fulfills
a variety of diverse functions including cell protection,
adhesion of bacteria to solid surfaces, and participate in
cell-cell interactions. Incorporation of EPS or EPSproducing cultures in dairy foods can provide
viscosifying, stabilizing, and water-binding functions.
These EPS may also be further used in mouth-feel, texture
and taste perception of fermented dairy products
(Vijayendra et al., 2008; Peant et al., 2005; Yamamoto
and Takano, 1996).
Guslandi, M., P. Giollo and P.A. Testoni, 2003. A pilot
trial of Saccharomyces boulardii in ulcerative colitis.
Eur. J. Gastroenterol. Hepatol., 15: 697-698.
Hyeong-Jun, L., K. So-Young and L. Wan-Kyu, 2004.
Isolation of cholesterol lowering lactic acid bacteria
from human intestine for probiotic use. J. Vet. Sci.,
5(4): 391-395.
Lee, Y.W., W.S. Roh and J.G. Kim, 1992. Benefits of
fermented milk in rats fed by hypercholesterolemic
diet (II). Korean J. Food Hyg., 7: 123-135.
Ljungh, A. and T. Wadstrom, 2006. Lactic acid bacteria
as probiotics. Curr. Issues Intest. Microbiol., 7:
73-89.
Mansour-Ghanaei, F., N. Dehbashi, K. Yazdanparast and
A. Shafaghi, 2003. Efficacy of saccharomyces
boulardii with antibiotics in acute amoebiasis. World
J. Gastroenterol., 9: 1832-1833.
Nagorska, K., K. Hinc, M.A. Strauch and
M. Obuchowski, 2008. Influence of the *-B stress
factor and yxaB, the gene for a putative
exopolysaccharide synthase under *-B control, on
Biofilm formation. J. Bacteriol., 190: 3546-3556.
Oberg, C.J., J.R. Broadbent and D.J. McMahon, 1998.
Applications of EPS production by LAB. J. Appl.
Microbiol., 150: 1187-1193.
Parvez, S., K.A. Malik, S. Ah Kang and H.Y. Kim, 2006.
Probiotics and their fermented food products are
beneficial for health. J. Appl. Microbiol., 100:
1171-1185.
Peant, B., G. Lapointe, C. Gilbert, D. Atlan, P. Ward and
D. Roy, 2005. Comparative analysis of the
exopolysaccharide biosynthesis gene clusters from
four strains of Lactobacillus rhamnosus.
Microbiology, 151: 1839-1851.
Ramos, A., I.C. Boels, W.M. de Vos and H. Santos, 2001.
Relationship between glycolysis and
exopolysaccharide biosynthesis in Lactococcus
lactis. Appl. Environ. Microbiol., 67: 33-41.
Salminen, M.K., S. Tynkkynen, H. Rautelin, M. Saxelin,
M. Vaara, P. Ruutu, S. Sarna, V. Valtonen and
A. Jarvinen, 2002. Lactobacillus bacteremia during
a rapid increase in probiotic use of Lactobacillus
rhamnosus GG in Finland. Clin. Infect. Dis., 35:
1155-1160.
Sanders, M.E. and J. Huis in’t Veld, 1999. Bringing a
probiotic-containing functional food to the market:
Microbiological, product, regulatory and labeling
issues. Antonie van Leeuwenhoek, 76: 293-315.
Searcy, R.L. and L.M. Bergquist, 1960. A new color
reaction for the quantification of serum cholesterol.
Clin. Chim. Acta, 5: 192-199.
Stiles, M.E. and W.H. Holzapfel, 1997. Lactic acid
bacteria of foods and their current taxonomy. Int. J.
Food Microbiol., 36: 1-29.
CONCLUSION
From the study we have selected 7 isolates to be
considered for further screening process to fulfill the need
of a probiotic in the food industry. All the selected
isolates were characterized to species level and found to
be L08: Lactobacillus pentosum, L10: L. jungurthi, L16:
L. reuteri, L18: L. fermentum, L29: L. plantarum, L43: L.
brevis, and L47. L. casei. Table 4 shows the
characteristics of the selected isolates.
ACKNOWLEDGMENT
We would like to thank the management of VIT
University, Vellore for providing us their premises for
carrying out this research work. Our heart felt thanks goes
to Dean, SBST, Late Prof. Kunthala Jayaraman, Advisors
and T. Jayashree (JRF) and other faculty for their valid
help.
REFERENCES
Bauer, A.W., M.M. Kirby, J.C. Sherris and M. Turk,
1966. Antibiotic susceptibility testing by a
standardized single disk method. Am. J. Clin. Pathol.,
45: 493-496.
Berg, R., 1996. The indigenous gastrointestinal
microflora. Trends Microbiol., 4: 430-435.
Dubois, M.K., A. Gilles, J.K. Hamilton, P.A. Rebers and
F. Smith, 1956. Colorimetric method for
determination of sugars and related substances. Anal.
Chem., 28(3): 350-356.
9
Br. J. Dairy Sci., 2(1): 5-10, 2011
Ulrich, S. and K.L. Friedrich, 1987. Identification of
Lactobacilli from meat and meat products. Food
Microbiol., 35: 1-27.
Vijayendra, S.V.N., G. Palanivel, S. Mahadevamma and
R.N. Tharanathan, 2008. Physico-chemical
characterization of an exopolysaccharide produced
by a non-ropy strain of Leuconostoc sp. CFR 2181
isolated from dahi, an Indian traditional lactic
fermented milk product. Carbohydrate Polymers, 72:
300-307.
Yamamoto, N. and T. Takano, 1996. Isolation and
characterization of a plasmid from Lactobacillus
helveticus CP53. Biosci. Biotechnol. Biochem., 60:
2069-2070.
Zhou, J.S., P.K. Gopal and H.S. Gill, 2000a. Potential
probiotic lactic acid bacteria Lactobacillus
rhamnosus (HN001), Lactobacillus acidophilus
(HN017) and Bifidobacterium lactis (HN019) do not
degrade gastric mucin in vitro. Int. J. Food
Microbiol., 63: 81-89.
Zhou, J.S., Q. Shu, K.J. Rutherfurd, J. Prasad, M. Birtles,
P.K. Gopal and H.S. Gill, 2000b. Safety assessment
of potential probiotic lactic acid bacterial strains
Lactobacillus rhamnosus HN001, Lb. acidophilus
HN017, and Bifidobacterium lactis HN019 in
BALB/c mice. Int. J. Food Microbiol., 56: 87-96.
10