Study of Lactobacillus as Probiotic Bacteria

Abstract

Because of inhibitory effect, selected probiotic lactobacilli may be used as biological preservative, so, the aim of this study was to present some data on lactobacillus as probiotic bacteria. Lactic acid bacteria were isolated from sausage. Each isolate of lactobacillus species was identified by biochemical tests and comparing their sugar fermentation pattern. Antibacterial activities were done by an agar spot, well diffusion and blank disk method. Enzyme sensitivity of supernatant fluid and concentrated cell free culture after treatment with α-amylase, lysozyme and trypsin was determined. The isolated bacteria were Lacto. plantarum, Lacto delbruekii, Lacto. acidophilus, Lacto. brevis. The isolated bacteria had strong activity against indicator strains. The antibacterial activity was stable at 100ºC for 10 min and at 56ºC for 30 min, but activity was lost after autoclaving. The maximum production of plantaricin was obtained at 25 - 30ºC at pH 6.5. Because, lactobacilli that used to process sausage fermentation are producing antimicrobial activity with heat stability bacteriocin, so, these bacteria may be considered to be a healthy probiotic diet. Lactobacilli originally isolated from meat products are the best condidates as probiotic bacteria to improve the microbiological safety of these foods.

Full text

Iranian J Publ Health, Vol. 33, No. 2, pp.1-7, 2004
1
Study of Lactobacillus as Probiotic Bacteria
*J Nowroozi
1
, M Mirzaii
1
, M Norouzi
2
1
Dept. of Medical Microbiology, Medical School, Iran University of Medical Sciences, Tehran, Iran
2
Dept. of Medical Virology, Medical School, Iran University of Medical Sciences, Tehran, Iran
Abstract
Because of inhibitory effect, selected probiotic lactobacilli may be used as biological preservative, so, the aim of this study
was to present some data on lactobacillus as probiotic bacteria. Lactic acid bacteria were isolated from sausage. Each isolate
of lactobacillus species was identified by biochemical tests and comparing their sugar fermentation pattern. Antibacterial
activities were done by an agar spot, well diffusion and blank disk method. Enzyme sensitivity of supernatant fluid and
concentrated cell free culture after treatment with α-amylase, lysozyme and trypsin was determined. The isolated bacteria
were Lacto. plantarum, Lacto delbruekii, Lacto. acidophilus, Lacto. brevis. The isolated bacteria had strong activity against
indicator strains. The antibacterial activity was stable at 100ºC for 10 min and at 56ºC for 30 min, but activity was lost after
autoclaving. The maximum production of plantaricin was obtained at 25 - 30ºC at pH 6.5. Because, lactobacilli that used to
process sausage fermentation are producing antimicrobial activity with heat stability bacteriocin, so, these bacteria may be
considered to be a healthy probiotic diet. Lactobacilli originally isolated from meat products are the best condidates as
probiotic bacteria to improve the microbiological safety of these foods.
Keywords: Lactobacillus, Probiotic, Sausage, Antibacterial activity, Iran
Introduction
Probiotic cultures have been associated histori-
cally with cultured of milks and dairy products,
from which there is substantial evidence for
positive effects on human health and general
well-being (1, 2). Several in vitro and in vivo
experiments on antagonism of different Lacto-
bacillus strains against Helicobacter pylori and
Clostridium difficile, Campylobacter jejuni, E.
coli were performed. All tested human Lacto-
bacillus strains were able to inhibit the growth
of all strains of anaerobic human gastrointesti-
nal pathogens (3, 4). In addition, bacteriocins
have properties such as antitumour and anticho-
lestrol activity. Chemical reactions associated
with reduction of nitrate, improvements inimmu
nological status and adsorption of vitamins B
group (5). The transity lactic acid bacteria in
the gastrointestinal tract are capable of deliver-
ing enzymes and other substances into the
intestine which possibly help to control intesti-
nal flora (6). Also, the antioxidative activity of
lactic acid bacteria is reported (7). Because of
inhibitory effect, selected probiotic lactobacilli
may be used as biological preservative, so, the
aim of this study was to present some data on
isolation, growth, and antimicrobial activity,
effect of pH, heat, and sensitivity to proteolytic
enzymes of lactobacillus as probiotic bacteria.
Materials and Methods
Each of the following experiments was repeated
thrice to get better results.
Isolation The isolation of lactic acid bacteria
from sausages bought randomly from shops
with different brand of factory was done by
using MRS (pepton, meat extract, yeast extract,
glucose, tween 80) medium. Briefly, 1g of sau-
sage was mixed and vortexed into MRS broth
medium, incubated at 37ºC for 24 h. Growth
from MRS broth cultures was used to streak on
MRS agar plate. Lactobacillus species of these
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*Corresponding author: P. O. BOX: 14155-6183, Fax: +98 21 8058719

J Nowroozi et al: Lactobacillus as…
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isolates were identified by comparing their
sugar fermentation patterns with the scheme de-
scribed in Bergey’s Manual of Systematic
Bacteriology (8).
Growth of bacteria Lactobacilli were grown
in MRS broth or MRS agar. One ml of an
overnight culture of lactobacillus was used to
inoculate 100 ml of MRS broth and incubation
was continued at 20, 25, 30, 35, 37, 40, 45ºC
for 24 h. Samples were removed at regular
intervals (30 min) for the determination of
turbidity (measured at 660 nm), culture pH and
antibacterial activity. The experiment was re-
peated with broth in which the initial pH was
adjusted to 2 to 12 with HCl or NaCl. Initial
and final pH of all samples was also measured.
Culture supernatant (200µl) was heated in a
boiling water bath for 10 min and cooled rap-
idly on ice. Serial twofold dilutions of the
heated supernatants were made in 0.2 N HCl,
and 10µl of each dilution was spotted on to
fresh, duplicate indicator lawns. Cultures were
incubated for 24 h.
Effect of different sugar and NaCl concentra-
tion on production of bacteriocin Isolated
lactobacillus was grown in MRS broth without
beef extract, supplemented with different con-
centration of glucose, xylose, sucrose, furoc-
tose, galactose, maltose and NaCl. Then, re-
maining activity against indicator strains was
assayed.
Preparation of culture supernatant The
bacteriocin producing strain was grown in MRS
broth for 24 h at 25ºC. A cell free solution was
obtained by centrifuge the culture, followed by
filtration of the supernatant through a 0.2 µl
pore size filter. The supernatant was adjusted to
pH 6.5 or dialyzed for 24 h against MRS broth
at 4ºC.
Mode of action one ml of cell free culture
supernatant of isolated lactobacilli was added to
10 ml of a fresh culture logarithmic phase of
indicator bacteria. Culture optical density were
determined (at 660 nm) at appropriate intervals.
Antimicrobial activity For detection of
antagonistic activities, an agar spot procedure,
well diffusion assay and blank disk method
were used. For the agar spot test, supernatant of
overnight cultures of lactobacillus strains were
spotted (1mm) onto the surface of BHI agar
plates of indicator strains and incubated for 24
h at 37ºC to allow colony develop. For the agar-
well diffusion assay, an overnight culture of the
indicator strain was used to inoculate agar
growth media at 37ºC. Wells of 5mm diameter
were cut into agar plates and 50 µl of culture
supernatant fluid containing antibacterial activ-
ity were added to each well. Supernatant fluid
was obtained by growing the inhibitory pro-
ducer strain overnight in MRS broth at 30ºC.
Cells were then removed by centrifugation and
the supernatant fluid placed in the wells and
allowed to diffuse into the agar for 24 h at 4ºC.
The plates were then incubated at optimum
growth temperature of the indicator strains and
examined after 24 h for inhibition zone. Five
sterile paper blank disks were placed on the
agar plate which was inoculated by indicator
strains and 20 µl of the filtered supernatant of
lactobacilli was applied. Plates were incubated
and observed for zones inhibition.
Indicator strains used as indicator organisms for
bacteriocin screening were Staphylococcus
aureus, Salmonella typhi, Yersinia enterocoli-
tica, Bacillus subtilis, Listeria monocytogenes
and other lactobacilli isolated from sausage
without antibacterial activity. The plates were
incubated at 30ºC for 24-48 h or until growth of
the test organism could be easily observed with
naked eye.
Sensitivity to pH and heat To test sensitivity
to pH, the supernatant was adjusted to pHs be-
tween 2 to 12 with HCl or NaOH and incu-
bated. To test heat stability, the supernatant
fluid was heated in boiling water for 10 min, at
56ºC for 15 min, or autoclaved at 121ºC for 15
min. In all cases, the activity remaining after
treatment was measured by spotting procedure.
This experiment repeated and the solutions
were kept at 4 and -20ºC for 4 weeks, then
antibacterial activity was measured. Sensitivity
Sensitivity proteolytic enzymes To test for

Iranian J Publ Health, Vol. 33, No. 2, pp.1-7, 2004
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enzyme sensitivity, cell free culture supernatant
fluid was treated for 1h at 30ºC with trypsin, α-
amylase, lysozyme at final concentration of
0.5mg/ml, 220 IU/mg/ml and 22IU/mg/ml, and
incubated at 37ºC for 1 h.Concentrated cell free
culture supernatants were heated at 100ºC for
20 min and the remaining activity was deter-
mined by spotted procedure.
Bacteriocin concentration one liter
lactobacilli culture was grown in MRS broth at
30ºC until the late logarithmic phase. The cell
removed by centrifugation for 12 min. at 4ºC,
and ammonium sulphate was gradually added
to achieve 40% saturation. The sample was kept
at 4ºC with stirring for 30 min. After centrifu-
gation for 30 min, the resulting pellet was
mixed and solubilized in 120 ml of 10 mM so-
dium phosphate buffer, pH 5.8. Then antimicro-
bial activity was measured against indicator
bacteria (9).
Results
A total of 28 lactic acid bacteria isolated from
sausage were tested for antimicrobial activity.
Only 4 strains (14.3%) of lactobacilli (Lacto.
plantarum, Lacto. delbruekii, Lacto. acidophi-
lus, Lacto. brevis) were shown to produce a
bacteriocin-like substance. Their sensitivity
varied greatly. Lacto. plantarum produced a
more heat stable bacteriocin than the other
isolated strains, which exhibited a broad spec-
trum of inhibitory activity. The antibacterial
activity of plantaricin was more potent than the
other isolated strains when sensitive strains
were in the logarithmic growth phase, including
cell lysis, as observed by decreased in optical
density. No bacteriocin activity was found in
cultures grown at 4 or 8ºC. However, bacterio-
cin production was observed at 20, 25, 30, 37,
40 and 45ºC. At all of these temperatures, the
maximum antimicrobial activity in the growth
medium was obtained in the late logarithmic
phase growth and early of stationary phase.
The amounts of bacteriocin produced at 25 and
30ºC were similar. The bacteriocin activity in
the supernatant was stable and no decrease in
activity was detected after 5 days at 25ºC. The
antibacterial activity was stable at 100ºC for 10
min and at 56ºC for 30 min, but all activity was
lost after autoclaving. The antibacterial activity
was not lost by freezing and thawing, and long
term storage at 4 and -20ºC. When the super-
natants of the cultures containing Lacto. planta-
rum were checked, a small zone of inhibition
was first observed on plates after 6 h at 25ºC
and larger zones of inhibition were detected
after 24 h.
Factors affecting bacteriocin activity The
antibacterial activity of bacteriocin was de-
stroyed by trypsin treatment, but was unaf-
fected by α-amylase and lysozyme.
The inhibitory activity remained stable over the
pH range 2 to10, but was lost after incubation at
pH 12, indicating its sensitivity to alkali treat-
ment. All activity was lost after autoclaving.
The antimicrobial properties of the Lacto-
lactobacillus strains tested were very variable.
Many of the strains showed weak or no inhibi-
tion of the pathogenic strains.
Only 4 strains (14.3%) inhibited the growth of
pathogenic bacteria broadly. The maximum
production of the bacteriocin was obtained at
25ºC at pH 6.5.
Bacteriocin production Maximum
production of bacteriocin was obtained in MRS
broth containing at least 1-2% glucose or xy-
lose. Also, MRS medium with 1% NaCl found
that, the antibacterial activity increased. The
inhibitory activity was maximal at the
beginning of the stationary phase and remained
stable long after growth had ceased, even in the
presence of the producer cells.
Zone inhibition of Staph.aureus against super-
natant of lactobacilli by agar spot method,
blank disk, and agar well diffusion assay are
shown in Figs 1, 2, 3.

J Nowroozi et al: Lactobacillus as…
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Fig. 1 : Zone inhibition of Staph. aureus against supernatant of Lacto. plantarum by
agar spot method.
a: Lacto. Plantarum, b: Lacto. delbruekii, c: Lacto. acidophilus, d: Lacto. brevis, e: Lacto. casei
Fig. 2: Zone inhibition of Staph. aureus against supernatant of Lacto. plantarum by
blank disk method.
a: Lacto. plantarum, b: Lacto. delbruekii, c: Lacto. acidophilus, d: Lacto. brevis, e: Lacto. casei

Iranian J Publ Health, Vol. 33, No. 2, pp.1-7, 2004
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Fig. 3 : Zone inhibition of Staph. aureus against supernatant of Lacto. plantarum by
agar well diffusion method.
a: Lacto. plantarum, b: Lacto. delbruekii, c: Lacto. acidophilus, d: Lacto. brevis, e: Lacto. casei
Discussion
Of 28 lactobacilli isolated from fermented
sausage, 4 (14.3%) lactobacilli had antibacterial
activity against indicator strains ( L. monocyto-
genes, Y. enterocolitica, Staph. aureus, B. sub-
tilis, S. typhi and other isolated lactobacilli with
no antibacterial activity), which were further
characterized. Their antimicrobial substances
inactivated by trypsin and designed as bacterio-
cin (plantaricin). Lacto. plantarum (plantaricin)
showed the broadest range of inhibitory action.
This is agreeing by the results of Kelley et al
(9) and not agrees elsewhere (10). The latter
reported Lacto. casei with a potent antimicro-
bial activity.
Since antibacterial activity decreased after
treatment with trypsin, but not affected with
lysozyme and α-amylase, so, the bacteriocins
have probably a pertinacious nature. This was
in accordance with Gonzalez et al (11). Also,
the protein nature of plantaricin K was
confirmed by its sensitivity to trypsin (12).
Bacteriocin of Lacto. plantarum on the basis of
its stability in the medium, its broad spectrum
of activity on some pathogenic and spoilage
food bacteria and its high potency of plantaricin
production is recommended as food preserva-
tive. Our results showed that bactericidal action
of the bacteriocin against indicator strains were
on logarithmic phase and early stationary phase
and cell lyses in actively growing cells, thereby
causing a decrease in culture optical density.
This was also confirmed by Gao et al (13).
In this study, production of plantaricin was best
in MRS broth, or in a medium containing
peptone, yeast extract, beef extract, glucose,
sodium acetate and Tween 80. Glucose could
be replaced by xylose without a decrease in the
amount of plantaricin, but other carbohydrates
resulted in less bacteriocin being produced.

J Nowroozi et al: Lactobacillus as…
6
Maximum production was coincided with onset
of logariyhmic phase and early of stationary
phase, and these conditions of low pH and high
cell number have also been found to be neces-
sary for the production of high levels of bac-
teriocins. Maximum production of plantaricin
KW30 (9) and bacteriocin of Lacto. delbrueckii
(14) were in MRS broth. Their results are simi-
lar to our results.
Our results showed that bacteriocin activity was
very stable under a series of different condi-
tions such as storage at room temperature for 5
days, 4ºC and -20ºC, and heating (100ºC for 10
min or 56ºC for 30 min). This is confirmed by
Rekhif et al (15).
In general, bacteriocin are from lactobacilli
specially Lacto. plantarum relatively heat stable
with promising inhibitory spectra of antimicro-
bial activities.
Their general heat stability is an advantage,
temperature stability being a very important
parameter if a bacteriocin is to be used as a
food preservative because many proc- essing
procedures involve a heating step.
However, the bacteriocins from Lacto. planta-
rum described in this paper appear quite prom-
ising as potential biopreservatives. Our results
are confirmed by some researchers (9, 11, 15).
Lactic acid bacteria originally isolated from
meat, meat products and dairy products are
probably the best candidates as probiotic bacte-
ria to improve the microbiological safety of
these foods. Since, they are well adapted to the
conditions in meats and dairy products and
should therefore be more competitive than from
other sources.
Interest in lactic acid bacteria is growing. Also,
bacteriocins produced by lactic acid bacteria are
great interest to the food fermentation industry
because they may inhibit the growth of many
food spoilage and pathogenic bacteria. There-
fore, an investigation of bacteriocins in lactic
acid bacteria may offer potential applicability in
food preservation.
Acknowledgements
We would like to thank to laboratory staff in
Dept. of Microbiology, Iran University of
Medical Sciences, Iran this study was a part of
Mr Mehdi Mirzaii thesis.
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