ISOLATION, CHARACTERIZATION AND MICROENCAPSULATION OF PROBIOTIC Lactobacillus curvatus G7 FROM CHICKEN CROP

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Abstract
The controlled release of bioactive substances to their site of action in the GIT is essential in modern drug and food industries. The major obstacles that probiotic bacteria should overcome are stomach acidity and bile salts. In this research a Lactobacillus curvatus strain was isolated from chicken crop; it was identified based on morphological and biochemical characteristics and tested for its probiotic properties. Furthermore; the survival of free and microencapsulated Lb curvatus in 1 % sodium alginate was evaluated in GIT-like conditions. The results showed that 27.87 % of the free cells were found to be resistant to acidic conditions (pH 2) after 1 hour of incubation, while only 2.09 % survived after 2 hours of incubation therefore the bacteria could not be capable of resisting in the stomach. Microencapsulation improved the viability particularly after 2 hours for the reason that 11.36 % of the cells survived after 2 hours. On the other hand, in bile salts, the percentage of survival of the free cells of Lb curvatus was 47 after 4 hours of incubation and decreased to 40 after 8 hours. However, the microencapsulated form resists more since 66 % of the cells survived after 4 hours and more than 52 % survived in bile salts after 8 hours. It appears evidently that cell entrapment in sodium alginate protects the bacteria from gastric and intestinal hostile conditions.
ISOLATION, CHARACTERIZATION AND MICROENCAPSULATION
OF PROBIOTIC Lactobacillus curvatus G7 FROM CHICKEN CROP
Houria Ouled-Haddar1,2, Tayeb idoui2, Mohamed Sifour1,2, Messaouda Guezira2, Messaouda Bouthabet 2
1 Laboratory of Molecular Toxicology, University of Jijel, Jijel, Algeria
2 Department of Molecular and Cell Biology, University of Jijel, Jijel, Algeria
Email: hrourou2002@gmail.com
Abstract: The controlled release of bioactive substances to their site of action in the
GIT is essential in modern drug and food industries. The major obstacles that probiotic
bacteria should overcome are stomach acidity and bile salts. In this research a
Lactobacillus curvatus strain was isolated from chicken crop; it was identified based on
morphological and biochemical characteristics and tested for its probiotic properties.
Furthermore; the survival of free and microencapsulated Lb curvatus in 1 % sodium
alginate was evaluated in GIT-like conditions. The results showed that 27.87 % of the
free cells were found to be resistant to acidic conditions (pH 2) after 1 hour of
incubation, while only 2.09 % survived after 2 hours of incubation therefore the bacteria
could not be capable of resisting in the stomach. Microencapsulation improved the
viability particularly after 2 hours for the reason that 11.36 % of the cells survived after
2 hours. On the other hand, in bile salts, the percentage of survival of the free cells of Lb
curvatus was 47 after 4 hours of incubation and decreased to 40 after 8 hours. However,
the microencapsulated form resists more since 66 % of the cells survived after 4 hours
and more than 52 % survived in bile salts after 8 hours. It appears evidently that cell
entrapment in sodium alginate protects the bacteria from gastric and intestinal hostile
conditions.
Keywords: Lactobacillus curvatus, probiotic, microencapsulation, chicken crop
INTRODUCTION
Probiotics are defined by the FAO as “live microorganisms which, when administered in adequate amounts,
confer a health benefit on the host(FAO report).
Currently, probiotics are being used extensively in veterinary to replace the use of antibiotics. In poultry farming,
probiotics are essentially used to provide beneficial microorganisms that were basically absent in chicken's digestive
tract, thus, the least can profit by favorable effects offered by the introduced microorganisms (Lutful Kabirm,
2009; Gournier-Château et al., 1994). The two main commercial preparations are targeting the crop and the
anterior small intestine as well as the caecum (Fuller et Turvey, 1997). The effects of some probiotic bacteria were
reported; they include modification of the microbial composition and metabolic activity of the intestinal flora,
inhibition of infective pathogens like Escherichia coli, Salmonella typhimurium and Staphylococcus aureus by
competitive exclusion, and enhancing the growth and development indexes in chicken (Higgins et al., 2010, Awad
et al. 2009, Reque et al. 2000). However, during GI passage, cultures are required to tolerate the low pH of the
stomach, and the antimicrobial activity of bile salts, for that reason, it is important to find methods for enhancing the
viability of microbial cells in the digestive tract, one of them is microencapsulation which consists of the technology
for packaging active materials in miniature, sealed capsules that can release their contents at controlled rates under
specific conditions according to Shahidi and Han (1993). Several studies have shown that microencapsulation of
bacteria with alginate at different concentrations or other gels protects them against acid stress, allowing the cells to
survive in the stomach and to be delivered in the intestine (Lee et al. 2004, Crittenden et al. 2006). Generally, most
of the researchers are in agreement that alginate is the most suitable material for encapsulating food ingredients even
though the recent studies are providing new improvements in capsule texture and rheology characteristics.
In the present study, a lactic acid bacterium from the crop content of chickens was isolated, identified and
assessed for its ability to inhibit the growth of some pathogenic bacteria and to attach to intestinal epithelium. In
addition, the tolerance of the bacterium to GIT-like conditions was evaluated before and after microencapsulation in
2 % sodium alginate.
MATERIALS AND METHODS
Isolation of lactic acid bacteria
A 10 g sample of the content of local chicken crop was serially diluted in normal saline, then; the appropriate
dilutions were plated on MRS agar and incubated for 24 hours at 37°C. The obtained colonies were cultured in MRS
broth and further purified.
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Test organisms
The following strains were used as test organisms for antimicrobial activity; Escherichia coli, Klebsiella spp.
from a local rabbit GIT, E. coli ATCC 25929 and Staphylococcus aureus.
Identification of LAB
The isolated strains were identified based on their morphological and biochemical properties according to
Bergey (1994). The tests included gram stain, catalase, arginine dihydrolase, acetoine production, citrate utilization,
growth in hypersaline solution, fermentation type and sugars fermentation. The obtained results were analyzed by
API-LAB program at the "Laboratoire de Biologie des Microorganismes et Biotechnologie" at Es-Senia University,
Oran.
Antibacterial activity
The antibacterial activity of Lb. curvatus culture, the cell-free supernatant and the NaOH neutralized supernatant
(pH 6) against the cited bacteria was evaluated according to Tagg et al. (1976) based on disc diffusion method.
Assay of the in vitro adherence of LAB to epithelial cells
The method described by Lin et al. 2007 was used for the assay of the in vitro adherence of LAB to epithelial
cells. Segment of chicken crop were opened and washed with sterilized phosphate-buffer saline (PBS, pH 7.2). It
was held in PBS at 4 °C for 30 min to remove the surface mucus and then washed three times with PBS. Epithelial
cells were scrapped into sterilized PBS. The cell suspension was examined by microscopy to ensure that
contaminated bacteria had been removed and the epithelial cell concentration was adjusted to approximately 5x104
cells/ml. The adherence of LAB strain to the epithelial cells was assayed as follows: the overnight culture of LAB in
MRS broth was centrifuged and the cell pellet was resuspended to approximately 1.108 CFU/ml in PBS (pH 7.2).
One milliliter of the bacterial suspension was mixed with 1 ml of the suspension of epithelial cells from chicken.
The mixture in a tube was rotated at 20 rev/min at 37 °C for 30 min. The adhesion was observed using light
microscopy (magnification fold, 100x) after stained with 0.5% crystal violet for 5 min (Lin et al. 2007).
Microencapsulation of LAB in 2% sodium alginate
Alginate (2 % w/v) capsules containing the Lb. curvatus cells were prepared by dissolving 2 g of sodium alginate
in 80 mL distilled water under constant mechanical stirring, and heating at 80ºC. The solution was autoclaved and
cooled to 40ºC to which 20 mL of a freshly prepared cell suspension was added and homogenized. The final
solution contained approximately 88.1011 UFC/mL. The mixture was injected through a needle into 100 mL of
autoclaved and pre-cooled 0.05M CaCl2 crosslinking bath. The resultant capsules were allowed to harden in the
cross-linking solution for 30 min, and then washed three times with distilled water (Boyaval et al., 1985).
Survival of LAB in acidic conditions
The viability of free and microencapsulated cells of Lb. curvatus in acidic conditions was tested by incubating
MRS broth (pH 2) inoculated with approximately 1010 UFC/ml (free or encapsulated cells) for 2 hours at 37° C. A
viable count on MRS agar was carried out at 1h intervals over the assay period after appropriate serial dilution in
normal saline. The plates were incubated at 37ºC for 48 h. For microencapsulated cells, the count was determined
after lysis of the capsules in 2M M phosphate buffer (pH7)
Tolerance to bile
The viability of free and microencapsulated cells of Lb. curvatus in bile conditions was studied by incubating
MRS broth supplemented with 0.3% bile salts with approximately 1010 UFC/ml (free or encapsulated cells) for 8
hours at 37° C. A viable count on MRS agar was carried out at 1h intervals over the assay period after appropriate
serial dilution in normal saline. The plates were incubated at 37ºC for 48 h. For microencapsulated cells, the count
was determined as described before.
RESULTS AND DISCUSSION
Eighteen strains were isolated on MRS medium from chicken crop, after biochemical identification it appeared
that most of them belonged to Lactobacillus curvatus. Lactobacillus curvatus J7 was chosen for further
investigations.
Antimicrobial activity test
The antimicrobial activity of the selected bacterium against some bacteria was evaluated in three ways in order
to determine the nature of the inhibitory element. The crude culture, the crude cell-free supernatant as well as the
neutralized supernatant were used to analyze the antagonistic effect; the results are shown in table 1.
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Table 1 Effect of different culture fractions on some test microorganisms.
Test microorganisms
Tested fractions
Inhibition zone diameter (mm)
Klebsiella
E. coli
Staphylococcus
aureus
Crude culture
21
18
17
Cell-free supernatant
19
20
22
Neutralized supernatant
12
14
15
The culture of Lb. curvatus showed a good inhibitory effect against the four tested strains, the inhibition zone
diameters are ranging from 17 mm for S. aureus to 21 mm for Klebsiella, in addition, the cell-free supernatant
displayed also an inhibitory effect whereas the neutralized supernatant did not lost the whole inhibitory activity
although the diameters of the zones are less important (from 12 for Klebsiella to 15 for S. aureus). Several
mechanisms have been reported to describe antagonistic action of probiotic bacteria such as competitive exclusion,
production of antimicrobial compounds, modulation of immune response, alternation of intestinal bacterial
metabolic activity, alteration of microecology of the animal intestine, and inhibition of bacterial translocation. The
production of antimicrobial agents could be easily demonstrated in vitro by the disc diffusion assay; they include
fatty acids, organic acids, hydrogen peroxide, and diacetyl, acetoin and the small, heat-stable inhibitory peptides
called ‘bacteriocins’(Soomro et al., 2002; Simova et al., 2009).
In our experiment, the probiotic Lb. curvatus decreased the growth of the tested microorganisms not only by the
production of lactic acid but other substances could be involved like bacteriocins or hydrogen peroxide this was
confirmed by the residual activity found in the neutralized supernantants.
In vitro adhesion test
The adhesion test of Lb. curvatus to epithelial cells was conducted as described before as it is one of the most
important criteria to select probiotic bacteria (Roy et al., 2006); the results shown in figure 1 indicated that the cells
of the lactic acid bacterium are adherent to the selected epithelial tissue.
Fig. 1 Adhesion of Lb. curvatus to epithelial cells (A: positive result, B: negative control).
As described by Lin et al. 2007, Lb. fermentum cells; isolated from chicken crop highly attach the epithelial
cells, which make them; in addition to the other properties; a good candidate to be selected as a probiotic.
The mechanism of adhesion of these cells is not completely understood, although it was suggested that some
lactic acid bacteria like Lb. plantarum and Lb. rhamnosus are capable of colonizing the lower digestive tract for a
long period resulting in the inhibition of pathogenic bacteria by competing to specific receptors required for
adherence (Robin and Rouchy, 2001; Roy et al., 2006).
Survival test
Microencapsulation of Lb. curvatus was conducted by using 1% sodium alginate; the microcapsules prepared by
extrusion technique were spherical and uniform in size (3 mm), each bead contains approximately 9.1012UFC. The
survival of free and microencapsulated cells in acidic pH of the stomach was evaluated by a 2 h in vitro SGJ
survival assay (Figure 2).
The viability of the free cells decreased intensively after the first hour of incubation in acidic conditions, it
reached approximately 28 %; moreover, only 2 % of the cells remained viable after 2 hours, however, the cells in a
microencapsulated state are slightly more resistant since after one hour, 40% of cells survived and after 2 hours, the
viability attained 11%.
A
B
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0
10
20
30
40
50
60
70
80
90
100
0 0,5 1 1,5 2 2,5
Percent viability
Incubation time (hours)
free
encapsulated
Fig. 2 Effect of acidic pH (2) on the survival of free and microencapsulated Lb. curvatus cells.
The chicken GIT contained a complex microbial community distributed unequally in its different compartments;
the normal flora consists mainly of lactic acid bacteria particularly lactobacilli, enterobacteria and other groups are
also found (Gabriel et al. 2005; Lin et al. 2007). Gizzard's microbial community is less abundant due to the high
acidity; the hostile conditions of the duodenum reduced as well the incidence of microbes, although some
lactobacilli, enterococci and coliforms were isolated (Fuller, 1984). Probiotics must then survive the transit
through the gizzard to exert beneficial effects; therefore, resistance to a low pH (2) for at least 2 hours is required for
a probiotic cell to be delivered effectively to the intestine. Several microencapsulation materials were used to protect
probiotic cells including sodium alginate, carraghenane, pectin, whey proteins… (Voo et al. 2011; Kailasapathy,
2002). However; alginate matrix system is the most widely used and investigated biopolymer for cell
bioencapsulation. It is biocompatible, and it can gel at mild condition with the presence of calcium cations. In a
related study; Lb. acidophilus and Lb. rhamnosus were significantly protected from stomach conditions (Ding and
Shah 2009), similarly; microencapsulated Lb. acidophilus and Bifidobacterium sp. showed 16 % and 16.7 %
increase in viability after incubation at pH 2 for 2 hours when compared to free cells (Vidhyalakshmien, 2009). Lee
and Heo (2000) showed that Bifidobacterium longum encapsulated in calcium alginate containing 2.0, 3.0, and
4.0% sodium alginate tolerated significantly incubation in a simulated gastric juice (pH 1.5) better than free cells.
The death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel
concentration and bead size.
Figure 2 shows that Lactobacillus curvatus was most likely to survive the passage through the stomach,
furthermore microencapsulation within alginate capsules resulted in an approximate 5.4-fold increase in the survival
of cells in pH 2 after 2 hours of incubation.
0
10
20
30
40
50
60
70
80
90
100
0246810
Percent viability
Incubation time (hours)
free
encapsulated
Fig. 3 Effect of bile salts (0.3%) on the survival of free and microencapsulated Lb. curvatus cells.
Viability of probiotic cells in the presence of bile salts was conducted as described by incubating the cells in
MRS medium supplemented with 0.3 % bile salts. Results showed in figure 3 indicated that the viable count of free
cells decreased by approximately 53 % after 4 hours of incubation, it decreased to reach 40 % after 8 hours with an
average cell concentration of about (152.1012 UFC/mL), moreover; the gel-enclosed cells resisted more, more than
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68 % of the cells were found to survive bile treatment for 4 hours; and more than 50 % tolerated the treatment after
8 hours.
Bile salts are the second barrier that probiotic cells should bypass to attain their site of action. In general, the
required concentration of bile salts considered necessary to screen for resistant strains for human and animal use is
0.3% (Pacheko et al. 2010, Lin et al. 2007). Several studies reported the improvement of cell viability when
exposed to bile salts by microencapsulation, Ding and Shah (2009) found that Lb. plantarum and Bifidobacterium
lactis type Bi-07 were slightly sensitive to bile toxicity (39 % of the cells survived the treatment); however,
microencapsulation in 3% alginate enhanced the viability by 2-fold. In a different study; L. bulgaricus KFRI 673, an
acid-sensitive strain was found to survive SGI exposure when protected in alginate microparticles coated with a high
molecular weight chitosan (Lee et al. 2004). Conversely, Bifidobacterium infantis, Lactobacillus casei and L.
acidophilus encapsulated in symbiotic beads composed of Hi-Maize starch (a prebiotic) and sodium alginate did not
demonstrate a significant increase in survival when subjected to in vitro high acid and bile salt conditions (Sultana
et al .2000).
In this study; Lb. curvatus cells were found to be relatively resistant to bile toxicity, in addition; the use of
alginate gel improved their tolerance, as approximately 1.3 fold increase in viability was observed after 8 hours of
treatment.
In conclusion, the isolated Lb. curvatus was found to present good probiotic properties, it displayed
antimicrobial activity against some selected pathogenic bacteria, and a substantial adhesion capacity. Furthermore,
the viability in GIT like conditions was increased by the alginate microencapsulation, which provides additional
evidence that alginate-based microparticles are suitable for food ingredient delivery.
REFERENCES
Awad W. A., Ghareeb K., Abdel-Raheem S. and Bohm J. 2009. Effects of dietary inclusion of probiotic and
symbiotic on growth performance, organ weights, and intestinal histomorphology of broiler chickens. Poult.
Sci. 88: 49-55.
Bergey D. H. and Holt J. G. 1994. Bergey's Manual of Determinative Bacteriology. 9th Ed. Lippincott Williams
& Wilkins. US.
Boyawal P., Lebrum A. and Goulet J. 1985. Etude de l’immobilisation de LB. helveticus dans des billes
d’alginate de calcium. Lait, 65:185.189.
Crittenden R., Weerakkody R., Sanguansri L. and Augustin M. 2006. Synbiotic microcapsules that enhance
microbial viability during nonrefrigerated storage and gastrointestinal transit. Appl. Environ. Microbiol. 72(3):
2280-2282.
Ding W. K. and Shah N. P. 2007. Acid, bile, and heat Tolerance of free and microencapsulated probiotic
bacteria. J. Food Sci. 72 (9): 446450.
Fuller R. and Turvey A. 1997. Bacteria associated with intestinal wall of the fowl. J. Appl. Bacteriol. 44: 75-
80.
Fuller R. 1984. Microbial activity in the alimentary tract of birds. Production and Nutrition. 43: 55-61.
Gabriel I., Mallet S. and Sibill P. 2005. La microflore digestive des volailles: facteurs de variation et
conséquences pour l'animal. INRA, Nouzilly, France: 309-222.
Gournier-Château N., Larpent J. P., Castillanos M.I. and Larpent J. L. 1994. Les probiotiques en alimentation
animale et humaine. Technique et Documentation. Lavoisier : 1-129.
Higgins J. P., Higgins S. E., Wolfenden A. D., Henderson S. N., Torres-Rodriguez A., Vicente J. L. Hargis B.
M. and Tellez G. 2010. Effect of lactic acid bacteria probiotic culture treatment timing on Salmonella enteritidis
in neonatal broilers. Poult. Sci. 89: 243-247.
FAO/OMS Rapport d’experts. 2001. Health and nutritional properties of probiotics in food including powder
milk with live lactic acid bacteria.
Kailasapathy K. 2002. Microencapsulation of probiotic Bacteria: technology and potential applications. Curr.
Iss. Intest. Microbiol ; 3: 39-48.
Lee K. I. and Heo T. R. 2000. Survival of Bifidobacterium longum immobilized in calcium alginate beads in
simulated gastric juices and bile salt solution. Appl. Environ. Microbiol. 66: 869-973.
TOJSAT
TOJSAT : The Online Journal of Science and Technology- January 2012, Volume 2, Issue 1
Copyright © TOJSAT www.tojsat.net
5
Lee J. S., Cha D. S. and Park H. J. 2004. Survival of freeze-dried Lactobacillus bulgaricus KFRI 673 in
chitosan-coated calcium alginate microparticles. J. Agri. Food Chem. 72:7300-7305.
Lin W. H., Yu B., Jang S.H. and Tsen H. Y. 2007. Different probiotic properties for Lactobacillus fermentum
strains isolated from swine and poultry. Anaerobe. 13: 107-113.
Lutful Kabir S. M. 2009. The role of probiotics in the poultry industry. Int. J. Mol. Sci. 10: 3531-3546.
Pacheco K. C., Del Toro G. V., Martinez F. R. and Duran-Paramo. 2010. Am. J. Agri. Biol. Sci. 5(1): 37-42.
Reque F. E., Pandey1., Franco S. G. and Soccol C. R. 2000. Isolation, identification and physiological study of
Lactobacillus fermentum LBP for use as probiotic in chickens. Braz. J. Microbiol. 31:303-307.
Robin J. M. and Rouchy A. 2001. Les probiotiques. Nutrithérapie INFO.
Roy D., Amiot J., Boutin Y. and Lamoureux M. 2006. Innocuité, Qualité et Efficacité des probiotiques.
Biotechnologie des cultures lactiques d’intérêt laitier et probiotique. Canada.
Shahidi F. and Han X. Q. 1993. Encapsulation of food ingredients. Crit. Rev. Food Sci. Nutr. 33(6): 501-547.
Simova E. D., Beshkova D. B. and Dimitrov Z. P. 2009. Characterization and antimicrobial spectrum of
bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products. J. Appl.
Microbiol., 106: 692-701.
Soomro A. H, Masud T. and Anwaar K. 2002. Role of lactic acid bacteria (LAB) in food reservation and
Human Health: A Review. Pak. J. Nutr., 1: 20-24.
Sultana K., Godward G., Reynolds N., Arumugasawamy R., Peiris P. and Kailasspathy K. 2000. Encapsulation
of probiotic bacteria with alginate- starch and evaluation of survival in simulated gastrointestinal conditions and
in yoghurt. Int. J. Food Microbiol. 62:47-55.
Tagg J. R., Dajani A. S. and Wannamaker L. W. 1976. Bacteriocins of Gram positive bacteria. Bacteriol. Rev.
40: 722-756.
Vidhyalakshmi R., Bhakyyaraj R. and Subhasree R. S. 2009. Encapsulation. « The future of Probiotics ». Adv.
Biol. Res. 3: 96-103.
Voo W., Ravindra P., Tey B. T. and Chan E. S. 2011. Comparison of alginate and pectin based beads for
production of poultry probiotic cells. J. Biosci. Bioeng. 111(3): 294-299.
TOJSAT
TOJSAT : The Online Journal of Science and Technology- January 2012, Volume 2, Issue 1
Copyright © TOJSAT www.tojsat.net
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    In the present study, a series of experiments were conducted to evaluate the ability of a combination of 3 ATCC lactobacilli (LAB3) or a commercially available probiotic culture (PROB) to reduce Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) in broiler chicks. Additionally, we varied the timing of PROB administration in relationship to Salmonella challenge and determined the influence on recovery of enteric Salmonella. In experiments 1 to 3, chicks were randomly assigned to treatment groups and were then challenged via oral gavage with Salmonella Enteritidis. Chicks were treated 1 h after Salmonella Enteritidis challenge with LAB3 or PROB. Twenty-four hours posttreatment, cecal tonsils were collected for recovery of enteric Salmonella. In experiments 4 to 7, day-of-hatch chicks were randomly assigned to treatment groups and were then treated with PROB via oral gavage and placed into pens. Chicks were challenged with Salmonella Enteritidis 24 h after treatment via oral gavage. At 24 h after Salmonella Enteritidis challenge, cecal tonsils were collected and recovery of enteric Salmonella was determined. In experiments 8 to 10, 1-d-old chicks were randomly assigned to treatment groups and were then challenged via oral gavage with Salmonella Enteritidis and placed into pens. Chicks were treated 24 h after challenge with PROB via oral gavage. Twenty-four hours post PROB treatment, cecal tonsils were collected and enriched as described above. It was found that PROB significantly reduced cecal Salmonella Enteritidis recovery 24 h after treatment as compared with controls or LAB3-treated chicks in experiments 1 to 3 (P<0.05). Administration of PROB 24 h before Salmonella Enteritidis challenge significantly reduced recovery of Salmonella Enteritidis in 2 out of 4 experiments and no reduction in cecal Salmonella Enteritidis was observed when chicks were challenged with Salmonella Enteritidis and treated 24 h later with PROB. These data demonstrate that PROB more effectively reduced Salmonella Enteritidis than LAB3, and the timing of PROB treatment affects Salmonella Enteritidis-associated reductions.
  • Article
    To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides. Seven LAB strains [Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121 degrees C for 15 min, stable at pH 2-10, sensitive to protease, insensitive to alpha-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N-terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins. Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics. The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori.