Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature

Aims: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α‐linolenic acid (α‐LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. Methods and Results: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1–60%). CLA conversion, occurring in three strains, was lower (2–5%). The LAI gene sequences of the ten LAI‐positive strains shared 75–99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6·2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5·5 (30°C) or 37°C (pH 6·2), LA was not converted and α‐LNA only slightly converted. Conclusions: LAB show strain‐dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. Significance and Impact of the Study: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.     

Anti‐listerial activity of bacteriocin‐producing Lactobacillus curvatus CWBI‐B28 and Lactobacillus sakei CWBI‐B1365 on raw beef and poultry meat

Aim: The study aimed to evaluate the effect of the bacteriocins produced by Lactobacillus sakei CWBI‐B1365 and Lactobacillus curvatus CWBI‐B28 on the growth and survival of Listeria monocytogenes in raw beef and poultry meat. Methods and Results: The sakacin P and sakacin G structural genes were identified in Lact. curvatus CWBI‐B28 and Lact. sakei CWBI‐B1365 using PCR amplification, respectively. The effect of the two bacteriocinogenic strains either alone or together, and that of the nonbacteriocin‐producing strain Lact. sakei LMG17302, on the growth of L. monocytogenes was evaluated in beef and poultry meat. In raw beef, the pathogenic bacteria were inhibited by the bacteriocinogenic strains. The bacteriocinogenic strains had no activity in raw chicken meat when inoculated separately, while they showed a clear anti‐Listeria effect when applied together. Conclusion: Sakacin G producing Lact. sakei and sakacin P producing Lact. curvatus may be applied in raw beef to inhibit L. monocytogenes. In poultry meat, the inhibition of L. monocytogenes could only be achieved by a combined application of these bacteriocin‐producing strains. Significance and Impact of the Study: In some meat products, the combined application of different class IIa bacteriocin producing lactic acid bacterium can enhance the anti‐listerial activity.     

Influence of a combination of two potential probiotic strains, Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502® on bowel habits of healthy adults

Aims: This study aims to investigate the effect of different kinds of food products enriched with a combination of two potential probiotic strains, Lactobacillus rhamnosus IMC 501^® and Lactobacillus paracasei IMC 502^®, on bowel habits of healthy adults. Methods and Results: Fifty healthy volunteers took part in a double‐blind placebo probiotic feeding study (25 fed probiotics, 25 fed placebo) for 12 weeks. Each volunteer ingested daily one or more food products enriched with a combination of the two potential probiotic strains (probiotic group) or the same food products without the probiotics (control group). Faecal samples were collected before, at the end and 2 weeks later the intervention period, and some of the main groups of faecal bacteria were enumerated by plate count and real‐time PCR. Questionnaires on bowel habits were submitted to volunteers. After the intervention, a significant increase in faecal lactobacilli and bifidobacteria were observed in the probiotic group, and stool frequency and stool volume were higher in the probiotic group than in the placebo group. Conclusions: Daily consumption of food products enriched with the two potential probiotic strains, Lact. rhamnosus IMC 501^® and Lact. paracasei IMC 502^®, contributes to improve intestinal microbiota with beneficial properties and enhances bowel habits of healthy adults. Significance and Impact of the Study: The study revealed that Lact. rhamnosus IMC 501^® and Lact. paracasei IMC 502^® exert a positive effect, in terms of improved bowel habits, on healthy adults.     

Boza, a natural source of probiotic lactic acid bacteria

Aims: To evaluate the probiotic properties of strains isolated from boza, a traditional beverage produced from cereals. Methods and Results: The strains survived low pH conditions (pH 3·0), grew well at pH 9·0 and were not inhibited by the presence of 0·3% (w/v) oxbile. Cytotoxicity levels of the bacteriocins, expressed as CC₅₀, ranged from 38 to 3776 μg ml^?1. Bacteriocin bacST284BZ revealed high activity (EC₅₀ = 735 μg ml^?1) against herpes simplex virus type 1. Growth of Mycobacterium tuberculosis was 69% repressed after 5 days in the presence of bacST194BZ. Various levels of auto‐cell aggregation and co‐aggregation with Listeria innocua LMG 13568 were observed. Adhesion of the probiotic strains to HT‐29 cells ranged from 18 to 22%. Conclusions: Boza is a rich source of probiotic lactic acid bacteria. All strains survived conditions simulating the gastrointestinal tract and produced bacteriocins active against a number of pathogens. Adherence to HT‐29 and Caco‐2 cells was within the range reported for Lactobacillus rhamnosus GG, a well‐known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Boza contains a number of different probiotic lactic acid bacteria and could be marketed as a functional food product.     

Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra

Aims: This study was designed to isolate and characterize the lactic acid microbiota of the musts and wines of a young denomination of origin area, Ribeira Sacra in north‐west Spain. Methods and Results: Over three consecutive years (2007, 2008 and 2009), we examined musts and wines from four cellars in different zones of the region. Through biochemical and genetic tests, 459 isolates of lactic acid bacteria (LAB) were identified as the following species: Lactobacillus alvei (0·7%), Lactobacillus brevis (1·7%), Lactobacillus frumenti (0·9%), Lactobacillus kunkeei (12%), Lactobacillus plantarum (6·5%), Lactobacillus pentosus (0·9%), Lactococcus lactis ssp. lactis (3%), Leuconostoc citreum (0·7%), Leuconostoc fructosum (synon. Lactobacillus fructosum) (3·7%), Leuconostoc mesenteroides ssp. mesenteroides (2·8%), Leuconostoc pseudomesenteroides (0·2%), Oenococcus oeni (59%), Pediococcus parvulus (7%) and Weisella paramesenteroides (synon. Leuconostoc paramesenteroides) (0·9%). Of these species, O. oeni was the main one responsible for malolactic fermentation (MLF) in all cellars and years with the exception of Lact. plantarum, predominant in 2007, in one cellar, and Lact. brevis, Lact. frumenti and Ped. parvulus coexisting with O. oeni in one cellar in 2009. Different strains (84) of LAB species (14) were identified by biochemical techniques (API strips, the presence of plasmids, enzyme activities and MLF performance) and molecular techniques (PCR). All assays were carried out with every one of the 459 isolates. To select candidates for use as culture starters, we assessed malolactic, β‐glucosidase and tannase activities, the presence of genes involved in biogenic amine production and plasmid content. Conclusions: A high diversity of LAB is present in the grape musts of Ribeira Sacra but few species are responsible for MLF; however, different strains of such species are involved in the process. As far as we are aware, this is the first report of Lact. frumenti thriving in wine. Significance and Impact of the Study: Information on LAB populations in must and wine is presented. A large collection of well‐characterized strains of LAB are available as starter cultures to winemakers.     

Inhibitory effect of oral Lactobacillus against oral pathogens

Aims: To determine the inhibitory effect of oral Lactobacillus against putative oral pathogens. Methods and Results: Total 357 strains comprising 10 species of oral Lactobacillus, Lactobacillus fermentum (195), Lactobacillus salivarius (53), Lactobacillus casei (20), Lactobacillus gasseri (18), Lactobacillus rhamnosus (14), Lactobacillus paracasei (12), Lactobacillus mucosae (12), Lactobacillus oris (12), Lactobacillus plantarum (11) and Lactobacillus vaginalis (10) were used as producer strains. Inhibitory effect against a panel of indicators, periodontitis‐ and caries‐related pathogens, was assessed. Most oral Lactobacillus was able to inhibit the growth of both periodontitis‐ and caries‐related pathogens. The strongest inhibitory activity was associated with Lact. paracasei, Lact. plantarum, Lact. rhamnosus, Lact. casei and Lact. salivarius. Lactobacillus SD1–SD6, representing the six species with the strong inhibitory effect, inhibited growth of Streptococcus mutans ATCC 25175 in the biofilm model. Also, it was demonstrated that growth of Strep. mutans was inhibited in a mixture with Lact. paracasei SD1. The inhibition was enhanced in acidic condition and 5% glucose. Conclusions: The results have shown that oral Lactobacillus SD1–SD6 showed a strong inhibitory effect against Strep. mutans and Streptococcus sobrinus, as well as, Gram‐negative periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Significance and Impact of the Study: The results indicated that Lactobacillus may be of benefit as probiotics for the prevention of oral diseases.     

Probiotic‐mediated competition,exclusion and displacement in biofilm formation by food‐borne pathogens

The objective of this study was to examine the inhibitory effect of probiotic strains on pathogenic biofilm formation in terms of competition, exclusion and displacement. Probiotic strains (Lactobacillus acidophilus KACC 12419, Lact. casei KACC 12413, Lact. paracasei KACC 12427 and Lact. rhamnosus KACC 11953) and pathogens (Salmonella Typhimurium KCCM 40253 and Listeria monocytogenes KACC 12671) were used to evaluate the auto‐aggregation, hydrophobicity and biofilm formation inhibition. The highest auto‐aggregation abilities were observed in Lact. rhamnosus (17·5%), Lact. casei (17·2%) and Lact. acidophilus (15·1%). Salm. Typhimurium had the highest affinity to xylene, showing the hydrophobicity of 53·7%. The numbers of L. monocytogenes biofilm cells during the competition, exclusion and displacement assays were effectively reduced by more than 3 log when co‐cultured with Lact. paracasei and Lact. rhamnosus. The results suggest that probiotic strains can be used as alternative way to effectively reduce the biofilm formation in pathogenic bacteria through competition, exclusion and displacement.

Significance and Impact of the Study

This study provides new insight into biofilm control strategy based on probiotic approach. Probiotic strains effectively inhibited the biofilm formation of Listeria monocytogenes through the mechanisms of competition, exclusion and displacement. These findings contribute to better understand the probiotic‐mediated competition, exclusion and displacement in biofilm formation by pathogens.     

Expression of fluorescent proteins in Lactobacillus rhamnosus to study host–microbe and microbe–microbe interactions

Probiotic Lactobacillus strains are widely used to benefit human and animal health, although the exact mechanisms behind their interactions with the host and the microbiota are largely unknown. Fluorescent tagging of live probiotic cells is an important tool to unravel their modes of action. In this study, the implementation of different heterologously expressed fluorescent proteins for the labelling of the model probiotic strains Lactobacillus rhamnosusGG (gastrointestinal) and Lactobacillus rhamnosusGR‐1 (vaginal) was explored. Heterologous expression of mTagBFP2 and mCherry resulted in long‐lasting fluorescence of L. rhamnosusGG and GR‐1 cells, using the nisin‐controlled expression (NICE) system. These novel fluorescent strains were then used to study in vitro aspects of their microbe–microbe and microbe–host interactions. Lactobacillus rhamnosusGG and L. rhamnosusGR‐1 expressing mTagBFP2 and mCherry could be visualized in mixed‐species biofilms, where they inhibited biofilm formation by Salmonella Typhimurium–gfpmut3 expressing the green fluorescent protein. Likewise, fluorescent L. rhamnosusGG and L. rhamnosusGR‐1 were implemented for the visualization of their adhesion patterns to intestinal epithelial cell cultures. The fluorescent L. rhamnosus strains developed in this study can therefore serve as novel tools for the study of probiotic interactions with their environment.     

Whey protein coating bead improves the survival of the probiotic Lactobacillus rhamnosus CRL 1505 to low pH

Aims: To evaluate the efficacy of a novel microencapsulation procedure using whey protein and pectin to improve the survival rate of Lactobacillus rhamnosus CRL 1505 to low pH and bile. Methods and Results: Lactobacillus rhamnosus CRL 1505 was encapsulated by ionotropic gelation using pectin (PE) and pectin–whey protein (PE–WP). Both types of beads (MC_PE/WP and MC_PE–WP/WP) were covered with a layer of whey protein by complex coacervation. The noncapsulated lactobacilli were not sensitive to bile salts but to acid. Both microparticles protected Lact. rhamnosus CRL 1505 at pH 2·0, but only MC_PE/WP was effective at pH 1·2. Conclusions: The combination of ionotropic gelation and complex coacervation techniques is efficient to obtain microcapsules of pectin covered with whey proteins. The MC_PE/WP beads were more stable than the MC_PE–WP/WP beads in simulated gastric conditions, thus offering better protection to Lact. rhamnosus CRL 1505 at low pH. Significance and Impact of the Study: Pectin beads with a whey protein layer (MC_PE/WP) could be used as probiotic carrier in functional foods of low pH (e.g. apple juice), thus protecting Lact. rhamnosus CRL 1505 against the stressful conditions of the gastric tract.     

The identification of a low molecular mass bacteriocin,rhamnosin A,produced by Lactobacillus rhamnosus strain 68

Aims: This study focuses on the isolation and characterization of a peptide with bacteriocin‐like properties isolated from Lactobacillus rhamnosus strain 68, previously identified by 16S rRNA gene sequencing and originating from human gastrointestinal flora. Methods and Results: The peptide was isolated from a supernatant of bacteria maintained under restrictive conditions by a combination of ethanol precipitation and reversed‐phase chromatography. The molecular mass of the peptide as assessed by mass spectrometry was 6433·8 Da. An isoelectric point of 9·8 was determined by 2D‐PAGE. The peptide designated rhamnosin A inhibited Micrococcus lysodeikticus ATCC 4698 but did not inhibit Lactobacillus plantarum 8014 or Lact. plantarum 39268. Inhibitory activity against M. lysodeikticus at concentrations used in this study was shown to be bacteriostatic rather than bacteriolytic or bactericidal. Rhamnosin A retained biological activity after heat treatment (95°C, 30 min) but was sensitive to proteolytic activity of pepsin and trypsin. Conclusions: The N‐terminal sequence of rhamnosin A, as determined by Edman degradation and in more detail by blast analysis, did not show identity with any currently available Lact. rhamnosus HN001‐translated protein sequences, nor any significant similarity with other sequences in the nonredundant protein sequence database. Being a small, heat‐stable, nonlanthionine‐containing peptide, rhamnosin A should be categorized as a class II bacteriocin. Significance and Impact of the Study: This study describes a partial bacteriocin sequence isolated from Lact. rhamnosus 68 and broadens our understanding of bacteriocins.     

Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis

Aims: To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. Methods and Results: Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 1·9 × 10⁶ CFU LAB g^?1) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1 : 1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8 h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact. murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact. plantarum, Lact. brevis and Lact. sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat‐to‐goat variations did not affect the LAB communities found in the rumen fluid. Conclusion: LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. Significance and Impact of the Study: Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract.     

Bioconversion of isoflavone glycosides to aglycones,mineral bioavailability and vitamin B complex in fermented soymilk by probiotic bacteria and yeast

Aim: To study the role of β‐glucosidase producing probiotic bacteria and yeast in the biotransformation of isoflavone glycosides to aglycones, mineral bioavailability and vitamin B complex in fermented soymilk. Methods and Results: Five isolates of probiotic lactic acid bacteria (LAB), Lactobacillus acidophilus B4496, Lactobacillus bulgaricus CFR2028, Lactobacillus casei B1922, Lactobacillus plantarum B4495 and Lactobacillus fermentum B4655 with yeast Saccharomyces boulardii were used to ferment soymilk to obtain the bioactive isoflavones, genistein and daidzein. High‐performance liquid chromatography was used to analyse the concentration of isoflavones. Bioactive aglycones genistein and daidzein after 24 and 48 h of fermentation ranged from 97·49 to 98·49% and 62·71 to 92·31% respectively with different combinations of LAB with yeast. Increase in bioavailability of minerals and vitamin B complex were also observed in fermented soymilk. Conclusions: LAB in combination with yeast S. boulardii has great potential for the enrichment of bioactive isoflavones, enhancing the viability of LAB strains, decreasing the antinutrient phytic acid and increasing the mineral bioavailability in soymilk fermentation. Significance and Impact of the Study: Fermentation of soymilk with probiotic organisms improves the bioavailability of isoflavones, assists in digestion of protein, provides more soluble calcium, enhances intestinal health and supports immune system. Increased isoflavone aglycone content in fermented soymilk improves the biological functionality of soymilk.     

Antibacterial and Antibiofilm Activity of Lactic Acid Bacteria Isolated from Traditional Artisanal Milk Cheese from Northeast China Against Enteropathogenic Bacteria

The present study aims to investigate the probiotic properties of novel strains of lactic acid bacteria isolated from traditional artisanal milk cheese from Northeast China and to explore their antibacterial activity against enteropathogenic bacteria. Of the 321 isolates, 86 exhibited survival in low pH, resistance to pancreatin, and tolerance to bile salts; of these, 12 inhibited the growth of more than seven enteropathogenic bacteria and exhibited antibiofilm activities against Staphylococcus aureus CMCC26003 and/or Escherichia coli CVCC230. Based on 16S ribosomal RNA sequence analysis, the 12 isolates were assigned to Lactobacillus plantarum (7), Lactobacillus helveticus (3), Pediococcus acidilactici (1), and Enterococcus faecium (1) species. In addition, 5 of the 12 strains were susceptible to most of the tested antibiotics. Furthermore, four strains with sensitivity to antibiotics showed significantly high levels of hydrophobicity similar to or better than the reference strain Lactobacillus rhamnosus GG. Moreover, three strains were confirmed safe through non-hemolytic activities and bacterial translocation. Overall, the selected Lact. plantarum 27053 and 27172 and Lact. helveticus 27058 strains can be considered potential probiotic strains and candidates for further application in functional food and prevention or treatment of gastrointestinal diseases.     

Peptide Extracts from Cultures of Certain Lactobacilli Inhibit Helicobacter pylori

Helicobacter pylori inhibition by probiotic lactobacilli has been observed in vitro and in vivo. Carefully selected probiotic Lactobacillus strains could therefore play an important role in the treatment of H. pylori infection and eradication. However, the underlying mechanism for this inhibition is not clear. The aim of this study was to examine if peptide extracts, containing bacteriocins or other antibacterial peptides, from six Lactobacillus cultures (Lactobacillus acidophilus La1, Lactobacillus amylovorus DCE 471, Lactobacillus casei YIT 9029, Lactobacillus gasseri K7, Lactobacillus johnsonii La1, and Lactobacillus rhamnosus GG) contribute to the inhibition of H. pylori. Peptide extracts from cultures of Lact. amylovorus DCE 471 and Lact. johnsonii La1 were most active, reducing the viability of H. pylori ATCC 43504 with more than 2 log units within 4 h of incubation (P < 0.001). The four other extracts were less or not active. When six clinical isolates of H. pylori were tested for their susceptibility towards five inhibitory peptide extracts, similar observations were made. Again, the peptide extracts from Lact. amylovorus DCE 471 and Lact. johnsonii La1 were the most inhibitory, while the three other extracts resulted in a much lower inhibition of H. pylori. Protease-treated extracts were inactive towards H. pylori, confirming the proteinaceous nature of the inhibitory substance.     

Probiotic Characteristics of <Emphasis Type="Italic">Lactobacillus curvatus</Emphasis> DN317, a Strain Isolated from Chicken Ceca

The probiotic characteristics of Lactobacillus curvatus DN317, a strain isolated from chicken ceca, were evaluated. This strain was selected for study from the isolated Lactobacillus strains because it has specific anti-microbial activity against Campylobacter jejuni ATCC 33560, Camp. jejuni NCTC 11168, Listeria monocytogenes ATCC 7644, and Bacillus subtilis ATCC 8633. Lact. curvatus DN317 showed an auto-aggregation percentage of 72% and presented the highest co-aggregation with Lact. monocytogenes ATCC 7644 (68%) compared to B. subtilis ATCC 8633 (45%), Camp. jejuni ATCC 33560 (36%), and Camp. jejuni NCTC 11168 (35%). The data revealed that Lact. curvatus DN317 could survive at 0.25% bile, maintain viability at pH 2.5 for 30 min, produce biosurfactants, and adhere to Caco-2 cells. Quantification of IL-6, IL-8, IL-10, and β-defensin 2 levels shows that Lact. curvatus DN317 induces an increase in IL-8 and β-defensin 2 secretion, while the levels of IL-6 and IL-10 do not change. Lact. curvatus DN317 showed high levels of esterase and cysteine arylamidase activities (5); moderate levels of esterase lipase, β-galactosidase, and α-galactosidase activities (4, 3); and weak levels of leucine arylamidase, valine arylamidase, and acid phosphatase activity (1). Various activities were obtained of α-chymotrypsin, β-glucuronidase, β-glucosidase, and N-acetyl-β-glucosaminidase, which have been associated with intestinal diseases. Lact. curvatus DN317 lowered the cholesterol level in MRS with and without bile. Antibiotic susceptibility tests indicated that DN317 was sensitive to ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, clindamycin, erythromycin, and vancomycin but was resistant to chloramphenicol and ciprofloxacin. These results suggest that Lact. curvatus DN317 could potentially function as a probiotic.     

Isolation and characterization of bacteriocin‐producing bacteria from the intestinal microbiota of elderly Irish subjects

Aims

To isolate and characterize bacteriocins produced by predominant species of lactic acid bacteria from faeces of elderly subjects.

Methods and Results

Screening over 70 000 colonies, from faecal samples collected from 266 subjects, using the indicator organisms Lactobacillus bulgaricus LMG 6901 and Listeria innocua DPC 3572, identified 55 antimicrobial‐producing bacteria. Genomic fingerprinting following ApaI digestion revealed 15 distinct strains. The antimicrobial activities associated with 13 of the 15 strains were sensitive to protease treatment. The predominant antimicrobial‐producing species were identified as Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus and Enterococcus spp. A number of previously characterized bacteriocins, including ABP‐118 and salivaricin B (from Lact. salivarius), enterocin B (Enterococcus faecium), lactacin B (Lact. acidophilus), gassericin T and a variant of gassericin A (Lact. gasseri), were identified. Interestingly, two antimicrobial‐producing species, not generally associated with intestinally derived microorganisms were also isolated: Lactococcus lactis producing nisin Z and Streptococcus mutans producing mutacin II.

Conclusion

These data suggest that bacteriocin production by intestinal isolates against our chosen targets under the screening conditions used was not frequent (0·08%).

Significance and Impact of the Study

The results presented are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine.     

Quantitative Appraisal of the Probiotic Attributes and In Vitro Adhesion Potential of Anti-listerial Bacteriocin-producing Lactic Acid Bacteria

Estimation of bile tolerance, endurance to gastric and intestinal environment and adhesion potential to intestinal cells are significant selection criteria for probiotic lactic acid bacteria (LAB). In this paper, the probiotic potential of native bacteriocin-producing LAB isolated previously from indigenous source has been determined through quantitative approaches. Among fifteen anti-listerial bacteriocin-producing native LAB, ten strains were found to be bile tolerant. The presence of bile salt hydrolase (bsh) gene in native Lactobacillus plantarum strains was detected by PCR and confirmed by nucleic acid sequencing of a representative amplicon. Interestingly, three native LAB strains exhibited significant viability in simulated gastric fluid, analogous to the standard LAB Lactobacillus rhamnosus GG, while an overwhelming majority of the native LAB strains demonstrated the ability to survive and remain viable in simulated intestinal fluid. Quantitative adhesion assays based on conventional plating method and a fluorescence-based method revealed that the LAB isolates obtained from dried fish displayed significant in vitro adhesion potential to human adenocarcinoma HT-29 cells, and the adhesion level was comparable to some of the standard probiotic LAB strains. The present study unravels putative probiotic attributes in certain bacteriocin-producing LAB strains of non-human origin, which on further in vivo characterization could find specific applications in probiotic food formulations targeted for health benefits.     

Isolation and phenotypic characterization of Lactobacillus casei and Lactobacillus paracasei bacteriophage-resistant mutants

Aims: To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC‐A. Methods and Results: Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD–PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage‐resistant mutants were tested against phages PL‐1, J‐1, A2 and MLC‐A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC‐A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. Conclusions: Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC‐A but also to other Lact. casei and Lact. paracasei phages. Significance and Impact of the Study: This study highlights isolation of spontaneous bacteriophage‐resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.     

Reduction in Listeria monocytogenes and spoilage bacteria on smoked catfish using X-ray treatments

Aims: To determine the efficacy of X‐ray processes in inactivating L. monocytogenes levels in smoked catfish during storage at 5°C and to determine the effects of X‐ray doses on controlling the growth of spoilage bacteria on smoked catfish during storage at 5°C for up to 5 weeks. Methods and Results: Smoked catfish fillets inoculated with L. monocytogenes were treated with 0·0–2·0 kGy X‐ray and stored at 5°C for 5 weeks. The negative controls (uninoculated/untreated) and uninoculated samples treated with the lowest (0·1 kGy) and highest (2·0 kGy) doses were stored at 5°C and tested for psychrotrophs count during the 5 weeks of storage. The initial L. monocytogenes population on smoked catfish was significantly (P < 0·05) reduced to undetectable level by a treatment of 1·0 kGy or higher. The initial psychrotrophs count on smoked catfish was significantly reduced from 4·7 CFU g^?1 to below the detectable level by a treatment with 2·0 kGy. Conclusions: Smoked catfish treated with 2·0 kGy X‐ray had no detectable L. monocytogenes throughout 35 days of storage at 5°C. A treatment with 2·0 kGy X‐ray also kept the levels of psychrotrophs in the smoked catfish within the acceptable level until 35 days. Significance and Impact of the Study: The results of this investigation indicate that X‐ray at 2·0 kGy can eliminate L. monocytogenes and extend the shelf life of smoked catfish stored at refrigeration temperature.     

Lactobacillus plantarum AS1 binds to cultured human intestinal cell line HT-29 and inhibits cell attachment by enterovirulent bacterium Vibrio parahaemolyticus

Aim: Lactobacillus plantarum AS1 was incubated with HT‐29 adenocarcinoma cell line to assess its adhesion potency and examined for its inhibitory effect on the cell attachment by an enterovirulent bacterium Vibrio parahaemolyticus. Methods and Results: Lactobacillus plantarum AS1 attached efficiently to HT‐29 cells as revealed by scanning electron microscopy and bacterial adhesion assay. Lactobacillus plantarum AS1 significantly reduced V. parahaemolyticus attached to HT‐29 cells by competition, exclusion and displacement mode. Lactobacillus plantarum AS1 seems to adhere to human intestinal cells via mechanisms that involve different combinations of carbohydrate and protein factors on the bacteria and eukaryotic cell surface. Conclusion: Strain Lact. plantarum AS1 inhibits the cell attachment of a pathogen V. parahaemolyticus by steric hindrance mechanism. Also, antibacterial factors such as bacteriocins, lactic acid and exopolysaccharides could be involved. Significance and Impact of the Study: The ability to inhibit the adhesion of V. parahaemolyticus to intestinal cell line warrants further investigation to explore the use of probiotic strain Lact. plantarum AS1 in the management of gastroenteritis caused by V. parahaemolyticus.