Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract

AIMS: The study of two human strains of Lactobacillus to be used as probiotics in the gastrointestinal tract. METHODS AND RESULTS: The Lactobacillus acidophilus UO 001 and Lact. gasseri UO 002, were resistant to the gastrointestinal conditions (pH 2 and 3, presence of pepsin, pancreatin or bile salts), the resistance was enhanced in the presence of skimmed milk. Additionally, adhered to Caco-2 cells through glycoproteins in Lact. gasseri and carbohydrates in the case of Lact. acidophilus. These strains are able to inhibit the growth of certain enteropathogens: Salmonella, Listeria and Campylobacter without interfering with the normal microbiota of the gastrointestinal tract, as stated by using the mixed culture and the spot agar test. Finally, strongly adherent Lact. gasseri were found to inhibit the attachment of Escherichia coli O111 to intestinal Caco-2 cells under the condition of exclusion. CONCLUSIONS: These results indicate that the two strains of Lactobacillus from human origin present important properties for survival in, and colonization of, the gastrointestinal tract, that give them potential probiotic. SIGNIFICANCE AND IMPACT OF THE STUDY: Two strains of Lactobacillus isolated from human vagina of healthy premenopausal women could be promising candidates to be used in the preparation of probiotic products and for their use as health-promoting bacteria.     

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS-PAGE of whole cell proteins and rRNA-targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.     

β-Galactosidase and 6-phospho-β-galactosidase activities in strains of the Lactobacillus acidophilus complex

β-Galactosidase (β-gal) and 6-phospho-β-galactosidase (P-β-gal) activities were measured in a total of 34 strains from Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gasseri and Lactobacillus johnsonii. The Lact. gasseri strains have P-β-gal but little or no β-gal activities. The strains from other species have β-gal but only very little P-β-gal activities.     

Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex

The Lactobacillus acidophilus complex includes Lact. acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact. acidophilus complex. A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and definitive method for the identification of Lact. acidophilus complex members.     

Antibacterial activity of Lactobacillus strains isolated from dry fermented sausages

One hundred strains of lactic acid bacteria isolated from dry cured sausages were tested for antagonistic activity against a set of test strains. Nine of 52 strains of Lactobacilus casei and three of 48 strains of Lact. plantarun produced inhibition zones against the indicator species. The substance excreted by Lact. casei CRL 705 was active against Lact. plantarum, Listeria monocytogenes, Staphylococcus aureus and a wide range of Gram-negative bacteria. The activity of the antibacterial compound from Lact. casei CRL 705 was destroyed by papain, trypsin and pepsin, but was resistant to heat (100°C for 20 min), lysozyme and catalase. The agent was produced during the growth cycle and when the concentrated and neutralized supernatant fluid was added to a fresh culture of sensitive cells it produced a rapid inactivation. A decrease in optical density (O.D.) over time, indicative of cell lysis, was also observed. These characteristics allowed us to identify the inhibitory compound as a bacteriocin which we termed Lactocin 705.     

Beta-galactosidase, phospho-beta-galactosidase and phospho-beta-glucosidase activities in lactobacilli strains isolated from human faeces

AIMS: Lactose intolerance, a serious health problem for Asians, can be solved using probiotic bacteria having high lactose hydrolysis activities. We determined the distribution of beta-galactosidase (beta-gal), phospho-beta-galactosidase (P-betagal) and phospho-beta-glucosidase (P-beta-glc) activities in species of lactic acid bacteria (LAB) isolated from human faeces to select strains for potential use in fermented dairy products, e.g. yogurt. METHODS AND RESULTS: The sugar substrates, o-nitrophenyl-beta-D- galactopyranoside 6-phosphate and o-nitrophenyl-beta-D-glucopyranoside 6-phosphate, were synthesized and used to measure respectively P-beta-gal and P-beta-glc activities. Sixty-five toluene-treated strains were examined for three lactase enzyme activities. Lactobacillus mucosae OLL2848 showed the highest beta-gal activity (107.09 U mg(-1) of protein) among the Lactobacillus strains from human faeces. Lactobacillus gasseri OLL2836 and OLL 2948 showed the highest P-beta-gal (46.58 U) and P-beta-glc (50.19 U)activity, respectively, with no beta-gal activity. CONCLUSIONS: The expression of P-beta-glc induced by lactose was characteristic of Lact. gasseri. Because this LAB is a major inhabitant of the human intestine. This enzyme is a key glycosidase involved in lactose utilization. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report describing the distribution of three glycosidase activities used in lactose metabolism in LAB isolated from human faeces for possible use in functional foods.     

The antimicrobial effect of organic acids, sour dough and nisin against Bacillus subtilis and B. licheniformis isolated from wheat bread

Prevention of growth in wheat bread for more than 6 d of approximately 10⁶ rope-producing Bacillus subtilis spores per gram of dough was achieved by addition of propionic or acetic acids at levels of 0·10% v/w (based on flour weight), or by addition of 15% sour dough fermented with Lactobacillus plantarum C11, Lact. brevis L62, Lact. plantarum ('vege-start 60'), Lact. plantarum (ch 20), Lact. maltaromicus (ch 15), or the commercial sour dough starter culture, Lact. sanfrancisco L99. These cultures resulted in an amount of total titratable acids above 10 in the sour dough and a pH value below 4·8 in the final bread. Bacteriocin-producing lactic acid bacteria added as starter cultures in wheat dough and nisin (Nisaplin) at levels up to 100 p.p.m. g⁻¹ flour had no effect against B. subtilis and B. licheniformis strains, despite the fact that nisin-producing strains of Lactococcus lactis ssp. lactis among 186 strains of lactic acid bacteria had demonstrated inhibitory activity against B. subtilis and B. licheniformis in an agar spot assay.     

Characterization of the aggregation promoting factor from Lactobacillus gasseri, avaginal isolate

Lactobacillus gasseri 2459, isolated from the human vagina, exhibits a strongautoaggregating phenotype. Filter-sterilized spent supernatants of this strain promote aggregationof Lact. plantarum LL441 and Enterococcus faecalis EF. Aggregation wasabolished upon exposure of the cells to proteases and, in the case of Ent. faecalis , tometaperiodate, which suggests the involvement of cell-surface proteins and glycoproteins,respectively, in the aggregation phenotype. In accordance with this, a 75 kDa surface protein, andpossibly another of approximately 94 kDa, appears in Lact. plantarum LL441 culturesincubated with Lact. gasseri culture supernatants. The diffusible aggregation promotingfactor was purified from stationary phase culture supernatants and determined to be a 2 kDahydrophilic peptide active at pH 3–4 and stable at neutral and acid pH. The activity wasresistant to heat, chymotrypsin, chelating agents, triton X-100 and reducing agents, but sensitiveto other proteases and SDS.     

Enzyme production by lactobacilli and the potential link with infective endocarditis

H.J. OAKEY, D.W.S. HARTY AND K.W. KNOX. 1995. Fifty-six strains of lactobacilli were examined for the production of glycosidases and proteases (arylamidases) that could be associated with the ability to grow in vivo and/or be a factor in the pathogenesis of endocarditis. The strains were from seven species, with an emphasis on Lactobacillus rhamnosus and Lact. paracasei subsp. paracasei , both of which have been associated with endocarditis and provided 12 of the 13 strains isolated from cases of the disease. Other species were Lact. acidophilus, Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris.
Commonly expressed glycosidase activities were α-D-galactosidase and β- N -acetyl-D-glucosaminidase followed by β-D-glucosidase and α-L-fucosidase. The combined production of β- N -acetyl-D-glucosaminidase and α-D-galactosidase was a feature of the endocarditis isolates. In contrast, β-D-galactosidase was produced by very few of the strains within species implicated in endocarditis but most of the strains of Lact. salivarius, Lact. fermentum and Lact. oris.
The most commonly produced arylamidases active against substrates employed for testing human blood clotting cascade were activated protein C(Ca)-like, activated factor X(Xa)-like and Hageman factor-like followed by kallikrein-like and chymotrypsin-like enzymes. Kallikrein-like enzyme activity was shown more frequently by strains from species commonly isolated from cases of endocarditis ( Lact. rhamnosus and Lact. paracasei subsp. paracasei ) than from other oral species ( Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris ).
The data indicate that some lactobacilli can produce enzymes that would enable the breakdown of human glycoproteins and the synthesis and lysis of human fibrin clots, characteristics which aid the colonization and survival of bacteria infecting an endocarditis vegetation.     

Oxalate consumption by lactobacilli: evaluation of oxalyl-CoA decarboxylase and formyl-CoA transferase activity in Lactobacillus acidophilus

AIMS: This study was undertaken to evaluate the oxalate-degrading activity in several Lactobacillus species widely used in probiotic dairy and pharmaceutical preparations. Functional characterization of oxalyl-CoA decarboxylase and formyl-CoA transferase in Lactobacillus acidophilus was performed in order to assess the possible contribution of Lactobacillus in regulating the intestinal oxalate homeostasis. METHODS AND RESULTS: In order to determine the oxalate-degrading ability in 60 Lactobacillus strains belonging to 12 species, a screening was carried out by using an enzymatic assay. A high variability in the oxalate-degrading capacity was found in the different species. Strains of Lact. acidophilus and Lactobacillus gasseri showed the highest oxalate-degrading activity. Oxalyl-CoA decarboxylase and formyl-CoA transferase genes from Lact. acidophilus LA14 were cloned and sequenced. The activity of the recombinant enzymes was assessed by capillary electrophoresis. CONCLUSIONS: Strains of Lactobacillus with a high oxalate-degrading activity were identified. The function and significance of Lact. acidophilus LA14 oxalyl-CoA decarboxylase and formyl-CoA transferase in oxalate catabolism were demonstrated. These results suggest the potential use of Lactobacillus strains for the degradation of oxalate in the human gut. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of probiotic strains with oxalate-degrading activity can offer the opportunity to provide this capacity to individuals suffering from an increased body burden of oxalate and oxalate-associated disorders.     

Selection of lactobacilli for chicken probiotic adjuncts

During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains (Salmonella enteritidis and Escherichia coli). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3.0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo. It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.     

Selection of lactobacilli for chicken probiotic adjuncts

During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains ( Salmonella enteritidis and Escherichia coli ). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3·0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo . It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci

P.S. COCCONCELLI, D. PORRO, S. GALANDINI AND L. SENINI. 1995. A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.     

Selection of Potential Probiotic Bacteria from Exclusively Breastfed Infant Faeces with Antagonistic Activity Against Multidrug-Resistant ESKAPE Pathogens

The past decade has brought a significant rise in antimicrobial resistance, and the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) have considerably aggravated a threat to public health, causing nosocomial infections worldwide. The objective of the current study was to isolate novel probiotic strain with antimicrobial activity against multidrug-resistant ESKAPE pathogens. For this purpose, eighteen breastfed infant faeces were collected and lactic acid bacteria (LAB) with antagonistic activity were isolated. Out of 102 anaerobic LAB isolated, only nine exhibited inhibitory activity against all ESKAPE pathogens. These selected nine isolates were further characterized for their probiotic attributes such as lysozyme tolerance, simulated gastrointestinal tolerance, cellular auto-aggregation and cell surface hydrophobicity. Bile salt deconjugation and cholesterol-lowering capacity was also determined. Among all nine, isolate LBM220 was found to possess superior probiotic potential. Confirmatory identification of isolate LBM220 was done by both 16S rRNA sequence analysis and mass spectrometric analysis using MALDI-TOF. Based on BLAST result, isolate LBM220 was identified as Lactobacillus gasseri. Phylogenetic analysis of Lactobacillus gasseri LBM220 [accession number MN097539] was performed. Also, detailed safety evaluation study of Lact. gasseri LBM220 showed the presence of intrinsic antibiotic resistance and the absence of hemolytic, DNase, gelatinase and toxic mucinolytic activity. Time kill assay was also performed to confirm the strong kill effect of Lact. gasseri LBM220 on all six multidrug resistant ESKAPE pathogens. Thus, Lact. gasseri LBM220 can be utilized and explored as potential probiotic with therapeutic intervention.

     

Isolation, properties and behaviour of tyramine-producing lactic acid bacteria from wine

Wines containing high levels of biogenic amines were investigated for the presence of tyramine-producing strains. Two different Lactobacillus brevis (IOEB 9809 and IOEB 9901) able to produce the amine were isolated. None of the isolated strains identified as Oenococcus oeni formed tyramine. In addition, other Lact. brevis and Lact. hilgardii strains from our collection (IOEB) and the American Type Culture Collection (ATCC) were strong tyramine producers. Lactobacillus brevis IOEB 9809 and Lact. hilgardii IOEB 9649 were found to produce tyramine and phenylethylamine simultaneously. The conditions that can influence tyramine formation in wine were evaluated for three strains of Lact. brevis (IOEB 9809 and IOEB 9901) and Lact. hilgardii (IOEB 9649). Tyrosine was the major factor affecting tyramine formation and was enhanced by the presence of sugars, mainly glucose. Tyrosine decarboxylase (TDC) activity greatly depended on the presence of the precursor, which suggested that tyrosine induced the TDC system. These results indicate that Lactobacillus could be the lactic acid bacteria responsible for tyramine production in wine.     

Characterization and selection of vaginal Lactobacillus strains for the preparation of vaginal tablets

AIMS: To characterize and select Lactobacillus strains for properties that would make them a good alternative to the use of antibiotics to treat human vaginal infections. METHODS AND RESULTS: Ten Lactobacillus strains belonging to four different Lactobacillus species were analysed for properties relating to mucosal colonization or microbial antagonism (adhesion to human epithelial cells, hydrogen peroxide production, antimicrobial activity towards Gardnerella vaginalis and Candida albicans and coaggregation with pathogens). The involvement of electrostatic interactions and the influence of bacterial metabolic state in the binding of lactobacilli to the cell surface were also studied. Adherence to epithelial cells varied greatly among the Lactobacillus species and among different strains belonging to the same Lactobacillus species. The reduction in surface negative electric charge promoted the binding of several Lactobacillus strains to the cell membrane whereas lyophilization reduced the adhesion capacity of many isolates. The antimicrobial activity of lactobacilli culture supernatant fluids was not directly related to the production of H2O2. CONCLUSIONS: Three strains (Lactobacillus brevis CD2, Lact. salivarius FV2 and Lact. gasseri MB335) showed optimal properties and were, therefore, selected for the preparation of vaginal tablets. The selected strains adhered to epithelial cells displacing vaginal pathogens; they produced high levels of H2O2, coaggregated with pathogens and inhibited the growth of G. vaginalis. SIGNIFICANCE AND IMPACT OF THE STUDY: The dosage formulation developed in this study appears to be a good candidate for the probiotic prophylaxis and treatment of human vaginal infections.     

Fermentation of fructans by epiphytic lactic acid bacteria

A total of 712 strains of lactic acid bacteria isolated from forage grasses were studied for their ability to ferment fructans of phlein- as well as inulin-type. Only 16 strains utilized phlein and eight of these also fermented inulin. They were identified as Lactobacillus paracasei subsp. paracasei, Lact. plantarum, Lact. brevis and Pediococcus pentosaceus . In the species Lact. paracasei subsp. paracasei , all strains gave positive results, whereas the other positive strains possessed unique properties within their own species. In all but two cases (strains of the species Lact. plantarum ), the phlein was more intensively fermented than the inulin, as indicated by a lower pH and a higher lactic acid concentration. On the basis of the outcome of this study it seems worthwhile to inoculate grasses of low sugar content before ensiling with an active strain that can ferment fructans.     

Isolation of cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and molecular comparison with dairy-related Lactobacillus helveticus strains

Aims:  To isolate cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and compare them with dairy-related Lactobacillus helveticus strains using molecular typing methods.
Methods and Results:  The number of thermophilic bacteria in seven commercial cheeses manufactured with mesophilic starters was estimated to be <10 CFU g⁻¹. Implementation of an enumeration step in the isolation method made it possible to isolate one thermophilic strain from each of five of seven cheeses. Comparing repetitive sequence PCR (rep-PCR) profiles of the isolates with dairy-related Lact. helveticus strains indicated that one isolate was a Lact. helveticus . Partial sequencing of 16S rRNA confirmed this, and the remaining four strains were identified as Lactobacillus delbrueckii , Lactobacillus fermentum and Enterococcus faecium . The rep-PCR profile of the isolated Lact. helveticus was identical to the rep-PCR profile of the Lact. helveticus adjunct culture used in the specific cheese, but their pulsed field gel electrophoresis profiles differed slightly.
Conclusion:  It was possible to isolate cultivable thermophilic bacteria from ripened cheeses manufactured with mesophilic starter and thermophilic adjunct cultures by using an enumeration step.
Significance and Impact of the Study:  Isolation of cultivable thermophilic bacteria from ripened cheeses made with mesophilic starters offers an original source for new dairy-relevant cultures.     

Antimicrobial potential of four Lactobacillus strains isolated from breast milk

AIMS: The antimicrobial potential of four lactobacilli (Lactobacillus salivarius CECT5713, Lactobacillus gasseri CECT5714, L. gasseri CECT5715 and Lactobacillus fermentum CECT5716), isolated from fresh human breast milk, was evaluated in this study and compared with Lactobacillus coryniformis CECT5711, a reuterin-producing strain isolated from an artisan goat's cheese. METHODS AND RESULTS: Agar diffusion tests, competitive adhesion assays and mucin expression assays were carried out in order to value the antibacterial properties of the lactobacilli strains. The antibacterial capability of the strains was tested in vivo by using a murine infection model with Salmonella choleraesuis. The results revealed that all the strains studied, displayed antibacterial properties against pathogenic bacteria. However, the antibacterial potential varied among the lactobacilli tested and, in fact, L. salivarius CECT5713 showed not only the best in vitro antibacterial activity, but also the highest protective effect against a Salmonella strain in the murine infection model. CONCLUSION: The four breast-milk lactobacilli, and particularly L. salivarius CECT5713, possess potent antibacterial activities that result in a higher protection against S. choleraesuis CECT4155 in a mouse infection model. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that lactobacilli from breast milk could contribute to an anti-infective protection in neonates and would be excellent candidates for the development of infant probiotic products.