Use of Group-Specific and RAPD-PCR Analyses for Rapid Differentiation of <Emphasis Type="Italic">Lactobacillus</Emphasis> Strains from Probiotic Yogurts

The increasing interest in probiotic lactobacilli implicates the requirement of techniques that allow a rapid and reliable identification of these organisms. In this study, group-specific PCR and RAPD-PCR analyses were used to identify strains of the Lactobacillus casei and Lactobacillus acidophilus groups most commonly used in probiotic yogurts. Group-specific PCR with primers for the L. casei and L. acidophilus groups, as well as L. gasseri/johnsonii, could differentiate between 20 Lactobacillus strains isolated from probiotic yogurts and assign these into the corresponding groups. For identification of these strains to species or strain level, RAPD profiles of the 20 Lactobacillus strains were compared with 11 reference strains of the L. acidophilus and L. casei group. All except one strain could be attributed unambigously to the species L. acidophilus, L. johnsonii, L. crispatus, L. casei, and L. paracasei. DNA reassociation analysis confirmed the classification resulting from the RAPD-PCR.     

Gut immune stimulation by non pathogenic Gram(+) and Gram(-) bacteria. Comparison with a probiotic strain

We analyzed the gut immune stimulation induced by Gram-positive bacteria: non probiotic Lactobacillus acidophilus CRL 1462 and Lactobacillus acidophilus A9; two potentially probiotic strains: L. acidophilus CRL 924 and Lactobacillus delbrueckii subsp. bulgaricus CRL 423; comparatively with a probiotic strain: Lactobacillus casei CRL 431. We also studied Gram-negative bacteria: Escherichia coli 129 and E. coli 13-7 in BALB/c mice. All the strains increased the number of IgA+ cells. We analyzed the cytokines IFNγ, TNFα, IL-17, IL-12, IL-6 and MIP-1α. The Gram(+) strains increased the number of IL-10+ cells. Gram(−) strains did not increase IL-10+ cells, but they increased the number of IL-12+ cells. The probiotic strain increased mainly IFNγ and TNFα. In the study of the receptors TLR-2, TLR-4 and CD-206, we demonstrated that only the probiotic strain increased the number of CD-206+ cells. All the Gram(+) strains increased the number of TLR-2+ cells and the Gram(−) strains of the TLR-4+ cells. The probiotic strain induced the release of IL-6 by a preparation enriched in intestinal epithelial cells (IEC). Gram(+) and Gram(−) bacteria activated different immune receptors and induced a different cytokine profile. The probiotic strain showed a great activity on the immune cells and the enriched population in IEC, activating mainly cells of the innate immune system.     

Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins

Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and β-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly β-casein, while degradation of α_s-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. β-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.     

Bacteria competing with the adhesion and biofilm formation by <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The activity of antagonistic substances produced by Pseudomonas aeruginosa and Lactobacillus acidophilus against the planktonic and sessile populations of Staphylococcus aureus strains was demonstrated. The strongest effects were caused by probiotic L. acidophilus strain — bacteriocin-like inhibitory substances (BLIS) positive. However, the S. aureus A3 growth, adhesion and biofilm formation was also limited by cell-free supernatant of L. acidophilus H-1 (BLIS negative). Moreover, competitive direct interactions were observed between staphylococci and the above bacteria, which influenced the formation of dualspecies aggregates on the surface.     

Supportive Role of Probiotic Strains in Protecting Rats from Ovariectomy-Induced Cortical Bone Loss

Osteoporosis is a major health problem that occurs as a result of an imbalance between bone formation and bone resorption. Different approaches have been established for treating osteoporosis. Recently, because of their health benefits and also low adverse reaction, probiotics have been receiving considerable attention. In this study, we compared the effectiveness of five probiotic strains, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, Bifidobacterium longum, and Bacillus coagulans, in protecting rats from ovariectomized (OVX)-induced bone loss. Forty-nine adult female Sprague-Dawley rats were allocated into seven groups as follows: group 1, control; group 2, OVX; group 3, OVX + Lactobacillus acidophilus; group 4, OVX + Lactobacillus casei; group 5, OVX + Bacillus coagulans; group 6, OVX + Bifidobacterium longum; and group 7, OVX + Lactobacillus reuteri. Probiotics were fed to OVX groups at the concentration of (1 × 10⁹ CFU/ml/day) for 4 weeks. Then, biochemical parameters, including vitamin D, calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP), were assessed. Dual-energy X-ray absorptiometry (DEXA) scans were used that assess bone mineral density (BMD), bone marrow concentration (BMC), and area of global, femur, spine, and tibia. Lactobacillus acidophilus and Lactobacillus casei significantly increased Ca and ALP and decreased P in treated groups. Lactobacillus casei, Lactobacillus reuteri, and Bifidobacterium longum increased vitamin D significantly. Lactobacillus acidophilus and Lactobacillus casei indicated the most effects on BMD. In terms of BMC, and bone area, Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus casei demonstrated the significant enhancement in OVX groups treated with. Among the probiotics used in this study, Lactobacillus acidophilus and Lactobacillus casei showed the most effects in terms of BMD, BMC, bone area, and biochemical parameters. It seems that probiotics effects on bone health are strain dependent, but further studies should be done to prove these findings.

     

Probiotic Lactobacillus fermentum UCO-979C biofilm formation on AGS and Caco-2 cells and Helicobacter pylori inhibition

The ability of the human isolate Lactobacillus fermentum UCO-979C to form biofilm and synthesize exopolysaccharide on abiotic and biotic models is described. These properties were compared with the well-known Lactobacillus casei Shirota to better understand their anti-Helicobacter pylori probiotic activities. The two strains of lactobacilli synthesized exopolysaccharide as detected by the Dubois method and formed biofilm on abiotic and biotic surfaces visualized by crystal violet staining and scanning electron microscopy. Concomitantly, these strains inhibited H. pylori urease activity by up to 80.4% (strain UCO-979C) and 66.8% (strain Shirota) in gastric adenocarcinoma (AGS) cells, but the two species showed equal levels of inhibition (~84%) in colorectal adenocarcinoma (Caco-2) cells. The results suggest that L. fermentum UCO-979C has probiotic potential against H. pylori infections. However, further analyses are needed to explain the increased activity observed against the pathogen in AGS cells as compared to L. casei Shirota.     

Antibacterial and phytochemical studies on Calotropis gigantia (L.) R. Br. latex against selected cariogenic bacteria

In vitro antibacterial potential of the chloroform, ethyl acetate, hexane, methanol and aqueous extracts of Calotropis gigantia (L.) R. Br. was evaluated by using five cariogenic bacteria, Actinomyces viscosus, Lactobacillus acidophilus, Lactobacillus casei, Streptococcus mitis and Streptococcus mutans. Agar well diffusion method and minimum inhibitory concentration (MIC) were used for this purpose. The chloroform extracted fraction of latex showed inhibitory effect against S. mutans and L. acidophilus with MIC value of 0.032 and 0.52 mg/mL, respectively. Qualitative investigation on structure elucidation of bioactive compound using IR, NMR and GC–MS techniques revealed the presence of methyl nonanoate, a saturated fatty acid.     

Rapid Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">acidophilus</Emphasis> by Restriction Analysis of the 16S–23S rRNA Intergenic Spacer Region and Flanking 23S rRNA Gene

A rapid molecular approach was developed for the initial identification of Lactobacillus acidophilus strains which are difficult to identify using a single biochemical test. The 16S–23S rRNA intergenic spacer regions and flanking 23S rRNA genes of 19 strains of lactobacilli were amplified and the nucleotide sequences and restriction site polymorphisms were analyzed. AluI was the most useful of the restriction enzymes analyzed and produced reproducible digestion profiles in the L. helveticus, L. plantarum, and L. casei groups, as well as in L. acidophilus. This restriction fragment length polymorphism method may be useful for the identification of L. acidophilus strains in dairy products.     

Antimicrobial effects of terpinen-4-ol against oral pathogens and its capacity for the modulation of gene expression

Abstract

This study evaluated the antibacterial activity of terpinen-4-ol against Streptococcus mutans and Lactobacillus acidophilus and its influence on gbpA (S. mutans) and slpA (L. acidophilus) gene expression. As measured by XTT assay, the concentrations of terpinen-4-ol that effectively inhibited the biofilm were 0.24% and 0.95% for S. mutans and L. acidophilus, respectively. Confocal microscopy revealed the presence of a biofilm attached to the enamel and dentin block surfaces with significant terpinen-4-ol effects against these microorganisms. The expression of the gbpA and slpA genes involved in adherence and biofilm formation was investigated using RT-PCR. Expression of these genes decreased after 15?min with 0.24% and 0.95% terpinen-4-ol in S. mutans and L. acidophilus, respectively. These findings demonstrate the antimicrobial activity of terpinen-4-ol and its ability to modulate the expression of gbpA and slpA genes, emphasizing the therapeutic capacity of terpinen-4-ol as an alternative to inhibit adherence in biofilm.     

Lipophilic 9,10‐Dehydrofukinone Action on Pathogenic and Non‐Pathogenic Bacterial Biofilms. Why Is This Main Volatile Metabolite in Senecio?

The effect of a natural sesquiterpene ketone, 9,10‐dehydrofukinone (DHF), on pathogenic Staphylococcus aureus and Pseudomonas aeruginosa strains isolated from chronic infectious processes, was the focus of the present study. Lipophilic DHF produced important antibacterial synergistic effects in association with ciprofloxacin (CPX) against two biofilm‐forming strains of S. aureus HT1 (FIC=0.21) and P. aeruginosa HT5 (FIC=0.05). Hence, this mixture constitutes an excellent strategy to combat these biofilm‐producing bacteria that overexpress drug efflux pumps as a resistance mechanism. Additionally, a substantial rise in beneficial Lactobacillus biofilm biomass was determined as a very significant finding of this association. Particularly, a non‐pathogenic biofilm increment of 119 % was quantified when the mixture was added to a probiotic L. acidophilus ATCC SD‐5212 culture. A surface activity enhanced in 71 % with respect to untreated L. acidophilus culture was also generated by the DHF and CPX association, and therefore, a glycoprotein synthesis induction mediated by the mixture is discussed. The results obtained could help in the development of new selective antibiotics. From an ecological standpoint, the present study strongly suggests that DHF is a polyfunctional organic molecule produced with a high yield in Senecio punae that exerts a positive impact on a non‐pathogenic plant bacterium L. plantarum CE105.     

Protoplast fusion between Lactobacillus casei and Lactobacillus acidophilus

Summary From the fusion between Lactobacillus casei and Lactobacillus acidophilus, 8 fusants were selected: Four were able to ferment maltose, lactose, galactose and mannose, but two had greater abilities of acid production than parents. Increased values of up to 7.6–8 % in -galactosidase activity were obtained from two when compared to that of L. acidophilus, whereas another 2 had activities of 800 and 548 nmol/mg protein/min comparable to that of L casei giving a value of 400 nmol/mg protein/min in phospho--galactosidase activity.     

In Silico Prediction and In Vitro Assessment of Multifunctional Properties of Postbiotics Obtained From Two Probiotic Bacteria

In this study, a global metabolite profile using Raman spectroscopy analysis was obtained in order to predict, by an in silico prediction of activity spectra for substance approach, the bioactivities of the intracellular content (IC) and cell wall (CW) fractions obtained from Lactobacillus casei CRL 431 and Bacillus coagulans GBI-30 strains. Additionally, multifunctional in vitro bioactivity of IC and CW fractions was also assessed. The metabolite profile revealed a variety of compounds (fatty acids, amino acids, coenzyme, protein, amino sugars), with significant probable activities (Pa > 0.7) as immune-stimulant, anti-inflammatory, neuroprotective, antiproliferative, immunomodulator, and antineoplastic, among others. Moreover, in vitro assays exhibited that both IC and CW fractions presented angiotensin-converting enzyme–inhibitory (> 90%), chelating (> 79%), and antioxidant (ca. 22–57 cellular antioxidant activity units) activities. Our findings based on in silico and in vitro analyses suggest that L. casei CRL 431 and B. coagulans GBI-30 strains appear to be promising sources of postbiotics and may impart health benefits by their multifunctional properties.

     

Tyramine degradation and tyramine/histamine production by lactic acid bacteria and Kocuria strains

Of 53 strains of lactic acid bacteria and Kocuria, screened for production or degradation of biogenic amines, 29 Kocuria varians and four strains of Enterococcus faecalisproduced tyramine and, at lower concentrations, histamine. In contrast, Lactobacillus strains that did not possess amino acid decarboxylase activity degraded tyramine. The greatest tyramine oxidase activity was present in the strains L. casei CRL705 (98% degradation) and CRL678 (93%) as well as in L. plantarum CRL681 (69%) and CRL682 (60%).     

Improved Viability of Microencapsulated Probiotics in a Freeze-Dried Banana Powder During Storage and Under Simulated Gastrointestinal Tract

Freeze-dried banana powder represents an ideal source of nutrients and has not yet been used for probiotic incorporation. In this study, microencapsulation by freeze drying of probiotics Lactobacillus acidophilus and Lactobacillus casei was made using whey protein isolate (WPI), fructooligosaccharides (FOS), and their combination (WPI + FOS) at ratio (1:1). Higher encapsulation yield was found for (WPI + FOS) microspheres (98%). Further, microcapsules of (WPI + FOS) were used to produce a freeze-dried banana powder which was analyzed for bacterial viability under simulated gastrointestinal fluid (SGIF), stability during storage at 4 °C and 25 °C, and chemical and sensory properties. Results revealed that (WPI + FOS) microcapsules significantly increased bacteria stability in the product over 30 days of storage at 4 °C averaging (≥ 8.57 log CFU/g) for L. acidophilus and (≥ 7.61 log CFU/g) for L. Casei as compared to free cells. Bacteria encapsulated in microspheres (WPI + FOS) were not significantly affected by the SGIF, remaining stable up to 7.05 ± 0.1 log CFU/g for L.acidophilus and 5.48 ± 0.1 log CFU/g for L.casei after 90 min of incubation at pH 2 compared to free cells which showed minimal survival. Overall, encapsulated probiotics enriched freeze-dried banana powders received good sensory scores; they can therefore serve as safe probiotics food carriers.

     

Heat-Killed Lactic Acid Bacteria Inhibit Nitric Oxide Production via Inducible Nitric Oxide Synthase and Cyclooxygenase-2 in RAW 264.7 Cells

Heat-killed lactic acid bacteria perform immunomodulatory functions and are advantageous as probiotics, considering their long product shelf-life, easy storage, and convenient transportation. In this study, we aimed to develop appropriate heat treatments for industrial preparation of probiotics with antioxidant activity. Among 75 heat-killed strains, Lactococcus lactis MG5125 revealed the highest nitric oxide inhibition (86.2%), followed by Lactobacillus acidophilus MG4559 (86.0%), Lactobacillus plantarum MG5270 (85.7%), Lactobacillus fermentum MG4510 (85.3%), L. plantarum MG5239 (83.9%), L. plantarum MG5289 (83.2%), and L. plantarum MG5203 (81.8%). Moreover, the heat-killed selected strains markedly inhibited lipopolysaccharide-induced nitric oxide synthase and cyclooxygenase-2 expression. The use of heat-killed bacteria with intact bio-functionality can elongate the shelf-life and simplify the food processing steps of probiotic foods, given their high stability. The antioxidant and immune-modulatory activities of the heat-killed strains selected in this study indicate a strong potential for their utilization probiotic products manufacturing.

     

Strain identification of probiotic Lactobacillus casei-related isolates with randomly amplified polymorphic DNA and pulsed-field gel electrophoresis methods

Typing of reference strains and isolates identified as Lactobacillus casei, Lactobacillus paracasei or Lactobacillus rhamnosus was carried out using randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) analyses. Strains of L. paracasei were mainly grouped in the same cluster as those of L. casei. The RAPD fingerprints of strains ATCC 393 and ATCC 15820 differ from those of the L. rhamnosus and L. paracasei/casei strains further supporting classification of these strains as a separate group. The RAPD profiling could be used for classification and discrimination of isolates belonging to the L. casei group.     

Lactobacillus acidophilus Autolysins Inhibit Helicobacter pylori In Vitro

Antibacterial activity of 17 strains of lactobacilli was tested against 10 strains of H. pylori. The inhibition observed was related to the acid production and the low pH attained. No relationship between CagA phenotype of H. pylori strains and tolerance to lactic acid was observed. In mixed cultures, L. acidophilus CRL 639 showed an autolytic behavior after 24 h of culture. At this moment, H. pylori CCUG17874 showed a decrease of 2 log-cycle, and no viable count was detected after 48 h. The bactericidal effect of L. acidophilus CRL 639 in mixed cultures is related to a proteinaceous compound released after cell lysis. Received: 19 June 2000 / Accepted: 12 July 2000     

Detection of polygalacturonases and pectin esterases in lactic acid bacteria

Of 80 strains of lactic acid bacteria tested, only Lactobacillus casei strains HNK10 and L1–8, Lactobacillus plantarum Lc5 and Lactococcus lactis NN01 produced polygalacturonases (EC 3.2.1.) and/or pectin-esterases (EC 3.1.1.). Crude extracellular extracts of strain L1–8 were able to clarify pectin.     

Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus

While the remarkable health effects of conjugated linoleic acid (CLA) catalyzed from α-linoleic acid by the enzyme linoleate isomerase (LI, EC 5.2.1.5) are well recognized, how widely this biochemical activity is present and the mechanisms of its regulation in lactic acid bacteria are unknown. Although certain strains of Lactobacillus acidophilus can enrich CLA in fermented dairy products, it is unknown if other strains share this capacity. Due to its immense economic importance, this work aimed to investigate genetic aspects of CLA production in L. acidophilus for the first time. The genomic DNA from industrial and type strains of L. acidophilus were subjected to PCR and immunoblot analyses using the putative LI gene of L. reuteri ATCC 55739 as probe. The CLA production ability was estimated by gas chromatography of the biomass extracts. The presumptive LI gene from L. acidophilus ATCC 832 was isolated and sequenced. The resulting sequence shared 71% identity with that of L. reuteri and at least 99% with reported sequences from other L. acidophilus strains. All the strains accumulated detectable levels of CLA and tested positive by PCR and immunoblotting. However, no apparent correlation was observed between the yields and the hybridization patterns. The results suggest that LI activity might be common among L. acidophilus and related species and provide a new tool for screening potential CLA producers.     

Molecular Discrimination of Lactobacilli Used as Starter and Probiotic Cultures by Amplified Ribosomal DNA Restriction Analysis

Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by PCR amplifications by using group-specific or species-specific 16S rDNA primers. The numerical analysis of the ARDRA patterns obtained by using CfoI, HinfI, Tru9I, and ScrFI was an efficient typing tool for identification of species of the L. acidophilus and L. casei complex. ARDRA by using CfoI was a reliable method for differentiation of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Finally, strains ATCC 393 and ATCC 15820 exhibited unique ARDRA patterns with CfoI and Tru9I restriction enzymes as compared with the other strains of L. casei, L. paracasei, and L. rhamnosus. Received: 30 August 2000 / Accepted: 2 October 2000