Identification and analysis of the function of surface layer proteins from three <Emphasis Type="Italic">Lactobacillus</Emphasis> strains

To identify and investigate the role of surface layer proteins (SLPs) on the probiotic properties of Lactobacillus strains, SLPs were extracted from Lactobacillus bulgaricus fb04, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM by 5 mol/L lithium chloride. The molecular masses of the four SLPs were approximately 45–47 kDa as analyzed by SDS-PAGE. Hydrophobic amino acids were the main components of the four SLPs. The secondary structure content of the four SLPs showed extensive variability among different strains. After the SLPs were removed from the cell surface, the autoaggregation ability, coaggregation ability, and gastrointestinal tolerability of the four lactobacilli were significantly reduced as compared with the intact cells (P?<?0.05). When exposed to bile salt stress, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM expressed more SLPs as determined by Bradford method. In conclusion, the four lactobacilli all possessed functional SLPs, which had positive contributions to the probiotic properties of the four Lactobacillus strains. This research could reveal the biological contributions of SLPs from Lactobacillus strains and offer a theoretical basis for the application of lactobacilli and their SLPs in food and pharmaceutical industries.     

In vitro assessment of safety and probiotic potential characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from water buffalo mozzarella cheese

The aim of this study was to evaluate the safety and probiotic potential characteristics of ten Lactobacillus spp. strains (Lactobacillus fermentum SJRP30, Lactobacillus casei SJRP37, SJRP66, SJRP141, SJRP145, SJRP146, and SJRP169, and Lactobacillus delbrueckii subsp. bulgaricus SJRP50, SJRP76, and SJRP149) that had previously been isolated from water buffalo mozzarella cheese. The safety of the strains was analyzed based on mucin degradation, hemolytic activity, resistance to antibiotics and the presence of genes encoding virulence factors. The in vitro tests concerning probiotic potential included survival under simulated gastrointestinal (GI) tract conditions, intestinal epithelial cell adhesion, the presence of genes encoding adhesion, aggregation and colonization factors, antimicrobial activity, and the production of the β-galactosidase enzyme. Although all strains presented resistance to several antibiotics, the resistance was limited to antibiotics to which the strains had intrinsic resistance. Furthermore, the strains presented a limited spread of genes encoding virulence factors and resistance to antibiotics, and none of the strains presented hemolytic or mucin degradation activity. The L. delbrueckii subsp. bulgaricus strains showed the lowest survival rate after exposure to simulated GI tract conditions, whereas all of the L. casei and L. fermentum strains showed good survivability. None of the tested lactobacilli strains presented bile salt hydrolase (BSH) activity, and only L. casei SJRP145 did not produce the β-galactosidase enzyme. The strains showed varied levels of adhesion to Caco-2 cells. None of the cell-free supernatants inhibited the growth of pathogenic target microorganisms. Overall, L. fermentum SJRP30 and L. casei SJRP145 and SJRP146 were revealed to be safe and to possess similar or superior probiotic characteristics compared to the reference strain L. rhamnosus GG (ATCC 53103).     

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.     

Comparative evaluation of automated ribotyping and RAPD-PCR for typing of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. occurring in dental caries

A group of 67 Lactobacillus spp. strains containing Lactobacillus casei/paracasei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus salivarius species isolated from early childhood caries and identified to the species level in a previous study (?vec et al., Folia Microbiol 54:53–58, 2009) was characterized by automated ribotyping performed by the RiboPrinter^® microbial characterization system and by randomly amplified polymorphic DNA fingerprinting (RAPD-PCR) with M13 primer to evaluate these techniques for characterization of lactobacilli associated with dental caries. Ribotyping revealed 55 riboprints among the analysed group. The automatic identification process performed by the RiboPrinter system identified 18 strains to the species level, however cluster analysis divided obtained ribotype patterns into individual clusters mostly corresponding to the species assignment of particular strains. RAPD-PCR fingerprints revealed by the individual Lactobacillus spp. showed higher variability than the ribotype patterns and the fingerprint profiles generated by the analysed species were distributed among one to four clusters. In conclusion, ribotyping is shown to be more convenient for the identification purposes while RAPD-PCR fingerprinting results indicate this method is a better tool for typing of Lactobacillus spp. strains occurring in dental caries.     

Selection of Potential Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> with Inhibitory Activity Against <Emphasis Type="Italic">Salmonella</Emphasis> and Fecal Coliform Bacteria

Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.     

<Emphasis Type="Italic">Lactobacillus casei</Emphasis> as a biocatalyst for biofuel production

Microbial fermentation of sugars from plant biomass to alcohols represents an alternative to petroleum-based fuels. The optimal biocatalyst for such fermentations needs to overcome hurdles such as high concentrations of alcohols and toxic compounds. Lactic acid bacteria, especially lactobacilli, have high innate alcohol tolerance and are remarkably adaptive to harsh environments. This study assessed the potential of five Lactobacillus casei strains as biocatalysts for alcohol production. L. casei 12A was selected based upon its innate alcohol tolerance, high transformation efficiency and ability to utilize plant-derived carbohydrates. A 12A derivative engineered to produce ethanol (L. casei E1) was compared to two other bacterial biocatalysts. Maximal growth rate, maximal optical density and ethanol production were determined under conditions similar to those present during alcohol production from lignocellulosic feedstocks. L. casei E1 exhibited higher innate alcohol tolerance, better growth in the presence of corn stover hydrolysate stressors, and resulted in higher ethanol yields.     

Probiotic Strawberry Yogurts: Microbiological,Chemical and Sensory Properties

This study was performed to determine the viability of Lactobacillus acidophilus and Bifidobacterium bifidum in yogurt made with strawberry marmalade (SM) and to examine the quality properties of probiotic yogurt. Acidity, pH, bacterial counts and sensory analysis of the yogurt samples were investigated on days 1, 3, 5, 7, 10 and 14 during storage at 4 °C. The survival rate of L. acidophilus was greater than that of B. bifidum. The viability of L. acidophilus decreased during the storage period, but B. bifidum numbers remained stable during the storage period. The highest L. acidophilus count (7.20 log cfu/g) was found in L. acidophilus + B. bifidum SM yogurt on day 1. The highest B. bifidum count (6.13 log cfu/g) was detected in yogurt containing L. acidophilus + B. bifidum SM yogurt on day 7. Yeast and mould counts of all yogurts increased during the storage period. Coliform bacteria and Staphylococcus aureus were not detected in the yogurt samples. The highest overall acceptance sensory score was observed in yogurts containing L. acidophilus. Considering the sensory and probiotic characteristics of all yogurt samples, this study suggested that strawberry yogurt with a suitable 5–7 day storage period can be produced with single L. acidophilus addition or single B. bifidum addition.     

The Role of Probiotics in the Treatment of Dysentery: a Randomized Double-Blind Clinical Trial

Diarrhea is considered as an important cause of morbidity and mortality, even though one of the main reasons of death following diarrhea is initiated by dysentery. In recent years, the consumption of probiotics has been proposed for the treatment of infectious diarrhea. Despite most of the studies on probiotics have focused on acute watery diarrhea, few studies in the field of dysentery have found beneficial effects of probiotics. This study is a randomized double-blind clinical trial. The patients were randomly placed into control and case groups. In the intervention group, the patients received probiotics in the form of Kidilact® sachet, which contained high amounts of 7-strain friendly bacteria strains of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Bifidobacterium infantis, Bifidobacterium breve, and Streptococcus thermophiles. On the other hand, the patients in the control group received placebo sachets on a daily basis for 5 days. It is notable that the treatment protocol of acute dysentery was done on both groups. The results of this study showed significant differences in the duration of blood in diarrhea between probiotic consumers (2.62 days) and the control group (3.16 days) (P value = 0.05). Additionally, significant differences in the average length of hospitalization in probiotic consumers (3.16 days) and control (3.66 days), (P value = 0.02) could be claimed that the consumption of probiotics is effective in reducing the duration of dysentery and diarrhea. The results of this study suggest that the use of probiotics can be effective in reducing the duration of blood in diarrhea. This study was also recorded in the Iran center of clinical trials registration database (IRCT2014060617985N1).     

Biofilm formation and antimicrobial sensitivity of lactobacilli contaminants from sugarcane-based fuel ethanol fermentation

Industrial ethanol fermentation is subject to bacterial contamination that causes significant economic losses in ethanol fuel plants. Chronic contamination has been associated with biofilms that are normally more resistant to antimicrobials and cleaning efforts than planktonic cells. In this study, contaminant species of Lactobacillus isolated from biofilms (source of sessile cells) and wine (source of planktonic cells) from industrial and pilot-scale fermentations were compared regarding their ability to form biofilms and their sensitivity to different antimicrobials. Fifty lactobacilli were isolated and the most abundant species were Lactobacillus casei, Lactobacillus fermentum and Lactobacillus plantarum. The majority of the isolates (87.8%) were able to produce biofilms in pure culture. The capability to form biofilms and sensitivity to virginiamycin, monensin and beta-acids from hops, showed inter- and intra-specific variability. In the pilot-scale fermentation, Lactobacillus brevis, L. casei and the majority of L. plantarum isolates were less sensitive to beta-acids than their counterparts from wine; L. brevis isolates from biofilms were also less sensitive to monensin when compared to the wine isolates. Biofilm formation and sensitivity to beta-acids showed a positive and negative correlation for L. casei and L. plantarum, respectively.     

Comparison of antibiotic resistance and copper tolerance of <Emphasis Type="Italic">Enterococcus</Emphasis> spp. and <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. isolated from piglets before and after weaning

In China, antimicrobials and copper are used extensively as growth-promoting agents for piglets. This study aimed to characterize the role of in-feed copper in the emergence of copper-tolerant and antibiotic-resistant Enterococcus and Lactobacillus isolates in Chinese pig farms. Feces of the same eight piglets from four litters at 7 and 55 days old and their mothers were traced in order to isolate Enterococcus spp. and Lactobacillus spp.. The minimum inhibitory concentrations of 10 antimicrobials and copper sulfate were determined using an agar dilution method. The feed levels of Cu²⁺ for lactating sows, suckling piglets, and weaned piglets were 6, 177, and 18 mg/kg, respectively. All the 136 Enterococcus isolates were sensitive to vancomycin; and the resistance rates to penicillin, enrofloxacin, and high level streptomycin resistance increased significantly after weaning. For the 155 Lactobacillus isolates, the resistance rates to ampicillin, chloramphenicol, tetracycline, and enrofloxacin were significantly higher in weaned piglets. The ratios of copper tolerant Enterococcus and Lactobacillus isolates both increased significantly after weaning (P < 0.05). A phenotypic correlation was observed after classifying the isolates into two groups (CuSO₄ MIC₅₀ < 16 or ≧16 for enterococci; CuSO₄ MIC₅₀ < 12 or ≧12 for lactobacilli) and comparing the antimicrobial-resistant percentage of two groups. On species level, a significant increase of E. faecalis to enrofloxacin was observed in line with the increase of copper MIC (P < 0.05). The findings revealed the changes of the antibiotic resistance and copper tolerance level of enterococci and lactobacilli between suckling and weaned piglets and demonstrated that there might be a strong association between in-feed copper and increased antibiotic resistance in enterococci and lactobacilli in Chinese intensive swine farms.     

Effect of Milk Fermented with Lactic Acid Bacteria on Diarrheal Incidence,Growth Performance and Microbiological and Blood Profiles of Newborn Dairy Calves

The effect of the administration of milk fermented with lactic acid bacteria to calves was evaluated. The strains included were: Lactobacillus murinus CRL1695, Lact. mucosae CRL1696, Lact. johnsonii CRL1693, and Lact. salivarius CRL1702, which were selected for their beneficial and functional properties and isolated from healthy calves in the northwestern region of Argentina. The trial was conducted on a dairy farm located in Tucumán (Holando-Argentino calves). A randomized controlled trial was performed in which 56 new-born animals were divided into two groups: the treated group (T) received the fermented milk for 60 days and the control group (C) only milk. The animals were fed a solid diet ad libitum. The treated group was given a daily dose of 1 × 10⁹CFU of the probiotic fermented milk while the control group was fed milk. Body weight and biometrical parameters were recorded between 15 and 60 days of age, and average daily gain was calculated with three samplings per animal throughout the trial. Rectal swabs and fecal and blood samples were also collected. Results showed the efficacy of the probiotic: lower morbidity and mortality of calves (morbidity was 69.20% in animals without the probiotic, and 46.15% in probiotic-treated animals, with P = 0.09; mortality in C was 34.61 and 7.69% in animals fed with ferment milk; P = 0.02).The calves fed with probiotic evidenced an improvement in nutritional parameters, body condition and weight gain (health index P = 0.01; average daily gain P = 0.03).Viable bacterial numbers showed no differences between the two experimental groups. Hematological parameters and serum proteins were not modified by the treatment. The results suggest that the fermented milk containing lactic acid bacteria can be a viable veterinary product for young calves due to its beneficial effects on health and growth.     

Characterization of a <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> strain with potential oral probiotic properties

Background

The microflora composition of the oral cavity affects oral health. Some strains of commensal bacteria confer probiotic benefits to the host. Lactobacillus is one of the main probiotic genera that has been used to treat oral infections. The objective of this study was to select lactobacilli with a spectrum of probiotic properties and investigate their potential roles in oral health.

Results

An oral isolate characterized as Lactobacillus brevis BBE-Y52 exhibited antimicrobial activities against Streptococcus mutans, a bacterial species that causes dental caries and tooth decay, and secreted antimicrobial compounds such as hydrogen peroxide and lactic acid. Compared to other bacteria, L. brevis BBE-Y52 was a weak acid producer. Further studies showed that this strain had the capacity to adhere to oral epithelial cells. Co-incubation of L. brevis BBE-Y52 with S. mutans ATCC 25175 increased the IL-10-to-IL-12p70 ratio in peripheral blood mononuclear cells, which indicated that L. brevis BBE-Y52 could alleviate inflammation and might confer benefits to host health by modulating the immune system.

Conclusions

L. brevis BBE-Y52 exhibited a spectrum of probiotic properties, which may facilitate its applications in oral care products.

     

Adaptation and Probiotic Potential of Lactobacilli,Isolated from the Oral Cavity and Intestines of Healthy People

The present study shows that, from 300 Lactobacillus strains isolated from the oral cavity and large intestine of 600 healthy people, only 9 had high antagonistic activity against pathogens and opportunistic pathogens. All antagonistic strains of lactobacilli have been identified by 16S rRNA sequencing and assigned to four species: Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus casei. In addition, these lactobacilli appeared to be nonpathogenic and had some probiotic potential: the strains produced lactic acid and bacteriocins, showed high sensitivity to broad-spectrum antibiotics, and were capable of forming biofilms in vitro. With the help of PCR and specific primers, the presence of genes for prebacteriocins in L. plantarum (plnEF, plnJ, plnN) and L. rhamnosus (LGG_02380 and LGG_02400) has been revealed. It was found that intestinal strains of lactobacilli were resistant to hydrochloric acid and bile. Lactobacilli isolated from the oral cavity were characterized by a high degree of adhesion, whereas intestinal strains were characterized by average adhesion. Both types of lactobacilli had medium to high rates of auto-aggregation and hydrophobicity and could coaggregate with pathogens and opportunistic pathogens. Additionally, the ability of the lactobacilli strains to produce gasotransmitters, CH₄, CO₂, C₂H₆, CO, and NH₃, has been revealed.     

Evaluation of the Diversity of Probiotic <Emphasis Type="Italic">Bacillus</Emphasis>, <Emphasis Type="Italic">Clostridium</Emphasis>, and <Emphasis Type="Italic">Bifidobacterium</Emphasis> Using the Illumina-Based Sequencing Method

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.     

Effect of a Probiotic Intake on Oxidant and Antioxidant Parameters in Plasma of Athletes During Intense Exercise Training

The aim of this study was to evaluate the effect of Lactobacillus rhamnosus IMC 501^® and Lactobacillus paracasei IMC 502^® on oxidative stress in athletes during a four-week period of intense physical activity. Two groups of twelve subjects each were selected for this analysis. The first group consumed a daily dose of a mixture of the two probiotic strains (1:1 L. rhamnosus IMC 501^® and L. paracasei IMC 502^®; ~10⁹ cells/day) for 4 weeks. The second group (control) did not consume any supplements during the 4 weeks. Blood samples collected immediately before and after the supplementation were analyzed, and plasma levels of reactive oxygen metabolites and biological antioxidant potential were determined. Faeces were also collected and analyzed before and at the end of the probiotic supplementation. Antioxidative activity and oxidative stress resistance of the two strains were determined in vitro. Results demonstrated that intense physical activity induced oxidative stress and that probiotic supplementation increased plasma antioxidant levels, thus neutralizing reactive oxygen species. The two strains, L. rhamnosus IMC 501^® and L. paracasei IMC 502^®, exert strong antioxidant activity. Athletes and all those exposed to oxidative stress may benefit from the ability of these probiotics to increase antioxidant levels and neutralize the effects of reactive oxygen species.     

Human milk and mucosal lacto- and galacto-<Emphasis Type="Italic">N</Emphasis>-biose synthesis by transgalactosylation and their prebiotic potential in <Emphasis Type="Italic">Lactobacillus</Emphasis> species

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-d-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants.     

Clinical strains of <Emphasis Type="Italic">Lactobacillus</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> and protect <Emphasis Type="Italic">Galleria mellonella</Emphasis> against experimental candidiasis

Candida albicans is the most common human fungal pathogen and can grow as yeast or filaments, depending on the environmental conditions. The filamentous form is of particular interest because it can play a direct role in adherence and pathogenicity. Therefore, the purpose of this study was to evaluate the effects of three clinical strains of Lactobacillus on C. albicans filamentation as well as their probiotic potential in pathogen-host interactions via an experimental candidiasis model study in Galleria mellonella. We used the reference strain Candida albicans ATCC 18804 and three clinical strains of Lactobacillus: L. rhamnosus strain 5.2, L. paracasei strain 20.3, and L. fermentum strain 20.4. First, the capacity of C. albicans to form hyphae was tested in vitro through association with the Lactobacillus strains. After that, we verified the ability of these strains to attenuate experimental candidiasis in a Galleria mellonella model through a survival curve assay. Regarding the filamentation assay, a significant reduction in hyphae formation of up to 57% was observed when C. albicans was incubated in the presence of the Lactobacillus strains, compared to a control group composed of only C. albicans. In addition, when the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela increased in all experimental groups. We concluded that Lactobacillus influences the growth and expression C. albicans virulence factors, which may interfere with the pathogenicity of these microorganisms.     

Assessment of the antifungal activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Pediococcus</Emphasis> spp. for use as bioprotective cultures in dairy products

Fungi are commonly involved in dairy product spoilage and the use of bioprotective cultures can be a complementary approach to reduce food waste and economic losses. In this study, the antifungal activity of 89 Lactobacillus and 23 Pediococcus spp. isolates against three spoilage species, e.g., Yarrowia lipolytica, Rhodotorula mucilaginosa and Penicillium brevicompactum, was first evaluated in milk agar. None of the tested pediococci showed antifungal activity while 3, 23 and 43 lactobacilli isolates showed strong antifungal activity or total inhibition against Y. lipolytica, R. mucilaginosa and P. brevicompactum, respectively. Then, the three most promising strains, Lactobacillus paracasei SYR90, Lactobacillus plantarum O_VI9 and Lactobacillus rhamnosus BIO_III28 at initial concentrations of 10⁵ and 10⁷ CFU/ml were tested as bioprotective cultures against the same fungal targets in a yogurt model during a 5-week storage period at 10 °C. While limited effects were observed at 10⁵ CFU/ml inoculum level, L. paracasei SYR90 and L. rhamnosus BIO_III28 at 10⁷ CFU/ml respectively retarded the growth of R. mucilaginosa and P. brevicompactum as compared to a control without selected cultures. In contrast, growth of Y. lipolytica was only slightly affected. In conclusion, these selected strains may be good candidates for bioprotection of fermented dairy products.     

Adaptation of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> to Thermal Stress Yields a Thermotolerant Variant Which Also Exhibits Improved Survival at pH 2

Loss in probiotic viability upon exposure to stressful storage and transport conditions has plagued the probiotic market worldwide. Lactobacillus acidophilus is an important probiotic that is added to various functional foods. It is known to be fairly labile and susceptible to temperature variations that it encounters during processing and storage which increases production cost. It has been repeatedly demonstrated that pre-exposure to sub-lethal doses of stress, particularly, temperature and pH, leads to improved survival of various probiotics when they subsequently encounter the same stress of a much greater magnitude. Attempts to adapt L. acidophilus to temperatures as high as 65 °C to arrive at a thermotolerant variant have not been reported previously. To improve viability at elevated temperatures, we gradually adapted the L. acidophilus NCFM strain to survival at 65 °C for 40 min. Following adaptation, the variant showed a 2-log greater survival compared to wild-type at 65 °C. Interestingly, this thermotolerant variant also demonstrated a 2-log greater stability compared to wild-type at pH 2.0. The improved pH and temperature stress tolerance exhibited by this variant remained unaltered even when the strain was lyophilized. Moreover, the thermotolerant variant demonstrated improved stability compared to wild-type when stored for up to a week at 37 and 42 °C. Probiotic properties of the variant such as adherence to epithelial cells and antibacterial activity remained unaltered. This strain can potentially help address the issue of significant loss in viable cell counts of L. acidophilus which is typically encountered during probiotic manufacture and storage.     

Influence of manufacturing processes on cell surface properties of probiotic strain <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> Lcr35<Superscript>®</Superscript>

The influence of the industrial process on the properties of probiotics, administered as complex manufactured products, has been poorly investigated. In the present study, we comparatively assessed the cell wall characteristics of the probiotic strain Lactobacillus rhamnosus Lcr35® together with three of its commercial formulations with intestinal applications. Putative secreted and transmembrane-protein-encoding genes were initially searched in silico in the genome of L. rhamnosus Lcr35®. A total of 369 candidate genes were identified which expressions were followed using a custom Lactobacillus DNA chip. Among them, 60 or 67 genes had their expression either upregulated or downregulated in the Lcr Restituo® packet or capsule formulations, compared to the native Lcr35® strain. Moreover, our data showed that the probiotic formulations (Lcr Lenio®, Lcr restituo® capsule and packet) showed a better capacity to adhere to intestinal epithelial Caco-2 cells than the native Lcr35® strain. Microbial (MATS) tests showed that the probiotic was an electron donor and that they were more hydrophilic than the native strain. The enhanced adhesion capacity of the active pharmaceutical ingredients (APIs) to epithelial Caco-2 cells and their antipathogen effect could be due to this greater surface hydrophilic character. These findings suggest that the manufacturing process influences the protein composition and the chemical properties of the cell wall. It is therefore likely that the antipathogen effect of the formulation is modulated by the industrial process. Screening of the manufactured products’ properties would therefore represent an essential step in evaluating the effects of probiotic strains.