An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.     

Ileal mucosal response to the same probiotic Lactobacillus strains is markedly different between suckling and adult mice

While evidence shows that probiotic supplementation exerts beneficial effects on developing children and animals, it is unclear whether it would exert equal or similar effects on adult human and animals. In this study, response to probiotic lactobacilli in ileal mucosa of suckling and adult mice was compared by evaluating gene expression profiles using DNA microarray. Two probiotic strains, Lactobacillus gasseri CP2305s and Lactobacillus plantarum CPA305C were used. Supplementation of probiotics for 7 days affected completely different genes in suckling and adult mice, regardless of the probiotic strain. The results suggested that ileal mucosal responses to probiotics are age stage specific.     

The probiotic properties and potential of vaginal Lactobacillus spp. isolated from healthy women against some vaginal pathogens

During the last decade, probiotic research has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic strains. The most studied probiotics belong to the genus Lactobacillus. In this study, 80 Lactobacillus spp. isolated from healthy women tolerated low pH and were able to grow in the presence of bile salts. RAPD PCR technique resulted in the identification of 38 different types. These isolates were then evaluated based on adhesion capacity, antibiotic susceptibility and tolerance in simulated gastrointestinal tract. Species-specific PCR and detection of bacteriocin-related genes were also surveyed. Among the isolates, five strains—Lacticaseibacillus rhamnosus NO21, Lacticaseibacillus casei NO1, Lactiplantibacillus plantarum NO4, Lactobacillus acidophilus NO7 and Lactobacillus gasseri NO38—presented acceptable antibiotic susceptibility pattern. Further analysis showed antimicrobial activity of Lacticaseibacillus culture against various bacterial pathogens and real-time PCR showed all five strains were able to prevent the colonization of bacterial pathogens. All five selected strains produced organic acids, hydrogen peroxide and were resistant to the spermicide. In addition, they lacked haemolytic activity with the ability of hydrophobicity, auto-aggregation and co-aggregation with pathogens. These results suggest that the vaginal microbiome could be a good source for the isolation of probiotics and the strains of this study may be considered as good probiotic candidates.     

The Effects of Limosilactobacillus reuteri LR-99 Supplementation on Body Mass Index,Social Communication,Fine Motor Function,and Gut Microbiome Composition in Individuals with Prader–Willi Syndrome: a Randomized Double-Blinded Placebo-Controlled Trial

Prader–Willi syndrome (PWS) is a rare genetic disorder associated with developmental delay, obesity, and neuropsychiatric comorbidities. Limosilactobacillus reuteri (Lactobacillus reuteri, Lact. reuteri) has demonstrated anti-obesity and anti-inflammatory effects in previous studies. In the present study, we aim to evaluate the effects of Lact. reuteri supplementation on body mass index (BMI), social behaviors, and gut microbiota in individuals with PWS. We conducted a 12-week, randomized, double-blind, placebo-controlled trial in 71 individuals with PWS aged 6 to 264 months (64.4 ± 51.0 months). Participants were randomly assigned to either receive daily Lact. reuteri LR-99 probiotic (6 × 10¹⁰ colony forming units) or a placebo sachet. Groupwise differences were assessed for BMI, ASQ-3, and GARS-3 at baseline, 6 weeks, and 12 weeks into treatment. Gut microbiome data was analyzed with the QIIME2 software package, and predictive functional profiling was conducted with PICRUSt-2. We found a significant reduction in BMI for the probiotic group at both 6 weeks and 12 weeks relative to the baseline (P < 0.05). Furthermore, we observed a significant improvement in social communication and interaction, fine motor function, and total ASQ-3 score in the probiotics group compared to the placebo group (P < 0.05). Altered gut microbiota was observed in the probiotic group to favor weight loss and improve gut health. The findings suggest a novel therapeutic potential for Lact. reuteri LR-99 probiotic to modulate BMI, social behaviors, and gut microbiota in Prader–Willi syndrome patients, although further investigation is warranted.

Trial registration Chinese Clinical Trial Registry: ChiCTR1900022646

     

Inhibitory effect of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> on periodontopathic and cariogenic bacteria

The interaction between Lactobacillus reuteri, a probiotic bacterium, and oral pathogenic bacteria have not been studied adequately. This study examined the effects of L. reuteri on the proliferation of periodontopathic bacteria including Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, and Tannerella forsythia, and on the formation of Streptococcus mutans biofilms. Human-derived L. reuteri strains (KCTC 3594 and KCTC 3678) and rat-derived L. reuteri KCTC 3679 were used. All strains exhibited significant inhibitory effects on the growth of periodontopathic bacteria and the formation of S. mutans biofilms. These antibacterial activities of L. reuteri were attributed to the production of organic acids, hydrogen peroxide, and a bacteriocin-like compound. Reuterin, an antimicrobial factor, was produced only by L. reuteri KCTC 3594. In addition, L. reuteri inhibited the production of methyl mercaptan by F. nucleatum and P. gingivalis. Overall, these results suggest that L. reuteri may be useful as a probiotic agent for improving oral health.     

Assessment and comparison of probiotic potential of four Lactobacillus species isolated from feces samples of Iranian infants

The probiotic potential of Lactobacillus species isolated from infant feces was investigated. For this study, the antibiotic susceptibility, tolerance in gut‐related conditions, antimicrobial activity, and ability to adhere to a human colorectal adenocarcinoma cell line (Caco‐2 cells) of four common Lactobacillus species (Lactobacillus paracasei [n = 15], Lactobacillus rhamnosus [n = 45], Lactobacillus gasseri [n = 20] and Lactobacillus fermentum [n = 18]) were assessed. Most isolates that which were sensitive to imipenem, ampicillin, gentamycin, erythromycin and tetracycline were selected for other tests. L. gasseri isolates had the greatest sensitivity to gastric and intestinal fluids (<10% viability). L. fermentum (FH5, FH13 and FH18) had the highest adhesion to Caco‐2 cells. The lowest antibacterial activity against pathogenic bacteria was shown by L. gasseri strains in spot tests. Furthermore, non‐adjusted cell‐free culture supernatants with low pH had greater antimicrobial activity, which was related to organic acid. The results showed that some isolates of L. rhamnosus and L. fermentum are suitable for use as a probiotic.     

Effects of Lactobacillus gasseri BNR17 on body weight and adipose tissue mass in diet-induced overweight rats

We investigated the weight-gain suppressive effect of Lactobacillus gasseri BNR17 isolated from human breast milk. Rats were fed a high-carbohydrate diet and administered BNR17 (BNR17 group) twice daily for twelve weeks. Changes were observed in body weight and white adipose tissue mass. The percent increase in body weight (P=0.0331) and fat pad mass (P<0.01) was significantly lower in the BNR17 group, and the FER was moderately lower (P=0.0769). These data suggest that BNR17 can prevent diet-induced overweight and may become an alternative method for treating weight problems and obesity.     

Protective Effect of the Intracellular Content from Potential Probiotic Bacteria against Oxidative Damage Induced by Acrylamide in Human Erythrocytes

The aim of this study was to assess the protective effect of the intracellular content obtained from potential probiotic bacteria against acrylamide-induced oxidative damage in human erythrocytes. First, the antioxidant properties of 12 potential probiotic strains was evaluated. Two commercial probiotic bacteria were included as reference strains, namely, Lactobacillus casei Shirota and Lactobacillus paracasei 431. Data showed that the intracellular content from four strains, i.e., Lactobacillus fermentum J10, Lactobacillus pentosus J24 and J26, and Lactobacillus pentosus J27, showed higher (P < 0.05) antioxidant capacity in most methods used. Thereafter, the intracellular content of such pre-selected strains was able to prevent the disturbance of the antioxidant system of human erythrocytes exposed to acrylamide, thereby reducing cell disruption and eryptosis development (P < 0.05). Additionally, the degree of oxidative stress in erythrocytes exposed to acrylamide was significantly (P < 0.05) reduced to levels similar to the basal conditions when the intracellular content of Lact. fermentum J10, Lact. pentosus J27, and Lact. paracasei 431 were employed. Hence, our findings suggest that the intracellular contents of specific Lactobacillus strains represent a potential source of metabolites with antioxidant properties that may help reduce the oxidative stress induced by acrylamide in human erythrocytes.

     

Biological characteristics and whole-genome analysis of the potential probiotic,Lactobacillus reuteri S5

Lactic acid bacteria are micro-organisms used for probiotic purposes and form major parts of human and mammalian intestinal microbiota, exerting important health-promoting effects on the host. Here, we evaluated Lactobacillus reuteri strain S5 isolated from the intestines of healthy white feather broilers. Lactobacillus reuteri S5 grew best after 20 h of incubation in MRS medium. Lactic acid production was 1·42 mmol l⁻¹ at 24 h, which was well tolerated. Activities of T-AOC, GSH-Px and T-SOD in the cell-free fermentation supernatant of L. reuteri S5 were higher than those in the bacteria, and the strain showed good hydrophobicity in vitro. The dominant carbon and nitrogen sources of L. reuteri S5 were glucose and soybean meal. A high-quality complete genome map of L. reuteri S5 was obtained using a Pacbio nanopore third-generation sequencing platform. The results showed that L. reuteri S5 possesses a complete primary metabolic pathway, encoding the main functional enzymes of the glycolysis pathway and pentose phosphate pathway. The genome contains genes encoding antioxidants and conferring tolerance to inorganic salt ions, acids and bile salts. This study shows that L. reuteri S5 is a probiotic strain with excellent probiotic characteristics and has great potential for the development of feed additives to promote animal health.     

Evaluation of Phytate-Degrading Lactobacillus Culture Administration to Broiler Chickens

Probiotics have been demonstrated to promote growth, stimulate immune responses, and improve food safety of poultry. While widely used, their effectiveness is mixed, and the mechanisms through which they contribute to poultry production are not well understood. Microbial phytases are increasingly supplemented in feed to improve digestibility and reduce antinutritive effects of phytate. The microbial origin of these exogenous enzymes suggests a potentially important mechanism of probiotic functionality. We investigated phytate degradation as a novel probiotic mechanism using recombinant Lactobacillus cultures expressing Bacillus subtilis phytase. B. subtilis phyA was codon optimized for expression in Lactobacillus and cloned into the expression vector pTRK882. The resulting plasmid, pTD003, was transformed into Lactobacillus acidophilus, Lactobacillus gallinarum, and Lactobacillus gasseri. SDS-PAGE revealed a protein in the culture supernatants of Lactobacillus pTD003 transformants with a molecular weight similar to that of the B. subtilis phytase. Expression of B. subtilis phytase increased phytate degradation of L. acidophilus, L. gasseri, and L. gallinarum approximately 4-, 10-, and 18-fold over the background activity of empty-vector transformants, respectively. Phytase-expressing L. gallinarum and L. gasseri were administered to broiler chicks fed a phosphorus-deficient diet. Phytase-expressing L. gasseri improved weight gain of broiler chickens to a level comparable to that for chickens fed a control diet adequate in phosphorus, demonstrating proof of principle that administration of phytate-degrading probiotic cultures can improve performance of livestock animals. This will inform future studies investigating whether probiotic cultures are able to provide both the performance benefits of feed enzymes and the animal health and food safety benefits traditionally associated with probiotics.     

Oral Administration of Lactobacillus Strains Isolated from Breast Milk as an Alternative for the Treatment of Infectious Mastitis during Lactation

In this study, 20 women with staphylococcal mastitis were randomly divided in two groups. Those in the probiotic group daily ingested 10 log₁₀ CFU of Lactobacillus salivarius CECT5713 and the same quantity of Lactobacillus gasseri CECT5714 for 4 weeks, while those in the control one only ingested the excipient. Both lactobacillus strains were originally isolated from breast milk. On day 0, the mean staphylococcal counts in the probiotic and control groups were similar (4.74 and 4.81 log₁₀ CFU/ml, respectively), but lactobacilli could not be detected. On day 30, the mean staphylococcal count in the probiotic group (2.96 log₁₀ CFU/ml) was lower than that of the control group (4.79 log₁₀ CFU/ml). L. salivarius CECT5713 and L. gasseri CECT5714 were isolated from the milk samples of 6 of the 10 women of the probiotic group. At day 14, no clinical signs of mastitis were observed in the women assigned to the probiotic group, but mastitis persisted throughout the study period in the control group women. In conclusion, L. salivarius CECT5713 and L. gasseri CECT5714 appear to be an efficient alternative for the treatment of lactational infectious mastitis during lactation.     

Inhibitory potential of lactobacilli against <Emphasis Type="Italic">Escherichia coli</Emphasis> internalization by HT 29 cells

A quantitative fluorescent in situ hybridization method was employed to evaluate the competitive inhibitory effect of three Lactobacillus strains (Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus plantarum) against Escherichia coli internalization in a model system of HT 29 cells. Furthermore, aggregation and adhesion abilities of the Lactobacillus strains were examined. All lactobacilli were able to attach to the HT 29 cells and aggregate with pathogens; however, the adhesion and aggregation degree was strain-dependent. L. reuteri possessed a high capacity of adhesion (6.80 ± 0.63; log CFU ± SEM per well), whereas lower capacities were expressed by L. gasseri (4.52 ± 0.55) and L. plantarum (4.90 ± 0.98). Additionally, L. reuteri showed the rapid or normal ability to aggregate with selected E. coli in comparison with remaining two lactobacilli, which showed only slow or negative aggregative reaction. Internalization of E. coli into the cell lines was markedly suppressed by L. reuteri, while L. gasseri and L. plantarum caused only a minimum anti-invasion effect. The fact that L. reuteri in our experiments showed an outstanding potential for adhering to the colon epithelial cell line, compared with the rest strains, suggested that one of the possible mechanisms of preventing pathogen adhesion and invasion is simple competitions at certain receptors and capability to block receptor binding sites, or that an avid interaction between L. reuteri and the host cell might be modulating intracellular events responsible for the E. coli internalization. Moreover, L. reuteri exhibited a strong ability to aggregate with E. coli, which could be another limiting factor of pathogen invasion.     

Characterization of Intestinal Lactobacillus reuteri Strains as Potential Probiotics

This study was conducted to evaluate the probiotic properties of Lactobacillus reuteri isolated from human infant feces (less than 3?months). Out of thirty-two representative L. reuteri strains isolated from the infant human feces, nine isolates (i.e. LR5, LR6, LR9, LR11, LR19, LR20, LR25, LR26 and LR34) showed survival in acid, bile and simulated stomach?Cduodenum passage conditions, indicating their high tolerance to gastric juice, duodenal juice and bile environments. The nine isolates did not show strong hydrophobic properties because the percentages of adhesion to the apolar solvent, n-hexadecane, did not exceed 40%, showing that their surfaces were rather hydrophilic. Functionality of these nine probiotic isolates was supported by their antagonistic activity and their ability to deconjugate bile salts. The safety of the nine indigenous L. reuteri isolates was supported by the absence of transferable antibiotic resistance determinants, DNase activity, gelatinase activity and hemolysis. The results obtained so far suggest that the nine strains are resistant to low pH, bile salts and duodenum juice, so they could survive when passing through the upper part of the gastrointestinal tract and fulfill their potential probiotic action in the host organism. According to these results, the L. reuteri strains isolated from human infant feces possess interesting probiotic properties that make them potentially good candidates for probiotics.     

Evaluation of Reuterin Production in Urogenital Probiotic Lactobacillus reuteri RC-14

Classified as a distinct species in 1980, Lactobacillus reuteri strains have been used in probiotic formulations for intestinal and urogenital applications. In the former, the primary mechanism of action of L. reuteri SD2112 (ATCC 55730) has been purported to be its ability to produce the antibiotic 3-hydroxypropionaldehyde (3-HPA), also known as reuterin. In the vagina, it has been postulated that probiotic Lactobacillus reuteri RC-14 does not require reuterin production but mediates a restoration of the normal microbiota via hydrogen peroxide, biosurfactant, lactic acid production, and immune modulation. The aim of the present study was to determine whether strain RC-14 produced reuterin. Using PCR and DNA dot blot analyses, numerous Lactobacillus species, including RC-14, were screened for the presence of the gene encoding the large subunit of glycerol dehydratase (gldC), the enzyme responsible for reuterin production. In addition, lactobacilli were grown in glycerol-based media and both high-performance liquid chromatography and a colorimetric assay were used to test for the presence of reuterin. L. reuteri RC-14 was determined to be negative for gldC sequences, as well as for the production of reuterin when cultured in the presence of glycerol. These findings support that the probiotic effects of L. reuteri RC-14, repeatedly demonstrated during numerous studies of the intestine and vagina, are independent of reuterin production.     

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.

     

Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus,Lactobacillus crispatus,Lactobacillus amylovorus,Lactobacillus gallinarum,Lactobacillus gasseri,and Lactobacillus johnsonii

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.     

Lactobacillus reuteri KT260178 Supplementation Reduced Morbidity of Piglets Through Its Targeted Colonization,Improvement of Cecal Microbiota Profile,and Immune Functions

Supplementing suckling piglets with Lactobacillus reuteri isolated from a homologous source improves L. reuteri colonization number in the gastrointestinal tract, which can have health benefits. This study investigated dietary L. reuteri supplementation on the growth and health—including immune status—of piglets, as well as its colonization. A total of 60 sows with similar parity and body weight were allocated into one of three groups after secretion (n = 20 each, with 10 neonatal piglets of each): untreated control, L. reuteri supplementation, and antibiotic treatment. The experimental duration was 28 days, from birth of piglets to their group transferred. For the first 7 days after birth, all neonatal piglets were fed by sows. Piglets in the L. reuteri supplementation group were administered with 1.0 ml L. reuteri fermentation broth containing 5.0 × 10⁷ CFU. From 7 to 28 days, piglets were given basal feed (control), basal feed supplemented with L. reuteri (1.0 × 10⁷ CFU/g), or aureomycin (150 mg/kg). L. reuteri colonization in the distal jejunum and ileum was increased in piglets in the L. reuteri-supplemented as compared to the control group after 28 days, as determined by fluorescence in situ hybridization and real-time PCR analysis. Total Lactobacillus and Bifidobacterium counts in the cecum were higher whereas total aerobic bacteria (Escherichia coli and Staphylococcus) counts were lower in the L. reuteri as compared to the control group. L. reuteri supplementation also improved body antioxidant status and immune function relative to control animals. Strain-specific L. reuteri administered to piglets colonizes the intestinal mucosa and improves cecal microbiota profile and whole-body antioxidant and immune status, leading to better growth and lower morbidity and mortality rates.

     

Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes

Lactobacillus gasseri K7 is a probiotic strain that produces bacteriocins gassericin K7 A and K7 B. In order to develop a real-time quantitative PCR assay for the detection of L. gasseri K7, 18 reference strains of the Lactobacillus acidophilus group and 45 faecal samples of adults who have never consumed strain K7 were tested with PCR using 14 pairs of primers specific for gassericin K7 A and K7 B gene determinants. Incomplete gassericin K7 A or K7 B gene clusters were found to be dispersed in different lactobacilli strains as well as in faecal microbiota. One pair of primers was found to be specific for the total gene cluster of gassericin K7A and one for gassericin K7B. The real-time PCR analysis of faecal samples spiked with K7 strain revealed that primers specific for the gene cluster of the gassericin K7 A were more suitable for quantitative determination than those for gassericin K7 B, due to the lower detection level. Targeting of the gassericin K7 A or K7 B gene cluster with specific primers could be used for detection and quantification of L. gasseri K7 in human faecal samples without prior cultivation. The results of this study also present new insights into the prevalence of bacteriocin-encoding genes in gastrointestinal tract.     

Effect of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> on the proliferation of <Emphasis Type="Italic">Propionibacterium acnes</Emphasis> and <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis>

While it is generally accepted that Propionibacterium acnes is involved in the development of acne, other bacteria including Staphylococcus epidermidis have also been isolated from the acne lesion. The interaction between Lactobacillus reuteri, a probiotic bacterium, and acnegenic bacteria is unclear. This study examined the effects of L. reuteri on the proliferation of P. acnes and S. epidermidis. Human-derived L. reuteri strains (KCTC 3594 and KCTC 3678) and rat-derived L. reuteri KCTC 3679 were used. All strains exhibited significant inhibitory effects on the growth of P. acnes and S. epidermidis. The proliferation of P. acnes was decreased by 2-log scales after incubation with L. reuteri for 24 h. In addition, the proliferation of S. epidermidis was decreased by 3-log scales after incubation with L. reuteri for 24 h, whereas the growth of L. reuteri was unaffected by P. acnes or S. epidermidis. Among the L. reuteri strains examined, L. reuteri KCTC 3679 had the strongest inhibitory effect on the growth of P. acnes and S. epidermidis, followed by L. reuteri KCTC 3594 and L. reuteri KCTC 3678. Interestingly, reuterin, an antimicrobial factor, was produced only by L. reuteri KCTC 3594. The most pronounced the antibacterial activities of L. reuteri were attributed to the production of organic acids. Overall, these results suggest that L. reuteri may be a useful probiotic agent to control the growth of bacteria involved in acne inflammation and prevent acne.     

Molecular Identification and Typing of Putative Probiotic Indigenous Lactobacillus plantarum Strain Lp91 of Human Origin by Specific Primed-PCR Assays

In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase (bsh) and collagen-binding protein (cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles.