Innovative method to grow the probiotic Lactobacillus reuteri in the omega3-rich microalga Isochrysis galbana.

Microalgae are natural sources of valuable bioactive compounds, such as polyunsaturated fatty acids (PUFAs), that show antioxidant, anti-inflammatory, anticancer and antimicrobial activities. The marine microalga Isochrysis galbana (I. galbana) is extremely rich in ω3 PUFAs, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Probiotics are currently suggested as adjuvant therapy in the management of diseases associated with gut dysbiosis. The Lactobacillus reuteri (L. reuteri), one of the most widely used probiotics, has been shown to produce multiple beneficial effects on host health. The present study aimed to present an innovative method for growing the probiotic L. reuteri in the raw seaweed extracts from I. galbana as an alternative to the conventional medium, under conditions of oxygen deprivation (anaerobiosis). As a result, the microalga I. galbana was shown for the first time to be an excellent culture medium for growing L. reuteri. Furthermore, the gas-chromatography mass-spectrometry analysis showed that the microalga-derived ω3 PUFAs were still available after the fermentation by L. reuteri. Accordingly, the fermented compound (FC), obtained from the growth of L. reuteri in I. galbana in anaerobiosis, was able to significantly reduce the adhesiveness and invasiveness of the harmful adherent-invasive Escherichia coli to intestinal epithelial cells, due to a cooperative effect between L. reuteri and microalgae-released ω3 PUFAs. These findings open new perspectives in the use of unicellular microalgae as growth medium for probiotics and in the production of biofunctional compounds.

Culture conditions affect Lactobacillus reuteri DSM 17938 ability to perform glycerol bioconversion into 3-hydroxypropionic acid.

The platform molecule 3-hydroxypropionic acid (3-HP) can be produced using Lactobacillus reuteri through a two-step bioprocess that involves a growth phase followed by a bioconversion phase. The bioproduction is performed by resting cells that convert glycerol into 3-HP and 1,3-propanediol in fed-batch mode. This work aimed at studying the effect of the growth conditions of L. reuteri DSM 17938 during the first step, on the glycerol bioconversion into 3-HP during the second step. A Plackett and Burman design was carried out to test, in controlled bioreactors, the effect of 11 growth conditions simultaneously, at fixed bioconversion conditions. The supplementation of the growth medium with vitamin B12 and cysteine displayed a negative effect on the 3-HP bioproduction. The addition of glucose, phytone peptone, Tween 80, 1,2-propanediol and betaine in the growth medium, together with a low temperature and an optimal pH of 6.0 during the growth phase increased the bioconversion duration from 56 h to 89 h at a glycerol feeding rate of 0.5 g·h-1. A validating experiment displayed that the 3-HP titer, 3-HP production yield and 3-HP specific production rate were significantly improved by 25 %, 150 % and 61 %, respectively.

Levan from Bacillus amyloliquefaciens JN4 acts as a prebiotic for enhancing the intestinal adhesion capacity of Lactobacillus reuteri JN101.

Improving intrinsic adhesion performance of the known probiotics facilitates their residence and colonization, and therefore exerts more beneficial effects on the human or animal host. In this study, through adaptive culture with levan, Lactobacillus reuteri JN101 achieved the same biomass and exhibited 2.6 times higher adhesion capacity to HT-29 cells than those grown with glucose. The mechanism study related to this adhesion enhancement showed that the elevated proportion of unsaturated fatty acids facilitated the bacterial cells to overcome repulsive forces to approach the intestinal epithelial cell. At the same time, and the greater amounts of cell membrane proteins, such as S-layer protein (3.2 folds), elongation factor Tu (2.6 folds) and phosphoglycerate kinase (2.4 folds) probably enhanced the complementary interactions to the receptor on the epithelial cell. These results presented here indicated levan could be used as a potential prebiotic to regulate the adhesion capacity of probiotics, and provide ground for developing the specific-probiotics oriented functional food.

Lactobacillus reuteri DSM 17938 Protects against Gastric Damage Induced by Ethanol Administration in Mice: Role of TRPV1/Substance P Axis.

This study aimed to evaluate the effect of Lactobacillus reuteri DSM 17938 (DSM) on ethanol-induced gastric injury, and if its possible mechanism of action is related to inhibiting the transient receptor potential vanilloid type 1 (TRPV1). We evaluated the effect of supplementing 108 CFU•g body wt-1•day-1 of DSM on ethanol-induced gastric injury. DSM significantly reduced the ulcer area (1.940 ± 1.121 mm²) with 3 days of pretreatment. The effects of DSM supplementation were reversed by Resiniferatoxin (RTX), TRPV1 agonist (3 nmol/kg p.o.). Substance P (SP) (1 µmol/L per 20 g) plus 50% ethanol resulted in hemorrhagic lesions, and DSM supplementation did not reverse the lesion area induced by administering SP. TRPV1 staining intensity was lower, SP, malondialdehyde (MDA) and nitrite levels were reduced, and restored normal levels of antioxidant parameters (glutathione and superoxide dismutase) in the gastric mucosa in mice treated with DSM. In conclusion, DSM exhibited gastroprotective activity through decreased expression of TRPV1 receptor and decreasing SP levels, with a consequent reduction of oxidative stress.

Arabinoxylan from Argentinian whole wheat flour promote the growth of Lactobacillus reuteri and Bifidobacterium breve.

Arabinoxylans are part of dietary fibre and have received attention given their emergent prebiotic character. Four arabinoxylans extracts were obtained from Argentinian soft and hard wheat. In vitro assays were performed to describe the extent to which the extracts from whole wheat flour support selective growth of Bifidobacterium breve and probiotic Lactobacillus reuteri ATCC23272 in a defined media. The prebiotic effect was evaluated by three quantitative scores: relative growth, prebiotic activity score and prebiotic index. For prebiotic index equation the growth of Bacteroides and Clostridium strains was compared to that of bifidobacteria and lactic acid bacteria. All the arabinoxylans extracts supported the growth of Lactobacillus and Bifidobacterium, reaching higher prebiotic activity score values than inulin (0·37 and 0·36 for Lactobacillus and Bifidobacterium respectively). AX2 from soft wheat and AX4 from hard showed similar prebiotic index value to commercial inulin (2·64, 2·52 and 2·22 respectively), and AX3 extract presented higher prebiotic index value (4·09) than the positive control and other prebiotic index reported for arabinoxylans. These extracts could be used as prebiotic, synbiotic compositions or novel food prototypes to treat dysbiosis associated with many diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work demonstrates that AX extracts from Argentinian soft and hard wheat promote efficiently the growth of probiotic strain L. reuteri ATCC23272 and B. breve 286, validated with three different parameters that consider the growth of representative strains of Bacteria genera found in the gut. The evaluation of AX extracts as a food supplement in a murine model could confirm their ability to modulate the microbiome. Novel food prototypes including AX and probiotics could relieve local symptoms and may act as psychobiotics with a beneficial effect on microbiome-brain axis.

Resilience of small intestinal beneficial bacteria to the toxicity of soybean oil fatty acids.

Over the past century, soybean oil (SBO) consumption in the United States increased dramatically. The main SBO fatty acid, linoleic acid (18:2), inhibits in vitro the growth of lactobacilli, beneficial members of the small intestinal microbiota. Human-associated lactobacilli have declined in prevalence in Western microbiomes, but how dietary changes may have impacted their ecology is unclear. Here, we compared the in vitro and in vivo effects of 18:2 on Lactobacillus reuteri and L. johnsonii. Directed evolution in vitro in both species led to strong 18:2 resistance with mutations in genes for lipid biosynthesis, acid stress, and the cell membrane or wall. Small-intestinal Lactobacillus populations in mice were unaffected by chronic and acute 18:2 exposure, yet harbored both 18:2- sensitive and resistant strains. This work shows that extant small intestinal lactobacilli are protected from toxic dietary components via the gut environment as well as their own capacity to evolve resistance.

Changes in the diversity and composition of gut microbiota of weaned piglets after oral administration of Lactobacillus or an antibiotic.

The gut microbiota plays important roles in the health and well-being of animals, and high-throughput sequencing facilitates exploration of microbial populations in the animal gut. However, previous studies have focused on fecal samples instead of the gastrointestinal tract. In this study, we compared the microbiota diversity and composition of intestinal contents of weaned piglets treated with Lactobacillus reuteri or chlortetracycline (aureomycin) using high-throughput sequencing. Nine weaned piglets were randomly divided into three groups and supplemented with L. reuteri, chlortetracycline, or saline for 10 days, and then the contents of three intestinal segments (jejunum, colon, and cecum) were obtained and used for sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. The microbiota diversity and composition in the jejunum were different from those in the colon and cecum among the three treatments. In the jejunum, treatment with L. reuteri increased the species richness of the microbiota, as indicated by the ACE and Chao1 indexes, compared with the chlortetracycline group, in which several taxa were eliminated. In the colon and cecum, relative abundances of the phylum Firmicutes and the genus Prevotella were higher in the chlortetracycline group than in the other groups. Distances between clustered samples revealed that the L. reuteri group was closer to the chlortetracycline group than the control group for jejunum samples, while colon and cecum samples of the L. reuteri group were clustered with those of the control group. This study provides fundamental knowledge for future studies such as the development of alternatives to antibiotics.

Evaluation of acrylamide-removing properties of two Lactobacillus strains under simulated gastrointestinal conditions using a dynamic system.

The aim of this study was to evaluate the capability of Lactobacillus reuteri NRRL 14171 and Lactobacillus casei Shirota to remove dietary acrylamide (AA) under simulated gastrointestinal conditions using a dynamic system. The effects of different AA levels or bacteria concentration on toxin removal by Lactobacillus strains were assessed. Thereafter, AA-removing capability of bacteria strains under either fasting or postprandial simulated gastrointestinal conditions was evaluated. Commercial potato chips were analyzed for their AA content, and then used as a food model. Average AA content (34,162µg/kg) in potato chips exceeded by ca. 34-fold the indicative values recommended by the EU. Toxin removal ability was dependent on AA content and bacterial cell concentration. A reduction on bacterial viability was observed in the food model and at the end of both digestive processes evaluated. However, bacteria survived in enough concentrations to remove part of the toxin (32-73%). Both bacterial strains were able to remove AA under different simulated gastrointestinal conditions, being L. casei Shirota the most effective (ca. 70% removal). These findings confirmed the risk of potato chips as dietary AA exposure for consumers, and that strains of the genus Lactobacillus could be employed to reduce the bioavailability of dietary AA.

Oral Microbiota Shift after 12-Week Supplementation with Lactobacillus reuteri DSM 17938 and PTA 5289; A Randomized Control Trial.

BACKGROUND: Lactobacillus spp. potentially contribute to health by modulating bacterial biofilm formation, but their effects on the overall oral microbiota remain unclear. METHODS AND FINDINGS: Oral microbiota was characterized via 454-pyrosequencing of the 16S rDNA hypervariable region V3-V4 after 12 weeks of daily Lactobacillus reuteri DSM 17938 and PTA 5289 consumption. Forty-four adults were assigned to a test group (n = 22) that received lactobacilli lozenges (108 CFU of each strain/lozenge) or a control group that received placebo (n = 22). Presence of L. reuteri was confirmed by cultivation and species specific PCR. Tooth biofilm samples from 16 adults before, during, and after exposure were analyzed by pyrosequencing. A total of 1,310,292 sequences were quality filtered. After removing single reads, 257 species or phylotypes were identified at 98.5% identity in the Human Oral Microbiome Database. Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, and Actinobacteria were the most abundant phyla. Streptococcus was the most common genus and the S. oralis/S. mitis/S. mitis bv2/S. infantis group comprised the dominant species. The number of observed species was unaffected by L. reuteri exposure. However, subjects who had consumed L. reuteri were clustered in a principal coordinates analysis relative to scattering at baseline, and multivariate modeling of pyrosequencing microbiota, and culture and PCR detected L. reuteri separated baseline from 12-week samples in test subjects. L. reuteri intake correlated with increased S. oralis/S. mitis/S. mitis bv2/S. infantis group and Campylobacter concisus, Granulicatella adiacens, Bergeyella sp. HOT322, Neisseria subflava, and SR1 [G-1] sp. HOT874 detection and reduced S. mutans, S. anginosus, N. mucosa, Fusobacterium periodicum, F. nucleatum ss vincentii, and Prevotella maculosa detection. This effect had disappeared 1 month after exposure was terminated. CONCLUSIONS: L. reuteri consumption did not affect species richness but induced a shift in the oral microbiota composition. The biological relevance of this remains to be elucidated. TRIAL REGISTRATION: ClinicalTrials.gov NCT02311218.

Effect of traditional leafy vegetables on the growth of lactobacilli and bifidobacteria.

Traditional leafy vegetables, apart from being a staple in the diet of most of sub-Saharan Africa, are an essential part of traditional medicine and are used daily by traditional healers in the region to treat a wide variety of ailments. In this study, a batch culture technique was used to investigate whether 25 infusions from 22 traditional leafy vegetables stimulated the growth of Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus reuteri and Bifidobacterium longum in pure culture. High performance liquid chromatography was used to determine the inulin content of the infusions. Sonchus oleraceus stimulated all four strains and Taraxacum officinale stimulated three strains. In total, 18 plants stimulated at least one of the four probiotic strains. The inulin content of the infusions varied between 2.5% and 3.6%, with Asparagus sprengeri containing the highest percentage. These results indicate that traditional leafy vegetables do stimulate the growth of the selected lactobacilli and bifidobacteria in pure culture and contain inulin. These infusions can now be tested for prebiotic potential using mixed culture systems or human hosts.

Investigation of probiotic bacteria as dental caries and periodontal disease biotherapeutics.

Oral diseases, specifically dental caries and periodontal disease, are characterised by increases in pathogenic microorganisms, increased demineralisation and increased inflammation and levels of inflammatory markers. Despite the therapeutic strategies, oral diseases have elevated prevalence rates. Recent work has demonstrated that probiotic bio-therapeutics can decrease oral pathogen counts, including caries-causing Streptococcus mutans and oral inflammation. The aim of this work was to investigate putative probiotic bacteria, selected for S. mutans inhibition and for their oral health-promoting characteristics. The probiotic bacteria were screened for S. mutans inhibition, probiotic bacteriocin activity, salivary pH modulation, probiotic nutrient (sucrose) competition, probiotic co-aggregation with S. mutans, bacterial attachment to oral epithelial keratinocytes, bacterial nitric oxide production and bacterial antioxidant activity. The results indicate that Lactobacillus reuteri strains NCIMB 701359, NCIMB 701089, NCIMB 702655 and NCIMB 702656 inhibited S. mutans to non-detectable levels (<10 cfu/ml). L. reuteri strains also demonstrated the highest antioxidant capacity of the tested strains (7.73-13.99 µM Trolox equivalents), suggesting their use as both caries and periodontal disease therapeutics. Although Lactobacillus fermentum NCIMB 5221 inhibited S. mutans at lower levels, it significantly buffered the pH (4.18) of saliva containing S. mutans, co-aggregated with S. mutans (10.09%), demonstrated high levels of sucrose consumption (138.11 mM) and successfully attached to gingival epithelial cells (11%). This study identified four L. reuteri strains and one L. fermentum strain to be further investigated as oral disease biotherapeutics.

Effect of tablets containing probiotic bacteria (Lactobacillus reuteri) on early caries lesions in adolescents: a pilot study.

The objective of the study was to investigate the effect of tablets containing probiotic lactobacilli on early caries lesions in adolescents with quantitative light-induced fluorescence (QLF). 36 healthy adolescents of both sexes (12-17 years of age) were enrolled and randomly allocated to a placebo-controlled trial with two parallel groups. The test group received two tablets daily containing two strains of Lactobacillus reuteri (DSM 17938 and ATCC PTA 5289) for a period of three months, while the control group got identical placebo tablets without live bacteria. The primary outcome was QLF-readings (change in fluorescence, ΔF and lesion area, mm2) at baseline and after 3 months, conducted at two buccal sites of each individual, pre-selected with clearly visible clinical signs of enamel demineralisation (white spots). Significantly more premolars were allocated to the placebo group, while the test group had more incisors (P<0.05). There were no statistically significant differences in fluorescence values between the groups, neither at baseline, nor at the follow-up. There was however a significant decrease in fluorescence over time in the test group, but not in the placebo group (P<0.05). No alterations of the lesion area (ΔA) were found in any group. The inter-examiner intra-class correlation coefficient-value for QLF-readings was excellent. No side- or adverse effects were reported during the intervention period. This pilot study found a significant decrease over time in the test group. However, no statistically significant differences in fluorescence values between the groups were found. Hence, the null hypothesis could not be rejected.

Privileged incorporation of selenium as selenocysteine in Lactobacillus reuteri proteins demonstrated by selenium-specific imaging and proteomics.

An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS ((78)Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine.

Effects of potato fiber and potato-resistant starch on biomarkers of colonic health in rats fed diets containing red meat.

UNLABELLED: The effects of red meat consumption with and without fermentable carbohydrates on indices of large bowel health in rats were examined. Sprague-Dawley rats were fed cellulose, potato fiber, or potato-resistant starch diets containing 12% casein for 2 wk, then similar diets containing 25% cooked beef for 6 wk. After week 8, cecal and colonic microbiota composition, fermentation end-products, colon structure, and colonocyte DNA damage were analyzed. Rats fed potato fiber had lower Bacteroides-Prevotella-Porphyromonas group compared to other diet groups. Colonic Bifidobacterium spp. and/or Lactobacillus spp. were higher in potato fiber and potato-resistant starch diets than in the cellulose diet. Beneficial changes were observed in short-chain fatty acid concentrations (acetic, butyric, and propionic acids) in rats fed potato fiber compared with rats fed cellulose. Phenol and p-cresol concentrations were lower in the cecum and colon of rats fed potato fiber. An increase in goblet cells per crypt and longer crypts were found in the colon of rats fed potato fiber and potato-resistant starch diets. Fermentable carbohydrates had no effect on colonic DNA damage. Dietary combinations of red meat with potato fiber or potato-resistant starch have distinctive effects in the large bowel. Future studies are essential to examine the efficacy of different types of nondigestible carbohydrates in maintaining colonic health during long-term consumption of high-protein diets. PRACTICAL APPLICATION: Improved understanding of interactions between the food consumed and gut microbiota provides knowledge needed to make healthier food choices for large bowel health. The impact of red meat on large bowel health may be ameliorated by consuming with fermentable dietary fiber, a colonic energy source that produces less harmful by-products than the microbial breakdown of colonic protein for energy. Developing functional red meat products with fermentable dietary fiber could be one way to promote a healthy and balanced macronutrient diet.

Persistence of Lactobacillus reuteri DSM17938 in the human intestinal tract: response to consecutive and alternate-day supplementation.

BACKGROUND: Probiotics may enhance gastrointestinal health and immune function. The efficacy of different probiotic dosing strategies on colonization and persistence of probiotics is undefined. OBJECTIVE: The authors assessed colonization and persistence of Lactobacillus reuteri (L. reuteri) DSM17938 (BioGaia AB, Stockholm, Sweden) after daily or alternate-day dosing. METHODS: Volunteers ate pudding with L. reuteri (10(9) CFU) daily (n = 9) or on alternate days (n = 9) over 7 days. Fecal samples were collected on dosing days (D1-7) and after dosing ended (D13-15 and D20-22) and were analyzed for the presence of L. reuteri. Results are reported in 3-day increments (D2-4, D5-7, D13-15, and D20-22). RESULTS: L. reuteri count rose in response to daily supplementation ([mean ± SD] D2-4: 4 × 104 ± 2 × 104 CFU, p < 0.01; D5-7: 10 × 104 ± 9 × 104 CFU, p < 0.01) and alternate-day supplementation (D2-4: 21 × 104 ± 20 × 104 CFU, p < 0.01; D5-7: 11 × 104 ± 15 × 104 CFU, p = 0.06) and fell in both groups 1 week after dosing ended (p < 0.01). Total volunteers with detectable L. reuteri 1 and 2 weeks after dosing ended was similar in response to daily feeding (4/9 and 2/9, respectively) and alternate-day feeding (3/9 and 2/9, respectively). L. reuteri count was higher D2-4 in response to alternate-day vs daily feeding (p < 0.05) but similar thereafter. CONCLUSIONS: Alternate-day probiotic intake achieves equivalent colonization to daily intake, but colonization declines rapidly once dosing stops. It is possible that, initially, responsiveness to probiotics may differ between individuals, but those differences do not persist with longer consumption.

Effect of glutathione on growth of the probiotic bacterium Lactobacillus reuteri.

Glutathione (GSH) is an abundant nonprotein thiol that plays numerous roles within the cell. Previously, we showed that Lactobacillus salivarius has the capacity to mount a glutathione-mediated acid-tolerance response. In the present work we provide evidence of a requirement for GSH by Lactobacillus reuteri and have studied the role of GSH during cell growth. Medium supplementation with 0.5 mM GSH as the sole sulfur source enhanced cell growth, resulting in an increase in glucose consumption, and increased cell GSH and protein contents compared with levels seen in the absence of supplementation. Moreover, L. reuteri showed enhanced amino acid consumption when grown with 0.5 mM GSH. These findings indicate that glutathione is a nutrient for bacterial growth.