Reuteri Culture Research Articles

Oxygen Transfer Effect on the Growth of Limosilactobacillus reuteri ATCC 53608 and on Its Metabolic Capacity

Limosilactobacillus reuteri is a probiotic microorganism used in the treatment of gastrointestinal disorders. The effect of oxygen transfer on cultures of L. reuteri ATCC 53608 at shake flask and stirred tank bioreactor scales was studied, using MRS and molasses-based media. At shake flask scale, in MRS medium, a maximum bacterial concentration of 2.01 ± 0.02 g L−1 was obtained; the oxygen transfer coefficient was 2.01 ± 0.04 h−1. Similarly, in a 7.5 L bioreactor, in MRS, a maximum bacterial concentration of 2.46 ± 0.16 g L−1 was achieved (kLa = 2.64 ± 0.06 h−1). In contrast, using a molasses-based medium, bacterial concentration reached 3.13 ± 0.17 g L−1 in the 7.5 L bioreactor. A progressive reduction in lactic acid concentration and yield was observed as the oxygen transfer coefficient increased, at shake flask scale. Also, the oxygen transfer coefficient strongly affected the growth of L. reuteri in shake flask and bioreactor and allowed us to successfully scale up L. reuteri culture, producing similar maximum bacterial concentrations in both scales (2.01 g L−1 and 2.46 g L−1 in MRS). This is the first study on oxygen transfer coefficients in L. reuteri, and it is a valuable contribution to the field as it provides important insights about how this organism tolerates oxygen and adapts its metabolism for larger biomass production.Graphical

Transcriptome Analysis of Glycerin Regulating Reuterin Production of Lactobacillus reuteri.

Reuterin can be produced from glycerol dehydration catalyzed by glycerol dehydratase (GDHt) in Lactobacillus reuteri and has broad application prospects in industry, agriculture, food, and other fields as it is active against prokaryotic and eukaryotic organisms and is resistant to proteases and lipases. However, high concentrations of glycerin inhibit reuterin production, and the mechanism behind this phenomenon is not clear. To elucidate the inhibitory mechanism of glycerol on reuterin synthesis in L. reuteri and provide reference data for constructing an L. reuteri culture system for highly effective 3-hydroxypropionaldehyde synthesis, we used transcriptome-sequencing technology to compare the morphologies and transcriptomes of L. reuteri cultured in a medium with or without 600 mM of glycerol. Our results showed that after the addition of 600 mM of glycerol to the culture medium and incubation for 10 h at 37 °C, the culture medium of L. reuteri LR301 exhibited the best bacteriostatic effect, and the morphology of L. reuteri cells had significantly changed. The addition of 600 mM of glycerol to the culture medium significantly altered the transcriptome and significantly downregulated the transcription of genes involved in glycol metabolism, such as gldA, dhaT, glpK, plsX, and plsY, but significantly upregulated the transcription of genes related to D-glucose synthesis.

ADHESIVE PROPERTIES OF BACTERIA UNDER THE INFLUENCE OF BIFIDOBACTERIUM BIFIDUM 1 AND LACTOBACILLUS REUTERI DSM 17938I CELL-FREE EXTRACTS

Increasing in the adhesive potential of probiotic bacteria on the one hand and weakening the adhesive ability of the pathobionts on the other is a pathogenetically based approach in the fight against intestinal infectious diseases and intestinal dysbiosis. At present postbiotic products are considered as one of the most promising means of influencing the intestinal microbiota. The aim of the work was to study the effect of cell-free extracts containing derivatives of Bifidobacterium bifidum 1 and Lactobacillus reuteri DSM 17938 on the adhesive properties of probiotic (L. reuteri DSM 17938 and B. bifidum 1) and opportunistic bacteria (Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922). Four cell-free extracts were investigated: L – obtained from the L. reuteri disintegrate; ML – obtained from the L. reuteri culture, cultivated in its own disintegrate; B – obtained from the B. bifidum disintegrate; MB – obtained from the B. bifidum culture, cultivated in its own disintegrate. The effect of cell-free extracts on the adhesive properties of microorganisms was studied by using Brilis method. ML, MB, and B extracts were found to be able to significantly increase the adhesive potential of B. bifidum and L. reuteri. Under their influence adhesion index calculated for bifidobacteria increased by 41.1 % (ML), 41.5 % (MB) and 50.6 % (B), and the microorganism adhesion index calculated for lactobacilli increased by 30.77 % (ML), 44.45 % (MB) and 44.97 % (B). Both studied probiotic bacteria moved up from the category of «moderately adhesive» into the category of «strongly adhesive» with microorganism adhesion index > 4.0. Lactobacilli also became «strongly adhesive» under the influence of L extract, while microorganism adhesion index increased by 37.9 %. Among the studied extracts, only L extract caused a significant increase in the adhesion index of the S. aureus test culture. The other extracts caused minor changes in the adhesive potential of the test cultures. Under their influence, there was a tendency to increase or decrease the adhesive ability of bacteria. Under the influence of MB extract, the S. aureus test culture passed from the «strongly adhesive» category to the «moderately adhesive» category, and the E. coli test culture transferred from the «moderately adhesive» category to the «weakly adhesive» category. The data obtained are of great importance when developing new metabiotics based on the B. bifidum 1 and L reuteri DSM 17938 probiotic strains derivatives.

The effects of cell-free extracts derived from probiotic strains Bifidobacterium bifidum and Lactobacillus reuteri on the proliferation and biofilm formation by Lactobacillus reuteri in vitro

The aim of the research was to investigate the ability of cell-free extracts, containing derivatives of probiotic strains Bifidobacterium bifidum and Lactobacillus reuteri , to influence the proliferation and biofilm formation by Lactobacillus reuteri in vitro ; to evaluate the prospects for the creation of new metabiotics and means of increasing the overall productivity of probiotic cell biomass based on them. Materials and methods . Cell-free extracts were obtained from probiotic strains B. bifidum 1 and L. reuteri DSM 17938 by the authors’ method. Probiotic strain L. reuteri DSM 17938 used as a test culture. The investigation of the proliferation and biofilm formation by L. reuteri was carried out by spectrophotometric method using a microtiter-plate reader “Lisa Scan EM” (Erba Lachema s.r.o., Czech Republic). Results . It has been established that the cell-free extract obtained from L. reuteri culture grown in its own disintegrate, supplemented with glycerol and glucose, is the only one of all studied, which stimulates both proliferation and biofilm formation by L. reuteri . The cell-free extracts, obtained from L. reuteri and B. bifidum disintegrates and from L. reuteri culture, grown in its own disintegrate, stimulate proliferation of test-culture to varying degrees (depending on the type of extract and its content in the culture medium), but have a significant inhibitory effect on the biofilm formation by L. reuteri . The extract, obtained from B. bifidum culture, grown in its own disintegrate, does not have a significant effect on proliferation and greatly suppresses the biofilm formation by L. reuteri . Conclusions . The results of the study allow us to positively evaluate the prospects for the creation of new metabiotics based on probiotic derivatives. Cell-free extract, obtained from L. reuteri culture, grown in its own disintegrate supplemented with glycerol and glucose can contribute to the survival and facilitate inoculation of the introduced probiotic in the gastrointestinal tract when used together. Derivative-containing extracts with a pronounced growth-stimulating effect can be the basis for creating the means of increasing the overall productivity of the probiotic cell biomass. The use of such means will increase the economic efficiency of probiotic cultures production. Obtained data induce further study of the biochemical composition, elucidation of the mechanism of cell-free extracts action and confirmation of their efficacy in vivo .

Staphylococcal proliferation and biofilm formation in vitro under the influence of cell-free extracts of probiotic origin

The paper presents the results of the study on proliferation and biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis under the influence of cell-free extracts obtained by the author’s method and containing derivatives of probiotic strains Вifidobacterium bifidum and Lactobacillus reuteri.The aim of this work was to study the ability of cell-free extracts containing derivatives of probiotics Bifidobacterium bifidum and Lactobacillus reuteri to influence proliferation and biofilm formation by staphylococci in vitro, to evaluate the prospects of their use for the correction of microecological disorders and adjuvant therapy of staphylococcal infection.Materials and methods. Cell-free extracts were obtained from commercial strains B. bifidum and L. reuteri by the authors’ method. Reference strain of S. aureus AТСС 25923 and clinical isolate of S. epidermidis were used as a test cultures. The investigation of the proliferation and biofilm formation by staphylococci was carried out by spectrophotometric method using a microtiter-plate reader “Lisa Scan EM” (Erba Lachema s.r.o., Czech Republic).Results. It has been established that the effect of cell-free extract on proliferation and biofilm formation depends on the type of extract and on the species of staphylococcus. Among the five studied extracts, only one significantly inhibits the proliferation and biofilm formation of both staphylococci species. It is the cell-free extract, obtained from L. reuteri culture, grown in its own disintegrate supplemented with glycerol and glucose. The proliferative activity of S. aureus is sensitive to the L. reuteri derivatives while the proliferative activity of S. epidermidis is sensitive to the B. bifidum derivatives. The filtrates of disintegrates have stimulatory effect, while the filtrates of cultures have inhibitory effect on the staphylococcal proliferation. The biofilm formation by S. aureus is significantly inhibited by B. bifidum derivatives and is stimulated by L. reuteri derivatives. The biofilm formation by S. epidermidis is stimulated by derivatives of bifidobacteria and does not change in the presence of derivatives of lactobacteria in the growth medium.Conclusions. Obtained results indicate a high bioregulatory potential of cell-free extracts of probiotic origin and the possibility of drugs development for microecological disorders correction on their basis. They also confirm that the method of obtaining probiotic derivatives with bacteriotropic activity through precursor-directed biosynthesis is promising. Cell-free extract, obtained from L. reuteri culture, grown in its own disintegrate supplemented with glycerol and glucose, exhibits pronounced anti-staphylococcal activity in vitro. After confirming efficacy in vivo, it can be recommended for the adjuvant therapy of staphylococcal infections.

Influence of cell-free extracts of Bifidobacterium bifidum and Lactobacillus reuteri on proliferation and biofilm formation by Escherichia coli and Pseudomonas aeruginosa

The development of new effective preparations for the correction of microecological disorders based on probiotic derivatives requires a comprehensive study of the biological activity of the latter. We studied the proliferative activity and biofilm formation by clinical isolates: Escherichia coli and Pseudomonas aeruginosa under the influence of cell-free extracts containing structural components and metabolites of the Bifidobacterium bifidum and Lactobacillus reuteri probiotic strains. Cell-free extracts were obtained from disintegrates and cultures of probiotics. Disintegrates were prepared by cyclic freezing-thawing of probiotic cell suspensions. The cultures were obtained by cultivating probiotic microorganisms in their own disintegrates. The obtained disintegrates and cultures were filtered. The proliferative activity of the test cultures was studied using the spectrophotometric microtiter plate method after an hour-long exposure in undiluted cell-free extracts and subsequent cultivation in a nutrient medium containing 30%vol of the studied extracts at 37 °C for 24 hours. The biofilm formation of the test cultures was studied with 30% vol content of cell-free extracts in the cultivation medium using the spectrophotometric microtiter plate method. All the studied extracts exerted a similar effect on the proliferative activity and biofilm formation by E. coli and P. aeruginosa. Exposure of the test cultures in all undiluted extracts during an hour led to a significant decrease in the optical density of the test samples: optical density of the test wells ranged from 36.5% to 49.8% of the control wells. The test cultures that were exposed to the extracts: filtrate of L. reuteri disintegrate (L), filtrate of В. bifidum disintegrate (B) and filtrate of В. bifidum culture, grown in В. bifidum disintegrate (MB) after dilution and subsequent cultivation over the next 24 hours completely restored the ability to proliferate. The proliferative activity of the test cultures that were exposed to the extracts: filtrate of L. reuteri culture, grown in L. reuteri disintegrate (ML) and filtrate of L. reuteri culture, grown in L. reuteri disintegrate supplemented with 0.8 M glycerol and 0.4 M glucose (MLG), was significantly inhibited after dilution and subsequent cultivation. The inhibition indices calculated for the ML extract were: 25.9% (E. coli) and 53.0% (P. aeruginosa). Inhibition indices calculated for the MLG extract were: 62.0% (E. coli) and 96.9% (P. aeruginosa). MLG extract had more pronounced inhibitory effect on the proliferation of the test cultures than ML extract. All the studied extracts exerted significant inhibitory effect on the biofilm formation of the test cultures. Analysis of the results of the study shows that cell-free extracts of L. reuteri culture grown in its disintegrate without supplementation or supplemented with glycerol and glucose have the highest antimicrobial activity and can be used as metabiotics to prevent overgrowth of potentially pathogenic bacteria, as well as inoculation and proliferation of pathogenic gram-negative bacteria in the gastrointestinal tract. They can be used alone or in combination with cellular probiotics to enhance their probiotic action. This study encourages further careful investigation of the biochemical composition of cell-free extracts and clarifying the mechanism of their action.

Bifidogenic properties of cell-free extracts derived from probiotic strains of Bifidobacterium bifidum and Lactobacillus reuteri

Comprehensive study of the biological activity of structural components and metabolites of “beneficial” microorganisms opens the prospects of efficient and rational use of their biotechnological potential in the correction of microecological and related disorders. The study tested proliferative activity and biofilm formation by Bifidobacterium bifidum probiotic strain under the influence of cell-free extracts containing structural components and metabolites of the probiotic strains of B. bifidum and Lactobacillus reuteri. Cell-free extracts were obtained by disintegrating suspensions of probiotic cells by cyclic freezing-thawing, cultivating probiotic microorganisms in their own disintegrates and subsequent filtration of the obtained disintegrates and cultures. The proliferative activity and biofilm formation of the probiotic test culture were studied by spectrophotometric microtiter plate method with 10%vol, 30%vol and 50%vol content of cell-free extracts in the cultivation medium. All investigated extracts showed a significant concentration-dependent stimulatory effect on the proliferative activity of B. bifidum. According to the degree of stimulatory effect on the B. bifidum proliferation, cell-free extracts arranged in ascending order: MLG (filtrate of L. reuteri culture, grown in L. reuteri disintegrate supplemented with 0.8 M glycerol and 0.4 M glucose) < MB (filtrate of В. bifidum culture, grown in В. bifidum disintegrate) < B (filtrate of В. bifidum disintegrate) < ML (filtrate of L. reuteri culture, grown in L. reuteri disintegrate) < L (filtrate of L. reuteri disintegrate). With the same content in the culture medium, filtrates of disintegrates had a more pronounced stimulatory effect than filtrates of cultures grown in their own disintegrates. Cell-free extracts from L. reuteri (L and ML) exerted a more pronounced stimulatory effect than cell-free extracts from B. bifidum. Not all studied cell-free extracts stimulated the biofilm formation by B. bifidum. The effect of cell-free extracts on this process depended on their type and concentration. Extract L had a predominantly inhibitory effect on biofilm formation by B. bifidum. The most pronounced stimulatory effect on biofilm formation by B. bifidum came from extract MLG. ML, B and MB extracts stimulated this process approximately equally. The detection of significant bifidogenic effect of the studied cell-free extracts may contribute to their pharmaceutical applications. Cell-free extracts can be used as metabiotics or prebiotics for increasing the survival of the injected probiotic, facilitating its inoculation in the gastrointestinal tract when used together. The obtained data encourage further careful study of the biochemical composition of cell-free extracts and efforts to clarify the mechanism of their action.

Application of the selected antifungal LAB isolate as a protective starter culture in pan whole-wheat sourdough bread

In vitro antifungal activity against Aspergillus niger was used to screen the lactic acid bacteria (LAB) isolated from whole wheat sourdough. Sequencing results of the PCR products led to the identification of Lactobacillus reuteri as the selected isolate. The LAB isolate had a proper anti-aflatoxigenic capability, and it reduced the amount of B1, B2, G1 and G2 aflatoxins, based on the results of HPLC analysis. n-Decanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid were also identified as the bioactive ingredients using gas chromatography/mass spectrometry in the antifungal fraction obtained from preparative thin-layer chromatography of the L. reuteri culture filtrate. Furthermore, it was revealed that the sourdough fermented with the selected LAB isolate had a significant influence (P < 0.05) on the quality of the produced pan bread in terms of hardness, specific volume and phytic acid content. The growth of A. niger on the produced bread was also remarkably decreased using the controlled sourdough (containing the selected LAB isolate) in comparison with the spontaneous sourdough and control bread. Based on these findings, techno-functional properties of the selected LAB isolate were clarified, and therefore this isolate can be used successfully as a potent protective starter culture for sourdough bread processing.

The level of beta defensin-2 in saliva and its expression in parotid gland epithelial cells after probiotic (Lactobacillus reuteri) induction to inhibit Streptococcus mutans in caries.

Objective:The aim of this study was to prove that administrating L. reuteri probiotics can increase the level of BD-2 saliva and BD-2 expression in the epithelial parotid glands of Wistar rats.Materials and Methods:Experimental design in this study was randomized control group post test only. Twenty-four white male Rattus norvegicus Wistar strain rats were divided into four groups. The negative control group included rats not induced by S. mutans whereas the positive control group included rats induced by S. mutans. The two treatment groups are as follows: treatment 1 (T1), the group that is induced for 14 days by L. reuteri and 7 days by S. mutans and treatment 2 (T2), the group which is induced simultaneously by S. mutans and L. reuteri for 14 days. L. reuteri culture at a concentration of 108 colony-forming unit/ml and S. mutans culture at a concentration of 1010 are induced in the oral cavity of the Wistar rats. The Elisa technique is used to examine the salivary level of BD-2, whereas the immunohistochemical technique is used to examine the BD-2 expression in the epithelial salivary glands.Results:The study shows the increasing levels of BD-2 and BD-2 expression in the epithelial parotid glands after the administration of L. reuteri probiotics. Besides, there is a relationship between the increasing expression of BD-2 in the epithelial parotid glands with the decreasing amount of S. mutans.Conclusion:Giving L. reuteri probiotic scan increases the level of saliva of BD-2 and the expression of BD-2 in the parotid glands.

English

As an alternative to traditional methods for controlling microorganisms in food, bioprotection using lactic acid bacteria is promising. Lactobacillus reuteri exhibits an ability to produce reuterin, an antimicrobial substance presenting effects against Gram-positive and Gram-negative bacteria, as well as molds, yeasts and protozoa. In the present study, the activity of L. reuteri culture and extract over psychrophilic microbiota of Litopenaeus vannamei shrimp kept under refrigeration was evaluated. Sterile extract of L. reuteri was able to reduce the counts of psychrophiles in 2 log cycles, while L. reuteri culture kept the initial count during the whole storage period. L. reuteri was efficient in controlling psychrophiles in shrimp suggesting its use as a food bioprotector. Key words: Lactobacillus reuteri, reuterin, antimicrobial activity.

The increasing of beta-defensin-2 level in saliva after probiotic Lactobacillus reuteri administration

Background: Commesal bacteria is an excellent inducer for beta defensin-2 (BD-2). Probiotics bacteria Lactobacillus reuteri (L. reuteri) as commensal bacteria may play the same role as an excellent inducer for BD-2. Beta defensin is natural antimicrobial peptides widely expressed in oral cavity, including in epithelium salivary gland. Streptococcus mutans (S. mutans) as the main of bacteria causing caries are sensitive to BD-2. Purpose: This research was aimed to determine whether administration of probiotic L. reuteri can increase salivary BD-2 level in Wistar rats. Methods: This research can be considered as a laboratory experimental research with a randomized control group post test only design. Twenty-four male Rattus norvegicus Wistar strain rats aged 3 months were used. They were randomly divided into four groups, namely two control groups (negative control group that was not induced and positive control group induced with S. mutans), and two treatment groups (K1: induced with L. reuteri for 14 days and S. mutans for 7 days, and K2: induced with L. reuteri and S. mutans simultaneously for 14 days). L. reuteri culture at a concentration of 10 8 CFU/ml and S. mutans culture at a concentration of 10 10 CFU/ml were induced into the oral cavity of Wistar rats. An examination of BD-2 level was then conducted by using Elisa techniques. results: There was significant difference of salivary BD-2 level among those treatment groups (p=0.001). BD-2 level in saliva was increased after the administration of L. reuteri. Conclusion: L. reuteri probiotic can increase salivary BD-2 level in Wistar rats.

Coexpression and secretion of endoglucanase and phytase genes in Lactobacillus reuteri.

A multifunctional transgenic Lactobacillus with probiotic characteristics and an ability to degrade β-glucan and phytic acid (phytate) was engineered to improve nutrient utilization, increase production performance and decrease digestive diseases in broiler chickens. The Bacillus subtilis WL001 endoglucanase gene (celW) and Aspergillus fumigatus WL002 phytase gene (phyW) mature peptide (phyWM) were cloned into an expression vector with the lactate dehydrogenase promoter of Lactobacillus casei and the secretion signal peptide of the Lactococcus lactis usp45 gene. This construct was then transformed into Lactobacillus reuteri XC1 that had been isolated from the gastrointestinal tract of broilers. Heterologous enzyme production and feed effectiveness of this genetically modified L. reuteri strain were investigated and evaluated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the molecular mass of phyWM and celW was approximately 48.2 and 55 kDa, respectively, consistent with their predicted molecular weights. Endoglucanase and phytase activities in the extracellular fraction of the transformed L. reuteri culture were 0.68 and 0.42 U/mL, respectively. Transformed L. reuteri improved the feed conversion ratio of broilers from 21 to 42 days of age and over the whole feeding period. However, there was no effect on body weight gain and feed intake of chicks. Transformed L. reuteri supplementation improved levels of ash, calcium and phosphorus in tibiae at day 21 and of phosphorus at day 42. In addition, populations of Escherichia coli, Veillonella spp. and Bacteroides vulgatus were decreased, while populations of Bifidobacterium genus and Lactobacillus spp. were increased in the cecum at day 21.