Analysis of antimicrobial and immunomodulatory substances produced by heterofermentative <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

Antimicrobial and immunomodulatory potential of various Lactobacillus reuteri strains is closely connected to their metabolite production profile under given cultivation conditions. We determined the in vitro production of antimicrobial substances such as organic acids, ethanol, and reuterin by four strains of L. reuteri (L. reuteri E, L. reuteri KO5, L. reuteri CCM 3625, and L. reuteri ATCC 55730). All studied L. reuteri strains showed the ability to produce lactic acid, acetic acid, and ethanol with concominant consumption of glucose and together with phenyllactic acid—a potent antifungal compound—with concominant consumption of phenylalanine. The reuterin production from glycerol was confirmed for all analyzed lactobacilli strains except L. reuteri CCM 3625. Production of organic acids, ethanol, and reuterin is significantly involved in antimicrobial activity of lactobacilli which was determined using the dual-culture overlay diffusion method against six indicator bacteria and five indicator moulds. In comparison to the referential L. reuteri ATCC 55730, the highest inhibition potential was observed against Escherichia coli CCM 3988 and Pseudomonas aeruginosa CCM 3955. Among analyzed indicators of moulds, the growth of Alternaria alternata CCM F-128 was the most inhibited by all four analyzed L. reuteri strains. Finally, the immunomodulatory potential of analyzed lactobacilli were proven by the determination of the in vitro production of biogenic amines histamine and tyramine. L. reuteri CCM 3625 was able to produce tyramine, and L. reuteri E and L. reuteri KO5 were able to produce histamine under given cultivation conditions.     

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.     

Colonization and Immunomodulation by Lactobacillus reuteri ATCC 55730 in the Human Gastrointestinal Tract

Lactobacillus reuteri ATCC 55730 is a probiotic (health-promoting) bacterium widely used as a dietary supplement. This study was designed to examine local colonization of the human gastrointestinal mucosa after dietary supplementation with L. reuteri ATCC 55730 and to determine subsequent immune responses at the colonized sites. In this open clinical investigation, 10 healthy volunteers and 9 volunteers with ileostomy underwent gastroscopy or ileoscopy and biopsy samples were taken from the stomach, duodenum, or ileum before and after supplementation with 4 × 10⁸ CFU of live L. reuteri ATCC 55730 lactobacilli per day for 28 days. Biopsy specimen colonization was analyzed using fluorescence in situ hybridization with a molecular beacon probe, and immune cell populations were determined by immunostaining. Endogenous L. reuteri was detected in the stomach of 1 subject and the duodenum of 3 subjects (out of 10 subjects). After L. reuteri ATCC 55730 supplementation, the stomachs of 8 and the duodenums of all 10 subjects were colonized. Three ileostomy subjects (of six tested) had endogenous L. reuteri at baseline, while all six displayed colonization after L. reuteri supplementation. Gastric mucosal histiocyte numbers were reduced and duodenal B-lymphocyte numbers were increased by L. reuteri ATCC 55730 administration. Furthermore, L. reuteri administration induced a significantly higher amount of CD4-positive T-lymphocytes in the ileal epithelium. Dietary supplementation with the probiotic L. reuteri ATCC 55730 induces significant colonization of the stomach, duodenum, and ileum of healthy humans, and this is associated with significant alterations of the immune response in the gastrointestinal mucosa. These responses may be key components of a mechanism by which L. reuteri ATCC 55730 exerts its well-documented probiotic effects in humans.     

Genomic and Genetic Characterization of the Bile Stress Response of Probiotic Lactobacillus reuteri ATCC 55730

Probiotic bacteria encounter various stresses after ingestion by the host, including exposure to the low pH in the stomach and bile in the small intestine. The probiotic microorganism Lactobacillus reuteri ATCC 55730 has previously been shown to survive in the human small intestine. To address how L. reuteri can resist bile stress, we performed microarray experiments to determine gene expression changes that occur when the organism is exposed to physiological concentrations of bile. A wide variety of genes that displayed differential expression in the presence of bile indicated that the cells were dealing with several types of stress, including cell envelope stress, protein denaturation, and DNA damage. Mutations in three genes were found to decrease the strain's ability to survive bile exposure: lr1864, a Clp chaperone; lr0085, a gene of unknown function; and lr1516, a putative esterase. Mutations in two genes that form an operon, lr1584 (a multidrug resistance transporter in the major facilitator superfamily) and lr1582 (unknown function), were found to impair the strain's ability to restart growth in the presence of bile. This study provides insight into the possible mechanisms that L. reuteri ATCC 55730 may use to survive and grow in the presence of bile in the small intestine.     

Production of 3-hydroxypropionaldehyde using a two-step process with Lactobacillus reuteri

3-Hydroxypropionaldehyde (3-HPA) produced by Lactobacillus reuteri is a broad-spectrum antimicrobial substance of glycerol conversion. The aim of the present work was to optimize 3-HPA production by Lb. reuteri ATCC 53608 using a two-step process. The first step was the production of Lb. reuteri cells in optimal conditions. Cells were then harvested by centrifugation and suspended in glycerol solution, which the resting cells bioconverted to 3-HPA. The effect of biomass concentration, temperature, glycerol concentration, anaerobic/micro-aerophilic conditions, and incubation time was studied for high 3-HPA production. 3-HPA accumulation was limited by the death of cells in contact with high concentrations of 3-HPA. However, a very high 3-HPA concentration of 235±3 mM was obtained after 45 min of incubation at 30°C in 400 mM glycerol for an initial free-cell concentration of 1.6±0.3×10¹⁰ viable cells/ml. A high viability was maintained at low temperatures in the range 5–15°C, but with a slightly lower yield of 3-HPA at 5°C compared with higher temperatures, up to 37°C. Successive 1-h incubations of Lb. reuteri cells in 200 mM glycerol at 15°C to tentatively reuse the cells resulted in decreasing 3-HPA concentrations at the end of each cycle, with two successful production cycles yielding high 3-HPA concentrations of 147±1 mM and 128±2 mM.     

Highly Hydrolytic Reuteransucrase from Probiotic Lactobacillus reuteri Strain ATCC 55730

Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the α-(1→4) glucosidic type (~70%). This reuteran also contains α-(1→6)- linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing α-(1→4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of α-(1→4) linkages reported to date.     

The cell surface of Lactobacillus reuteri ATCC 55730 highlighted by identification of 126 extracellular proteins from the genome sequence

Bioinformatical analyses of a draft genome sequence of the commensal bacterium Lactobacillus reuteri ATCC 55730 revealed 126 genes encoding putative extracellular proteins. The function, localization and distribution in bacterial species were predicted. Interestingly, few proteins possessed LPXTG motifs or C-terminal transmembrane anchors. Instead eight proteins were putatively anchored by GW repeats and several secreted proteins were likely to be re-associated to the surface. The majority of the extracellular proteins were widely distributed, i.e., found universally or in gram-positive bacteria, but 24 were only detected in L. reuteri. Further, the number of transporters was lower, while the number of enzyme was higher than in related species.     

Transferability of a tetracycline resistance gene from probiotic <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> to bacteria in the gastrointestinal tract of humans

The potential of Lactobacillus reuteri as a donor of antibiotic resistance genes in the human gut was investigated by studying the transferability of the tetracycline resistance gene tet(W) to faecal enterococci, bifidobacteria and lactobacilli. In a double-blind clinical study, seven subjects consumed L. reuteri ATCC 55730 harbouring a plasmid-encoded tet(W) gene (tet(W)-reuteri) and an equal number of subjects consumed L. reuteri DSM 17938 derived from the ATCC 55730 strain by the removal of two plasmids, one of which contained the tet(W) gene. Faecal samples were collected before, during and after ingestion of 5 × 10⁸ CFU of L. reuteri per day for 14 days. Both L. reuteri strains were detectable at similar levels in faeces after 14 days of intake but neither was detected after a two-week wash-out period. After enrichment and isolation of tetracycline resistant enterococci, bifidobacteria and lactobacilli from each faecal sample, DNA was extracted and analysed for presence of tet(W)-reuteri using a real-time PCR allelic discrimination method developed in this study. No tet(W)-reuteri signal was produced from any of the DNA samples and thus gene transfer to enterococci, bifidobacteria and lactobacilli during intestinal passage of the probiotic strain was non-detectable under the conditions tested, although transfer at low frequencies or to the remaining faecal bacterial population cannot be excluded.     

Feed Fermentation with Reuteran- and Levan-Producing Lactobacillus reuteri Reduces Colonization of Weanling Pigs by Enterotoxigenic Escherichia coli

This study determined the effect of feed fermentation with Lactobacillus reuteri on growth performance and the abundance of enterotoxigenic Escherichia coli (ETEC) in weanling piglets. L. reuteri strains produce reuteran or levan, exopolysaccharides that inhibit ETEC adhesion to the mucosa, and feed fermentation was conducted under conditions supporting exopolysaccharide formation and under conditions not supporting exopolysaccharide formation. Diets were chosen to assess the impact of organic acids and the impact of viable L. reuteri bacteria. Fecal samples were taken throughout 3 weeks of feeding; at the end of the 21-day feeding period, animals were euthanized to sample the gut digesta. The feed intake was reduced in pigs fed diets containing exopolysaccharides; however, feed efficiencies did not differ among the diets. Quantification of L. reuteri by quantitative PCR (qPCR) detected the two strains used for feed fermentation throughout the intestinal tract. Quantification of E. coli and ETEC virulence factors by qPCR demonstrated that fermented diets containing reuteran significantly (P < 0.05) reduced the copy numbers of genes for E. coli and the heat-stable enterotoxin in feces compared to those achieved with the control diet. Any fermented feed significantly (P < 0.05) reduced the abundance of E. coli and the heat-stable enterotoxin in colonic digesta at 21 days; reuteran-containing diets reduced the copy numbers of the genes for E. coli and the heat-stable enterotoxin below the detection limit in samples from the ileum, the cecum, and the colon. In conclusion, feed fermentation with L. reuteri reduced the level of colonization of weaning piglets with ETEC, and feed fermentation supplied concentrations of reuteran that may specifically contribute to the effect on ETEC.     

Phosphoketolase pathway dominates in Lactobacillus reuteri ATCC 55730 containing dual pathways for glycolysis

Metabolic flux analysis indicated that the heterofermentative Lactobacillus reuteri strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of L. reuteri ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-1,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro ³¹P NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.     

Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs

Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri.     

Metabolic pathway of α‐ketoglutarate in Lactobacillus sanfranciscensis and Lactobacillus reuteri during sourdough fermentation

Aim: To identify metabolites of α‐ketoglutarate (α‐KG) in Lactobacillus sanfranciscensis and Lactobacillus reuteri in modified MRS and sourdough. Methods and Results: Lactobacillus sanfranciscensis and L. reuteri were grown with additional α‐KG in mMRS and in wheat sourdough. In mMRS, α‐KG was used as an electron acceptor and converted to 2‐hydroxyglutarate (2‐OHG) by both organisms. Production of 2‐OHG was identified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography (GC). Crude cell extracts of L. sanfranciscensis and L. reuteri grown with or without α‐KG exhibited OHG dehydrogenase activity of 6·3 ± 0·3, 2·3 ± 0·9, 1·2 ± 0·2, and 1·1 ± 0·1 mmol l^?1 NADH (min x mg protein)^?1, respectively. The presence of phenylalanine and citrate in addition to α‐KG partially redirected the use of α‐KG from electron acceptor to amino group acceptor. In wheat sourdoughs, α‐KG was predominantly used as electron acceptor and converted to 2‐OHG. Conclusions: Lactobacillus sanfranciscensis and L. reuteri utilize α‐KG as electron acceptor. Alternative use of α‐KG as amino group acceptor occurs in the presence of abundant amino donors and citrate. Significance and Impact of the Study: The use of α‐KG as electron acceptor in heterofermentative lactobacilli impacts the formation of flavour volatiles through the transamination pathway.     

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.     

3株罗伊氏乳杆菌生物学特性的分析比较

【目的】对3株罗伊氏乳杆菌的生物学特性进行分析比较,为后期生产应用提供一定的参考。【方法】对实验室保藏的3株罗伊氏乳杆菌的生长曲线、pH曲线、耐受人工胃液能力、耐受猪胆盐能力、黏附能力、抑菌能力和对抗生素的耐药性等特性进行了分析比较。【结果】3株菌生长趋势大致相同;3株菌对人工胃液均具有良好的耐受性,且可以有效地抑制大肠杆菌和金黄色葡萄球菌的生长;菌株L0和L2对高胆盐的环境耐受性较差,菌株L1则对高胆盐环境具有极强的耐受性;菌株L1和L2具有很强的黏附能力;3株菌对20种抗生素表现出不同的耐受性。【结论】菌株L1的生物学特性明显优于其他两株菌株,有利于后期的生产应用。     

Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus

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

Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine

Aims:  To investigate the abilities of various probiotic bacteria to produce volatile sulfur compounds (VSCs) relevant to food flavour and aroma.
Methods and Results:  Probiotic strains ( Lactobacillus acidophilus NCFM, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC55730 and L. reuteri BR11), Lactobacillus delbrueckii ATCC4797, L. plantarum ATCC14917 and Lactococcus lactis MG1363 were incubated with either cysteine or methionine. Volatile compounds were captured, identified and quantified using a sensitive solid-phase microextraction (SPME) technique combined with gas chromatography coupled to a pulsed flame photometric detector (SPME/GC/PFPD). Several VSCs were identified including H₂S, methanethiol, dimethyldisulfide and dimethyltrisulfide. The VSC profiles varied substantially for different strains of L. plantarum and L. reuteri and it was found that L. reuteri ATCC55730 and L. lactis MG1363 produced the lowest levels of VSCs ( P  < 0·05). Levels of VSCs generated by bacteria were found to be equivalent to, or higher than, that found in commercial cheeses.
Conclusions:  Several probiotic strains are able to generate considerable levels of VSCs and substantial variations in VSC generating potential exists between different strains from the same species.
Significance and Importance of the Study:  This study demonstrates that probiotic bacteria are able to efficiently generate important flavour and aroma compounds and therefore has implications for the development of probiotic containing foods.     

Isolation and Characterization of Human Intestinal Bacteria Capable of Transforming the Dietary Carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed in meat products during cooking. Although the formation of hazardous PhIP metabolites by mammalian enzymes has been extensively reported, research on the putative involvement of the human intestinal microbiota in PhIP metabolism remains scarce. In this study, the in vitro conversion of PhIP into its microbial derivate, 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3′,2′:4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1), by fecal samples from 18 human volunteers was investigated. High-performance liquid chromatography analysis showed that all human fecal samples transformed PhIP but with efficiencies ranging from 1.8 to 96% after 72 h of incubation. Two PhIP-transforming strains, PhIP-M1-a and PhIP-M1-b, were isolated from human feces and identified by fluorescent amplified fragment length polymorphism and pheS sequence analyses as Enterococcus faecium strains. Some strains from culture collections belonging to the species E. durans, E. avium, E. faecium, and Lactobacillus reuteri were also able to perform this transformation. Yeast extract, special peptone, and meat extract supported PhIP transformation by the enriched E. faecium strains, while tryptone, monomeric sugars, starch, and cellulose did not. Glycerol was identified as a fecal matrix constituent required for PhIP transformation. Abiotic synthesis of PhIP-M1 and quantification of the glycerol metabolite 3-hydroxypropionaldehyde (3-HPA) confirmed that the anaerobic fermentation of glycerol via 3-HPA is the critical bacterial transformation process responsible for the formation of PhIP-M1. Whether it is a detoxification is still a matter of debate, since PhIP-M1 has been shown to be cytotoxic toward Caco-2 cells but is not mutagenic in the Ames assay.     

Lactobacillus reuteri Prevents Diet-Induced Obesity,but not Atherosclerosis,in a Strain Dependent Fashion in Apoe?/? Mice

Objective

To investigate whether the specific strains of Lactobacillus reuteri modulates the metabolic syndrome in Apoe−/− mice.

Methods

8 week-old Apoe−/− mice were subdivided into four groups who received either L. reuteri ATCC PTA 4659 (ATCC), DSM 17938 (DSM), L6798, or no bacterial supplement in the drinking water for 12 weeks. The mice were fed a high-fat Western diet with 0.2% cholesterol and body weights were monitored weekly. At the end of the study, oral glucose and insulin tolerance tests were conducted. In addition, adipose and liver weights were recorded along with analyses of mRNA expression of ileal Angiopoietin-like protein 4 (Angptl4), the macrophage marker F4/80 encoded by the gene Emr1 and liver Acetyl-CoA carboxylase 1 (Acc1), Fatty acid synthase (Fas) and Carnitine palmitoyltransferase 1a (Cpt1a). Atherosclerosis was assessed in the aortic root region of the heart.

Results and Conclusions

Mice receiving L. reuteri ATCC gained significantly less body weight than the control mice, whereas the L6798 mice gained significantly more. Adipose and liver weights were also reduced in the ATCC group. Serum insulin levels were lower in the ATCC group, but no significant effects were observed in the glucose or insulin tolerance tests. Lipogenic genes in the liver were not altered by any of the bacterial treatments, however, increased expression of Cpt1a was found in the ATCC group, indicating increased β-oxidation. Correspondingly, the liver trended towards having lower fat content. There were no effects on inflammatory markers, blood cholesterol or atherosclerosis. In conclusion, the probiotic L. reuteri strain ATCC PTA 4659 partly prevented diet-induced obesity, possibly via a previously unknown mechanism of inducing liver expression of Cpt1a.     

3-Hydroxypropionaldehyde: applications and perspectives of biotechnological production

3-Hydroxypropionaldehyde (3-HPA) forms, together with HPA-hydrate and HPA-dimer, a dynamic, multi-component system (HPA system) used in food preservation, as a precursor for many modern chemicals such as acrolein, acrylic acid, and 1,3-propanediol (1,3-PDO), and for polymer production. 3-HPA can be obtained both through traditional chemistry and bacterial fermentation. To date, 3-HPA has been produced from petrochemical resources as an intermediate in 1,3-PDO production. In vivo, glycerol is converted in one enzymatic step into 3-HPA. The 3-HPA-producing Lactobacillus reuteri is used as a probiotic in the health care of humans and animals. The biotechnological production of 3-HPA from renewable resources is desirable both for use of 3-HPA in foods and for the production of bulk chemicals. The main challenge will be the efficient production and recovery of pure 3-HPA.     

Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10⁷) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.