R. Švejstil

Raffinose-Series Oligosaccharides in Soybean Products

Abstract Soybean foods forming a substantial part of Asian diet have still more expanded into European diet. Raffinose-series oligosaccharides (RSO) are important constituents of soya beans and they can be found also in soybean products. These oligosaccharides can be considered potentially prebiotic for their capability of influencing the composition of the host’s intestinal microbiota. The aim of the present paper was to determine the oligosaccharide content in various soybean products. Enzymatic assay has been used for the determination of oligosaccharides. RSO have been found in all tested samples and their content varied from 0.66 g per 100 g in soybean beverage to 5.59 g per 100 g in first clear soybean flour. Generally, the highest content of RSO has been detected in soybean flour in the average amount of 4.83 g per 100 g. There was no statistically significant difference observed in the amount of oligosaccharides in all four types of soybean flour (P < 0.01). Considerably high amounts of RSO have been found in sweet soybean bars and textured soy protein. Foods as soybean flour and soybean bar ‘Sójový suk’ seem to be effective natural sources of prebiotic oligosaccharides for humans.

Galliscardovia ingluviei gen. nov., sp. nov., a thermophilic bacterium of the family Bifidobacteriaceae isolated from the crop of a laying hen (Gallus gallus f. domestica)

Bacteria with potential probiotic applications are not yet sufficiently explored, even for animals with economic importance. Therefore, we decided to isolate and identify representatives of the family Bifidobacteriaceae, which inhabit the crop of laying hens. During the study, a fructose-6-phosphate phosphoketolase-positive strain, RP51T, with a regular/slightly irregular and sometimes an S-shaped slightly curved rod-like shape, was isolated from the crop of a 13 -month-old Hisex Brown hybrid laying hen. The best growth of the Gram-stain-positive bacterium, which was isolated using Bifidobacterium-selective mTPY agar, was found out to be under strictly anaerobic conditions, however an ability to grow under microaerophilic and aerobic conditions was also observed. Sequencing of the almost complete 16S rRNA gene (1444 bp) showed Alloscardovia omnicolens CCUG 31649T and Bombiscardovia coagulans BLAPIII/AGVT to be the most closely related species with similarities of 93.4 and 93.1 %, respectively. Lower sequence similarities were determined with other scardovial genera and other representatives of the genus Bifidobacterium. Taxonomic relationships with A. omnicolens and other members of the family Bifidobacteriaceaewere also demonstrated, based on the sequences of dnaK, fusA, hsp60 and rplB gene fragments. Low sequence similarities of phylogenetic markers to related scardovial genera and bifidobacteria along with unique features of the bacterial strain investigated within the family Bifidobacteriaceae(including the lowest DNA G+C value (44.3 mol%), a unique spectrum of cellular fatty acids and polar lipids, cellular morphology, the wide temperature range for growth (15-49 °C) and habitat) clearly indicate that strain RP51T is a representative of a novel genus within the family Bifidobacteriaceae for which the name Galliscardovia ingluviei gen. nov., sp. nov. (RP51T=DSM 100235T=LMG 28778T=CCM 8606T) is proposed.

Bifidobacterium apri sp. nov., a thermophilic actinobacterium isolated from the digestive tract of wild pigs (Sus scrofa)

Fresh samples of intestinal contents of three wild pigs originating from the Central Bohemia region were examined for the presence of bifidobacterial strains. During the study, we isolated many fructose-6-phosphate phosphoketolase-positive, strictly anaerobic, irregular rod-shaped bacterial isolates. Three of them were preliminarily identified as representing a novel species of the genus Bifidobacterium because their 16S rRNA gene sequence similarity with the closest relatives of thermophilic bifidobacteria (Bifidobacterium boum DSM 20432T, Bifidobacterium thermophilum DSM 20210T, Bifidobacterium thermacidophilumsubsp. porcinum LMG 21689T, Bifidobacterium thermacidophilumsubsp. thermacidophilum DSM 15837T) was in the range of 97.9 - 98.4 %. All three bacterial isolates had identical 16S rRNA, dnaJ1, fusA, gyrB and rplB gene sequences. Isolate RP115T was chosen as a representative of the bacterial group and DNA G+C content (mol%) determination, biochemical tests and analyses of physiological and morphological characteristics, habitat and chemotaxonomic traits (peptidoglycan structure, cellular fatty acids and polar lipids profile) were performed. The DNA-DNA hybridization analyses of RP115T and species representing the group of thermophilic bifidobacteria revealed values in the range from 33 to 53 %. This fact, together with relatively low sequence similarities of particular phylogenetic markers among examined bacterial strains and the phenotyping and chemotaxonomy results obtained, indicated that the evaluated bacterial isolate should be classified as representing a separate taxon within the specific group of thermophilic bifidobacteria. The name Bifidobacterium apri (of boar) sp. nov. has been proposed for the representative strain RP115T (=CCM 8605T=DSM 100238T=LMG 28779T).

Alloscardovia venturai sp. nov., a fructose 6-phosphate phosphoketolase-positive species isolated from the oral cavity of a guinea-pig (Cavia aperea f. porcellus)

A slightly irregular, short rod-shaped bacterial strain, MOZIV/2T, showing activity of fructose 6-phosphate phosphoketolase was isolated from the oral cavity of a home-bred guinea-pig. Based on comparative 16S rRNA gene sequence analyses, its closest relatives were Alloscardovia omnicolens DSM 21503T and Alloscardovia criceti DSM 17774T with 96.0 and 95.6 % pairwise similarities, respectively. Completeness of the compared sequences was 97.3 and 96.9 %, respectively. Growth was found only under anaerobic conditions. Activities of α- and β-gluco(galacto)sidases were detected in strain MOZIV/2T, which is characteristic for almost all members of the family Bifidobacteriaceae. Sequencing of other molecular markers (fusA, gyrB and xfp) revealed low gene sequence similarities to A. omnicolens DSM 21503T ranging from 72.7 to 87.5 %. Strain MOZIV/2T differed from other species within the genus Alloscardovia by the presence of C18 : 1ω9t. In addition, much higher proportions of C8 : 0, C11 : 0, C12 : 0, C14 : 1, C16 : 1 and C17 : 0 fatty acids were found in cells of strain MOZIV/2T. The peptidoglycan structure was of type A4α [l-Lys(l-Orn)-d-Asp], which is consistent with its classification within the genus Alloscardovia. The DNA G+C content (45.8 mol%) was lower than those found in other alloscardovia. Phylogenetic studies and evaluation of phenotypic characteristics including the results of biochemical, physiological and chemotaxonomic analyses confirmed the novel species status for strain MOZIV/2T, for which the name Alloscardovia venturai sp. nov. is proposed. The type strain is MOZIV/2T (=DSM 100237T=CCM 8604T=LMG 28781T).

Lactobacillus caviae sp nov., an obligately heterofermentative bacterium isolated from the oral cavity of a guinea pig (Cavia aperea f. porcellus)

A Gram-stain-positive, facultatively anaerobic, and catalase- and oxidase-negative bacterial strain designated MOZM2T, having 98.4 % 16S rRNA gene sequence identity with Lactobacillus reuteri DSM 20016T, was isolated from a swab of the oral cavity of a home-bred guinea pig. Comparative analyses based on the hsp60, pheS and tuf genes confirmed L. reuteri as its closest relative species, with calculated sequence similarities of 92.8, 88.8 and 96.9 %, respectively. DNA-DNA hybridisation revealed a 42 % degree of genetic similarity between the novel strain and L. reuteri DSM 20016T. Strain MOZM2T degrades carbohydrates via the 6-phosphogluconate/phosphoketolase pathway, evidenced by its production of gas from glucose and the end products of hexose catabolism. Comparative analysis of the cellular fatty acid profiles determined significant differences between MOZM2T and L. reuteri DSM 20016T in their proportions of C8 : 0, C14 : 1, C17 : 0, C18 : 2ω6t and C20 : 0 fatty acids. Results of genotypic analyses also demonstrated differences between these two strains. They also differed in DNA G+C content, and some biochemical and physiological characteristics. We therefore believe that the examined bacterial isolate should be considered as a new taxon within the group of obligately heterofermentative lactobacilli. The species name Lactobacillus caviae sp. nov. is proposed, of which the type strain is MOZM2T (=CCM 8609T=DSM 100239T=LMG 28780T).

Mupirocin-mucin agar for selective enumeration of Bifidobacterium bifidum.

Bifidobacterium bifidum is a bacterial species exclusively found in the human intestinal tract. This species is becoming increasingly popular as a probiotic organism added to lyophilized products. In this study, porcine mucin was used as the sole carbon source for the selective enumeration of B. bifidum in probiotic food additives. Thirty-six bifidobacterial strains were cultivated in broth with mucin. Only 13 strains of B. bifidum utilized the mucin to produce acids. B. bifidum was selectively enumerated in eight probiotic food supplements using agar (MM agar) containing mupirocin (100 mg/L) and mucin (20 g/L) as the sole carbon source. MM agar was fully selective if the B. bifidum species was presented together with Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, and Bifidobacterium longum subsp. longum species and with lactic acid bacteria (lactobacilli, streptococci). Isolated strains of B. bifidum were identified using biochemical, PCR, MALDI-TOF procedures and 16S rRNA gene sequencing. The novel selective medium was also suitable for the isolation of B. bifidum strains from human fecal samples.

Effect of dietary lupin (Lupinus albus) on the gastrointestinal microbiota composition in broiler chickens and ducks

Geigerová M., Švejstil R., Skřivanová E., Straková E., Suchý P. (2017): Effect of dietary lupin (Lupinus albus) on the gastrointestinal microbiota composition in broiler chickens and ducks. Czech J. Anim. Sci., 62, 369–376. The purpose of the study was to evaluate the amount of raffinose-series oligosaccharides (RSO) in soybean meal (SBM), whole white lupin seed meal (WLM), sunflower meal (SFM), and rapeseed oil meal (ROM) and to determine whether partial or complete dietary WLM replacement affected the numbers of bacteria in selected groups in the microbiota of broiler chickens and ducks without inducing any weight loss. Total counts of anaerobes, lactobacilli, bifidobacteria, and Escherichia coli in caecal samples from both ducks and broiler chickens, as well as in a crop chyme, in broiler chickens, were determined. Live weights before slaughter were determined. Both broiler chickens and ducks were fed a control diet with SBM (L0) or diet containing 50% or 100% WLM as a substitute for SBM (groups L50 and L100, respectively). In comparison with SBM, WLM contained significantly higher amounts of RSO, and the amounts of oligosaccharides in SFM (1.73 ± 0.26 g/100 g) and ROM (1.79 ± 0.14 g/100 g) were negligible compared to those in WLM (8.26 ± 0.14 g/100 g) and SBM (6.96 ± 0.21 g/100 g). The inclusion of lupin in chicken diets did not significantly affect the monitored bacterial groups in crop chyme, but a complete replacement of SBM with WLM (L100 group) in chicken diets significantly (P ≤ 0.05) increased the counts of lactobacilli in caecal samples. Partial (L50 group) and complete (L100 group) lupin supplementation in the duck diet significantly (P ≤ 0.05) increased counts of lactobacilli and bifidobacteria by at least one order of magnitude. E. coli counts in poultry were not affected by changes in diet. The results of our study indicate that partial dietary replacement of SBM with WLM did not significantly affect the live weight of broiler chickens and ducks, but that complete replacement of SBM with WLM may lead to weight loss.

Assessment of the synbiotic properites of human milk oligosaccharides and Bifidobacterium longum subsp. infantis in vitro and in humanised mice

The mode of delivery plays a crucial role in infant gastrointestinal tract colonisation, which in the case of caesarean section is characterised by the presence of clostridia and low bifidobacterial counts. Gut colonisation can be modified by probiotics, prebiotics or synbiotics. Human milk oligosaccharides (HMOs) are infant prebiotics that show a bifidogenic effect. Moreover, genome sequencing of Bifidobacterium longum subsp. infantis within the infant microbiome revealed adaptations for milk utilisation. This study aimed to evaluate the synbiotic effect of B. longum subsp. infantis, HMOs and human milk (HM) both in vitro and in vivo (in a humanised mouse model) in the presence of faecal microbiota from infants born by caesarean section. The combination of B. longum and HMOs or HM reduced the clostridia and G-bacteria counts both in vitro and in vivo. The bifidobacterial population in vitro significantly increased and produce high concentrations of acetate and lactate. In vitro competition assays confirmed that the tested bifidobacterial strain is a potential probiotic for infants and, together with HMOs or HM, acts as a synbiotic. It is also able to inhibit potentially pathogenic bacteria. The synbiotic effects identified in vitro were not observed in vivo. However, there was a significant reduction in clostridia counts in both experimental animal groups (HMOs + B. longum and HM + B. longum), and a specific immune response via increased interleukin (IL)-10 and IL-6 production. Animal models do not perfectly mimic human conditions; however, they are essential for testing the safety of functional foods.