Bifidobacterium carbohydrases-their role in breakdown and synthesis of (potential) prebiotics

There is an increasing interest to positively influence the human intestinal microbiota through the diet by the use of prebiotics and/or probiotics. It is anticipated that this will balance the microbial composition in the gastrointestinal tract in favor of health promoting genera such as Bifidobacterium and Lactobacillus. Carbohydrates like non-digestible oligosaccharides are potential prebiotics. To understand how these bacteria can grow on these carbon sources, knowledge of the carbohydrate-modifying enzymes is needed. Little is known about the carbohydrate-modifying enzymes of bifidobacteria. The genome sequence of Bifidobacterium adolescentis and Bifidobacterium longum biotype longum has been completed and it was observed that for B. longum biotype longum more than 8% of the annotated genes were involved in carbohydrate metabolism. In addition more sequence data of individual carbohydrases from other Bifidobacterium spp. became available. Besides the degradation of (potential) prebiotics by bifidobacterial glycoside hydrolases, we will focus in this review on the possibilities to produce new classes of non-digestible oligosaccharides by showing the presence and (transglycosylation) activity of the most important carbohydrate modifying enzymes in bifidobacteria. Approaches to use and improve carbohydrate-modifying enzymes in prebiotic design will be discussed.     

Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates

Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these dietary interventions. In this study the consumption of galactooligosaccharides was studied for 22 isolates of Bifidobacterium longum subsp. infantis, one of the most representative species in the infant gut microbiota. In general all isolates showed a vigorous growth on these oligosaccharides, but consumption of larger galactooligosaccharides was variable. Bifidobacterium infantis ATCC 15697 has five genes encoding β-galactosidases, and three of them were induced during bacterial growth on commercial galactooligosaccharides. Recombinant β-galactosidases from B. infantis ATCC 15697 displayed different preferences for β-galactosides such as 4′ and 6′-galactobiose, and four β-galactosidases in this strain released monosaccharides from galactooligosaccharides. Finally, we determined the amounts of short chain fatty acids produced by strain ATCC 15697 after growth on different prebiotics. We observed that biomass and product yields of substrate were higher for lactose and galactooligosaccharides, but the amount of acids produced per cell was larger after growth on human milk oligosaccharides. These results provide a molecular basis for galactooligosaccharide consumption in B. infantis, and also represent evidence for physiological differences in the metabolism of prebiotics that might have a differential impact on the host.     

Selection of probiotics and prebiotics for synbiotics and confirmation of their in vivo effectiveness

Influence of fructan-type oligosaccharides (as prebiotics) on growth and acidifying activity of Bifidobacterium strains (as probiotics) was studied in vitro, using minimal nutrition media. The selected synbiotic pairs of stimulated bifidobacteria strains and oligosaccharides enhancing their growth were studied in vivo to determine the effect of probiotics, prebiotics and synbiotics on microecology of gut. Daily, >10⁹ live cells of bifidobacteria strains and/or 5% (w/w) of oligofructose in the diet were orally administered to Wistar rats. After a 14-day feeding experiment, the numbers of faecal bifidobacteria, aerobic and facultatively anaerobic bacteria, coliforms, and spores of anaerobic and aerobic bacteria were determined using microbiological methods. In vitro studies showed that the majority of Bifidobacterium species utilised FOS and low-polymerised inulins, but only 18 out of 30 strains tested (mostly of B. longum and B. animalis species) were stimulated. Incorporation of oligofructose into the diet stimulated the proliferation of faecal bifidobacteria by 1.6 log cfu/g in comparison to the control. B. longum KN29.1 and KNA1 selected in vitro, slightly increased the faecal bifidobacteria live cell number by about 0.6 log cfu/g under in vivo conditions, whereas B. animalis KSp4 was not effective. But administration of bifidobacteria together with the prebiotic (as synbiotics) improved the bifidogenic effect by 1.4 log cfu/g of faeces. Supplementation of diet had almost no effect on the other determined groups of gut microflora. The obtained results showed the selective stimulation of faecal bifidobacteria by probiotics, prebiotic and synbiotics, a great bifidogenic effectiveness of oligofructose, confirmed proper selection of synbiotics and showed their higher effectiveness in relation to probiotics.     

Non-digestible oligosaccharides with prebiotic properties

The search for functional foods or functional food ingredients, i.e. foods or food ingredients that can enhance health, is beyond any doubt one of the leading trends in today's food industry. In this context, probiotics, i.e. living microbial food supplements, and prebiotics, i.e. non-digestible food ingredients, receive much attention. Both popular concepts target the gastrointestinal microbiota. While in the Western world, intake of probiotics has been recommended for long, prebiotics in general, and non-digestible oligosaccharides in particular, have only recently received attention. This review deals with production and characterization of non-digestible oligosaccharides and focuses on their role in promoting health and treating diseases. Attention is paid to the effects of non-digestible oligosaccharides on constipation, mineral absorption, lipid metabolism, cancer prevention, hepatic encephalopathy, glycemia/insulinemia, and immunomodulation.     

Progress in genomics, metabolism and biotechnology of bifidobacteria

Members of the genus Bifidobacterium were first described over a century ago and were quickly associated with a healthy intestinal tract due to their numerical dominance in breast-fed babies as compared to bottle-fed infants. Health benefits elicited by bifidobacteria to its host, as supported by clinical trials, have led to their wide application as probiotic components of health-promoting foods, especially in fermented dairy products. However, the relative paucity of genetic tools available for bifidobacteria has impeded development of a comprehensive molecular understanding of this genus. In this review we present a summary of current knowledge on bifidobacterial metabolism, classification, physiology and genetics and outline the currently available methods for genetically accessing and manipulating the genus.     

Prebiotics: Present and future in food science and technology

Because of its resident microbiota, the human colon is one of the body’s most metabolically active organs. The use of diet to fortify certain gut flora components is a popular current aspect of functional food sciences and prebiotics have a significant role. Prebiotics are selectively fermented ingredients that allow specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health. Improved techniques for analysis of the gut microflora, new food manufacturing biotechnologies, and increased understanding of the metabolism of prebiotic inulin and oligosaccharides by probiotics are facilitating development. Such developments are leading us to the time when we will be able to rationally develop prebiotics for specific functional properties and health outcomes. Thus, this review will focus on the progress of prebiotics in food science and technology in understanding the important role of prebiotics in health, beginning at the rationale of gut microflora and interactions with prebiotics. Furthermore, the classification criteria, food applications and safety assessment of prebiotics as food ingredient is also discussed.     

Introduction to pre- and probiotics

During the last 2–3 decades, attempts for improving the human health status, are focusing on ways for modulating the indigenous intestinal flora by live microbial adjuncts, now called “probiotics”. Comprising ca. 65% of the functional food world market, probiotics represent the major and still growing segment of this huge market, estimated to exceed a total volume of US $ 75 billion. The most typical active components of probiotic products are lactic acid bacteria, including bifidobacteria, lactobacilli and enterococci. Health claims related to probiotics are numerous, but include maintenance of normal/healthy intestinal flora and protection against infections, alleviation of lactose intolerance, and stimulation of the immune system. Strains with proven beneficial effects may be consumed in relatively high numbers in products, and are collectively called probiotics. In addition. bifidobacteria and lactobacilli, typical inhabitants of the human GIT, are considered beneficial, and may be stimulated by non-digestible food ingredients such as oligosaccharides, collectively called prebiotics. Probably aimed at the two “target regions” of the GIT, pre- and probiotics may be combined in a food product, called a synbiotic. Confirmed health claims are discussed and reference will be made to open questions and research challenges.     

Improved growth of bifidobacteria by cocultivation with Lactococcus lactis subspecies lactis

Cultivation of bifidobacteria in milk is a difficult and industrially valuable task. In this paper, we report the finding of a novel technique to improve the growth of bifidobacteria in dairy products and the results of mechanism studies. The growth of bifidobacteria in skim milk medium was found to be stimulated upon cocultivation with certain strains of Lactococcus lactis ssp. lactis. Bifidobacterium growth-stimulating (BGS) activity was observed on a wide range of bifidobacterial species. Bifidobacterium growth-stimulating activity was associated with the ability to grow in skim milk medium and the presence of a cell wall-anchored proteinase (PrtP) in Lc. lactis ssp. lactis. Studies on one strain, Lc. lactis ssp. lactis MCC857, showed that crude PrtP extracts and casein hydrolysates exhibited BGS activity. The casein hydrolysate BGS activity was found in the low molecular weight fraction by HPLC separation. A combination of 2 AA, Met and Leu, was found to account for a large portion of the casein hydrolysate BGS activity. In conclusion, this cocultivation system is highly efficient and industrially applicable for the production of fermented milk with high cell counts of bifidobacteria.     

Short communication: Genotype-phenotype association analysis revealed different utilization ability of 2'-fucosyllactose in Bifidobacterium genus

The oligosaccharide 2'-fucosyllactose (2'FL) in human breast milk selectively promotes the proliferation of bifidobacteria. One hundred fifty-one Bifidobacterium strains were evaluated for their capacity to utilize 2'FL based on the combination of phenotype and genotype association analysis. Through genotype analysis, 37 strains were predicted to have the ability to use 2'FL, including Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum ssp. longum, Bifidobacterium longum ssp. infantis, and Bifidobacterium dentium, whereas Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium pseudocatenulatum, and Bifidobacterium angulatum could not use 2'FL. For in vitro utilization, there were noteworthy differences for 2'FL usage among different species, which were 100% consistent with genotype prediction. The results indicated that 2'FL utilization ability differed even within the same species, and Bifidobacterium followed the currently well-known pathway to utilize 2'FL, which could provide guidance to develop personalized prebiotics for different bifidobacteria via gene-trait matching analysis.     

Intestinal survival and persistence of probiotic Lactobacillus and Bifidobacterium strains administered in triple-strain yoghurt

The aim of the present study was to investigate the intestinal survival and persistence of probiotic strains Lactobacillus F19, Lactobacillus acidophilus NCFB 1748, and Bifidobacterium animalis subsp. lactis Bb-12 consumed in a yoghurt (ABC product), and also their effect on the intestinal microbiota. Based on the results of culture studies and strain-level analysis by randomly amplified polymorphic DNA (RAPD) fingerprinting Lactobacillus F19 and B. animalis subsp. lactis Bb-12 survived well through the human gastrointestinal tract; they were detected in reasonable numbers in the faeces of 100% and 79% of the study subjects, respectively. Ingestion of the probiotic yoghurt increased transiently the numbers of bifidobacteria and lactobacilli. For lactobacilli the increase was due to the detection of the ingested probiotic strains in faeces, while in bifidobacteria the increase was likely caused by the increase of indigenous bifidobacteria since the ingested Bifidobacterium strain did not comprise the predominant part of bifidobacterial population during the intervention. Probiotic strains were infrequently detected in mucosal biopsy samples. The present study indicates that developing probiotic food products with multiple probiotic strains is feasible.     

谷物难消化碳水化合物益生元功效

肠道菌群结构与人体健康密切相关,而饮食又是影响肠道菌群组成重要因素。谷物中一些难消化碳水化合物可作为益生元,选择性刺激人体肠道中乳酸杆菌和双歧杆菌生长,改善人体肠道菌群结构。该文对肠道菌群与机体慢性疾病关系及几种谷物难消化碳水化合物益生功效进行探讨与概述。     

Genomics and ecological overview of the genus Bifidobacterium

Members of the genus Bifidobacterium are high G + C Gram positive bacteria belonging to the phylum Actinobacteria, and represent common inhabitants of the gastro-intestinal tract (GIT) of mammals, birds and certain cold-blooded animals. The overall microbial population that resides in the GIT, referred to as the “gut microbiota”, is an extremely complex community of microorganisms whose functions are believed to have a significant impact on human physiology. Different ecological relationships between bifidobacteria and their host can be developed, ranging from opportunistic pathogenic interactions (e.g. in the case of Bifidobacterium dentium) to a commensal or even health-promoting relationship (e.g. in the case of Bifidobacterium bifidum and Bifidobacterium breve species). Among the known health-promoting or probiotic microorganisms, bifidobacteria represent one of the most dominant group and some bifidobacterial species are frequently used as the probiotic ingredient in many functional foods. However, despite the generally accepted importance of bifidobacteria as constituents of the human microbiota, there is only limited information available on their phylogeny, physiology and genetics. Moreover, host-microbiota interactions and cross-talk between different members of the gut microbiota are far from completely understood although they represent a crucial factor in the development and maintenance of human physiology and immune system. The aim of this review is to highlight the genetic and functional features of bifidobacteria residing in the human GIT using genomic and ecology-based information.     

Potential use of whey concentrate and prebiotics as carrier agents to protect Bifidobacterium-BB-12 microencapsulated by spray drying

Bifidobacterium BB-12 was microencapsulated by spray drying using liquid whey or whey retentate obtained from nanofiltration and inulin or polydextrose. The microcapsules were then characterized and the viability of the bifidobacteria was determined for 90 days at 4 °C and at − 20 °C. All the microcapsules showed high count of bifidobacteria, low moisture content, and low water activity. The microcapsules showed similar morphologies and particle sizes. The density of the microcapsules decreased with the increase of the total solids of the feed solutions. The highest hygroscopicity was noted in the microcapsules produced with polydextrose. The time of dissolution in water was longer than it was in oil for all the samples. The presence of prebiotics in the microcapsules increased the L* values and decreased the b* values; meanwhile, the samples produced with whey retentate showed lower a* values. The results of the thermal analysis suggested that prebiotics improved the stability of the microcapsules.     

Development of functional ingredients for gut health

Microbial reactions in the gut have an essential role not only in gut health, but in general human health. The gut is the site of active fermentation of non-digestible diet components, as well as bioconversions and absorption of plant-derived compounds, such as phenolics. When developing nutritionally designed foods that promote health through gut microbial reactions, three different types of food ingredients can be used: living micro-organisms (probiotics), non-digestible carbohydrates (dietary fiber and prebiotics) and bioactive plant secondary metabolites (e.g. phenolics).     

Prebiotics and their long-term influence on the microbial populations of the mouse bowel

Various prebiotics were orally administered to mice and the evolution of different microbial populations was studied. The administration of prebiotics significantly increased lactobacilli and bifidobacteria in the large bowel content. Ingestion of prebiotics specifically lowered microbial populations of sulphite-reducing clostridia. Xylo-oligosaccharides (XOS) increased lactobacilli by 10 fold and produced the highest counts of bifidobacteria. In XOS-treated mice, levels of sulphite-reducing clostridia decreased significantly. Prebiotics slightly reduced the amount of aerobic bacteria and significantly increased the number of anaerobes in both the small and the large bowel. These effects of prebiotics were reverted by the basal diet.     

Microencapsulation of bifidobacteria by spray drying in the presence of prebiotics

This study was conducted to evaluate the viability and the physical properties of Bifidobacterium BB-12 microencapsulated by spray drying with partial replacement of reconstituted skim milk (RSM), as encapsulating agent, with the prebiotics inulin, oligofructose, and oligofructose-enriched inulin (at a ratio of 1:1, 200 g L^?1 total concentrations). The viable cell counts of the microcapsules were determined during storage for 180 days at 4 °C and at ? 18 °C. The physical characterization included analysis of morphology, particle size, moisture content, water activity, dissolution, hygroscopicity, color, and thermal properties. All the microcapsules produced in this study showed a high survival rate of bifidobacteria during storage at the temperatures evaluated. The microcapsules produced with inulin and those produced with oligofructose-enriched inulin showed higher initial counts. However, blending oligofructose-enriched inulin with RSM and blending oligofructose with RSM resulted in better protection of bifidobacteria during storage. All microcapsules showed similar morphologies and particle sizes, between 14.45 and 18.78 μm. The partial replacement with prebiotics decreased moisture content and water activity of the microcapsules. The time of dissolution in water was higher for the microcapsules produced with inulin, while the microcapsules produced with oligofructose were more hygroscopic. The value of a* increased in the microcapsules produced with prebiotics. The results of the thermal analysis suggest a higher stability of the microcapsules produced with prebiotics than those produced with RSM only.     

Characterization of bifidobacteria as starters in fermented milk containing raffinose family of oligosaccharides from lupin as prebiotic

The characterization of six bifidobacteria strains used as starters in the manufacture of fermented milk, containing the raffinose family of oligosaccharides (RFOs) as prebiotics, was carried out by applying in vitro methodologies. Selection criteria were the specific growth rate (μ), generation time (T_g), titratable acidity in milk, and enzymatic activities. Strain growth and acidification rate were determined in milk with and without 2% RFOs. Different growth rates, acidification rates and enzymatic patterns were observed among tested bifidobacterial strains. Bifidobacteria with higher levels of α-galactosidase activity showed growth and acidification rate enhancement when RFOs were added compared with lactose as sole carbon source. Based on the results, Bifidobacterium lactis Bb-12 was selected for further investigation as it possessed the enzymes required to enhance growth and metabolic activity in the presence of RFOs from lupins.     

Prebiotics as functional foods: A review

Prebiotics are short chain carbohydrates that are non-digestible by digestive enzymes in humans and selectively enhance the activity of some groups of beneficial bacteria. In the intestine, prebiotics are fermented by beneficial bacteria to produce short chain fatty acids. Prebiotics also render many other health benefits in the large intestine such as reduction of cancer risk and increase calcium and magnesium absorption. Prebiotics are found in several vegetables and fruits and are considered functional food components which present significant technological advantages. Their addition improves sensory characteristics such as taste and texture, and enhances the stability of foams, emulsions and mouthfeel in a large range of food applications like dairy products and bread. This contribution reviews bioactives from food sources with prebiotic properties. Additionally, food application of bioactive prebiotics, stimulation of the viability of probiotics, health benefits, epidemiological studies, and safety concerns of prebiotics are also reviewed.     

Host-microflora correlations in infant nutrition.

The intestinal microflora represents an enormous cell mass and has a high metabolic capacity. The symbiotic efficacy of these microbes in humans is still a matter of discussion. Of particular interest, from a biogenetic point of view, are potential symbiotic relations between the bifidobacterial microflora and the breast-fed infant. Our group has conducted studies related to this topic; they were aimed at determining the dimension of microbial assimilation by the host. Our studies with 15N-labeled bifidobacteria have shown that the bifidobacterial microflora is capable of upgrading nonessential nitrogen such as urea nitrogen for the synthesis of microbial protein. Oral single pulse labelings with 15N-labeled bifidobacteria were absorbed to approximately 90% and retained in the infant's protein pool to approximately 70%. These findings demonstrate the high intensity of the substrate flow from the microflora to the host. This might become important under conditions of marginal food protein intake or during periods of accelerated growth.     

Alpha-Galactosides: Antinutritional Factors or Functional Ingredients?

This review focuses on updated information about alpha-galactosides, their chemical structure, biosynthesis, plant physiological functions, occurrence in foods, positive and negative physiological effects in animals, changes during food processing, and their potential application as prebiotics in the food industry. Although alpha-galactosides are considered as the main flatus-causing factors, they are also involved in several important functions during plant and seed development and beneficially stimulate the growth and activity of living bifidobacteria and lactobacilli in the human colon. We focus here also on legumes as a source of this kind of prebiotics as potential health promoters.