In vitro fermentability of human milk oligosaccharides by several strains of bifidobacteria

This study was conducted to investigate the catabolism and fermentation of human milk oligosaccharides (HMO) by individual strains of bifidobacteria. Oligosaccharides were isolated from a pooled sample of human milk using solid-phase extraction, and then added to a growth medium as the sole source of fermentable carbohydrate. Of five strains of bifidobacteria tested (Bifidobacterium longum biovar infantis, Bifidobacterium bifidum, Bifidobacterium longum biovar longum, Bifidobacterium breve, and Bifidobacterium adolescentis), B. longum bv. infantis grew better, achieving triple the cell density then the other strains. B. bifidum did not reach a high cell density, yet generated free sialic acid, fucose and N-acetylglucosamine in the media, suggesting some capacity for HMO degradation. Thin layer chromatography profiles of spent fermentation broth suggests substantial degradation of oligosaccharides by B. longum bv. infantis, moderate degradation by B. bifidum and little degradation by other strains. While all strains were able to individually ferment two monosaccharide constituents of HMO, glucose and galactose, only B. longum bv. infantis and B. breve were able to ferment glucosamine, fucose and sialic acid. These results suggest that as a potential prebiotic, HMO may selectively promote the growth of certain bifidobacteria strains, and their catabolism may result in free monosaccharides in the colonic lumen.     

Novel bifidobacterial glycosidases acting on sugar chains of mucin glycoproteins

Bifidobacterium bifidum was found to produce a specific 1,2-alpha-L-fucosidase. Its gene (afc A) has been cloned and the DNA sequence was determined. The Afc A protein consisting of 1959 amino acid residues with a predicted molecular mass of 205 kDa can be divided into three domains; the N-terminal function-unknown domain (576 aa), the catalytic domain (898 aa), and the C-terminal bacterial Ig-like domain (485 aa). The recombinant catalytic domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and sugar chains of glycoproteins. The primary structure of the catalytic domain exhibited no similarity to those of any glycoside hydrolases but showed similarity to those of several hypothetical proteins in a database, which resulted in establishment of a novel glycoside hydrolase family (GH family 95). Several bifidobacteria were found to produce a specific endo-alpha-N-acetylgalactosaminidase, which is the endoglycosidase liberating the O-glycosidically linked galactosyl beta1-->3 N-acetylgalactosamine disaccharide from mucin glycoprotein. The molecular cloning of endo-alpha-N-acetylgalactosaminidase was carried out on Bifidobacterium longum based on the information in the database. The gene was found to comprise 1966 amino acid residues with a predicted molecular mass of 210 kDa. The recombinant protein released galactosyl beta1-->3 N-acetylgalactosamine disaccharide from natural glycoproteins. This enzyme of B. longum is believed to be involved in the catabolism of oligosaccharide of intestinal mucin glycoproteins. Both 1,2-alpha-L-fucosidase and endo-alpha-N-acetylgalactosaminidase are novel and specific enzymes acting on oligosaccharides that exist mainly in mucin glycoproteins. Thus, it is reasonable to conclude that bifidobacteria produce these enzymes to preferentially utilize the oligosaccharides present in the intestinal ecosystem.     

Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods

Due to the increasing use of bifidobacteria in probiotic products, it is essential to establish a rapid method for the qualitative and quantitative assay of the bifidobacteria in commercial products. In this study, partial sequences of the tuf gene for 18 Bifidobacterium strains belonging to 14 species were determined. Alignment of these sequences showed that the similarities among these Bifidobacterium species were 82.24% to 99.72%. Based on these tuf gene sequences, 6 primer sets were designed for the polymerase chain reaction (PCR) assay of B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. longum subsp. infantis, B. longum subsp. longum, and the genus of Bifidobacterium, respectively. These Bifidobacterium species are common probiotic species present in dairy and probiotic products. When each target Bifidobacterium spp. was assayed with the designed primers, PCR product with expected size was generated. In addition, for each target species, more than 70 bacterial strains other than the target species, including strains of other Bifidobacterium species, strains of Lactobacillus spp., Enterococcus spp., and other bacterial species, all generated negative results. PCR assay with primers specific to B. animalis subsp. lactis and B. longum subsp. longum confirmed the presence of these Bifidobacterium species in commercial yogurt products. In addition, for each product, enumeration of the bifidobacteria cells by culture method with BIM-25 agar and the quantitative real-time PCR showed similar cell counts. Such results indicated that within 15-d storage (4 °C) after manufacture, all the bifidobacteria cells originally present in yogurt products were viable and culturable during the storage.     

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.     

Production of oligosaccharides in yogurt containing bifidobacteria and yogurt cultures

Yogurts were prepared by using yogurt cultures combined to mixed cultures of bifidobacteria (Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum) and by adding a preincubation step (1.5 h at 50 degrees C) with bifidobacteria to the conventional method of manufacture in order to produce oligosaccharides. The survival of bifidobacteria was drastically affected during storage of yogurts, except for products containing B. animalis, in which viable counts remained at >10(6) cfu/g after 28 d of storage at 4 degrees C. Oligosaccharides with a degree of polymerization of 3 were produced during the preincubation step (0.31 to 0.68%), and the amount in the final products varied according to the species of bifidobacteria inoculated during the preincubation step or the concentration of bifidobacteria used as second inoculum during the fermentation process. In fact, the higher concentration of oligosaccharides measured at the end of the fermentation process (0.72%) and the 28 d-storage period (0.67%) was obtained for yogurts containing B. infantis. However, yogurts containing B. breve showed higher beta-galactosidase activities and had lower lactose concentrations after the fermentation process and the storage period than the other yogurts. The use of a mixed cultures of bifidobacteria (B. animalis, B. infantis, or B. breve) thus allows the production of yogurts in which bifidobacteria can survive in relatively high cell numbers and contain appreciable amount of oligosaccharides.     

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.     

Viability of microencapsulated bifidobacteria in simulated gastric juice and bile solution

Microencapsulated cells of Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 were prepared by spray drying the cell suspension containing the test organism and 10% (w/w) of the carrier material of either gelatin, soluble starch, skim milk or gum arabic. Survival of these microencapsulated and free cells of bifidobacteria in simulated gastric juice (pH 2.0 and 3.0) and bile solution (0.5% and 2.0%) was then examined. B. infantis CCRC 14633 was more susceptible than B. longum B6 to the simulated gastric environment and bile solution tested. Microencapsulated bifidobacteria exhibited a lower population reduction than free cells during exposure to simulated gastric environment and bile solution. This phenomenon was most pronounced when the test organism was exposed to gastric juice at pH 2.0 or 2.0% bile solution. Moreover, it was also observed that the protective effect exerted by encapsulation with spray drying varied with the carriers used and the strains of bifidobacteria.     

Selection of phytate-degrading human bifidobacteria and application in whole wheat dough fermentation

Lately, whole wheat products are highly recommended from their healthy properties. However, the presence of phytic acid (InsP(6)) could partly limit their benefits because it decreases the mineral bioavailability due to its chelating properties. The objective of this work was to select strains with high phytate-degrading activity from human feces, and evaluate their suitability for the bread making process. Twenty-three different bifidobacterial strains (13 from infants and 10 from adults) were isolated, belonging to the species Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium catenulatum. The phosphatase and phytase activities of these strains were evaluated as well as their ability to degrade InsP(6) during growth. Then, the fermentative ability of the strain showing the highest phytate-degrading activity (B. longum. BIF307) was determined in whole wheat breadmaking. The use of the selected bifidobacterial strain as starter during whole wheat fermentation resulted in bread with similar technological quality than the control (in absence of bifidobacteria) and crumb with lower levels of inositol phosphates. Therefore, the used of the selected Bifidobacterium strain in whole wheat breadmaking process could provide potential nutritional benefits by decreasing the antinutrient content of the product.     

Molecular and microbiological analysis of caecal microbiota in rats fed with diets supplemented either with prebiotics or probiotics

The potential health-improving effects of both a prebiotic and a probiotic infant formula have been evaluated in a rat model. Two groups of 10 rats were fed with either prebiotics containing fructo-oligosaccharides or probiotics containing viable Bifidobacterium lactis and Streptococcus thermophilus. The composition of their caecal microbiota was analyzed both by classical plate count of the main bacterial groups and by PCR amplification of a V3 fragment of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE). Both diets induced a significant reduction of clostridia and Bacteroides spp. compared to a control diet, whereas prebiotics were also able to reduce the number of coliforms and to increase the presence of bifidobacteria. DGGE analysis showed a significant increase of 16S rRNA gene fragments in rats fed with either probotics or prebiotics. Nineteen bands were sequenced and most of them showed similarity to cultured bacteria. Detection of Bifidobacterium spp. by this technique using genus-specific primers only permitted these bacteria to be detected in prebiotics-fed rats, whereas the use of Lactobacillus group-specific primers gave similar results in rats fed with any diet, in agreement with the plate count results.     

Exopolysaccharides Produced by Lactic Acid Bacteria and Bifidobacteria as Fermentable Substrates by the Intestinal Microbiota

The functional food market, including products formulated to maintain a “healthy” gut microbiota, i.e. probiotics and prebiotics, has increased enormously since the end of the last century. In order to favor the competitiveness of this sector, as well as to increase our knowledge of the mechanisms of action upon human health, new probiotic strains and prebiotic substrates are being studied. This review discusses the use of exopolysaccharides (EPS), both homopolysaccharides (HoPS) and heteropolysaccharides (HePS), synthesized by lactic acid bacteria and bifidobacteria as potential prebiotics. These extracellular carbohydrate polymers synthesized by some gut inhabitants seem to be resistant to gastrointestinal digestion; these are susceptible as well to biodegradability by the intestinal microbiota depending on both the physicochemical characteristics of EPS and the pool of glycolytic enzymes harbored by microbiota. Therefore, although the chemical composition of these HoPS and HePS is different, both can be fermentable substrates by intestinal inhabitants and good candidates as prebiotic substrates. However, there are limitations for their use as additives in the food industry due to, on the one hand, their low production yield and, on the other hand, a lack of clinical studies demonstrating the functionality of these biopolymers.     

Stability of bifidobacteria in powdered formula

Two stability tests of bifidobacteria in powdered formula were conducted. In a strain comparison test, three kinds of bifidobacterial powders; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63 powders, admixed in follow-up formula were used. In a commercial product evaluation, powdered formulas for toddlers containing bifidobacteria sold in the Indonesian market were analysed. When the inactivation rate constant of each sample, which was used as an index of the loss rate, was determined from the stability tests, B. longum was the most stable strain. The mean inactivation rate constant of commercial products was significantly lower than those obtained in strain comparison, although the same strains ( B. longum BB536 and B. breve M-16V) were used. A possible reason was the lower water activity of commercial products compared to the follow-up formula. Also, higher storage temperature yielded lower stability in all strains or samples, which obeys the Arrhenius theory.     

Viability During Storage of Selected Probiotic Lactobacilli and Bifidobacteria in a Yogurt-like Product

ABSTRACT: Multiple species cultures, including 2 strains of Streptococcus thermophilus and Lactobacillus acidophilus NCFM plus 1 strain each of Bifidobacterium longum and Lactobacillus casei , were used to make yogurt-like products. The lactobacilli and bifidobacteria were tested for growth in the products and subsequent viability during refrigerated storage. During fermentation, L. casei Com-5 actually declined in numbers, while L. casei E5 and E10 increased about 2 fold. Numbers of B. longum S9 increased about 3 fold while B. longum Com-4 did not increase. During storage, L. acidophilus NCFM appeared stable in all mixtures and both strains of bifidobacteria decreased. Lactobacillus casei E5 and E10 were more stable than was L. casei Com-5.     

Survival of bifidobacteria after spray-drying

To investigate the survival of bifidobacteria after spray-drying, Bifidobacterium infantis CCRC 14633, B. infantis CCRC 14661, B. longum ATCC 15708, B. longum CCRC 14634 and B. longum B6 were first spray-dried with different carrier media including 10% (w/w) gelatin, gum arabic and soluble starch. B. infantis CCRC 14633 and B. longum were also determined in skim milk. It was found that survival of bifidobacteria after spray-drying varied with strains and is highly dependent on the carriers used. Among the test organisms, B. longum B6 exhibited the least sensitivity to spray-drying and showed the highest survival of ca. 82.6% after drying with skim milk. Comparisons of the effect of carrier concentrations revealed that spray-drying at 10% (w/w) gelatin, gum arabic or soluble starch resulted in the highest survival of bifidobacteria. In addition, among the various outlet-air temperatures tested, bifidobacteria showed the highest survival after drying at 50 degrees C. Elevation of outlet-air temperature caused increased inactivation of bifidobacteria. However, the inactivation caused by increased outlet-air temperature varied with the carrier used, with the greatest reduction observed using soluble starch and the least with skim milk.     

一株Bifidobacterium longum CCFM760的生理特性、安全性评价和基因分析

长双歧杆菌(Bifidobacterium longum CCFM760)是一株从江苏省无锡市滨湖区两岁健康幼女粪便分离筛选得到的具有良好缓解便秘功效的益生菌。作者对其生长产酸能力、人工模拟肠胃液耐受性、抑菌能力、低聚糖利用能力、抗生素耐受能力和总超氧化物歧化酶(SOD)活性进行了检测。该菌具有良好的生长产酸能力和人工模拟肠胃液耐受性,对大肠杆菌和金黄色葡萄球菌有明显的抑制作用,能显著利用7种低聚糖,对14种抗生素敏感,培养液中总SOD活性达到(99.14±9.91)U/mL。此外,对该菌进行基因组草图测序,并从基因组角度解析了其低聚糖代谢能力、抗生素敏感性以及抗氧化能力。以上结果表明,B.longum CCFM760是一株安全的、具有实际应用潜力的益生菌。     

A Bifidobacterium-based synbiotic product to reduce the transmission of C. jejuni along the poultry food chain

With the ban of dietary antimicrobial agents, the use of probiotics, prebiotics and synbiotics has attracted a great deal of attention in order to improve intestinal health and control food-borne pathogens, which is an important concern for the production of safe meat and meat products. Recently, Campylobacter jejuni has emerged as a leading bacterial cause of food-borne gastroenteritis in humans, and epidemiological evidences indicate poultry and poultry products as the main source of human infection. This work aimed at the development of a synbiotic mixture capable of modulating the gut microbiota of broiler chickens to obtain an increase of the beneficial bacteria (i.e. bifidobacteria, lactobacilli) and a competitive reduction of C. jejuni. The prebiotic compound used in the mixture was chosen after an in vivo trial: a fructooligosaccharide and a galactooligosaccharide were separately administered to broilers mixed with normal feed at a concentration of 0.5% and 3%, respectively. Quantitative PCR on DNA extracted from fecal samples revealed a significant (p<0.05) increase of Bifidobacterium spp. in broilers treated with the galactooligosaccharide, coupled to a decrease (p<0.05) of Campylobacter spp. The galactooligosaccharide was then combined with a probiotic Bifidobacterium strain (B. longum subsp. longum PCB133), possessing in vitro antimicrobial activity against C. jejuni. The strain was microencapsulated in a lipid matrix to ensure viability into the feed and resistance to stomach transit. Finally, the synbiotic mixture was administered to broiler chickens for 14 days mixed with normal feed in order to have an intake of 10(9)CFU of PCB133/day. Bifidobacterium spp., Lactobacillus spp., Campylobacter spp., B. longum subsp. longum and C. jejuni were quantified in fecal samples. PCB133 was recovered in feces of all animals. C. jejuni concentration in poultry feces was significantly (p<0.05) reduced in chickens administered with the synbiotic mixture. This study allowed to highlight the positive effect of the synbiotic approach for C. jejuni reduction in broiler chickens, which is of fundamental importance for the safety of poultry meat consumers.     

Prebiotics and the Bioavailability of Minerals and Trace Elements

Promising evidence exists for a stimulatory influence of prebiotic carbohydrates (non-digestible oligosaccharides (NDO) and lactulose) on the bioavailability of minerals and trace elements, although most information is available from rat experiments. Because the number of human studies is limited, additional research is needed, especially studies that focus on the long-term effects of prebiotics on bone metabolism. The hypothesized mechanisms suggest that prebiotics that result in a strong pH reduction, a pronounced osmotic effect, a stimulation of the exchange of protons, an increased level of butyrate and calbindin, and an enlarged surface area in the colon may be promising candidates for increasing the absorption of minerals and trace elements. Because the place of colonic fermentation seems to be of influence in these mechanisms, the use of mixtures of different types of prebiotics is of special interest. Probiotics are involved in most prebiotic-related mechanisms, possibly mediated in part by the production of polyamines. Therefore, the combined use of pre- and probiotics may yield promising results. More detailed information on the present state-of-the-art concerning the effects of prebiotics on the bioavailability of minerals and trace elements in different life stages, discussed by study design and its underlying mechanisms, can be found in this review.     

成人粪便中长双歧杆菌长亚种的分离鉴定及多位点序列分型分析

为了对成人粪便中分离的长双歧杆菌长亚种进行多位点序列分型分析,采用改良MRS培养基从健康成人粪便中分离的长双歧杆菌长亚种,通过生理生化试验结合16S rDNA和热应激蛋白60(heat-shock protein,hsp60)同源性分析鉴定分离株,利用同源性分析及多位点序列分型(multilocus sequence typing,MLST)技术进一步分析长双歧杆菌长亚种的基因多态性。从12个健康成人体内分离得到24株厌氧的细菌菌株,经形态学观察、生理生化试验、16S rDNA及hsp60同源性分析发现,其中14株分离株为长双歧杆菌长亚种(Bifidobacterium longum subsp. longum)。MLST结果表明:14株长双歧杆菌长亚种可分为10种基因型,且均与Bifidobacterium longum subsp. longum ATCC 15707为不同基因型。健康成人粪便中分离的长双歧杆菌长亚种具有较大的基因多态性。     

Uncoupling of growth and acids production in Bifidobacterium ssp

Kinetics of batch cultivation of four species of Bifidobacterium in milk were examined in detail. Bifidobacteria could grow well in milk inoculated with cultures prepared in a synthetic medium. Cessation of growth occurred, however, in pH-controlled batch cultures, although incomplete utilization of lactose was observed. Lactate and acetate accumulation caused limitation on growth of bifidobacteria leading to an uncoupling of biomass and product formation. From 70 to 75% of both final lactate and acetate concentrations were produced during the stationary growth phase of Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum cultivated in milk, whereas Bifidobacterium infantis produced less acetate or lactate during this phase.     

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.