Assessment of adhesion properties of novel probiotic strains to human intestinal mucus

Potential new probiotic strains Lactobacillus brevis PELI, L. reuteri ING1, L. rhamnosus VTT E-800 and L. rhamnosus LC-705 were assessed for their adhesion properties using the human intestinal mucus model. The effect on the adhesion of exposure to acid and pepsin and to milk were tested to simulate gastric and food processing conditions, and the effect of different growth media on adhesion was tested. The properties of the four strains were compared to the well-investigated probiotic L. rhamnosus strain GG. Three of the tested strains showed significant adhesion properties in the mucus model, while L. brevis PELI had intermediate adhesion and L. rhamnosus LC-705 adhered poorly. Pretreatment with different milks decreased the adhesion and low pH and pepsin treatment reduced the adhesion of all tested strains except L. rhamnosus LC-705. No competitive exclusion of pathogenic Salmonella typhimurium or Escherichia coli SfaII was observed. The results indicate that major differences exist between tested proposed probiotic strains. The growth media and the food matrix significantly affect the adhesive ability of the tested strains. This has previously not been taken into account when selecting novel probiotic strains.     

Competitive exclusion of enteropathogens from human intestinal mucus by Bifidobacterium strains with acquired resistance to bile--a preliminary study

The ability to inhibit the adhesion and to displace selected pathogens from human intestinal mucus of two Bifidobacterium strains with acquired resistance to bile, were assessed and compared with those of their bile sensitive original strains. A preliminary characterization of the macromolecules involved in the adhesion was also carried out. The inhibition of adhesion and the displacement of enteropathogens previously adhered were found to be specific, depending on the strains used. The cholate-resistant strain Bifidobacterium bifidum M6dCo, that adhered more to mucus than its original, was able to inhibit the adhesion and to displace pathogens from mucus significantly more than its original cholate-sensitive strain B. bifidum M6. Contrary to this, two strains showing similar adhesion levels, B. bifidum A1 and its bile resistant derivative B. bifidum A1dOx, did not display any differences. Different molecules appear to be involved in the adhesion of the strains B. bifidum M6 and B. bifidum M6dCo. These differences in the cellular surface may explain the differences in competitive exclusion observed between both strains.     

In vitro analysis of probiotic strain combinations to inhibit pathogen adhesion to human intestinal mucus

Competition with pathogens for adhesion and colonization of the mucosal surfaces are possible protective mechanisms of probiotics. In this study we evaluated the ability of commercial strains (Lactobacillus rhamnosus GG, L. rhamnosus LC705, B. breve 99 and Propionibacterium freudenreichii ssp. shermanii JS) each strain alone and in different combinations to inhibit, displace and compete with selected pathogens in order to test the influence of the presence of the other probiotic in the adhesion pathogens to immobilized mucus. The ability to inhibit the adhesion or to displace adhered pathogens was variable depending on both the probiotic combination and the pathogen tested. Our results demonstrate that different probiotic combinations were able to enhance the inhibition percentages to pathogen adhesion to intestinal mucus. All probiotic combinations tested in this study showed inhibition abilities against pathogen infection with values which were over 40% for some pathogens tested. Combinations had specific properties and inhibition abilities against some or all of the tested pathogens. These results suggest that combinations of probiotics strains could be useful and more effective in inhibition of pathogen adhesion. The inhibition and displacement profiles were very different suggesting that different mechanisms are implied in both processes. Selection of new probiotic combinations should be conducted for specific target pathogens or pathogen associated microbiota aberrancies.     

Chemical, physical and enzymatic pre-treatments of probiotic lactobacilli alter their adhesion to human intestinal mucus glycoproteins

Intestinal mucus glycoproteins extracted from faeces of healthy adult subjects were used as a substratum for bacterial adhesion to investigate the effects of physical, chemical and enzymatic pre-treatments of the bacteria on their adhesion. The strains studied were Lactobacillus acidophilus 1 (LCI, Nestlé), L. rhamnosus strain GG (ATCC 53103), L. rhamnosus LC-705, and L. casei strain Shirota (Yakult, Yakult Ltd). Hereafter the strains are referred to as LA1, LGG, LC-705, and Shirota, respectively. Strains LA1 and LGG adhered greatly whereas the adhesion of strains LC-705 and Shirota to intestinal mucus glycoproteins was low. Adhesion of LA1 and LGG was reduced by boiling, autoclaving and by pepsin and trypsin treatments suggesting that the bacterial protein structures are essential for their adhesion. Treatment in ethanol and in propanol prior to adhesion significantly increased the adhesion of LA1 and LC-705, respectively. Adhesion of Shirota strain was not altered by any of the treatments.     

Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells: adhesion properties, competition against enteropathogens and modulation of IL-8 production

The human intestinal microbiota plays a pivotal role in human nutrition and health by promoting the supply of nutrients, preventing pathogen colonization and shaping and maintaining normal mucosal immunity. The depletion of the individual microbiota can result in a higher susceptibility to enteropathogenic bacteria infection. In order to reduce this risk, the use of food supplements containing probiotic bacteria has been recently addressed. In this paper, we investigate the protective role toward enteropathogen infection of probiotic strains belonging to Lactobacillus and Bifidobacterium. According to our experimental data, Lactobacillus acidophilus Bar13, L. plantarum Bar10, Bifidobacterium longum Bar33 and B. lactis Bar30 were effective in displacing the enteropathogens Salmonella typhimurium and Escherichia coli H10407 from a Caco-2 cell layer. Moreover, L. acidophilus Bar13 and B. longum Bar33 have been assessed for their immunomodulatory activity on IL-8 production by HT29 cells. Both strains showed the potential to protect enterocytes from an acute inflammatory response. These probiotic strains are potential candidates for the development of new functional foods helpful in counteracting enteropathogen infections.     

Adhesion of selected Bifidobacterium strains to human intestinal mucus and the role of adhesion in enteropathogen exclusion

The ability of potential probiotic strains to adhere to the intestinal mucosa and exclude and displace pathogens is of utmost importance for therapeutic manipulation of the enteric microbiota. The ability of seven selected human bifidobacterial strains and five human enteropathogenic strains to adhere to human intestinal mucus was analyzed and compared with that of four strains isolated from chicken intestines. The adhesion of the bifidobacterial strains ranged from 3 to 16% depending on the strain. Bifidobacterium strains of animal origin adhered significantly better than did strains of human origin. Of the pathogenic bacteria, Escherichia coli NCTC 8603 had the highest adhesion value (20%), Salmonella Typhimurium ATCC 29631, Enterobacter sakazakii ATCC 29544, and Clostridium difficile ATCC 9689 had adhesion values ranging from 10 to 15%, and Listeria monocytogenes ATCC 15313 had the lowest adhesive value (3%). The ability of these bifidobacteria to inhibit pathogen adhesion and to displace pathogens previously adhering to mucus was also tested. The inhibition of pathogens adhesion by these bifidobacterial strains was variable and clearly strain dependent. In general, bifidobacterial strains of animal origin were better able to inhibit and displace pathogens than were human strains. Preliminary characterization of bacterial adhesion was accomplished using different pretreatments to explore adhesion mechanisms. The results indicate that different molecules are implicated in the adhesion of bifidobacteria to the human intestinal mucus, constituting a multifactorial process.     

Short communication: effect of exopolysaccharide isolated from "viili" on the adhesion of probiotics and pathogens to intestinal mucus

The strong ropy character of the Scandinavian fermented milk viili is conferred by the exopolysaccharides (EPS) produced by lactococcal strains. These biopolymers can be responsible for some health benefits. We have assessed the influence of the EPS fraction isolated from commercial viili on the adhesion of some probiotics and pathogens to human intestinal mucus. Concentrations of viili EPS greater than 0.1 mg/mL promoted a decrease in adherence of Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus GG and this effect was dose-dependent. However, no modifications were detected on the adhesion levels of the pathogenic strains tested at a concentration of 1 mg/mL of EPS. Results obtained in the present work should be considered in the design of new probiotic products.     

Ability of Bifidobacterium strains with acquired resistance to bile to adhere to human intestinal mucus

The hydrophobicity and capacity to adhere to human intestinal mucus of Bifidobacterium strains with acquired resistance to bile were assessed and compared with those of their more sensitive original strains. The resistant variants used were previously obtained [Int. J. Food Microbiol. 82 (2003) 191; Int. J. Food Microbiol. 94 (2004) 79] by progressive adaptation of originally more sensitive strains to gradually increasing concentrations of bile. In five out of the seven groups of original and bile-resistant variants tested the resistant strains showed higher adhesion levels to human mucus (range between 1.4- and 4-fold) than their corresponding original strains. However, in the presence of physiologic concentrations of bile (0.3%, w/v) the adhesion level of all Bifidobacterium strains dropped between 7% and 74%, depending on the strain. In spite of this, the adhesion capability of three bile-resistant variants remained higher than that of their originals. Hydrophobicity evidenced considerable variability; in four out of the seven bile-resistant strains it was higher than in the original strains, although no direct correlation between adhesion and hydrophobicity could be established. It was concluded that the acquisition of bile resistance by our Bifidobacterium strains promoted changes in hydrophobicity and in the adhesion of these microorganisms to human intestinal mucus.     

Bacillus strains as human probiotics: characterization,safety, microbiome,and probiotic carrier

Food Science and Biotechnology - Both spore and vegetative forms of Bacillus species have been used as probiotics, and they have high stability to the surrounding atmospheric conditions such as...     

双歧杆菌对仔猪小肠黏液糖蛋白的粘附研究

通过扫描电镜观察细菌在仔猪肠黏膜的粘附状况，发现肠道茵群与黏膜表面的黏液层有密切联系。采用放射性代谢标记方法研究所拥有的双歧杆菌与仔猪肠黏膜黏液糖蛋白的粘附，发现青春双歧杆菌的粘附能力最强，为44．61%；其次是短双歧杆菌，为33．20％；婴儿双歧杆菌和长双歧杆菌分别是18．40％和11．78％。结果表明，所实验的糖、初乳粉以及婴儿双歧杆菌、长双歧杆菌、大肠杆菌和沙门氏茵上清均能导致青春双歧杆菌粘附下降，而青春双歧杆菌和短双歧杆菌上清却有促进其粘附的作用。     

人体肠道益生菌的作用及其应用

益生菌在人体肠道中定殖,并通过一系列代谢活动调节肠道微生态平衡,增强人体消化、免疫等功能.为了正确认识和应用益生菌从而达到增强人体健康的的益生作用,综述了近年来对益生菌功能的研究情况,及其在临床和食品上的主要应用,并对益生菌未来的研究方向提出了个人的观点和展望.     

Quality characteristics of yogurts fermented with short-chain fatty acid-producing probiotics and their effects on mucin production and probiotic adhesion onto human colon epithelial cells

Probiotics can ferment nondigestible carbohydrates and produce short-chain fatty acids (SCFA; acetate, propionate, and butyrate) in the human colon. In this study, the levels of SCFA were determined in the following yogurts fermented with different combinations of probiotics: (1) cocultures of Streptococcus thermophilus and Lactobacillus bulgaricus (control, C); (2) S. thermophilus, L. bulgaricus, and Bifidobacterium bifidum (C-Bb); (3) S. thermophilus, L. bulgaricus, and Lactobacillus acidophilus (C-La); and (4) S. thermophilus, L. bulgaricus, and Lactobacillus gasseri (C-Lg). Results showed that the acetate levels were significantly higher in C-Bb, C-La, and C-Lg yogurts than in C yogurt. Fermentation and physicochemical characteristics of all yogurts were identical. Treatment of mucus-secreting colon epithelial cells (HT29-MTX) with C-Bb, C-La, and C-Lg yogurt supernatants resulted in an increase in the expression of MUC2 and CDX2 and the production of mucin proteins. The adhesion of probiotics onto HT29-MTX cells increased following treatment with C-Bb, C-La, and C-Lg yogurt supernatants. Our data suggest that a yogurt diet rich in acetate improves the protective function of the intestinal epithelium.     

Screening probiotics from Lactobacillus strains according to their abilities to inhibit pathogen adhesion and induction of pro-inflammatory cytokine IL-8

Probiotics can be screened according to their abilities to inhibit pathogen adhesion and inhibit the production of pro-inflammatory cytokines. Eleven Lactobacillus strains isolated from traditional fermented dairy foods in Xinjiang, China, were studied for their potential to inhibit adhesion of Escherichia coli to intestinal epithelial cells and to inhibit E. coli–induced production of interleukin (IL)-8 by intestinal epithelial cells. The results showed that the 11 strains could inhibit adhesion of E. coli to Caco-2 cell monolayers and inhibit the induction of IL-8 production by E. coli in HT-29 cells. The inhibiting activities of the Lactobacillus strains against E. coli adhesion and IL-8 induction were strain-specific and not positively correlated, whereas the excluding activity of the strains against E. coli adhesion and their coaggregation with E. coli were positively correlated. The effector molecules of the strains with probiotic potential should be identified to explain the mechanism behind these observations.     

保持益生菌食品中益生菌活性的技术方法

益生菌食品是功能性食品中发展极快的一个领域。益生菌要对人体产生益生作用,必须在被食用时达到一定的活细胞数,并能在胃肠道中保持活性。如何保持益生菌食品中益生菌的活性,是食品工业面临的难题和研究热点。本文综述了目前国际上保持益生菌食品中益生菌活性的各种技术方法及研究思路,并对今后的研究方向进行了展望。     

两株具有潜在益生作用的人源链球菌的安全性评价

对分离于健康儿童口咽部的2株对金黄色葡萄球菌等常见致病菌具有广谱拮抗作用的甲型链球菌菌株1-4和4-2的安全性进行评价,为其作为呼吸道益生菌产品的研发和制备提供安全性依据.通过给予SPF级小鼠口服含有菌株1-4和4-2不同剂量的菌液的急性毒性实验以及耐药性的测定,观察动物是否出现死亡,急性毒性体征,细菌移位生长等情况....     

日本益生菌、益生元市场与应用概况

为了大力推广益生菌和益生元,介绍了日本益生菌制品、益生元、灭活细胞粉及对它们的管理情况。其中,日本厚生劳动省批准了65种特定保健食品,允许使用了近20种菌株．同时,各种功能性低聚糖已广泛使用在400多种食品、保健食品以及宠物饲料中,并说明了除益生菌活菌体外,益生菌或肠道正常菌群的灭活细胞,在大量存在下也可和活菌体一样具有免疫增强作用和发挥益生元的功能的作用。     

Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells

The survival of four strains of lactic acid bacteria in human gastric juice, in vivo and in vitro, and in buffered saline, pH 1 to 5, has been investigated. The strains studied include two Lactobacillus acidophilus strains, Lactobacillus bulgaricus, and Streptococcus thermophilus. In addition, the adhesion of these strains to freshly collected human and pig small intestinal cells and to pig large intestinal cells has been studied and the effect of milk on both survival and adhesion tested. As a result of these investigations, an in vitro test system for screening potential cultures for use as human dietary adjuncts can be developed. The ability to survive in gastric juice and to adhere varied significantly for the strains tested; L. acidophilus ADH survived and adhered better than the others while S. thermophilus survived and adhered poorly. For all strains, both survival and adhesion was enhanced by milk. As all strains adhered to some extent to both human and pig intestinal cells, the adhesion mechanism is probably a nonspecific attachment as opposed to other reported specific Lactobacillus adhesion to gastric tissue. From the survival and adhesion data it seems feasible to obtain elevated levels of viable Lactobacillus sp. in human intestine by careful selection of the bacterial strains ingested. Furthermore, the in vitro methods used here should be valuable to screen potential strains. The data presented here can then be correlated with human in vivo studies monitoring the beneficial effect of ingestion of these Lactobacillus.     

Adhesion and competitive inhibition and displacement of human enteropathogens by selected lactobacilli

Competition with pathogens for adhesion and colonization of the mucosal surfaces are possible protective mechanisms of probiotics. Here, we assessed the adhesive properties and the ability to inhibit the adhesion and to displace pathogens of three selected Lactobacillus strains using Caco-2 cells and a human intestinal mucus model. The adhesion levels showed a great variability, ranging between 1% and 9.7% in the mucus model and between 7.7 and 61.1 bacterial cells/microscopy view in the Caco-2 model. A good correlation between both models was observed. The ability to inhibit the adhesion or to displace adhered pathogens was variable depending on both the lactobacilli and the pathogen tested. The inhibition and displacement profiles were very different suggesting that different mechanisms are implied in both processes. A very high specificity in the inhibition of the adhesion and displacement of enteropathogens by lactobacilli was observed indicating the need for a case-by-case assessment in order to select strains with the ability to inhibit or displace a specific pathogen.     

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion, which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and R1 stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS R1 also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10–24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS R1 and with the increases in Bacteroides in cultures with both microbial EPS (R1 and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids.