In vitro evaluation of Lactobacillus gasseri strains of infant origin on adhesion and aggregation of specific pathogens

Numerous Lactobacillus species are members of the normal healthy human intestinal microbiota, and members of the Lactobacillus family predominate among the current marketed probiotic strains. Most of the current commercial probiotic strains have not been selected for specific applications but rather have been chosen based on their technological properties. Often the ability of such strains to temporarily colonize the gastrointestinal tract may be lacking, and the interactions with intestinal microbiota are few. Furthermore, the competitive exclusion properties of potential probiotic bacteria are strain specific and vary greatly. Thus, it is highly desirable that new candidate probiotic isolates originate from the healthy target population. In this study, seven newly isolated strains of Lactobacillus gasseri originating from feces of a healthy newborn child were evaluated for their ability to adhere to intestinal mucus, to autoaggregate and coaggregate with the model pathogens Cronobacter sakazakii (ATCC 29544) and Clostridium difficile (1296). All the bacterial strains, single or in combination, in viable and nonviable forms, were able to autoaggregate. The coaggregation with C. sakazakii or C. difficile was higher (P < 0.05) in nonviable than in the viable forms. Single L. gasseri strains showed similar adhesion abilities to intestinal colon mucus. The seven L. gasseri strains when combined were also able to significantly compete with, displace, and inhibit the adhesion of C. sakazakii and C. difficile in the mucus model. This study demonstrates that the studied L. gasseri strains fulfill the basic adhesion and aggregation properties for probiotics and could be considered for potential future use in children.     

Adhesion properties and competitive pathogen exclusion ability of bifidobacteria with acquired acid resistance

The adhesion properties of Bifidobacterium longum and Bifidobacterium catenulatum strains with an acquired resistance to acid and their ability to competitively exclude Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, Enterobacter sakazakii, and Clostridium difficile from adhering to human intestinal mucus were evaluated and compared with the results when the same experiments were run with the original acid-sensitive strains. In half of the four studied cases, the acid-resistant derivative showed a greater ability to adhere to human intestinal mucus than the original strain. The ability of bifidobacteria to inhibit pathogen adhesion to mucus was not generally improved by the acquisition of acid resistance. In contrast, three of the four acid-resistant strains showed a greater ability to displace preadhered pathogens than the original strains, especially preadhered Salmonella Typhimurium and C. difficile. Overall, the induction of acid resistance in bifidobacteria could be a strategy when selecting strains with enhanced stability and improved surface properties that favor their potential functionality as probiotics against specific pathogens.     

Exopolysaccharides produced by probiotic strains modify the adhesion of probiotics and enteropathogens to human intestinal mucus

Exopolysaccharides (EPSs) are exocellular polymers present in the surface of many bacteria, including Lactobacillus and Bifidobacterium. The genome sequence of several strains revealed the presence of EPS-encoding genes. However, the physiological role that EPSs play in the bacterial ecology still remains uncertain. In this study, we have assessed the effect of EPSs produced by Lactobacillus rhamnosus GG, Bifidobacterium longum NB667, and Bifidobacterium animalis IPLA-R1 on the adhesion of probiotic and enteropathogen strains to human intestinal mucus. The EPS fraction GG had no significant effect on the adhesion of L. rhamnosus GG and B. animalis IPLA-R1. However, the EPS fractions NB667 and IPLA-R1 significantly reduced the adherence of both probiotic strains. In contrast, the three EPS fractions increased the adhesion of Enterobacter sakazakii ATCC 29544 and Escherichia coli NCTC 8603. Higher adherence of Salmonella enterica serovar Typhimurium ATCC 29631 and Clostridium difficile ATCC 9689 was detected in the presence of the EPS fractions GG and NB667. In general, these effects were obtained at EPS concentrations of up to 5 mg/ml, and they were EPS dose dependent. The competitive exclusion of probiotics in the presence of EPS could suggest the involvement of these biopolymers in the adhesion to mucus. The increase in the adherence of enteropathogens could be explained if components of the pathogen surface are able to bind to specific EPSs and the bound EPSs are able to adhere to mucus. To the best of our knowledge, this is the first work reporting the effect of EPSs from probiotics on bacterial adhesion properties.     

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.     

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.     

Performance of bifidobacteria in oat-based media

Thirteen Bifidobacterium strains were isolated form the faeces of healthy elderly subjects. The ability of the isolated strains to acidify and survive in an oat-based medium was assessed and compared to a commercial starter Bifidobacterium strain. Since adhesion to the intestinal mucosa is one of the main selection criteria for potential probiotics, the influence of the oat medium on the ex vivo adhesion of the isolates to both human colonic tissue and to mucus from elderly subjects was assessed. Seven of the fourteen strains survived the 24-h fermentation, six of these acidified the medium to below pH 5. Supplementation of the medium with soy protein had a negative effect on survival and the acidification. The strains isolated from the elderly subjects were found to adhere in high levels to immobilised intestinal mucus from elderly. However, their adhesion to colonic tissue was significantly lower. Prior exposure to the oat-based medium increased the adhesion of two of the strains.     

Adhesion and aggregation properties of probiotic and pathogen strains

Autoaggregation has been correlated with adhesion, which is known to be a prerequisite for colonization and infection of the gastrointestinal tract by many pathogens. The coaggregation properties of probiotic strains with pathogens as well as their ability to displace pathogens are of importance for therapeutic manipulation of the aberrant intestinal microbiota. Consequently, the ability to aggregate and coaggregate are desirable properties for probiotics in health-promoting foods. Aggregation assays and bacterial adhesion to hydrocarbons (BATH test) demonstrated significant differences in cell surface properties among the tested commercial probiotic strains. Hydrophobicity increased when the cells were heat-inactivated. All probiotic strains tested showed aggregation abilities with the pathogen strains tested, but the results were strain-specific and dependent on time and incubation conditions. Our results indicate that the ability to autoaggregate, together with cell-surface hydrophobicity and coaggregation abilities with pathogen strains can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human or animal use.     

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.     

Respiratory burst activity in activated and unstimulated isolated bovine blood neutrophils during experimentally induced Escherichia coli mastitis.

The respiratory burst activity, measured as H2O2 production, of isolated bovine polymorphonuclear leucocytes (PMN) was evaluated during experimentally induced Escherichia coli mastitis by means of flow cytometry in cells activated by phorbol 12-myristate 13-acetate (PMA) and in unstimulated cells. As expected, a significantly reduced respiratory burst activity was observed in PMA-activated PMN 18 h after intramammary inoculation with Escherichia coli. At this time only 75% of the PMA-activated PMN showed a respiratory burst, but with a higher intensity than that measured before and later after infection with Esch. coli. In addition, an increase in the respiratory burst activity was observed in unstimulated blood PMN during a short period at 18 h after infection, when up to 30% of the unstimulated PMN had a respiratory burst activity. The increase in the respiratory burst intensity of PMA-activated PMN and the spontaneously augmented production of reduced oxygen species by the unstimulated PMN during infection with Esch. coli might indicate the production of a natural stimulator of burst activity in circulation, most probably originating from the inflamed udder.     

The effect of prebiotics on adherence of probiotics

Prebiotics are generally considered to promote the function or viability of probiotics via their fermentation, but their effect on the adherence of probiotics is still unclear. In this study, we examined the effect of 4 commercially available prebiotics [Orafti GR, Orafti P95, and Orafti Synergy (Beneo GmbH, Mannheim, Germany), and Vivinal (Friesland Foods Domo, Amersfoort, the Netherlands)] and 3 simple saccharides (glucose, galactose, and lactose) on the adherence of 5 probiotic type strains, 2 lactococci starter cultures, and 5 potential dairy probiotic strains from the Culture Collection of Dairy Microorganisms (Tábor, Czech Republic). Adherence was tested in microtiter plates on the following types of substrate: polystyrene alone and polystyrene coated with either porcine mucus or cocultures of the human colon cell lines Caco2 and HT29-MXT (1:9 ratio of HT29-MXT:Caco2). Adherence was evaluated as a change in fluorescence in the well of a microtiter plate. The most commonly observed effect (with a few exceptions) of prebiotics was decreased adherence of the tested strains observed on all types of substrate. The tested saccharides, which are part of the residual compounds of the used prebiotics, had a very similar effect—eliciting a decrease in adherence ability in the majority of the probiotic strains.     

益生性乳酸菌黏附性研究进展

综述了益生性乳酸菌的功能作用,及乳酸菌在人体肠道中的黏附、定居、抑菌等与人体肠道黏膜组成的主要化学成分的相关性。详述了近些年来有关乳酸菌之所以能在人体内定居的黏附性的研究,如黏附素、黏附受体、影响黏附素的影响因子等,以期有助于高黏附益生性乳酸菌株的选育及其益生菌制剂制备。     

The Effect of Digestive Enzymes on the Adhesion of Probiotic Bacteria In Vitro

ABSTRACT: Specific probiotics have several clinically proven health effects. Adhesion to the intestinal mucosa is considered important for many of these effects. In the current study, the effect of digestive enzymes and bile on the adhesion to intestinal mucus of 5 selected probiotics was studied. All of the digestive enzymes affected the adhesion of at least some of the tested strains. Bile was observed to reduce the adhesion of all strains tested. When the strains were sequentially exposed to 3 enzyme preparations and bile, the level of adhesion was reduced for all strains. The results suggest that the selection criterion "adhesion" for probiotics should be further refined.     

Characterisation of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11 and its non-EPS producing derivative strains as potential probiotics

Traditional fermented foods are the best source for the isolation of strains with specific traits to act as functional starters and to keep the biodiversity of the culture collections. Besides, these strains could be used in the formulation of foods claimed to promote health benefits, i.e. those containing probiotic microorganisms. For the rational selection of strains acting as probiotics, several in vitro tests have been proposed. In the current study, we have characterized the probiotic potential of the strain Lactobacillus paraplantarum BGCG11, isolated from a Serbian soft, white, homemade cheese, which is able to produce a "ropy" exopolysaccharide (EPS). Three novobiocin derivative strains, which have lost the ropy phenotype, were characterized as well in order to determine the putative role of the EPS in the probiotic potential. Under chemically gastrointestinal conditions, all strains were able to survive around 1-2% (10(6)-10(7)cfu/ml cultivable bacteria) only when they were included in a food matrix (1% skimmed milk). The strains were more resistant to acid conditions than to bile salts and gastric or pancreatic enzymes, which could be due to a pre-adaptation of the parental strain to acidic conditions in the cheese habitat. The ropy EPS did not improve the survival of the producing strain. On the contrary, the presence of an EPS layer surrounding the strain BGCG11 hindered its adhesion to the three epithelial intestinal cell lines tested, since the adhesion of the three non-ropy derivatives was higher than the parental one and also than that of the reference strain Lactobacillus rhamnosus GG. Aiming to propose a potential target application of these strains as probiotics, the cytokine production of peripheral blood mononuclear cells (PBMC) was analyzed. The EPS-producing L. paraplantarum BGCG11 strain showed an anti-inflammatory or immunosuppressor profile whereas the non-ropy derivative strains induced higher pro-inflammatory response. In addition, when PBMC were stimulated with increasing concentrations of the purified ropy EPS (1, 10 and 100μg/ml) the cytokine profile was similar to that obtained with the EPS-producing lactobacilli, therefore pointing to a putative role of this biopolymer in its immune response.     

Potential probiotic characteristics of Lactobacillus and Enterococcus strains isolated from traditional dadih fermented milk against pathogen intestinal colonization

Traditional fermented buffalo milk in Indonesia (dadih) has been believed to have a beneficial impact on human health, which could be related to the properties of the lactic acid bacteria (LAB) involved in its fermentation process. In previous studies, it was discovered that strains of dadih lactic isolates possessed some beneficial properties in vitro. In the present study, the adhesion capacity of specific LAB isolates from dadih to intestinal mucus was analyzed. Further, the ability to inhibit model human pathogens and displace them from mucus was assessed. The adhesion of tested LAB strains was strain-dependent and varied from 1.4 to 9.8%. The most adhesive Lactobacillus plantarum strain was IS-10506, with 9.8% adhesion. The competition assay between dadih LAB isolates and pathogens showed that a 2-h preincubation with L. plantarum at 37 degrees C significantly reduced pathogen adhesion to mucus. All tested LAB strains displaced and inhibited pathogen adhesion, but the results were strain-specific and dependent on time and pathogen strains. In general, L. plantarum IS-10506 showed the best ability against pathogen adhesion.     

益生菌对2型糖尿病小鼠的调节作用

目的:研究益生菌对2型糖尿病小鼠的降糖效果以及潜在的降糖机制。方法:以实验室34株益生菌为研究对象,利用α-葡萄糖苷酶抑制活性筛选具有降糖功效的菌株,并通过体外表面疏水性、自聚性衡量菌株益生特性,将筛选出的目标益生菌应用于动物模型,探究其降糖功效及潜在机制。采用高脂饮食结合链脲佐菌素方法建立2型糖尿病小鼠模型,所有小鼠连续灌胃8周后,检测其血糖水平、葡萄糖耐受量、糖化血红蛋白、胰岛素水平及胰岛素抵抗状况、血清炎症因子水平、胰高血糖素样肽-1以及粪便中短链脂肪酸含量。结果:在体外筛选实验中,副干酪乳杆菌J5和干酪乳杆菌K11有着良好的抑制α-葡萄糖苷酶活性和肠道黏附能力。动物实验中,干酪乳杆菌K11能够显著降低小鼠的血糖水平(P<0.05),改善糖耐量受损以及胰岛素抵抗(P<0.05);显著降低小鼠血清中肿瘤坏死因子-α和白细胞介素-6含量;明显提高血清中胰高血糖素样肽-1水平和粪便中短链脂肪酸的含量(P<0.05)。结论:干酪乳杆菌K11可显著调节2型糖尿病小鼠的血糖,作用机制可能与其调节肠道菌群产物短链脂肪酸、促进胰高血糖素样肽-1分泌并调节炎症因子有关。     

In vitro probiotic evaluation of potential antioxidant lactic acid bacteria isolated from idli batter fermented with Piper betle leaves

Lactic acid bacteria isolated from food sources can be introduced as probiotics because of their health‐promoting and nonpathogenic characteristics. Eight lactic acid bacteria from idli batter fermented with Piper betle leaves were selected to screen the antioxidant potential and biofilm‐forming ability. Five isolates exhibited good antioxidant potential and biofilm formation and showed antagonistic activity against the pathogenic biofilm‐forming Staphylococcus aureus. Further, in vitro probiotic properties of five isolates exhibited excellent acid tolerance, bile tolerance, simulated gastrointestinal juice tolerance, auto‐aggregation and co‐aggregation and hydrophobicity and showed resistance to antibiotics such as ciprofloxacin, nalidixic acid, norfloxacin and colistin. Isolates also showed positive bile salt hydrolase activity, cholesterol assimilation, β‐galactosidase production and mucin binding ability. The isolate KJBB10 exhibited good adhesion and pathogen exclusion percentage with human colon cancer cells (HCT‐15 and HT‐29). Hence, the isolates KJBB10, KJBB56 and KJBC06 can be used as beneficial probiotic starter cultures for the formulation of functional foods.     

Probiotic properties of lactic acid bacteria isolated from Mukeunji, a long-term ripened kimchi

Lactic acid bacteria (LAB) isolated from mukeunji was screened for characterization of beneficial properties required as probiotics. Among the isolates from mukeunji, 5 LAB strains were selected due to their high levels of tolerance to simulated gastric fluid. These strains were identified by using SDS-PAGE of whole cell protein pattern and 16S rDNA sequencing. Subsequently, it was found that 2 isolates, Lactobacillus brevis C1 and Lactobacillus plantarum G4 among the 5 isolates were highly resistant to bile cytotoxicity in terms of survival rate, 162.2 and 114.3%, respectively. According to the results of in vitro adhesion assay, Lb. brevis C1, Lb. plantarum G4, and Lactobacillus sakei N1 had higher numbers of adhesion to Caco-2 epithelium cells than that of Lactobacillus rhamnosus GG, a well known probiotic. None of the isolates produced a carcinogenic enzyme, β-glucuronidase. In addition, Lb. brevis C1 and Lb. plantarum G4 exhibited high levels of DPPH scavenging capacity. These results suggest that 2 isolates from mukeunji, Lb. brevis C1 and Lb. plantarum G4 may have potential for food products as probiotics because they showed high levels of bile acid tolerance and probiotic properties.     

Health and growth of veal calves fed milk replacers with or without probiotics

Four experiments with 1-wk-old veal calves were conducted to assess the influence of probiotics on growth and health indicators. In experiments 1 and 2, the liquid probiotic supplements were administered daily from experimental d 1 to 15. The treatment period in experiments 3 and 4 was extended to 56 d. The probiotics used were a multispecies probiotic (MSPB) containing different probiotic species of human origin, or a calf-specific probiotic (CSPB) containing 6 Lactobacillus species isolated from calf feces and selected on the basis of a combination of characteristics.When the data for the 4 experiments were pooled, the probiotics enhanced growth rate during the first 2 wk. During the 8-wk experimental period, average daily gain and feed efficiency were significantly improved in the probiotic-treated groups. The MSPB-induced increase in weight gain was greater when the control calves were considered less healthy based on a health score (an index of diarrhea and therapeutic treatments). Probiotic treatment tended to diminish mortality. The CSPB treatment reduced the incidence of diarrhea and the fecal counts of coliforms. When therapeutic treatment was intensive in the control calves, the ingestion of probiotics reduced the percentage of calves that required therapy and the amount of treatments needed against digestive or respiratory diseases. There was no clear difference in the efficiency of the MSPB and CSPB preparations. Further research is necessary to identify underlying mechanisms and to evaluate the potential of probiotics to improve respiratory health in veal calf production.     

Subtractive screening for probiotic properties of lactobacillus species from the human gastrointestinal tract in the search for new probiotics

ABSTRACT:  In the search for new probiotics, 61 Lactobacillus spp. isolates, belonging to 12 species and isolated as dominant lactic acid bacteria from the feces of healthy humans, were subjected to a subtractive system of in vitro analyses, which included desirable and undesirable traits. Twenty-four isolates were able to grow in 2% bovine bile, of which 13 grew in acidified broth at pH 3.5 in acidified cysteine-containing MRS broth. Intrinsic resistance to certain antimicrobial agents (cefoxitin, metronidazole, vancomycin) was observed in most isolates, but atypical resistances to erythromycin, clindamycin, or tetracycline were also found in 5 strains. Undesirable traits such as α-chymotrypsin or N-acetyl-β-glucosaminidase activities were not detected, but low β-glucuronidase and moderate β-glucosidase activities were recorded in 2 strains. Two Lactobacillus gasseri and 2 Lactobacillus paracasei selected strains inhibited several intestinal pathogens in an agar spot test, including strains of Escherichia coli , Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus . They also adhered to human Caco-2 and HT-29 epithelial cells in a manner comparable to Lactobacillus rhamnosus strain GG, and were unable to degrade pig gastric mucin in a plate assay. Together, these results suggest these 4 strains to be good probiotic candidates, concluding that the subtractive screening devised in this work could be a valuable tool in large-scale surveys for probiotics.