乳酸菌在肠道内黏附定殖的研究进展

乳酸菌是一类无芽孢的革兰氏阳性菌,能利用碳水化合物发酵产酸。由于其具有安全高效,易获取的优势,在食品、医疗及动物生产方面得到广泛运用。在动物肠道内,它利用糖类物质,生成酸性代谢物,降低肠道pH,改善肠道微生物环境。要实现乳酸菌在动物体内的良好定殖,首先要实现黏附,而黏附过程主要分为特异性结合与非特异性结合。黏附机制的相关研究中黏附素理论被普遍认可,包含了粘附蛋白学说、磷璧酸学说及细菌表面分子学说。本文就乳酸菌的黏附相关因子,体外黏附模型及黏附的影响因素做了阐述。     

Assuring the continued safety of lactic acid bacteria used as probiotics

Lactic acid bacteria have long been used to improve the safety of foods through fermentation. Some fermented products were also early used for their perceived health benefits, which lead to the development of probiotics as we now know them. Probiotics mainly belong to the genera Lactobacillus and Bifidobacterium. Most members of these genera are not considered pathogens or even opportunistic pathogens. Nevertheless, rare cases of Lactobacillus and Bifidobacterium infection have been reported, possibly even associated with the consumption of probiotic products. Such cases are extremely rare and the subjects always had severe underlying conditions most often affecting the immune system. There does not seem to be any risk for the general population. Safety assessments can be performed and many possible tests exist. It is, however, not certain these tests will prevent rare case of Lactobacillus infection in certain high-risk patients. The benefits of probiotic use should be weighed against the possible small risk. Such an evaluation will, in most cases, be favourable and should therefore not discourage consumption of probiotics. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Screening lactic acid bacteria from swine origins for multistrain probiotics based on in vitro functional properties

Lactic acid bacteria originated from swine feces and intestines were selected for potential probiotics based on their bile-salt resistance, low pH tolerance, potential adhesion to epithelial cells and especially functional properties, including production of antimicrobial substances, bile-salt hydrolase (BSH) and amylolytic activity. Results showed 7 isolates with antimicrobial activity, 5 with BSH activity and 3 with amylolytic activity were preliminarily selected from 485 lactic acid bacteria based on their highest potential with functional properties in vitro. The 15 isolates were further assayed on the essential characteristics as potential probiotics. All isolates were fully tolerant to 0.3% bile salts and 11 of them were able to resist pH 3 for 3 h without loss of viable cells. The eleven isolates were then evaluated on their adhesion capability. Wide variation in the hydrophobic character and specific adhesion efficiency was observed and three isolates G1-1, G22-2 and G8-5, with respective antimicrobial, BSH and amylolytic activities were finally selected. In addition, the three isolates were compatible in the coexistence assay. Isolate G1-1 was identified as Lactobacillus salivarius by API system and a 16S rRNA gene sequence analysis. Both G8-5 and G22-2 showed the closest homology to Lactobacillus reuteri according to their 16S rRNA gene sequences (99%). From the study, the three Lactobacilli strains were shown to share the functional properties necessary for probiotics use in animal additives. Their compatibility with respective in vitro activities was expected to show enhanced in vivo efficacy after combination for multistrain probiotics use.     

On the screening of hydrogen peroxide-generating lactic acid bacteria

The published plate methods for the detection of hydrogen peroxide-producing lactic acid bacteria, which employ horseradish peroxidase and a chromogen, clearly fail to detect all the organisms that produce H₂O₂. Whilst keeping the same principle, the use of a novel growth medium and of the chromogen tetramethyl-benzidine allows the detection of H₂O₂ production by strains previously classified as non-producers.     

In vitro screening of probiotic lactic acid bacteria and prebiotic glucooligosaccharides to select effective synbiotics

Probiotics and prebiotics have been demonstrated to positively modulate the intestinal microflora and could promote host health. Although some studies have been performed on combinations of probiotics and prebiotics, constituting synbiotics, results on the synergistic effects tend to be discordant in the published works. The first aim of our study was to screen some lactic acid bacteria on the basis of probiotic characteristics (resistance to intestinal conditions, inhibition of pathogenic strains). Bifidobacterium was the most resistant genus whereas Lactobacillus farciminis was strongly inhibited. The inhibitory effect on pathogen growth was strain dependent but lactobacilli were the most effective, especially L. farciminis. The second aim of the work was to select glucooligosaccharides for their ability to support the growth of the probiotics tested. We demonstrated the selective fermentability of oligodextran and oligoalternan by probiotic bacteria, especially the bifidobacteria, for shorter degrees of polymerisation and absence of metabolism by pathogenic bacteria. Thus, the observed characteristics confer potential prebiotic properties on these glucooligosaccharides, to be further confirmed in vivo, and suggest some possible applications in synbiotic combinations with the selected probiotics. Furthermore, the distinctive patterns of the different genera suggest a combination of lactobacilli and bifidobacteria with complementary probiotic effects in addition to the prebiotic ones. These associations should be further evaluated for their synbiotic effects through in vitro and in vivo models.     

Stimulatory effect of honey on multiplication of lactic acid bacteria under in vitro and in vivo conditions

The effect of honey and sucrose on lactic acid bacteria in vitro and in rat gut was studied to determine whether these organisms were affected differently by honey compared with sucrose. Under in vitro conditions, the number of Lactobacillus acidophilus and Lactobacillus plantarum counts increased 10-100 fold in the presence of honey compared with sucrose. Feeding of honey to rats also resulted in significant increase in counts of lactic acid bacteria. Although there was no significant difference in the counts of lactic acid bacteria in the small and large intestines of different groups, the honey-fed group showed a significant increase (P<0.05) in counts over the control and sucrose-fed animals. The results support the fact that consumption of honey has a beneficial effect on the physiological constitution of animals fed with it.     

In vitro growth characteristics of five candidate aquaculture probiotics and two fish pathogens grown in fish intestinal mucus

The selection of probiotics for aquaculture is usually based on their antagonism towards pathogens. However, other criteria such as growth, attachment to intestinal mucus and production of beneficial compounds should also be considered. We suggest a protocol for the isolation and selection of potential probiotic bacteria based on their in vitro growth characteristics and propose a ranking index (RI) to screen potential aquaculture probionts. We suggest that the lag period and doubling time are the most important criteria for the comparison of growth curves, hence the RI is based on the doubling time (t(d)) and lag period (lambda) obtained from the growth profile of each bacterium. Bacteria were isolated from the gut of the common clownfish, Amphiprion percula, and screened for antagonistic activity towards seven aquatic pathogens. All five candidate probiotics showed antagonism to various aquatic pathogens. When grown in intestinal fish mucus no probiotic had a RI higher than the two tested pathogens (Aeromonas hydrophila and Vibrio alginolyticus). However, candidate probiont AP1 had a faster specific growth rate (micro) (0.05) than the pathogens (0.049 and 0.047 respectively), while AP5 grown in marine broth had a shorter lag period than the pathogens. Strategies to increase probiotic concentration include the inoculation of high concentrations and the preconditioning of these bacteria to reduce the lag period. It should be tested whether or not such strategies will allow the probiotic bacteria to dominate initially and thereby gain a competitive advantage. This could become an important aspect under in vivo conditions where both attachment and nutrient supply differ from that found in in vitro studies.     

Identification and characterization of starter lactic acid bacteria and probiotics from Columbian dairy products

AIMS: Considering the significant rise in the probiotic market in Columbia, and given the lack of reports concerning the microbial population and strain performance in products from different producers, this study aims at determining the number of viable starter bacteria and probiotics in bio-yoghurts available at the Columbian market, identifying the species and analysing the performance of the isolated strains in bile acid resistance, antagonistic activity against pathogens, and adherence capacity to human intestinal epithelial cells. METHODS AND RESULTS: Seven bio-yoghurts were analysed for the bacterial species present. Species identification was carried out using 16S rRNA gene targeted PCR. The cultured bacteria were tested for bile acid resistance, adherence to a human intestinal epithelial cell line, and antagonism against the pathogen Salmonella enterica serovar Typhimurium. A total of 17 different strains were identified. Based on plate counting, all bio-yoghurts have at least total viable cells of approximately 10(7) CFU ml(-1). Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were the most frequently isolated bacteria. Viable Bifidobacterium was only recovered from one product. However, after PCR analysis, DNA of this genus was confirmed in five out of seven products. Major differences were found for S. typhimurium antagonism. The adherence capacity to Caco-2 cells was observed in 10 of the isolated strains. In general, low survival to simulated gastric juice was observed. CONCLUSIONS: Some of the isolated strains have probiotic potential, although not all of them were present in the advised amount to exert beneficial health effects. However, the full correct scientific name of the isolated bacteria and their viable counts were not included on the product label. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the identification and functionality of starter bacteria and probiotics present in dairy products on the Columbian market.     

耐乙酸乳杆菌的筛选及其产乳酸特性

【背景】耐受乙酸的乳酸菌是传统谷物醋醋酸发酵过程中产生乳酸及其风味衍生物的重要功能微生物。【目的】从镇江香醋醋醅中分离鉴定具有耐乙酸特性的乳酸菌,并评价不同条件下该菌株的产乳酸能力。【方法】利用4%(体积比)乙酸含量的MRS培养基分离耐乙酸乳酸菌;对其进行16S rRNA基因鉴定、基因组测序、形态观察以及生理生化特性研究;考察不同乙酸浓度、葡萄糖浓度、发酵温度和时间对菌株产乳酸能力的影响。【结果】分离得到一株可耐受6%乙酸的乳杆菌Lactobacillus sp. JN500903;在厌氧静置、接种量5%、乙酸浓度5%、葡萄糖浓度40 g/L、发酵温度37°C、发酵时间10 d条件下,该菌株乳酸产量为16.1 g/L。【结论】乳杆菌JN500903能够耐受6%乙酸浓度,具有在酸性环境下合成乳酸的能力,有一定的应用潜力。     

Intestinal mucus alters the ability of probiotic bacteria to bind aflatoxin B1 in vitro

Several probiotics are known to bind aflatoxin B(1) (AFB(1)) to their surfaces and to adhere to intestinal mucus. In this study, preincubation of two probiotic preparations with either AFB(1) or mucus reduced the subsequent surface binding of mucus and AFB(1), respectively, in a strain-dependent manner.     

The adhesion of lactic acid bacteria to the intestinal epithelium of agricultural animals

The lactic acid bacteria isolated from different ecological niches are established to be able to adhere in vitro to the intestine epithelium of pigs and cattle. The studied strains of enterococci have a higher index of adhesiveness (5.7-2.24) as compared to lactic acid bacilli (3.34-1.08). Detection of the lactic acid bacteria ability to adhere to intestine enterocytes of agricultural animals served as a premise to use those bacteria when constructing preparations of the directed action on the basis of living cultures.     

清香型白酒中乳酸菌和酵母菌的相互作用

【目的】探究清香型白酒中不同乳酸菌和酵母菌的相互作用,了解不同菌株的发酵性能,为更深入地认识白酒发酵机理、实现发酵过程优化提供理论基础。【方法】利用程序控温和固态发酵模拟清香型白酒酿造环境,测定纯培养和共培养中菌株的理化指标、活菌数以及主要代谢产物的变化。【结果】Saccharomyces cerevisiae YJ1糖消耗快产乙醇和酯类物质多,Lactobacillus plantarum JMRS4糖消耗快产酸较多。共培养中乳酸菌对Saccharomyces cerevisiae YJ1的生长和产乙醇抑制较大,对Candida aaseri MJ7产乙醇几乎无影响。乳酸菌对Pichia kudriavzevii MJ14的生物量和乙醇代谢抑制作用较小,还对其产己酸乙酯、乙酸乙酯和异戊醇等代谢产物有促进作用;而反过来Pichia kudriavzevii MJ14对3株乳酸菌产乳酸均有抑制作用,对产乙酸则有促进作用。【结论】建立了一种固态培养方法,结合清香型白酒发酵温度变化规律,有效模拟了实际发酵环境。Pichia kudriavzevii MJ14在与乳酸菌共培养中受到的抑制较小并能有效抑制乳酸菌产乳酸,Saccharomyces cerevisiae YJ1能代谢产生多种风味物质,对清香型白酒酿造有重要意义。     

乳酸菌与酵母菌共生机理的研究进展

早期研究者从营养角度分析乳酸菌与酵母菌的共生机制,发现混合培养过程中乳酸菌与酵母菌不仅存在互补机制,代谢产物相互之间能够产生促进或抑制作用。随着Lux S/AI-2介导的群体感应现象(QS)的发现与发展,目前研究者多从群体感应角度来探讨乳酸菌与酵母菌种间的信息交流模式。本文着重从营养及信号分子这两个角度阐述目前乳酸菌与酵母菌共生机理的研究进展。     

两种乳酸菌胞外多糖对人体粪便菌群的影响

目的 在体外利用7名志愿者的新鲜粪便作为来源,选择菊粉作为对照,研究干酪乳杆菌LC2W与鼠李糖乳杆菌Y37胞外多糖对人体肠道菌群的影响.方法 利用PCR-DGGE(变性梯度凝胶电泳)方法分析样品中菌群组成,高效液相色谱分析培养前后胞外多糖组分变化.结果 与菊粉相比,粪便菌群在添加乳杆菌胞外多糖培养后,Parabacteroides类群细菌明显增加,4名志愿者粪便样品中出现明显的双歧杆菌条带,两种乳杆菌胞外多糖对粪便菌群的影响差异无统计学意义;而添加菊粉培养后,Bacteroides类群细菌的条带明显增加.两种乳杆菌胞外多糖中主要是大分子量的部分被利用.结论 干酪乳杆菌LC2W与鼠李糖乳杆菌Y37的胞外多糖在体外有调节粪便菌群的作用.     

Assessment of the probiotic potential of lactic acid bacteria isolated from kefir grains: evaluation of adhesion and antiproliferative properties in in vitro experimental systems

The main objective of this study was to isolate lactic acid bacteria from kefir grains and investigate their probiotic potential. In this study, 48 bacterial strains were isolated from kefir grains, whereas 39 strains were categorized to the genus Lactobacillus. Evaluation of the probiotic potential of the isolated stains was performed, including resistance to low pH, tolerance to pepsin, pancreatin and bile salts, and antibiotic resistance. In addition, evaluation of adhesion and antiproliferative properties in in vitro experimental systems was also conducted. Strains SP2 and SP5 that displayed the best performance in the conducted in vitro tests were selected for further studies. Firstly, genotypic identification of the two strains was performed by partial 16S rRNA gene sequencing, BLAST analysis, and species-specific multiplex PCR assay. The two strains were confirmed to be Pediococcus pentosaceus SP2 and Lactobacillus paracasei SP5. Then, the adhesion properties of the two strains were examined in vitro. Both strains displayed substantial adherence capacity to HT-29 human colon cancer cells. Moreover, a significant decrease of HT-29 cell growth after treatment with viable P. pentosaceus SP2 or L. paracasei SP5 was recorded. In addition, downregulation of anti-apoptotic genes and over-expression of cell cycle–related genes was recorded by real-time PCR analysis. Treatment with conditioned media of the two strains also caused significant reduction of cancer cell proliferation in a time- and concentration-dependent manner. P. pentosaceus SP2 and L. paracasei SP5 displayed the best probiotic properties that exerted substantial adherence on human colon cancer cells as well as significant anti-proliferative properties.     

Metabolic and functional properties of lactic acid bacteria in the gastro-intestinal ecosystem: a comparative in vitro study between bacteria of intestinal and fermented food origin

Metabolic and functional properties of probiotic lactic acid bacteria (LAB) in the human gastro-intestinal ecosystem may be related to certain beneficial health effects. In this study, lactobacilli of either intestinal or fermented food origin were compared in their capability to survive low pH and bile, in their metabolic activity in the presence of bile salts and mucins, as well as in their potential to attach to enterocyte-like CaCO-2 cells. Food fermenting bacteria especially strains of the species Lactobacillus plantarum showed high tolerance to the consecutive exposure to hydrochloric acid (pH 1.5-2.5) and cholic acid (10 mM). Growth in and deconjugation of glycocholic (5 mM) and taurocholic acids (5 mM), as demonstrated for all lactobacilli of intestinal origin, was detected for food fermenting strains of the species L. plantarum, but not L. paracasei and L. sakei. Degradation of mucins was not observed for lactobacilli. Adhesion to the intestinal epithelial cell line CaCO-2 was demonstrated for several food fermenting bacterial strains in vitro. Soluble factors in the spent culture supernatants from intestinal and fermented food lactobacilli but not staphylococci cross reacted and synergized with cell wall components to promote adhesion to CaCO-2 cells. A competitive role of fecal bacteria on the adhesion of lactobacilli to CaCO-2 cells was demonstrated. In conclusion we have shown that metabolic and functional properties of intestinal lactobacilli are also found in certain bacteria of fermented food origin.     

Classification and characterization of lactic acid bacteria isolated from the intestines of common carp and freshwater prawns

Fifty-four isolates of lactic acid bacteria were obtained from the intestines of the common carp (Cyprinus carpio) and freshwater prawns (Macrobrachium rosenbergii) in Nakorn-Pathom Province, Thailand. All isolates were Gram-positive and catalase-negative cocci that did not produce gas from glucose and formed dl or L(+) lactic acid only. Most isolates were able to grow in broth at pH 9.6, in 6.5% NaCl (w/v) and 40% (w/v) bile. These isolates were divided into six groups (A-F) by sugar fermentation patterns. Strains in the groups A, B, C, and D showed intergroup DNA homology values of above 73.8%, indicating that these groups were composed of a single species. Following phylogenetic analysis, strains E 1, E 7, and E 26 from groups A, E, and F were placed in the clusters of the genera Lactococcus, Pediococcus, and Enterococcus, respectively. The type strains of Lactococcus garvieae, Pediococcus acidilactici, and Enterococcus faecium were the most closely related species with E 1, E 7, and E 26 in the phylogenetic tree, respectively. The DNA-DNA hybridization results indicated that strains in groups A (including groups B, C, and D), E, and F could be identified as belonging to the species Lactococcus garvieae, Pediococcus acidilactici, and Enterococcus faecium, respectively. Lactococcus garvieae was the dominant member of the population, accounting for 90.7% of the isolates.     

Differences in sperm migration through cervical mucus in vitro relates to sperm colonization of the oviduct and fertilizing ability in goats

Sperm migration in estrous cervical mucus can be used to measure the ability of spermatozoa to migrate through the genital tract. The relationship of this test with the sperm colonization of the isthmus, and its impact on fertility has not been evaluated in goats. Our objectives were to determine the differences among spermatozoa of different bucks in their ability to penetrate homologous cervical mucus in vitro and to determine the relationship between sperm displacement through cervical mucus and the ability of spermatozoa to colonize the oviduct and penetrate IVM oocytes, in vivo. Sperm migration in cervical mucus was assessed in flat capillary tubes with a phase contrast microscope. In the first experiment, fresh semen was used to establish differences between males in the ability of their spermatozoa to migrate in cervical mucus. In the second experiment, goats in estrus were inseminated with fresh spermatozoa from males with significant differences in mucus migration ability, and sperm numbers were evaluated at the UTJ. In the third experiment, the fertilization efficiency of IVM oocytes transferred to the oviduct of estrous females inseminated with semen from the same males as earlier, was used to assess the relationship between the mucus migration test and the in vivo fertilization performance of their spermatozoa. Spermatozoa from different males varies significantly in sperm migration efficiency in cervical mucus (15.5a +/- 1.2; 14.9a +/- 1.4; 17.5ab +/- 1.2; 17.0ab +/- 1.5; 19.7b +/- 1.2; 20.1b +/- 1.4 mm; media +/- S.E.M. for males A-F, respectively, P < 0.05). Spermatozoa from males with different mucus migration efficiency values produced different sperm populations at the oviduct reservoir of inseminated females (1,233 +/- 92.3 versus 28.8 +/- 17.0 spermatozoa of males with high and low relative migration efficiency, respectively, P < 0.02). Spermatozoa from males with different mucus migration efficiency values have different fertilization rates of IVM oocytes transferred to oviduct (47/96 (49.0%) versus 25/91 (27.5%) for males with high and low relative migration efficiency, respectively, P < 0.05). Cumulative results suggest that sperm migration in cervical mucus is related to the ability of spermatozoa to colonize the oviduct and to fertilize matured oocytes in vivo.     

Antagonistic activity of lactic acid bacteria as probiotics against selected bacteria of the Enterobaceriacae family in the presence of polyols and their galactosyl derivatives

Probiotic lactic acid bacteria were grown on erythritol, xylitol, sorbitol or lactitol and produced various derivatives: gal-erythritol, gal-xylitol, and gal-sorbitol as prebiotics. Galactosyl derivatives of erythritol, xylitol and sorbitol were metabolised by Lactobacillus spp. This resulted in their antagonistic activity against the test microflora. No activity was observed in the presence of xylitol and erythritol. Gal-sorbitol obtained by enzymatic transglycosylation from lactose had the same abilities of inducing the antagonistic activity of lactic acid bacteria that lactitol had.