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.     

Ice-cream as a probiotic food carrier

Ice-creams are food products showing potential for use as probiotic vehicles, with the added advantage of being appreciated by people belonging to all age groups and social levels. However, the development of ice-creams containing probiotic bacteria requires the overcoming of certain technological intrinsic requirements related to their processing stages. The aim of the present paper was to review the technological parameters involved in the production of probiotic ice-creams. Although the application of probiotics in cheeses, and especially in fermented milks, has been widely explored in the literature, ice-cream is a relatively innovative matrix for the application of probiotics, and thus a review about its potential as probiotic food carrier could be very helpful.     

Two antioxidative lactobacilli strains as promising probiotics

Two antioxidative strains tentatively identified as Lactobacillus fermentum, E-3 and E-18, were isolated from intestinal microflora of a healthy child. Survival time of these strains in the presence of reactive oxygen species (ROS), like hydrogen peroxide, superoxide anions and hydroxyl radicals, was significantly increased compared with a non-antioxidative strain, and also was quite similar to a highly ROS resistant strain of Salmonella typhimurium. E-3 and E-8 contain a remarkable level of glutathione, express Mn-SOD, which is important for the prevention of lipid peroxidation, and secrete hydrogen peroxide. Their significant antimicrobial activity combined with antioxidative properties may serve as defensive principles in the intestinal microbial ecosystem and overcome exo- and endogenous oxidative stress.     

Molecular characterization of the probiotic strain Bacillus cereus var. toyoi NCIMB 40112 and differentiation from food poisoning strains

Bacillus cereus var. toyoi strain NCIMB 40112 (Toyocerin), a probiotic authorized in the European Union as feed additive for swine, bovines, poultry, and rabbits, was characterized by DNA fingerprinting applying pulsed-field gel electrophoresis and multilocus sequence typing and was compared with reference strains (of clinical and environmental origins). The probiotic strain was clearly characterized by pulsed-field gel electrophoresis using the restriction enzymes Apa I and Sma I resulting in unique DNA patterns. The comparison to the clinical reference strain B. cereus DSM 4312 was done with the same restriction enzymes, and again a clear differentiation of the two strains was possible by the resulting DNA patterns. The use of the restriction enzymes Apa I and Sma I is recommended for further studies. Furthermore, multilocus sequence typing analysis revealed a sequence type (ST 111) that was different from all known STs of B. cereus strains from food poisoning incidents. Thus, a strain characterization and differentiation from food poisoning strains for the probiotic strain was possible.     

以凹土为载体的新型益生菌制剂的制备

以提纯凹土作为益生菌载体,研究了不同吸附条件对嗜酸乳杆菌、粪链球菌和保加利亚乳杆菌吸附率的影响。结果表明,将10mL菌体(菌悬液浓度为109CFU/mL)与3mL凹土(34.08mg/mL)混合,在pH6.5,35℃吸附40min,三种菌体的最大吸附率分别为89.08%、84.24%和89.42%。吸附后经冷冻干燥制备新型益生菌制剂,嗜酸乳杆菌、粪链球菌和保加利亚乳杆菌活菌数可达2.98×109、4.33×109和6.21×109CFU/g,存活率分别为95.1%、92.2%和95.9%。新型益生菌制剂在人工模拟肠胃环境下释放嗜酸乳杆菌、粪链球菌和保加利亚乳杆菌的活菌数分别在肠环境下4、6和4h达到最大,并且在3~15h内均能维持在108~109CFU/g之间。      

Enterococci as probiotics and their implications in food safety

Enterococci belong to the lactic acid bacteria (LAB) and they are of importance in foods due to their involvement in food spoilage and fermentations, as well as their utilisation as probiotics in humans and slaughter animals. However, they are also important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics and possess virulence factors such as adhesins, invasins, pili and haemolysin. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some can harbour virulence traits, but it is also thought that virulence is not the result of the presence of specific virulence determinants alone, but is rather a more intricate process. Specific genetic lineages of hospital-adapted strains have emerged, such as E. faecium clonal complex (CC) 17 and E. faecalis CC2, CC9, CC28 and CC40, which are high risk enterococcal clonal complexes. These are characterised by the presence of antibiotic resistance determinants and/or virulence factors, often located on pathogenicity islands or plasmids. Mobile genetic elements thus are considered to play a major role in the establishment of problematic lineages. Although enterococci occur in high numbers in certain types of fermented cheeses and sausages, they are not deliberately added as starter cultures. Some E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations. Such probiotics are administered to treat diarrhoea, antibiotic-associated diarrhoea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity. In animals, enterococcal probiotics are mainly used to treat or prevent diarrhoea, for immune stimulation or to improve growth. From a food microbiological point of view, the safety of the bacteria used as probiotics must be assured, and data on the major strains in use so far indicate that they are safe. The advantage of use of probiotics in slaughter animals, from a food microbiological point of view, lies in the reduction of zoonotic pathogens in the gastrointestinal tract of animals which prevents the transmission of these pathogens via food. The use of enterococcal probiotics should, in view of the development of problematic lineages and the potential for gene transfer in the gastrointestinal tract of both humans and animals, be carefully monitored, and the advantages of using these and new strains should be considered in a well contemplated risk/benefit analysis.     

益生菌Lactobacillus casei Zhang发酵特性的评价

以L.acidophilus NCFM,L.rhamnosus GG,L.casei Shirota和Bifidobacterium animalis Bb12为参照,对益生菌L.casei Zhang的发酵特性进行了研究.结果表明:发酵结束时,添加L.casei Zhang的发酵乳具有较高的pH值和较低的滴定酸度.4℃贮藏28 d后,L.casei Shirota和Bifidobacterium animalis Bb12的蛋白水解能力下降,而L.asei Zhang,L addophilusNCFM和L.rhamnosus GG蛋白水解能力不断上升.尽管Lcasei Zhang在脱脂乳中的发酵速度低于对照益生菌.但贮藏期内其活菌数缓慢上升并于贮藏结束时达到最高.L.casei Zhang完全具备益生菌所应有的发酵特性,因此其适合应用于发酵乳的开发.     

Potential probiotic characterization and effect of encapsulation of probiotic yeast strains on survival in simulated gastrointestinal tract condition

The present study is focused on probiotic characterization of four yeasts viz. Pichia barkeri VIT-SJSN01, Yarrowia lipolytica VIT-ASN04, Wickerhamomyces anomalus VIT-ASN01 and Saccharomyces cerevisiae VIT-ASN03 isolated from food samples based on their auto-aggregation, co-aggregation ability and haemolytic activity. All the yeast strains showed good self-adhering and co-adhering potentiality with a value index of greater than 85%. None of the strains exhibited haemolysis which confirmed their non-pathogenic nature. Yeast strains were encapsulated in sodium alginate, sodium alginate coated with chitosan and sodium alginate-gelatinized with starch. Size and morphology of the beads and capsules were determined using SEM analysis. Encapsulation output and viability under storage condition was investigated. It was found that probiotic yeasts encapsulated in sodium alginate beads, chitosan coated beads and microcapsules showed better survival to simulated gastrointestinal conditions compared to free cells.     

两株乳杆菌益生特性的体外研究

从健康个体口腔中分离得到了两株乳酸菌，经API细菌鉴定系统鉴定分别为植物乳杆菌Lactobacillus plantarum HO-69与Lactobacillus plantarum HO-160，并对两株植物乳杆菌的特性进行了研究。抑菌试验表明，两株菌的代谢产物具有较好的抑菌活性；两株菌对于人工消化液具有良好的耐受性，HO-69生长的最高胆汁质量分数为0．4％，HO-160为0.3％；两菌均具有较高的表面疏水性与酸碱电荷。     

人母乳源乳酸菌的筛选、鉴定及益生活性的初步研究

本研究从人母乳中分离乳酸菌。采用透明圈法从产后10³0 d的人母乳样品中筛选到一株产酸能力较强的乳酸菌。对筛选菌株进行形态学观察、生理生化实验及16S r DNA序列鉴定。结果发现,该菌株为革兰氏阳性,短杆状,无芽孢;生理生化特性符合鼠李糖乳杆菌的基本特征;16S r DNA序列及进化树分析进一步确认该菌株为鼠李糖乳杆菌,命名为鼠李糖乳杆菌Z5。在p H3.0的条件下模拟人工胃肠液共消化6 h后该菌存活率仍达90%以上,在0.15 g/L胆盐质量浓度培养6 h,菌株存活率为76.14%;抑菌实验发现,菌株培养液对沙门氏菌及大肠杆菌的生长有明显的抑制作用,抑菌圈直径达20 mm以上。细胞黏附实验发现,该菌株可有效黏附到Caco-2细胞表面,对Caco-2细胞的平均黏附数为7.54×105cfu/m L。从人母乳中获得了这株具有潜在益生性质的鼠李糖乳杆菌,为明确母乳中益生菌的功效及开发新型益生菌产品研究提供了良好的实验材料。      

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....     

Quality characteristics of kefir as a carrier for probiotic Lactobacillus rhamnosus GG

Kefir was prepared using kefir grains of different origins (US, Australia and India) and starter culture. The sensory scores for kefir made using starter culture and US kefir grains were significantly higher than for the other samples (P < 0.05). The index of viscosity was significantly higher (P < 0.05) in kefir prepared using Indian kefir grains. Shear stress–shear rate plots indicated yield stress in all kefirs. All samples exhibited shear‐thinning behaviour. The average count of Lactobacillus rhamnosus GG in kefir after incubation was 7.09 log CFU/g. No significant difference (P > 0.05) was observed in quality characteristics in kefir after the addition of LGG, indicating that it could be a potential probiotic carrier.     

二株嗜热链球菌益生特性的体外研究

对嗜热链球菌ST1和嗜热链球菌KS1的部分益生性及功能特性进行了研究。考察了ST1和KS1对酸和胆盐的耐受性,结果表明,ST1和KS1在pH值为3.0条件下培养3h的存活率分别为67.17%和67.49%,在pH值为4.0或质量分数为0.3%胆盐环境中仍可以生长,表现出较高的存活力。通过抑菌实验表明ST1和KS1可以抑制常见的致病菌金黄色葡萄球菌、大肠杆菌、鼠伤寒沙门氏菌和福氏志贺氏菌。在对抗生素敏感实验中,ST1对其中的3种抗生素有耐药性,而KS1对6种抗生素有耐药性。研究还发现ST1和KS1还具有较高的降胆固醇的能力,胆固醇的去除率分别为65.14%和74.49%。     

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。     

Lactobacillus fermentum strains from human breast milk with probiotic properties and cholesterol-lowering effects

Food Science and Biotechnology - The study aimed to identify two lactic acid bacteria from human breast milk and evaluate for their probiotic potential. Lactobacillus fermentum strains showed...     

Potential probiotic Lactobacillus strains from fermented sausages: Further investigations on their probiotic properties

A rational selection of probiotic microorganisms is an important challenge and requires the definition of fundamental information about the physiology and genetics of candidate strains. In this study, selected Lactobacillus (Lact.) strains already characterized in a previous study for their capability to resist low pH and to grow in conditions simulating the intestinal environment, were further investigated to explore their probiotic properties, such as the adhesion capability to intestinal human Caco-2 cell lines and their growth behaviour in the presence of various prebiotic carbohydrates. At first 25 Lactobacillus strains were characterized by pulsed field gel electrophoresis using the endonuclease NotI. Among them, 13 strains belonging to the Lact. plantarum-group were identified at species level by a multiplex PCR assay. Subsequently 11 Lactobacillus strains showing different PFGE restriction pattern and the best acid- and bile-resistances, were chosen to investigate their in vitro adhesion capability to human intestinal epithelial cells and their fermentation properties of five prebiotic substances (FOS, Inulin, IMO, GOS and lactulose) at a concentration of 2%. The 11 strains analysed in this study possessed good adhesion capability to Caco-2 cell layers and, in particular, the eight strains belonging to the Lact. plantarum-group showed the higher final number of viable adhering cells. Moreover a species-related fermentative behaviour was pointed out and the strain Lact. paracasei EL7 was the only one able to grow in the presence of all prebiotics tested. In conclusion the strains of Lactobacillus studied in this research could be further investigated to assess possible in vivo human health benefits.     

Persistence of probiotic strains in the gastrointestinal tract when administered as capsules, yoghurt, or cheese

Most clinical studies of probiotics use freeze-dried, powdered bacteria or bacteria packed in capsules. However, probiotics are commercially available in various food matrices, which may affect their persistence in the gastrointestinal tract. The objective of the study was to compare oral and faecal recovery during and after administration of a combination of Lactobacillus rhamnosus GG and LC705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium animalis subsp. lactis Bb12 as capsules, yoghurt, or cheese. This randomized, parallel-group, open-label trial (n = 36) included a 4-week run-in, 2-week intervention, and 3-week follow-up period. Participants consumed 10¹⁰ cfu/day of probiotic combination and provided saliva and faecal samples before, during, and after the intervention. Strain-specific real-time PCR was used to quantify the strains.L. rhamnosus GG was the only probiotic strain regularly recovered in saliva samples. During the intervention period it was recovered in the saliva of 88% of the volunteers at least once. No difference was found between the yoghurt and cheese groups. At the end of the intervention, L. rhamnosus GG and LC705 counts were high in faecal samples of all product groups (8.08 and 8.67 log₁₀ genome copies/g, respectively). There was no matrix effect on strain quantity in faeces or the recovery time after ceasing the intervention. For P. freudenreichii subsp. shermanii JS and B. animalis subsp. lactis Bb12, a matrix effect was found at the end of the intervention (P < 0.01 and P < 0.001, respectively) and in the recovery time during follow-up (P < 0.05 for both). Yoghurt yielded the highest faecal quantity of JS and Bb12 strains (8.01 and 9.89 log₁₀ genome copies/g, respectively). The results showed that the administration matrix did not influence the faecal quantity of lactobacilli, but affected faecal counts of propionibacteria and bifidobacteria that were lower when consumed in cheese. Thus, the consumption of probiotics in yoghurt matrix is highly suitable for studying potential health benefits and capsules provide a comparable means of administration when the viability of the strain in the capsule product is confirmed.     

Molecular and toxigenic characterization of Bacillus cereus and Bacillus thuringiensis strains isolated from commercial ground roasted coffee

Thirty samples of roasted ground coffee beans from 10 different commercial brands were analyzed to investigate the occurrence and levels of Bacillus cereus and Bacillus thuringiensis strains. Strains were evaluated for their genetic diversity by repetitive element sequence polymorphism PCR (Rep-PCR) and for their toxigenic profiles, i.e., the presence of hblA, hblC, hblD, nheA, nheB, nheC, cytK, ces, and entFM. Survival and multiplication of B. cereus sensu lato in the ready-to-drink coffee was determined to evaluate this beverage as a possible vehicle for B. cereus infection. B. cereus was detected in 17 (56.7%) of the 30 samples, and B. thuringiensis was detected in 8 (26.7%) of the 30 samples. Five samples did not produce any characteristic growth. The most common gene, entFM, was detected in 23 strains (92%). The NHE complex (nheA, nheB, and nheC genes) was found in 19 strains (76%). The HBL complex (hblA, hblC, and hblD) was found in 16 strains (64%). All strains were negative for ces. The cytK gene was found in 16 strains (64%). The computer-assisted cluster analysis of Rep-PCR profiles using a clustering criterion of 80% similarity revealed four main clusters. Cluster 1 was the predominant and comprised three B. thuringiensis strains with 100% similarity, cluster 2 comprised two B. cereus strains (100% similarity), cluster 3 comprised two B. thuringiensis strains (90% similarity), and cluster 4 comprised one B. thuringiensis strain and one B. cereus strain (85% similarity). The cluster analysis of fingerprints generated by Rep-PCR revealed a high genetic diversity among the B. cereus strains, suggesting that the contamination could have originated from different sources. In our experiments, when sugar was added and the beverage was kept in thermic bottles there was a significant increase in B. cereus sensu lato levels, which may increase the risk of food poisoning. These results highlight the need for additional studies on this subject to better evaluate coffee as a food poisoning vehicle.     

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.