Isolation and characterization of Lactobacillus species having potential for use as probiotic cultures for dogs

ABSTRACT:  The need to control pathogenic microorganisms in the intestinal tract of dogs is a growing concern. There is interest in using probiotics such as species of Lactobacillus to help control canine intestinal infections. For successful use as a probiotic, the bacterial species should be of canine intestinal origin since these species exhibit host specificity. Serial dilutions of freshly voided dog feces were plated on Lactobacillus selection (LBS) agar to isolate the cultures. Isolates were identified based on Gram stain, catalase test, and fermentation patterns using API 50 CH kits. All potential isolates were compared for bile resistance based on relative ability to grow in broth containing 0.3% Oxgall, the ability to inhibit Salmonella Typhimurium in associative broth cultures, and the production of reuterin. Of the lactobacilli isolated, Lactobacillus reuteri was the dominant species. However, some cultures of L. acidophilus also were isolated. We found variations among the isolates of L. reuteri and L. acidophilus with respect to bile tolerance. In general, isolates of L. reuteri appeared to be more bile resistant than were isolates of L. acidophilus . There were also variations in the ability to inhibit growth of S. Typhimurium. Some isolates of L. reuteri produced reuterin while others did not.     

苦荞蛋白对益生菌生长及其胆酸盐耐受能力的影响

本文以水溶性苦荞蛋白为研究对象,通过其对益生菌(植物乳杆菌、嗜酸乳杆菌)生长和益生菌胆酸盐耐受能力的影响,以及体外模拟肠道环境中的益生菌生长实验,探明了苦荞蛋白对益生菌生长及生存能力的影响。实验结果表明:添加不同浓度的苦荞蛋白(1、3和5 mg/m L),植物乳杆菌和嗜酸乳杆菌培养液的光密度值(OD600)分别提高12.33%、25.84%、35.31%和15.37%、26.66%、30.57%;同时,苦荞蛋白使植物乳杆菌和嗜酸乳杆菌对胆酸盐(胆酸钠(SC)、甘氨胆酸钠(SGC))的耐受作用显著提高(p0.05),其光密度值(OD600)分别增加了1.64、8.67和4.11、8.30倍。进一步的体外模拟培养实验表明苦荞蛋白对肠道中乳酸杆菌的生长作用显著提高(p0.05)。综上所述,苦荞蛋白可提高乳酸杆菌在胆酸盐中的生长并促进肠道中乳酸杆菌的生长,为苦荞蛋白在食品中的应用提供了理论依据。     

Importance of Bile Tolerance of Lactobacillus acidophilus Used as a Dietary Adjunct

Cultures of lactobacilli identified as Lactobacillus acidophilus from the intestinal contents of young calves varied in their ability to grow in broth containing .3% oxgall compared with control broth. Frozen concentrated cultures were prepared from a strain exhibiting low tolerance to bile and from a strain exhibiting high tolerance to bile. Plate counts were comparable from the concentrated cultures before and after frozen storage on lactobacilli MRS agar with and without .15% oxgall. In a feeding trial involving newborn dairy calves supplementation of the diet with the more bile resistant strain of Lactobacillus acidophilus caused greater increases of numbers of facultative lactobabilli in the upper small intestines than did the strain exhibiting lower resistance to bile. It was not possible to determine whether the lactobacilli would prevent intestinal infections in the calves challenged with enteropathogenic Escherichia coli. This portion of the study failed as the challenge with Escherichia coli did not cause infections even in control animals.     

Short communication: Lactose enhances bile tolerance of yogurt culture bacteria

Lactose is an energy source for culture bacteria. Bile tolerance is an important probiotic property. Our aim was to elucidate the effect of lactose on bile tolerance of yogurt starter culture Lactobacillus bulgaricus LB-12 and Streptococcus thermophilus ST-M5. Bile tolerance of pure cultures was determined using 0.3% oxgall in MRS THIO broth (Difco, Becton Dickinson, Sparks, MD) for L. bulgaricus and 0.3% oxgall in M17 broth (Oxoid, Basingstoke, UK) for Strep. thermophilus. Lactose was added to both broths at 0 (control), 1, 3, and 5% (wt/vol) broth. Dilutions were plated hourly for 12 h. Experiments were replicated 3 times. At 2, 4, and 12 h of incubation, lactose incorporated at all amounts, 1, 3, and 5% (wt/vol), showed higher counts of Strep. thermophilus ST-M5 compared with the control. Lactose use at 5% (wt/vol) significantly enhanced bile tolerance of both L. bulgaricus and Strep. thermophilus compared with control.     

Acid, bile, and heat tolerance of free and microencapsulated probiotic bacteria

ABSTRACT:  Eight strains of probiotic bacteria, including Lactobacillus rhamnosus , Bifidobacterium longum, L. salivarius, L. plantarum , L. acidophilus , L. paracasei , B. lactis type Bl-O4, and B. lactis type Bi-07, were studied for their acid, bile, and heat tolerance. Microencapsulation in alginate matrix was used to enhance survival of the bacteria in acid and bile as well as a brief exposure to heat. Free probiotic organisms were used as a control. The acid tolerance of probiotic organisms was tested using HCl in MRS broth over a 2-h incubation period. Bile tolerance was tested using 2 types of bile salts, oxgall and taurocholic acid, over an 8-h incubation period. Heat tolerance was tested by exposing the probiotic organisms to 65 °C for up to 1 h. Results indicated microencapsulated probiotic bacteria survived better ( P < 0.05) than free probiotic bacteria in MRS containing HCl. When free probiotic bacteria were exposed to oxgall, viability was reduced by 6.51-log CFU/mL, whereas only 3.36-log CFU/mL was lost in microencapsulated strains. At 30 min of heat treatment, microencapsulated probiotic bacteria survived with an average loss of only 4.17-log CFU/mL, compared to 6.74-log CFU/mL loss with free probiotic bacteria. However, after 1 h of heating both free and microencapsulated probiotic strains showed similar losses in viability. Overall microencapsulation improved the survival of probiotic bacteria when exposed to acidic conditions, bile salts, and mild heat treatment.     

Assimilation of cholesterol by yeast strains isolated from infant feces and Feta cheese

Eight yeast strains isolated from infant feces and the traditional Greek Feta cheese, selected for their probiotic properties, were tested along with a commercially available strain of Saccharomyces boulardii for their ability to remove cholesterol from a growth medium (yeast extract glucose peptone broth) supplemented with 0.3% Oxgall. The amount of cholesterol removed during 72 h of growth at 37 degrees C revealed significant variations among the yeast strains examined. Two isolates from infant feces, namely Saccharomyces cerevisiae KK1 and Isaatchenkia orientalis KK5.Y.1 and one isolate from Feta cheese, namely S. cerevisiae 832, along with the commercial strain S. boulardii, were able to remove cholesterol from the growth medium after 48 h of incubation at 37 degrees C. However, Saccharomyces strains proved to be able to remove cholesterol even after 24 h of growth at 37 degrees C. The cholesterol removed from the growth medium was not metabolically degraded but was rather assimilated into the yeast cells. The ability to assimilate cholesterol in vitro and to tolerate low pH levels, gastric juice, and bile indicate that S. cerevisiae 832, and especially S. cerevisiae KK1 and I. orientalis KK5.Y.1 (being more bile and gastric juice tolerant because of their human origin) may be promising candidate strains for use as probiotics.     

Amylolytic Cultures of Lactobacillus acidophilus : Potential Probiotics to Improve Dietary Starch Utilization

ABSTRACT: The amylase activity of twenty strains of Lactobacillus species isolated from intestinal contents of pigs was studied in vitro. Hydrolysis of starch was measured by following the disappearance of starch in a broth medium during the growth of all three strains. The amount of enzyme activity varied among strains. Three strains (A-4, L9 and L23) were selected for further study based on their high amylolytic activity in a broth containing soluble starch as the only carbohydrate source. Strains A-4 and L23 were identified as L. acidophilus and strain L9 was identified as L. fermentum. Among the three cultures, L. acidophilus L23 was selected as a potential candidate for use as a probiotic for improving digestion of starch in pigs based on its growth and hydrolysis of raw starch. The enzyme activity of L. acidophilus L23 was associated with the cell envelope and that of L. fermentum L9 was extracellular. The enzyme activity of L. acidophilus A4 was found in both locations. The enzyme activity of L. acidophilus L23 was inducible rather than constitutive.     

Bacteriocin involved in premature death of Lactobacillus acidophilus NCFM during growth at pH 6

Lactobacillus acidophilus NCFM maintained at pH 6 during growth in lactobacilli MRS broth appeared to exhibit premature death. However, during extended incubation, the culture reinitiated growth. Spent broth collected from the culture when it began the premature death was very toxic to the culture, but growth did occur during extended incubation of the assay tubes. When several successive subcultures with extended incubation were made in MRS broth containing added inhibitory spent broth, a strain (identified as L. acidophilus) was isolated that was resistant to the inhibitor. This strain produced a bacteriocin that was inhibitory to the growth of L. acidophilus NCFM and the predominating strains isolated from it. The producer strain grew more slowly than did the predominating strains in culture NCFM. The results show that when L. acidophilus NCFM, a mixed strain culture, was grown at pH 6, the inhibitory strain was able to grow sufficiently to produce enough bacteriocin to destroy the predominating strain(s) in the culture.     

酸菜来源植物乳杆菌体外去除胆固醇特性

对4株酸菜来源具有潜在益生特性的植物乳杆菌降胆固醇特性进行研究.研究发现其降胆固醇能力受胆盐浓度和胆盐种类的影响,当牛胆盐的添加浓度为3g/L时,降胆固醇能力达到最高(9.67mg/g～21.32mg/g[干重]);在培养基中添加不同种类的胆盐,降胆固醇能力不同,依次为牛磺胆酸钠＞混合胆盐＞甘氨胆酸钠.其产生的游离胆酸的量与胆固醇移除能力呈正相关,且以在牛磺胆酸钠存在下最佳.     

降胆固醇乳酸菌的筛选及其在内蒙古发酵香肠中的应用

通过对乳酸菌降胆固醇生物学特性及对发酵肉制品中胆固醇降解作用进行研究,结果表明,从内蒙古传统肉肠中分离筛选得到的8 株乳酸菌中,菌株X3-2B有较强的胆固醇降解能力。且在MRS培养基中添加3 g/L胆盐、20 g/L胆固醇和 20 g/L葡萄糖时菌株X3-2B的胆固醇降解能力最大,在不同培养基中发酵不同时间菌株X3-2B对胆固醇的降解能力显著高于标准菌株。在以菌株X3-2B为发酵剂的发酵香肠中,其胆固醇含量显著低于对照组。故菌株X3-2B可作为一株降胆固醇性能较好的肉制品发酵剂。     

Mechanisms of cholesterol removal by lactobacilli under conditions that mimic the human gastrointestinal tract

Five strains of lactobacilli were studied for their ability to remove cholesterol in vitro under conditions that mimic the human gastrointestinal tract. The highest assimilation of cholesterol was observed in media supplemented with oxgall and the lowest in the presence of taurocholic acid. Scanning electron micrographs showed that cholesterol was adhered to the cellular surface of lactobacilli cells. Resting and dead cells were able to remove cholesterol although in small amounts. Additionally, inhibition of cholesterol micelles formation was observed in the presence of bile salts. All strains were able to deconjugate bile salts, where higher deconjugation was observed in the presence of sodium glycocholate compared with other bile salts studied. All strains also exhibited bile salt hydrolase activity and most strains showed higher substrate specificity towards glycine-conjugated bile than towards taurine-conjugated bile. The results indicated that lactobacilli could remove cholesterol in vitro via various mechanisms, and may exert such hypocholesterolaemic effects in vivo.     

Functional characteristics of Lactobacillus spp. from traditional Maasai fermented milk products in Kenya

In this study functional characteristics of 23 representative Lactobacillus strains isolated from the Maasai traditional fermented milk 'Kule naoto' were determined. The Lb. acidophilus group strains showed resistance to gastric juice and bile. In addition, some Lb. acidophilus strains expressed bile salt hydrolase activity, and had ability to assimilate cholesterol in vitro. In-vitro adhesion to HT29 MTX cells of up to 70% was recorded. Lb. fermentum strains showed almost 100% survival under simulated stomach acidic conditions and physiological salt concentrations of bile salts, hydrophobicity values were over 80%. Most strains of the Lb. casei and Lb. acidophilus groups showed aggregation abilities of above 50%. Many strains expressed a protective effect against N-methyl-N'-nitro-N-nitrosoguanidine induced DNA damage according to the 'comet assay' and none was virulent. The antibiotic minimum inhibitory concentration of selected strains was established. According to these results, the Lactobacillus spp associated with 'Kule naoto', contain potentially probiotic (functional) strains.     

Bile tolerance, taurocholate deconjugation, and binding of cholesterol by Lactobacillus gasseri strains

Bile tolerance, deconjugation of sodium taurocholate, and the cholesterol-binding ability of 28 strains of Lactobacillus gasseri were examined. There was significant variation among strains in growth in media containing bile and also variation in the ability to bind cholesterol. Cultures grown for 12 h at 37 degrees C bound significantly more cholesterol than did cells from a 48-h incubation. Variation among strains in the ability to deconjugate sodium taurocholate was not significantly different. Maximal deconjugation of sodium taurocholate was achieved with the cells during the stationary phase of growth (12 h). Statistical analysis showed no significant correlation between bile tolerance and sodium taurocholate deconjugation, bile tolerance and cholesterol-binding ability, or sodium taurocholate deconjugation and cholesterol-binding ability.     

4株乳酸菌对模拟胃肠环境的耐受性及生长特性研究

对4株来源于自然发酵肉制品中的乳酸菌进行耐酸耐胆盐性能的测定,选取在pH3.0和含0.3 g/100 mL胆盐的MRS培养基中存活率均超过50%的2株菌（M12、M23）,进一步研究其在模拟胃肠液中的耐受性及MRS培养基中的生长特性。结果表明,这2株乳酸菌对人工模拟胃肠道环境有较好的耐受性,经过人工模拟胃液处理2 h后,存活率均＞50%,经人工模拟肠液处理10 h后仍可存活;这2株菌具有较强的增殖及产酸能力,24 h内均已进入稳定生长期,随着菌体数目的增加,培养基pH值快速降低,在肉制品的发酵温度范围内均能较好地生长。因此,这2株菌可作为潜在的益生菌菌株应用于肉制品的发酵。     

Relationship between bile tolerance and the presence of a ruthenium red staining layer on strains of Lactobacillus acidophilus.

Seventeen strains of Lactobacillus acidophilus were evaluated to determine the relationship between bile tolerance and the presence of an outer polysaccharide layer exterior to the cell wall when viewed by transmission electron microscopy. Bile tolerance is necessary for survival of lactobacilli in the intestinal tract, and the polysaccharide layer may be responsible for adherence to human intestinal tissue. These two factors may be the basis for use of L. acidophilus as a dietary adjunct. Ten strains exhibited a ruthenium red-stained outer polysaccharide layer. Three of the 10 strains had extremely dense layers, which may indicate stronger adherence properties. Seven strains did not contain a ruthenium red-stained outer layer; however, six strains that did not have the stained layer were resistant to 1.0% bile concentration. Fourteen strains were tolerant to 1% bile, one strain was tolerant to 6% bile, and two strains were sensitive to bile. No relationship between bile tolerance and the presence of the ruthenium red-stained outer polysaccharide layer was apparent.     

Bile salt hydrolase activity of three strains of Lactobacillus acidophilus

Three strains of Lactobacillus acidophilus, two from human intestinal origin (016 and L1) and one from porcine intestinal origin (ATCC 43121), were tested for their bile salt deconjugation activity. The L. acidophilus ATCC 43121 had more deconjugating activity of both sodium glycocholate and sodium taurocholate at pH 6.5 than did either L. acidophilus 016 or L1. The activity of intracellular bile salt hydrolase found in strain ATCC 43121 was 14-fold higher than that in either of the other two strains. The optimum pH for deconjugation of sodium glycocholate was between 4 and 5.5 for all three strains. For deconjugation of sodium taurocholate, the optimum pH was between 3.5 and 4.5 for strains L1 and ATCC 43121 and was between pH 5 and 6 for strain O16. The molecular mass of the enzyme in all three strains of L. acidophilus was estimated to be 126 kDa by Sephadex G-200 gel filtration. All three strains exhibited more bile salt hydrolase activity towards sodium glycocholate than towards sodium taurocholate.     

高效同化胆固醇乳酸菌菌株的筛选及在食品中的应用

从发酵食品中分离筛选能高效同化胆固醇的乳酸菌,以开发低胆固醇系列产品。从25个来源不同的样品中,分离筛选出8株乳酸菌,与活化出的8株已知乳酸菌共同在特殊培养基中培养,最后筛选出2株具有较高同化胆固醇能力的菌株。结果表明,嗜酸乳杆菌和植物乳杆菌应用于食品中,对食品风味影响较小,可大量应用。     

Effects of bile salt deconjugation by probiotic strains on the survival of antibiotic-resistant foodborne pathogens under simulated gastric conditions

This study was designed to evaluate the effects of bile acid deconjugation by probiotic strains on the antibiotic susceptibility of antibiotic-sensitive and multiple antibiotic-resistant Salmonella Typhimurium and Staphylococcus aureus. Eight probiotic strains, Bifidobacterium longum B6, Lactobacillus acidophilus ADH, Lactobacillus brevis KACC 10553, Lactobacillus casei KACC 12413, Lactobacillus paracasei ATCC 25598, Lactobacillus rhamnosus GG, Leuconostoc mesenteroides KACC 12312, and Pediococcus acidilactici KACC 12307, were used to examine bile acid tolerance. The ability to deconjugate bile acids was evaluated using both thin-layer chromatography and high-performance liquid chromatography. The antibiotic susceptibility testing was carried out to determine the synergistic inhibitory activity of deconjugated bile acids. L. acidophilus, L. brevis, and P. acidilactici showed the most tolerance to the conjugated bile acids. P. acidilactici deconjugated glycocholic acid and glycodeoxycholate from 3.18 and 3.09 mM to the detection limits, respectively. The antibiotic susceptibility of selected foodborne pathogens was increased by increasing the concentration of deconjugated bile acids. The study results are useful for understanding the relationship between bile acid deconjugation by probiotic strains and antibiotic susceptibility in the presence of deconjugated bile acids, and they may be useful for designing new probiotic-antibiotic combination therapy based on bile acid deconjugation.     

Ability of neonatal human Lactobacillus isolates to remove cholesterol from liquid media

The activity of a number of human intestinal strains of Lactobacillus acidophilus , including newly isolated neonatal strains, have been examined for their ability to remove cholesterol from liquid medium. In common with other authors we find that cholesterol will co-precipitate with deconjugated bile acids as pH falls to values less than 5.0. But we also report that for some strains removal of cholesterol can occur in the absence of bile and that co-precipitation at low pH values cannot account for all cholesterol removed. The activity is unlikely to be as a result of bacterial cholesterol oxidase or dehydrogenase but may be one of physical association with the bacterial cell surface.     

Characterization and purification of a bacteriocin produced by a potential probiotic culture, Lactobacillus acidophilus 30SC

Lactobacillus acidophilus 30SC was tested for its potential as a probiotic culture. The strain exhibited good acid tolerance in an artificial gastric solution as well as bile resistance in media containing 0.3% bile acids. The strain produced a heat-stable antimicrobial compound that was shown to be proteinaceous in nature and, therefore, referred to as a bacteriocin. The bacteriocin was active over a wide pH range and inhibited a number of gram-positive bacteria including Listeria ivanovii and pathogenic strains. The bacteriocin was purified by 50% ammonium sulfate precipitation followed by hydrophobic interaction column chromatography. The SDS-PAGE of the active fractions resulted in a single band with estimated molecular mass of 3.5 kDa. These results demonstrate the potential of L. acidophilus 30SC as a probiotic culture that can be utilized in the manufacturing of dairy foods and dietary supplements.