In vitro screening of lactobacilli with antagonistic activity against Helicobacter pylori from traditionally fermented foods

Helicobacter pylori may cause stomach diseases such as chronic gastritis, peptic ulcer, and gastric cancer, and several studies reported that lactobacilli have inhibitory effects on H. pylori. In this study, 38 Lactobacillus strains were screened for anti-H. pylori activity using in vitro methods, including survivability under the simulated gastric conditions, agar plate diffusion, urease activity, coaggregation, autoaggregation, and hydrocarbon analysis. The results indicate that 2 Lactobacillus strains showed potential anti-H. pylori activity in vitro. Lactobacillus plantarum 18 had the largest zone of inhibition and markedly reduced the urease activity of H. pylori. Lactobacillus gasseri Chen had higher coaggregation rate (58.15%) and hydrophobicity (59.27%) compared with the other strains. Further research is needed to verify the activities of these strains against H. pylori.     

The defense function of the digestive tract lactoflora

Lactobacilli can belong to the gut flora or to the flora associating with gastrointestinal epithelial surfaces. They play an important role within the microbial defense mechanisms taking part in the colonization resistance and in the decontamination of the host. Lactobacilli affect the immune response. They accelerate the revival of the small intestinal epithelial cells. Those strains of lactobacilli with distinct antagonistic properties with lysozyme activity and resistance against lysozyme often develop a compact glycocalyx. They are endowed with a high adhesion capacity. The intestinal lactoflora consists of several species with different defense properties.     

Antagonistic Activity of Spent Culture Supernatants of Lactic Acid Bacteria against Helicobacter Pylori Growth and Infection in Human Gastric Epithelial AGS Cells

ABSTRACT:  We investigated the bactericidal activity and exclusion effect of 10 strains of lactic acid bacteria (LAB) isolated from different commercial food products and infant feces against  Helicobacter pylori  ( H. pylori ) in human gastric epithelial AGS cells. Antagonistic activity of spent culture supernatants (SCS) from LAB (LAB-SCS) was tested, and the content of organic acids in SCS was analyzed with high-performance liquid chromatography (HPLC). In addition, the bactericidal activities of LAB-SCS were estimated by a time-kill assay and by measuring the exclusion effect of LAB-SCS against  H. pylori  in AGS cells. The results showed that SCS from certain strains with higher concentrations of organic acids dramatically decreased the viability of  H. pylori . We also proved that the organic acids could inhibit  H. pylori  adhesion and invasion of AGS cells. Furthermore, the concentration and speciation of organic acids in SCS after fermentation of LAB are important factors in the inhibition of  H. pylori  infection. In addition, the  in vitro  methods used in this study might provide for the rapid screening of potential probiotics with anti- H. pylori  activity in the dairy industry.     

具有拮抗幽门螺杆菌作用的植物乳杆菌特性研究

幽门螺杆菌是螺旋形或是S形的微需氧革兰氏阴性菌,是慢性活动性胃炎、消化性溃疡发病的主要病因,并与胃恶性肿瘤(胃癌、胃粘膜相关淋巴样组织淋巴瘤)关系密切。本文探讨了具有拮抗幽门螺杆菌作用的植物乳杆菌的生物特性,结果表明L.plantarum 18在低酸条件下可以生长,耐受pH2.5的人工胃液,能够抑制多种幽门螺杆菌的生长。该菌株生长比较快,4h进入生长对数期,14h后进入稳定期;最适生长pH为5～7,最适生长温度为30～37℃,最适接种量为1%～2%(V/V),可在0.2%的胆盐中生长。     

Antagonistic activity against Helicobacter pylori infection in vitro by a strain of Enterococcus faecium TM39

Lactic acid bacteria (LAB) strains from infant feces were screened for anti-Helicobacter pylori use. In the beginning, we selected the strains based on their capability to adhere to the human intestinal epithelial cell (Int-407), colonial enterocyte-like Caco-2 cell, human cervical epithelioid carcinoma cell (HeLa), and human gastric carcinoma cell (TSGH 9201). Then, acid and bile salt tolerance of these LAB strains was evaluated. In addition, the ability of these LAB strains to inhibit the growth of H. pylori and to expel H. pylori cells from TSGH 9201 were studied. The spent culture supernatant (SCS) of a selected strain TM39, i.e., TM39-SCS, significantly inhibited the viability of H. pylori in vitro. It also inhibited the urease activity of H. pylori in vitro. For these antagonistic effects, in addition to pH and lactic acid, some factors in TM39-SCS might play the major role. Treatment of H. pylori with the SCS or cells of strain TM39 significant reduced its binding to TSGH 9201 cells. Although strain TM39 is identified as Enterococcus faecium, it is not vancomycin resistant and is proved to be safe through the invasion study and a 28-day feeding study with Wistar rats.     

抗幽门螺杆菌乳酸菌的筛选

通过测定干酪乳杆菌（Lactobacillus casei）的自凝聚、疏水性及抑菌实验来筛选粘附性高、对幽门螺杆菌（Helicobacter pylori）有强抑制作用的益生菌株,并通过干酪乳杆菌与幽门螺杆菌的交互凝集和对幽门螺杆菌尿素酶活性的抑制进一步探讨其抑菌机制。结果表明,在4株干酪乳杆菌中,菌株JY300-8的自凝聚百分比在24 h达到71.7%,比菌株JY、JY160-15、JY300-10分别高19.7%、28.8%、29.6%（P＜0.05）;疏水性百分比为72.3%,比菌株JY、JY160-15、JY300-10分别高4.1%、8.0%、7.5%（P＜0.05）。同时,菌株JY300-8在24 h时的共凝集百分比为64.4%,较菌株JY提高了5.3%;而且菌株JY300-8对幽门螺杆菌尿素酶活性的抑制率达到62.3%。结果表明,在4株干酪乳杆菌中,菌株JY300-8具有良好的益生性能,对胃癌的生物致病菌具有良好的抑制效果,可用于后续胃癌动物模型的研究和抗胃癌的益生菌制剂的开发。     

Nonstarter lactobacilli isolated from soft and semihard Argentinean cheeses: genetic characterization and resistance to biological barriers

Nonstarter lactic acid bacteria isolated from Argentinean cheeses were identified and characterized by focusing on their resistance to biological barriers, along with other physiological features of potential interest, in the search for future probiotic organisms. Lactobacilli were enumerated and isolated from semihard and soft cheeses made with multistrain Streptococcus thermophilus starters. Lactobacilli counts in 1-week-old cheeses were between 10(5) and 10(7) CFU/g and then reached 10(7) CFU/ g in all 1-month samples, while streptococci were always above 10(9) CFU/g. A total number of 22 lactobacilli isolates were retained, identified, and characterized by in vitro tests. Species identity was determined by carbohydrate metabolism and species-specific PCR assays. Genetic diversity was explored by random amplified polymorphic DNA (RAPD) PCR analysis. The Lactobacillus strains were assigned to the species L. casei, L. plantarum, L. rhamnosus, L. curvatus, L. fermentum, and L. perolens. All the strains studied tolerated 25 ppm of lysozyme, and most of them showed resistance to 0.3% bile. After incubation in gastric solution (pH 2.0), counts decreased by several log units, ranging from 3.2 to 7.0. The strains were able to grow in the presence of bile salts, but only three isolates were capable of deconjugation. The nonstarter lactobacilli that were assayed fermented the prebiotic substrates (especially lactulose and inulin). Some strains showed high cell hydrophobicity and beta-galactosidase activity, as well as inhibitory activity against pathogenic bacteria. It was concluded that most of the lactobacilli isolated in this study demonstrated resistance to biological barriers and physiological characteristics compatible with probiotic properties, which make them suitable for further research in in vivo studies aimed at identifying new probiotic organisms.     

益生乳杆菌的筛选研究

以分离保存的15株乳杆菌为研究对象,从中筛选益生乳杆菌两株。通过检测乳杆菌的疏水性和自聚性,得到表面疏水性和自聚性均较高的菌株为Ind-3、CH10和M8。其中,乳杆菌CH10和M8在模拟胃肠环境下活菌数均达到106mL-1以上。通过灌胃乳杆菌CH10和M8,表明小鼠体重增加明显高与对照组,且实验组小鼠粪便中的β-半乳糖苷酶活性明显高于对照组。灌胃两株乳杆菌后,小鼠肠道内双歧杆菌与乳杆菌活菌数提高了,且肠杆菌、肠球菌和产气荚膜梭菌的生长繁殖受到不同程度的抑制,而对照组无明显变化。结果表明筛选出的两株乳杆菌可以促进小鼠对营养物质的吸收,提高肠道内有益菌的数量,抑制有害菌的生长繁殖,或许能够改善寄主肠道的微生态环境。     

酱香型白酒醇类活性成分抗幽门螺杆菌损伤胃黏膜上皮细胞活性的研究

为研究酱香型白酒醇类活性成分(active alcohols,AAs)抑制幽门螺杆菌(Helicobacter pylori)损伤胃黏膜上皮(GES-1)细胞活性及潜在的机制,检测了AAs抑制H.pylori的最小半数抑菌浓度(50%minimun inhibitory concentration,MIC 50)及苯乙醇(AA-29)抑制H.pylori损伤GES-1细胞活性;分析了AA-29对H.pylori脲酶活性的影响;通过RT-PCR分析了AA-29对H.pylori脲酶基因簇(ureA、ureB、ureE、ureH、ureI和nixA)、外膜蛋白基因(babA)和毒力基因(cagA和vacA)转录的影响;采用Western blotting检测了AA-29对cagA和vacA表达的影响;ELISA及流式检测AA-29对H.pylori诱导的GES-1细胞TNF-α、IL-1β、IL-6及细胞凋亡的影响。结果表明:AA-29活性最佳,可显著抑制H.pylori生长,其MIC 50(72 h)为105.2μg/mL;100μg/mL的AA-29可显著抑制H.pylori的脲酶活性;RT-PCR结果显示,AA-29可抑制H.pylori脲酶基因簇中的ureA、ureB、nixA及babA和cagA的转录及cagA的表达;AA-29可抑制H.pylori诱导的GES-1细胞TNF-α、IL-1β、IL-6分泌及细胞凋亡。酱香型白酒中的醇类化合物具有一定的抗H.pylori活性,其中AA-29可通过抑制H.pylori脲酶相关基因的表达并抑制H.pylori脲酶活性;AA-29可降低H.pylori babA、cagA转录及cagA的表达以减轻H.pylori对胃黏膜上皮细胞的损伤。     

Exfoliation of Helicobacter pylori from gastric mucin by glycopolypeptides from buttermilk

In the human stomach, Helicobacter pylori, an ulcer pathogenic bacterium, colonizes the gastric mucosal layer primarily. The ability of glycopolypeptides (GPP) prepared from buttermilk to exfoliate H. pylori bound to gastric mucin was investigated. The GPP were prepared from buttermilk by digestion with trypsin, papain, pancreatin, bromelain, or pepsin. Helicobacter pylori ATCC 43504T and 43579 adhered more strongly to all of the GPP tested than to whole buttermilk, the soluble fraction of buttermilk, gastric mucin prepared from mouse stomach, or commercial pig gastric mucin. The GPP digested with trypsin, papain, or pancreatin were significantly more adherent. When the GPP concentration was 10 mg/mL, bound H. pylori ATCC 43504T, 43579, and 5 clinical isolates were exfoliated markedly from immobilized porcine gastric mucin following treatment with GPP digested with trypsin or pancreatin. This ability of GPP did not correlate with sialic acid content, indicating that sialic acid content is not important in the exfoliation of this microorganism. Such an ability may depend on the structure or number of sugar chains, or the position of sialic acid. We conclude that GPP promote the exfoliation of H. pylori bound to gastric mucin and prevent the de novo adherence of this microorganism. As such, GPP are a promising food material for preventing H. pylori infection.     

Influence of combinations of Lactobacillus lactis and potassium sorbate on growth of psychrotrophs in raw milk

Cells of Lactobacillus lactis from frozen concentrated cultures were added (1 X 10(8)/ml) to refrigerated raw milk to determine their effect on growth of psychrotrophs during storage of the milk at 5 or 7 degrees C. Some strains of Lactobacillus lactis were significantly inhibitory toward the psychrotrophs in refrigerated raw milk at both temperatures. Two selected strains of Lactobacillus lactis were added along with potassium sorbate (.1 and .2%) to refrigerated raw milk stored at 5 degrees C to determine their combined effect on growth of the psychrotrophs. The combinations of cells of lactobacilli and sorbate were more inhibitory than either lactobacilli or sorbate alone. The measurement of hydrogen peroxide produced by strains of Lactobacillus lactis indicated that the strains most antagonistic toward psychrotrophs also were the more active producers of hydrogen peroxide. Addition of sorbate had no apparent effect on hydrogen peroxide production by the cultures during refrigerated storage.     

Antibiosis of some lactic acid bacteria including Lactobacillus acidophilus toward Listeria monocytogenes

Eleven strains of lactic acid bacteria were tested by the 'spot' on the 'lawn' method for their antagonistic activity against four strains of Listeria monocytogenes. Four out of the five strains of lactic acid bacteria most antagonistic toward the pathogen were those cultures known to produce bacteriocins. Four other strains of lactic acid bacteria were not antagonistic against Listeria by this method. Seventeen inhibition zones of the pathogen were obtained at 25 degrees C as compared to 10 at 32 degrees C. Lactobacillus acidophilus strains NU-A and 88, growing in the presence of L. monocytogenes in milk prevented the latter from attaining populations it would have in pure culture (P less than 0.01). 10(1.4)-10(3.5) lower numbers were noted. L. acidophilus in most cases exhibited a bacteriostatic effect toward the pathogen except for strain 88 which appeared to have a bactericidal effect (P less than 0.01) against Listeria strain OH. The lactobacilli reduced the pH of the milk to 4.7 over a 24 h period, showing that acid played a role in the observed antibiosis.     

Technological characterization of the natural lactic acid bacteria of artisanal Turkish White Pickled cheese

The aim of this study was to characterize the lactic acid bacteria (LAB) isolated from White Pickled cheeses produced with traditional methods; and to improve the quality of cheesemaking with a selection of bacterial cultures from artisanal White cheeses. LAB were isolated and identified from 30 White Pickled cheese samples collected from various cities in Turkey. Also, the numbers of several microbial groups (total aerobic mesophilic bacteria, LAB, enterococci, coliforms, moulds and yeasts) of cheese samples were enumerated. Lactobacilli, lactococci and enterococci were the most abundant microbial groups. The numbers of Enterococcus and Lactobacillus isolates were higher than those of the other LAB. Enterococcus faecalis (24.43%), Enterococcus faecium (17.61%) and Lactobacillus fermentum (19.88%) isolates were the most frequently isolated species. Lactococcus strains showed the highest acidifying activity, followed by Enterococcus and Lactobacillus strains. Proteolytic activity of Enterococcus faecalis strains was higher than that of the other enterococci species, except Enterococcus avium strains. Within lactobacilli strains, the highest mean proteolytic activity was that of Lactobacillus bifermentans, Lactobacillus brevis and Lactobacillus casei strains.     

Modulation of Th1/Th2 balance by Lactobacillus strains isolated from Kimchi via stimulation of macrophage cell line J774A.1 in vitro

Lactobacilli isolated from Kimchi, a Korean traditional food, were tested for their capacity to modulate the T helper (Th) 1/Th2 balance. Ovalbumin (OVA)-sensitized mouse splenocytes were cultured with 26 strains of lactobacilli; the highest IL-12 induction and lowest IL-4 production were then observed in 4 strains, including Lactobacillus plantarum CJLP55, CJLP56, CJLP133, and CJLP136. These strains produced a larger amount of IL-12, which enhances differentiation and activation of Th1 cells, in macrophage cell-lines more than positive control strains L. casei KCTC 3109(T) and L. rhamnosus GG, although they also induced production of IL-10, which is a suppressor of IL-12. Indeed, CJLP133-stimulated macrophages induced production of more Th1 cytokine IFN-γ and less Th2 cytokine IL-4 than KCTC 3109(T) and GG in co-cultivation with T cells. These findings suggest that lactobacilli from Kimchi may modulate the Th1/Th2 balance via macrophage activation in the hypersensitive reaction caused by Th2 cells. PRACTICAL APPLICATION: Allergic reactions including asthma and atopy are caused by predominance of Th2 response over Th1 response. Lactobacilli isolated from fermented foods such as yogurt, cheese, and Kimchi showed health-promoting activities. The present study indicated that several lactobacilli strains from Kimchi may reduce allergic reactions through macrophage-mediated induction of Th1 response.     

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.     

Lactobacilli as multifaceted probiotics with poorly disclosed molecular mechanisms

Lactic acid bacteria and more particularly lactobacilli have been used for the production of fermented foods for centuries. Several lactobacilli have been recognized as probiotics due to their wide range of health-promoting effects in humans. However, little is known about the molecular mechanisms underpinning their probiotic functions. Here we reviewed the main beneficial effects of lactobacilli and discussed, when the information is available, the molecular machinery involved in their probiotic function.Among the beneficial effects, lactobacilli can improve digestion, absorption and availability of nutrients. As an example, some strains are able to degrade carbohydrates such as lactose or α-galactosides that may cause abdominal pain. Furthermore, they can hydrolyze compounds that limit the bioavailability of minerals like tannin and phytate due to tannin acylhydrolase and phytase activities. In addition, it was shown that some lactobacilli strains can improve mineral absorption in Caco-2 cells. Lactobacilli can also contribute to improve the nutritional status of the host by producing B group vitamins. More recently, the role of lactobacilli in energy homeostasis, particularly in obese patients, is the object of an increased interest.Lactobacilli are also involved in the prevention of diseases. They have potential to prevent carcinogenesis through the modulation of enzymes involved in the xenobiotic pathway, and may prevent cardiovascular diseases such as hypertension through the production of a bioactive peptide that may have angiotensin converting enzyme inhibitor activity. Lactobacilli are increasingly studied for the treatment of inflammatory bowel diseases and exhibit interesting potential in the reduction of pain perception. The ability of some strains to bind to intestinal cells, their pathogen-associated molecular patterns and the metabolites they produce confer interesting immunomodulatory effects. Finally, pathogenic fungi, virus or bacteria can be inhibited by probiotics. They can reinforce the intestinal barrier, simply occupying the ecological niche, or they can have an active role by synthesizing various metabolites inhibiting pathogen development.Lactobacilli have a long standing history with foods and humans but comparatively, their history as probiotics is recent. Their effects are investigated in in vitro and in vivo models leading sometimes to contradictory or controversial results that make necessary final demonstrations through clinical trials. Researches on molecular mechanisms involved in the beneficial effects reviewed here are necessary for a better understanding of these effects, but could also lead to the development of molecular tools to help the screening of the probiotic potential of lactobacilli that are common inhabitants of numerous fermented foods around the world.     

Numbers and strains of lactobacilli in some probiotic products

The numbers and types of Lactobacillus strains in probiotic feed or food products for sale in Europe were compared with the information stated on the product labels. The labels of all 10 products gave information on the genus and species of Lactobacillus, and seven gave information on the numbers of a specified strain(s). Lactobacilli were recovered using a recently validated method, developed as part of the European Community Project SMT4 CT98-2235 for the official control of probiotic microorganisms used as feed additives. Polymerase chain reaction (PCR)-based methods were used to assess the accuracy of labelling with regard to genus and species, and pulsed-field gel electrophoresis (PFGE) was used to identify strains. Five products were mislabelled with respect to the numbers and three with respect to species of lactobacilli. In four cases, the specified strains were not detected. Four fermented milks sold under three trademarks contained the same strain, which was named differently on each label. As safety and functionality of probiotics are strain dependent, these results demonstrated the need to control lactobacilli present in commercially probiotic human food products, not only at the species but also at the strain level, to ensure their quality and protect the consumer. The usefulness of the official methods developed for animal feed additives to identify lactobacilli in probiotic foods for humans was demonstrated.     

干酪乳杆菌LC2W对两种不同胃癌细胞的黏附作用

选取人胃癌细胞株MKN-45和HGC-27,采用形态学观察和显微计数的方法比较菌液浓度、悬浮液、pH值对干酪乳杆菌LC2W(Lactobacillus casei LC2W)黏附细胞的情况。结果表明,LC2W对两种细胞均具有黏附性,对MKN-45的黏附性更强;这种黏附作用具有菌悬液浓度依赖性。当加入的菌悬液浓度达到一定数量时(109 mL-1),平均每个细胞黏附的乳杆菌数量趋于饱和;悬浮介质对Lb.caseiLC2W的黏附性具有明显的影响,其中悬浮于耗尽培养上清液(SCS)的Lb.casei LC2W表现出最好的黏附性;悬浮介质的pH值对Lb.casei LC2W的黏附性也有影响,pH=4.0时黏附性最好。将不同悬浮介质的pH值调整到pH=7.0后,SCS仍能很好的促进LC2W的黏附作用,表明SCS中存在某种促进黏附的物质。由此推测,Lb.casei LC2W对胃癌细胞的黏附机理可能为黏附素与受体的特异性结合。     

吡嗪类化合物抗幽门螺杆菌活性及机制研究

该实验研究了吡嗪类化合物（PYRs）对幽门螺杆菌（Hp）的抗菌活性及作用机制。采用PYRs处理Hp菌液,观察Hp的活性及形态变化;利用反转录-聚合酶链式反应（RT-PCR）分析PYRs对Hp基因及相关毒力蛋白表达的影响;检测PYRs预处理Hp对胃黏膜上皮细胞（GES-1）活性、细胞凋亡的影响。结果表明,PYR-19（2,5-二甲基-3-正戊基吡嗪）可抑制Hp增殖,Hp外膜蛋白基因（BabA）和毒力基因（CagA和VacA）的转录及毒力蛋白（CagA和VacA）的表达,Hp诱导的GES-1细胞炎性细胞因子分泌及对GES-1细胞的损伤,减轻Hp对胃黏膜上皮细胞的损伤能力。     

Survival of lactic acid bacteria from fermented milks in an in vitro digestion model exploiting sequential incubation in human gastric and duodenum juice

In the present study, the survival of 9 lactic acid bacteria (5 Lactococcus strains, 3 Lactobacillus strains, and 1 strain of Enterococcus hirae), was investigated in vitro under conditions similar to human digestion using human gastric and duodenal juices. The tolerance of the bacteria was also tested with traditional methods using acidic conditions and bile salts. The strains were subjected to a model digestive system comprising sequential incubation in human gastric and duodenal juices, in a 2-step digestion assay at 37°C, simulating the human upper gastrointestinal tract with human gastric juices at pH 2.5 and human duodenal juices at pH 7. The bacterial strains were tested either as washed cells from culture media or in fermented milk. The initial in vitro testing in acid and bile salts showed that Lactobacillus strains and the E. hirae strain displayed a significantly higher acid tolerance than the lactococci. The lactobacilli and the Enterococcus numbers increased, whereas the lactococci decreased at least 1 log during the bile salt treatment. The Lactobacillus strains showed the highest survival rate in the model digestive system when washed bacterial cultures were used with a minor log reduction, whereas the lactococci numbers were reduced by at least log 4. However, when using fermented milks in the model digestion system it was demonstrated that the Enterococcus strain and 2 strains of Lactococcus lactis ssp. cremoris benefited significantly from the presence of the fermented milk as food matrix, with log numbers >log 7 and 5, respectively, after digestion of the fermented milk. The analyses reported comprise a comprehensive in vitro testing regimen suitable for evaluation of the survival of candidate probiotic bacteria in human digestion as an initial prescreen to clinical trials.