Lactobacilli have been consistently associated with dental caries for decades;however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit iden...Lactobacilli have been consistently associated with dental caries for decades;however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on sociodemographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children’s medical history, and a questionnaire survey. Combined non-stimulated saliva and supragingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, ex- traction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cario- genic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.展开更多
Eighty sorts of herbs were fermented by Lactobaccilli (f-ESH). This material was proved by as safe in animal safety experiment. This work was aimed to revise the traditional way of hot water extraction to the fermenta...Eighty sorts of herbs were fermented by Lactobaccilli (f-ESH). This material was proved by as safe in animal safety experiment. This work was aimed to revise the traditional way of hot water extraction to the fermentation, in order to use up the original material and searching new activity as well. We tried to prove the new activity by fermentation for immune-competent cells in the host that administrated both of original remedy and the new fermented sample. In rodents, immune-compromised host was prepared with cancer chemotherapeutic agent. Our observations showed that the antigen stimulated animal increased the antibody producing cell for heterologous erythrocyte by compromised host by f-ESH. Moreover, by administration of f-ESH to immno-suppressed hosts, these samples regulated lymphocyte in number and functions, macrophage activities, and regulating anti-oxidative activity by phagocytic cells. The anti-oxidative assay was carried out ex-vivo system by peritoneal excaudate cell that we propose as suitable system for evaluating antioxidative assay. With these evidences, the original COF only augmented the level of lymphocytes in number, while f-ESH regulated the ratio of granulocytes and lymphocyte. In clinical study with 20 healthy volunteers, granulocyte and lymphocyte ratio was also resulted as neutral in peripheral white blood cells, 1, 2 and 3 weeks after the administration of f-ESH. We discussed the significance and mechanism of cleving complement components Fb by f-ESH in this text. Fb. The new cleavage of complement was directly evident by immune-electrophoretic method. In conclusion, this modification of food materials introduced new style of food intake resulted in the significant regulation such as antibody producing cells and anti-oxidative activity for phagocytic cells comparing traditional processing by hot water extraction.展开更多
The in vitro screening of lactobacilli isolates from uttapam batter, fermented supplementing with Piper betle L. leaves, was performed in order to select potent isolates for probiotic use. Their resistance to simulate...The in vitro screening of lactobacilli isolates from uttapam batter, fermented supplementing with Piper betle L. leaves, was performed in order to select potent isolates for probiotic use. Their resistance to simulated gastric and intestinal juices as well as their adhesion to epithelial intestinal HCT-15 and vaginal HeLa cell lines were assessed and also evaluated for their immunomodulatory ability in ex vivo condition. Isolates were able to adhere as well as reduce pathogen adhesion to monolayer cell lines’ surfaces and reduced production of pro-inflammatory cytokine TNF-α in LPS treated PBMCs, while enhanced that of anti-inflammatory cytokine IL-10. The isolates exhibited properties of auto-aggregation, co-aggregation, hydrophobicity, bile salt hydrolase activity and strong antimicrobial activity against pathogenic bacteria. Thus, the different lactobacilli isolates displayed potent probiotic and immunomodulatory properties among which AJ7 and AJ82 had a great potential and may have applications in fermented foods as immunomodulatory probiotic additives.展开更多
Objective To investigate the indigenous lactobacilli from the vagina of pregnant women and to screen the isolates with antagonistic potential against pathogenic microorganisms.Methods The strains were isolated from pr...Objective To investigate the indigenous lactobacilli from the vagina of pregnant women and to screen the isolates with antagonistic potential against pathogenic microorganisms.Methods The strains were isolated from pregnant women's vagina and identified using the API50CH system.The ability of the isolates to produce hydrogen peroxide was analyzed semi-quantitatively using the TMB-HRP-MRS agar.The antagonistic effects of the isolates on pathogenic microorganisms were determined with a double layer agar plate.Results One hundred and three lactobacilli strains were isolated from 60 samples of vaginal secretion from healthy pregnant women.Among them,78 strains could produce hydrogen peroxide,in which 68%,80%,80%,and 88% had antagonistic effects against Candida albicans CMCC98001,Staphylococcus aureus CMCC26003,Escherichia coli CMCC44113,and Pseudomonas aeruginosa CMCC10110,respectively.Conclusion The recovery of hydrogen peroxide-producing lactobacilli decreases with the increasing pregnant age and time.The most commonly isolated species from vagina of Chinese pregnant women are Lactobacillus acidophilus and Lactobacillus crispatus.Most of L.acidophilus and L.crispatus produce a high H2O2 level.展开更多
Microbiota in the gastrointestinal tract (GIT) of piglets during weaning transition can experience a sharp change which could result in growth reduction and diarrhea of weaned piglets. Dietary manipulations can play a...Microbiota in the gastrointestinal tract (GIT) of piglets during weaning transition can experience a sharp change which could result in growth reduction and diarrhea of weaned piglets. Dietary manipulations can play an important role in attenuating such changes caused by weaning stress. Therefore, ileal and colonic contents of weaned piglets were used as inocula, mannan oligosaccharide (MOS) or sugar beet pulp (SBP) was supplied as single energy sources to investigate effects of MOS or SBP on the shifts of gastro-intestinal microflora and lactobacilli populations. The universal bacteria- and lactobacilli-specific PCR/denaturing gradient gel electrophoresis (DGGE), cloning and sequencing techniques were used. DGGE profiles of the universal bacteria showed that great changes were found in the position, numbers and intensity of dominant bands after fermentation. The similarity of bacterial community between ileum and colon was increased to 85-97% by MOS or SBP treatment after fermentation from the similarity with 20% before fermentation. MOS treatment significantly increased the bacterial diversity and band number in both ileal and colonic fermentation (P<0.05). SBP treatment significantly increased the bacterial diversity and band number in colon (P<0.05). It implies that some species were enriched by the addition of MOS or SBP to increase the similarity and diversity of bacterial community in weaned piglets. Five specific bands appearing in MOS or SBP treatment group after fermentation were cloned and sequenced, the changes of species related to Prevotella and Ruminococcus were observed. Two bands related to uncultured bacterium with 98% similarity were detected by MOS or SBP treatment. However, there were no effects on the similarity, diversity index and lactobacilli species revealed by MOS or SBP treatment. These results imply that MOS or SBP could have beneficial effects on the weaning piglets by stablizing microbiota in the GIT microflora.展开更多
AIM: To investigate whether the stimulation of peripher- al blood mononuclear cells (PBMNC) with the cell debris and cell extraction of different probiotic strains is similar or species specifi c. METHODS: Three strai...AIM: To investigate whether the stimulation of peripher- al blood mononuclear cells (PBMNC) with the cell debris and cell extraction of different probiotic strains is similar or species specifi c. METHODS: Three strains of bifi dobacteria, 4 strains of lactobacilli, and E. coli nissle were sonicated and centri- fuged in order to divide them into cell extract and cell debris. PBMNC were separated by density gradient and incubated for 36 h with either the cell debris or the cell extract of single strains of probiotic bacteria in doses from 102 to 108 CFU/mL. Cell supernatants were taken and interleukin (IL)-10, IL-1β, and tumor necosis factor (TNF)-α were determined by ELISA. RESULTS: Depending on the species super-family, the strains had different stimulation patterns. Except for both L. casei strains, the cell extract of bifidobacteriaand lactobacilli had less stimulating capacity than cell debris, whereas the cell extract of E. coli nissle had simi- lar stimulating properties to that of the cell debris of the strain and significantly more stimulating capacity than that of bifi dobacteria and lactobacilli. The cell debris of bifi dobacteria stimulated more cytokine release than the cell debris of lactobacilli. The cell debris of lactobacilli did not have a stimulating capacity when lower concentra- tions were used. Neither cell extraction nor cell debris had an inhibitory effect on the production of the tested cytokines by stimulated PBMNC. CONCLUSION: The incubation of probiotic strains, which have been used in clinical trials for inflammatory diseases, with immunocompetent cells leads to different species specifi c reactions. High IL-10 response to cell de- bris of bifi dobacteria and E. coli nissle can be found. This corresponds to positive effects of bifi dobacteria and E. coli nissle in clinical trials for inflammatory bowel disease compared to negative outcomes obtained with lactoba- cilli.展开更多
Considering the importance of the poultry industry and the increasing interest in alternative growth promoters,probiotics are considered as a potential candidate for use in the poultry industry.In this study,Lactobaci...Considering the importance of the poultry industry and the increasing interest in alternative growth promoters,probiotics are considered as a potential candidate for use in the poultry industry.In this study,Lactobacillus species were isolated from 21 rectal swabs of 11 healthy 6-day-old and 10 healthy 21-day-old chickens and their fecal and feed samples.The isolates were characterized and their probiotic characteristics,including resistance to gastric acid and bile salts,biofilm formation and adherence to epithelium or mucus,amylase and protease activity and production of inhibitory compounds,were assessed.From 31 acid and bile resistant lactobacilli,only 2 Lactobacillus brevis and 1 Lactobacillus reuteri strains showed significant probiotic properties.These isolates indicated detectable attachment to Caco-2 cells and significant antibacterial activities against Gram-positive and Gram-negative pathogens.Additionally,phenotypic and genotypic diversity of lactobacilli isolates were studied by Phene Plate(PhP)system(PhP-LB)and random amplified polymorphic DNA(RAPD)-PCR,respectively.PhP-LB results of 24 L.brevis isolates showed a high phenotypic variation among the isolates.In comparison,results of RAPD-PCR highlighted a low diversity.Therefore,it seems that combination of the 2 techniques(PhP and RAPD-PCR)could result in a significant discriminatory power than each of them used alone.展开更多
将含有乳酸杆菌Lactobacillus casei Zhang的悬浮液分组饲喂小鼠,然后分别用E.coliO157和K88攻毒观察发病情况。攻毒4d后取对照组和喂菌组未死亡小鼠的肠内容物,用选择性培养基分离纯化大肠杆菌和乳酸菌,提取分离到菌株的总DNA,进行ERIC...将含有乳酸杆菌Lactobacillus casei Zhang的悬浮液分组饲喂小鼠,然后分别用E.coliO157和K88攻毒观察发病情况。攻毒4d后取对照组和喂菌组未死亡小鼠的肠内容物,用选择性培养基分离纯化大肠杆菌和乳酸菌,提取分离到菌株的总DNA,进行ERIC-PCR扩增分析。发现灌服L.casei Zhang可以降低攻毒后小鼠的死亡率,在停止饲喂乳酸菌的第4d从小鼠肠道内分离到L.casei Zhang,从饲喂组未分离到E.coliO157和K88,喂L.casei Zhang使小鼠肠道中大肠杆菌总数极显著低于对照组。表明所饲喂的乳杆菌可以在小鼠肠道内定殖并对小鼠起到保护作用。展开更多
基金supported by the National Institute of Dental and Craniofacial Research(R01DE019455).
文摘Lactobacilli have been consistently associated with dental caries for decades;however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on sociodemographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children’s medical history, and a questionnaire survey. Combined non-stimulated saliva and supragingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, ex- traction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cario- genic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.
文摘Eighty sorts of herbs were fermented by Lactobaccilli (f-ESH). This material was proved by as safe in animal safety experiment. This work was aimed to revise the traditional way of hot water extraction to the fermentation, in order to use up the original material and searching new activity as well. We tried to prove the new activity by fermentation for immune-competent cells in the host that administrated both of original remedy and the new fermented sample. In rodents, immune-compromised host was prepared with cancer chemotherapeutic agent. Our observations showed that the antigen stimulated animal increased the antibody producing cell for heterologous erythrocyte by compromised host by f-ESH. Moreover, by administration of f-ESH to immno-suppressed hosts, these samples regulated lymphocyte in number and functions, macrophage activities, and regulating anti-oxidative activity by phagocytic cells. The anti-oxidative assay was carried out ex-vivo system by peritoneal excaudate cell that we propose as suitable system for evaluating antioxidative assay. With these evidences, the original COF only augmented the level of lymphocytes in number, while f-ESH regulated the ratio of granulocytes and lymphocyte. In clinical study with 20 healthy volunteers, granulocyte and lymphocyte ratio was also resulted as neutral in peripheral white blood cells, 1, 2 and 3 weeks after the administration of f-ESH. We discussed the significance and mechanism of cleving complement components Fb by f-ESH in this text. Fb. The new cleavage of complement was directly evident by immune-electrophoretic method. In conclusion, this modification of food materials introduced new style of food intake resulted in the significant regulation such as antibody producing cells and anti-oxidative activity for phagocytic cells comparing traditional processing by hot water extraction.
文摘The in vitro screening of lactobacilli isolates from uttapam batter, fermented supplementing with Piper betle L. leaves, was performed in order to select potent isolates for probiotic use. Their resistance to simulated gastric and intestinal juices as well as their adhesion to epithelial intestinal HCT-15 and vaginal HeLa cell lines were assessed and also evaluated for their immunomodulatory ability in ex vivo condition. Isolates were able to adhere as well as reduce pathogen adhesion to monolayer cell lines’ surfaces and reduced production of pro-inflammatory cytokine TNF-α in LPS treated PBMCs, while enhanced that of anti-inflammatory cytokine IL-10. The isolates exhibited properties of auto-aggregation, co-aggregation, hydrophobicity, bile salt hydrolase activity and strong antimicrobial activity against pathogenic bacteria. Thus, the different lactobacilli isolates displayed potent probiotic and immunomodulatory properties among which AJ7 and AJ82 had a great potential and may have applications in fermented foods as immunomodulatory probiotic additives.
基金China Scholarship Council (2003836007)863 Program of China (2008AA10Z337)973 Program of China (2005DKA21202-1)
文摘Objective To investigate the indigenous lactobacilli from the vagina of pregnant women and to screen the isolates with antagonistic potential against pathogenic microorganisms.Methods The strains were isolated from pregnant women's vagina and identified using the API50CH system.The ability of the isolates to produce hydrogen peroxide was analyzed semi-quantitatively using the TMB-HRP-MRS agar.The antagonistic effects of the isolates on pathogenic microorganisms were determined with a double layer agar plate.Results One hundred and three lactobacilli strains were isolated from 60 samples of vaginal secretion from healthy pregnant women.Among them,78 strains could produce hydrogen peroxide,in which 68%,80%,80%,and 88% had antagonistic effects against Candida albicans CMCC98001,Staphylococcus aureus CMCC26003,Escherichia coli CMCC44113,and Pseudomonas aeruginosa CMCC10110,respectively.Conclusion The recovery of hydrogen peroxide-producing lactobacilli decreases with the increasing pregnant age and time.The most commonly isolated species from vagina of Chinese pregnant women are Lactobacillus acidophilus and Lactobacillus crispatus.Most of L.acidophilus and L.crispatus produce a high H2O2 level.
基金funded by the National Basic Research Program of China (973 Program,2004CB117500)
文摘Microbiota in the gastrointestinal tract (GIT) of piglets during weaning transition can experience a sharp change which could result in growth reduction and diarrhea of weaned piglets. Dietary manipulations can play an important role in attenuating such changes caused by weaning stress. Therefore, ileal and colonic contents of weaned piglets were used as inocula, mannan oligosaccharide (MOS) or sugar beet pulp (SBP) was supplied as single energy sources to investigate effects of MOS or SBP on the shifts of gastro-intestinal microflora and lactobacilli populations. The universal bacteria- and lactobacilli-specific PCR/denaturing gradient gel electrophoresis (DGGE), cloning and sequencing techniques were used. DGGE profiles of the universal bacteria showed that great changes were found in the position, numbers and intensity of dominant bands after fermentation. The similarity of bacterial community between ileum and colon was increased to 85-97% by MOS or SBP treatment after fermentation from the similarity with 20% before fermentation. MOS treatment significantly increased the bacterial diversity and band number in both ileal and colonic fermentation (P<0.05). SBP treatment significantly increased the bacterial diversity and band number in colon (P<0.05). It implies that some species were enriched by the addition of MOS or SBP to increase the similarity and diversity of bacterial community in weaned piglets. Five specific bands appearing in MOS or SBP treatment group after fermentation were cloned and sequenced, the changes of species related to Prevotella and Ruminococcus were observed. Two bands related to uncultured bacterium with 98% similarity were detected by MOS or SBP treatment. However, there were no effects on the similarity, diversity index and lactobacilli species revealed by MOS or SBP treatment. These results imply that MOS or SBP could have beneficial effects on the weaning piglets by stablizing microbiota in the GIT microflora.
基金Supported by a grant from "Trainig and Mobility of Researchers" program, RX-CT98-0240
文摘AIM: To investigate whether the stimulation of peripher- al blood mononuclear cells (PBMNC) with the cell debris and cell extraction of different probiotic strains is similar or species specifi c. METHODS: Three strains of bifi dobacteria, 4 strains of lactobacilli, and E. coli nissle were sonicated and centri- fuged in order to divide them into cell extract and cell debris. PBMNC were separated by density gradient and incubated for 36 h with either the cell debris or the cell extract of single strains of probiotic bacteria in doses from 102 to 108 CFU/mL. Cell supernatants were taken and interleukin (IL)-10, IL-1β, and tumor necosis factor (TNF)-α were determined by ELISA. RESULTS: Depending on the species super-family, the strains had different stimulation patterns. Except for both L. casei strains, the cell extract of bifidobacteriaand lactobacilli had less stimulating capacity than cell debris, whereas the cell extract of E. coli nissle had simi- lar stimulating properties to that of the cell debris of the strain and significantly more stimulating capacity than that of bifi dobacteria and lactobacilli. The cell debris of bifi dobacteria stimulated more cytokine release than the cell debris of lactobacilli. The cell debris of lactobacilli did not have a stimulating capacity when lower concentra- tions were used. Neither cell extraction nor cell debris had an inhibitory effect on the production of the tested cytokines by stimulated PBMNC. CONCLUSION: The incubation of probiotic strains, which have been used in clinical trials for inflammatory diseases, with immunocompetent cells leads to different species specifi c reactions. High IL-10 response to cell de- bris of bifi dobacteria and E. coli nissle can be found. This corresponds to positive effects of bifi dobacteria and E. coli nissle in clinical trials for inflammatory bowel disease compared to negative outcomes obtained with lactoba- cilli.
基金support by a grant(No.90007454)from Iran National Science Foundation.
文摘Considering the importance of the poultry industry and the increasing interest in alternative growth promoters,probiotics are considered as a potential candidate for use in the poultry industry.In this study,Lactobacillus species were isolated from 21 rectal swabs of 11 healthy 6-day-old and 10 healthy 21-day-old chickens and their fecal and feed samples.The isolates were characterized and their probiotic characteristics,including resistance to gastric acid and bile salts,biofilm formation and adherence to epithelium or mucus,amylase and protease activity and production of inhibitory compounds,were assessed.From 31 acid and bile resistant lactobacilli,only 2 Lactobacillus brevis and 1 Lactobacillus reuteri strains showed significant probiotic properties.These isolates indicated detectable attachment to Caco-2 cells and significant antibacterial activities against Gram-positive and Gram-negative pathogens.Additionally,phenotypic and genotypic diversity of lactobacilli isolates were studied by Phene Plate(PhP)system(PhP-LB)and random amplified polymorphic DNA(RAPD)-PCR,respectively.PhP-LB results of 24 L.brevis isolates showed a high phenotypic variation among the isolates.In comparison,results of RAPD-PCR highlighted a low diversity.Therefore,it seems that combination of the 2 techniques(PhP and RAPD-PCR)could result in a significant discriminatory power than each of them used alone.
