Cloning of Tap Ⅱ Chalcone Isomerases(CHI1 A) Gene and Construction of Lactococcus Lactis Expression Vector

[Objective]The aim was to clone TapⅡchalcone isomerases（CHI1 A） from soybean specially and construct expression vector of PNZ8149-CHI1 A,and then transform it into Lactococcus Lactis NICE systers.[Method]Chalcone isomerases（CHI1 A） was cloned by RTPCR method,and it was sequenced after cloning into pMD18-T vectors,and recombined to expression vector PNZ8149-CHI1 A,then it was transformed into Lactococcus Lactis NZ3900[Result]The sequencing results indicated that the cloned fragment of CHI1 A contained 670 nucleotides,and shared a sequence homology of 92% with that from Genbank accession number AF595413（CHI1 A）.CHI1 A was transformed into NICE expression system successfully by identification of PCR and digestion.[Conclusion]The foundation of using the microorganism fermentation method to produce flavonoids was laid by construction of efficient induction expression vector with chalcone isomerases CHI1 A.

Reconstruction of a Food-grade Expression Vector in Lactococcus Lactis

[Objective]In order to improve expression vector of L.lactis at food grade and widen application range of original system.[Method] Taking pNZ8149 as basis,the promoter PnisA of pNZ8149 was inserted L.lactis MG1363 and SPusp45 of unknown secretory protein in downstream.Through PCR technology,specific primers were used to delete restriction sites between promoter sequence and signal peptide gene sequence and ensure better distance between SD sequence and start codon to construct secreting expression vector pNZS.The reporter gene gus was recombined into multiple cloning site of pNZS to construct pNZS-gus and L.lactis was transformed by electroporation.10 ng/ml nisin was used for induction culture,then culture solution was conducted GUS staining test.[Result]The new constructed L.lactis N3900/pNZS-gus system could express active GUS protein and GUS protein could be secreted out of cell.[Conclusion]The successful construction of this system lays foundation for secretion expression study of protein and oral vaccine research.

Diversity and Phylogenetic Analysis of Lactic Acid in Forage of Xinjiang Uigur Autonomous Region

[Objective] The aim was to conduct preliminary investigation and diversity analysis of lactic acid bacteria resources in forage from Turpan of Xinjiang. [Method] The lactic acid bacteria in the three kinds of forage ingredients in Xinjiang were isolated by using plate separation method and screened by MRS＋CaCO3 solid medium. Morphological, physiological and biochemical identification and 16S rDNA gene sequence analysis were carried out to the isolated eighty strains of lactic acid bacteria, to explore its taxonomic status. [Result] Twenty strains of lactic acid bacteria were obtained from alfalfa, forty-one from wheat, and nineteen from corn. The physiological and biochemical identification and 16S rDNA gene sequence analysis results showed that the eighty strains of lactic acid bacteria belonged to two genera, namely, Lactobacillus, Enterococcus; 7 species, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus paracasei, Entercoccus faecium, Entercoccus durans, Lactobacillus plantarum, Entercoccus hirae. Lactobacillus casei and Entercoccus faecium were ubiquitous in the three kinds of forage ingredients. Besides these two lactic acid bacteria, there were Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus plantarum in wheat, Lactobacillus plantarum, Lactobacillus paracasei, Entercoccus hirae, Entercoccus durans in alfalfa, Lactobacillus plantarum, Entercoccus durans in corn. [Conclusion] There is a big diversity of lactic acid bacteria in different forage from Turpan of Xinjiang, in which Lactobacillus casei, Entercoccus faecium are the key bacteria for forage fermentation.

Breeding of Ferment Bacteria and Control of Methanol and Higher Alcohols in Jujube Wine

Jujube wine is a health drink with local characteristics and is worth pro- moting. Jujube wine fermentation can be divided into alcoholic fermentation and mal- olactic fermentation. Yeast is the key of alcoholic fermentation while lactic acid bac- teria is the key of malolactic fermentation, and therefore the breeding of yeast and lactic acid bacteria is crucial for the quality of jujube wine. Besides, the control of methanol is a major problem in production, and the control of higher alcohol is also difficult. Thus, we summarized the research related with the breeding of yeast and lactic acid bacteria, and the control of methanol and higher alcohols, and proposed that breeding specialized yeast and lactic acid bacteria was the future research di- rection. Moreover, the production mechanism of methanol and higher alcohols was investigated, and the content of methanol and higher alcohols was effectively con- trolled on the basis of quality guarantee, providing references for the production technology of jujube wine.

Effects of Bacteria and Accessories on Chicken Manure Compost

[Objective] In order to study the influence of bacteria and accessories of chicken manure compost.[Method] The experiment was conducted to investigate the changes of temperature,the quantification of Escherichia coli and the odor during chicken manure compost by adding sawdust,rice husk,yeast and lactic acid bacteria respectively.[Result] The results showed that：（1） The yeast group reached the highest fermentation temperature 67.6 ℃,which was 6.9 ℃ higher than that of lactic acid bacteria group,and the fermentation time of yeast group kept over 55 ℃for 16 days,which was 5 days longer than that of lactic acid bacteria group,both of which were better than that of control group（55.9 ℃,5 days）;（2）The highest fermentation temperature of sawdust group was 2.2 ℃ higher than that of rice husk group,and the fermentation time of sawdust group kept over 55 ℃ was 3 days longer than that of rice husk group;（3） The quantification of E.coli reduced from10^5 to 10^2per gram in both of the yeast group and the lactic acid bacteria group.The odor of the yeast group and the lactic acid bacteria group disappeared in the seventh day and the eighth day respectively.[Conclusion] The results showed that the adding of yeast and sawdust was the best condition for chicken manure composting in this experiment.

Properties and Propagation of Enzyme Preparation for Green Corn Stover Silage

Based on the facts that the adhered lactic acid bacteria on raw corn stovers are rare and the fermentation and silage quality of corn stovers are poor, we carried out this research to study the utilization prospects of additives containing lactic acid bacteria and enzymes in preparation of green corn stover silage. The lactic acid bacteria and enzymes were screened out and propagated. The isolated Lactobacil us species included Lactococcus lactis （Enterococcus, Leuconostoc, Labto-coccus, Streptococcus, etc.） and Lactobacil us. The additives would not only improve the silage quality, but also reduce the fermentation losses.

爱的乳酸菌在作怪

我又一次让自己在梦境中重复了那一日的悲伤，是的，只是梦境，我只是用重复的梦魇来折磨自己。醒来后，仍旧是泪流满面，我翻过身，找出硌得我生疼的那个玉坠，微黄的，雕成佛手模样的小小一粒，很精致。

Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria

AIM： To investigate the correlation between in vitro and in vivo immunomodulation potential of the probiotic strain and its ability to prevent experimental colitis in mice. METHODS： In vitro immunomodulation was assessed by measuring interleukJn （IL）-12p70, IL-10, tumor necrosis factor alpha （TNFα） and interferon 7 （IFNγ） release by human peripheral blood mononuclear cells （PBMCs） after 24 h stimulation with 13 live bacterial strains. A murine model of acute TNBS-colitis was next used to evaluate the prophylactic protective capacity of the same set of strains. RESULTS： A strain-specific in vivo protection was observed. The strains displaying an in vitro potential to induce higher levels of the anti-inflammatory cytokine IL-10 and lower levels of the inflammatory cytokine IL-12, offered the best protection in the in vivo colitis model. In contrast, strains leading to a low IL-10/IU12 cytokine ratio could not significantly attenuate colitis symptoms, CONCLUSION：These results show that we could predict the in vivo protective capacity of the studied lactic acid bacteria （LAB） based on the cytokine profile we established in vitro. The PBHC-based assay we used may thus serve as a useful primary indicator to narrow down the number of candidate strains to be tested in murine models for their anti-inflammatory potential.

A probiotic treatment containing Lactobacillus,Bifidobacterium and Enterococcus improves IBS symptoms in an open label trial

Objective： To evaluate the efficacy and safety of live combined Bifidobacterium, Lactobacillus and Enterococcus capsules in treatment of irritable bowel syndrome, Methods： Eighty-fve patients [male 32, female 53; age （45.31±11,72） years] were given live combined Bifidobacterium, Lactobacillus and Enterococcus capsules 1260 mg/d t.i.d. ×4 weeks. Syndrome scales were used to evaluate the efficacy in gastrointestinal syndrome. Fecal flora was also measured before and after the treatment. Six bacteria were cultured and the colony forming units were counted in stool. SPSS was used for data analysis. Results： Seventy-four patients finished the follow-up. No side-effect was found. For treatment of irritable bowel syndrome, the effective rate of live combined Bifidobacterium, Lactobacillus and Enterococcus capsules was 56.8% in the second week, 74.3% in the fourth week and 73.0% in the sixth week. Single symptom was improved, especially in abdominal pain and stool character. The probiotica containing live combined Bifidobacterium, Lactobacillus and Enterococcus could increase bifidobacterium count （P〈0.01） and lactobacillus count （P〈0.05）; decrease bacteroides count （P〈0.05） and enterococci count （P〈0.01）; No obvious changes were observed in clostridium diffcile colonitis and enterobacteriaceae （P〉0.05）. Conclusion： The result of the study indicated that the administration of live combined Bifidobacterium, Lactobacillus and Enterococcus improved the symptom of irritable bowel syndrome and that there was a gradual increase of this effect. Thereafter conditions remained stable for 2 weeks. That improvement may be associated with alterations in gastrointestinal flora.

Protective effect of selenium-enriched lactobacilluson CCI_4-induced liver injury in mice and its possible mechanisms

AIM： To study the protective effects and mechanisms of Se-enriched lactobacillus on liver injury caused by carbon tetrachloride （CCl4） in mice. METHODS： Seventy-two ICR mice were randomly divided into four groups： normal group, CCl4-induced model group, low Se-enriched lactobacillus treatment group （L-Se group）, and high Se-enriched lactobacillus treatment group （H-Se group）. During a 3-wk experimental period, the common complete diet was orally provided daily for normal group and model group, and the mice in L-Se and H-Se groups were given a diet with 2 and 4 mg of organoselenium from Se-enriched lactobacillus per kg feed, respectively. From the 2nd wk of experiment, the model group, L-Se group, and H-Se group received abdominal cavity injection of olive oil solution containing 500 mL/L CCl4 （0.07 mL/100 g body mass） to induce liver injury, and the normal group was given olive oil on every other day for over 2 wk. In the first 2 wk post injection with CCl4, mice in each group were killed. The specimens of blood, liver tissue, and macrophages in abdominal cavity fluid were taken. Then the activities of the following liver tissue injury-associated enzymes including glutathione peroxidase （GSH-Px）, superoxide dismutase （SOD）, alanine aminotransferase （ALT） and aspartate aminotransferase （AST） as well as malondialdehyde （MDA） content were assayed. Changes of phagocytic rate and phagocytic index in macrophages were observed with Wright-Giemsa stain. Plasma TNF-α level was measured by radioimmunoassay. The level of intracellular free Ca^2＋ （[Ca^2＋]i） in hepatocytes was detected under a laser scanning confocal microscope. RESULTS： During the entire experimental period, the AST and ALT activities in liver were greatly enhanced by CCl4 and completely blunted by both low and high doses of Se-enriched lactobacillus. The Se-enriched lactobacillus- protected liver homogenate GSH-Px and SOD activities were higher or significantly higher than those in model group and were close to those in normal group. CCl4 significantly increased MDA content in liver homogenates, while administration of Se-enriched lactobacillus prevented MDA elevation. Phagocytic rate and phagocytic index of macrophages decreased after CCl4 treatment compared to those in normal control, but they were dramatically rescued by Se-enriched lactobacillus, showing a greatly higher phagocytic function compared to model group. CCl4 could significantly elevate plasma TNF-α and hepatocyte [Ca^2＋]i level, which were also obviously prevented by Se-enriched lactobacillus. CONCLUSION： Se-enriched lactobacillus can intervene in CCl4-induced liver injury in mice by enhancing macrophage function activity to keep normal and beneficial effects, elevating antioxidant-enzyme activities and reducing lipid peroxidation reaction, inhibiting excessive release of TNF-α, preventing the dramatic elevation of [Ca^2＋]i in hepatocytes.

Probiotic Leuconostoc mesenteroides ssp. cremoris and Streptococcus thermophilus induce IL-12 and IFN-γ production

AIM： To investigate the capacity of potentially probiotic strains from six bacterial genera to induce cytokine production alone or in combinations in order to identify potential enhancing or synergistic effects in order to select probiotic bacteria for in vivo purposes.METHODS： Cytokine production in human peripheral blood mononuclear cells （PBMC） in response to stimulation with eleven different potentially probiotic bacterial strains from Streptococcus, Lactobacillus, Bifidobacterium, Lactococcus, L euconostoc a n d Propionibacterium genera was analysed. Production and mRNA expression of TNF-α, IL-12, IFN-y and IL-10 were determined by ELISA and Northern blotting, respectively.RESULTS： All tested bacteria induced TNF-α production. The best inducers of Thl type cytokines IL-12 and IFN-y were Streptococcus and Leuconostoc strains. All BiHdobacterium and Propionibacterium strains induced higher IL-IO production than other studied bacteria. Stimulation of PBMC with any bacterial combinations did not result in enhanced cytokine production suggesting that different bacteria whether gram-positive or gram- negative compete with each other during host cell interactions.CONCLUSION： The probiotic S. thermophilus and Leuconostoc strains are more potent inducers of Thl type cytokines IL-12 and IFN-γ than the probiotic Lactobacillus strains. Bacterial combinations did not result in enhanced cytokine production.

Preventative effects of a probiotic, Lactobacillus salivarius ssp. salivarius, in the TNBS model of rat colitis

AIM： To investigate the intestinal anti-inflammatory effect and mechanism of a probiotic Lactobacillus salivarius ssp. salivariusCECT5713 in the TNBS model of rat colitis. METHODS： Female Wistar rats （180-200 g） were used in this study. A group of rats were administered orally the probiotic L. salivarius ssp. salivarius （5 × 10^8 CFU suspended in 0.5 mL of skimmed milk） daily for 3 wk. Two additional groups were used for reference, a non-colitic and a control colitic without probiotic treatment, which received orally the vehicle used to administer the probiotic. Two weeks after starting the experiment, the rats were rendered colitic by intracolonic administration of 10 mg of TNBS dissolved in 0.25 mL of 500 mL/L ethanol. One week after colitis induction, all animals were killed and colonic damage was evaluated both histologically and biochemically. The biochemical studies performed in colonic homogenates include determination of myeloperoxidase （MPO） activity, glutathione （GSH） content, leukotriene B4 （LTB4） and tumor necrosis factor α （TNF-α） levels, as well as inducible nitric oxide synthase （iNOS） expression. In addition, the luminal contents obtained from colonic samples were used for microbiological studies, in order to determine Lactobacilli and Bifidobacteria counts. RESULTS： Treatment of colitic rats with L. salivarius ssp. salivarius resulted in amelioration of the inflammatory response in colitic rats, when compared with the corresponding control group without probiotic treatment. This antiinflammatory effect was evidenced macroscopically by a significant reduction in the extent of colonic necrosis and/or inflammation induced by the administration of TNBS/ethanol （2.3±0.4 cm vs3.4±0.3 cm in control group, P〈0.01） and histologically by improvement of the colonic architecture associated with a reduction in the neutrophil infiltrate in comparison with non-treated colitic rats. The latter was confirmed biochemically by a significant reduction of colonic MPO activity （105.3±26.0 U/g vs 180.6±21.9 U/g, P〈0.05）, a marker of neutrophil infiltration. The beneficial effect was associated with an increase of the colonic GSH content （1 252±42 nmol/g vs i 087±51 nmol/g, P〈0.05）, which is depleted in colitic rats, as a consequence of the oxidative stress induced by the inflammatory process. In addition, the treatment of colitic rats with L. salivarius resulted in a significant reduction of colonic TNF-（~ levels （509.4±68.2 pg/g vs782.9±60.1 pg/g, P〈0.01） and in a lower colonic iNOS expression, when compared to TNBS control animals without probiotic administration. Finally, treated colitic rats showed higher counts of Lactobacilli species in colonic contents than control colitic rats, whereas no differences were observed in Bifidobacteria counts. CONCLUSION： Administration of the probiotic L. salivarius ssp. salivarius CECT5713 facilitates the recovery of the inflamed tissue in the TNBS model of rat colitis, an effect associated with amelioration of the production of some of the mediators involved in the inflammatory response in the intestine, such as cytokines, including TNF-α and NO. This beneficial effect could be ascribed to its effect on the altered immune response that occurs in this inflammatory condition.

Lactobacilli inhibit interleukin-8 production induced by Helicobacter pylori lipopolysaccharide-activated Toll-like receptor 4

AIM： To investigate the effect of Lactobacillus bulgaricus （LBG） on the Toll-like receptor 4 （TLR4） pathway and interleukin-8 （IL-8） production in SGC-7901 cells treated with Helicobacter pyloriSydney strain 1 lipopolysaccharide （HpyloriSS1-LPS）. METHODS： SGC-7901 cells were treated with HpyloriSS1-LPS in the presence or absence of pretreatment for 1 h with viable LBG or supernatant recovered from LBG culture MRS broth （LBG-s）. Cellular lysates were prepared for Western blot with anti-TLR4, anti-transforming growth factor β-activated kinase 1 （TAK1）, anti-phospho-TAK1, anti-nuclear factor κB （NF-κB）, anti-p38 mitogen-activated protein kinase （p38MAPK）, and anti-phospho-p38MAPK antibodies. The amount of IL-8 in cell culture medium was measured by ELISA. RESULTS： H pyloriSS1-LPS up-regulated the expression of TLR4, stimulated the phosphorylation of TAKI, subsequently enhanced the activation of NF- κB and the phosphorylation of p38MAPK in a time- dependent manner, leading to augmentation of IL-8 production in SGC-7901 cells. Viable LBG or LBG-s pretreatment attenuated the expression of TLR4, inhibited the phosphorylation of TAK1 and p38MAPK, prevented the activation of NF-κB, and consequently blocked IL-8 production.CONCLUSION： H py/oriSS1-LPS induces IL-8 production through activating TLR4 signaling in SGC-7901 cells and viable LBG or LBG-s prevents H pyloriSS1-LPS-mediated IL-8 production via inhibition of the TLR4 pathway.

Probiotic bacteria change Escherichia coli-induced gene expression in cultured colonocytes:Implications in intestinal pathophysiology

AIM: To investigate the change in eukaryotic gene expression profile in Caco-2 cells after infection with strains of Escherichia coli and commensal probiotic bacteria. METHODS: A 19 200 gene/expressed sequence tag gene chip was used to examine expression of genes after infection of Caco-2 cells with strains of normal flora E. coli,Lactobacillus plantarum,and a combination of the two. RESULTS: The cDNA microarray revealed up-regulation of 155 and down-regulation of 177 genes by E. coli . L. plantarum up-regulated 45 and down-regulated 36 genes. During mixed infection,27 genes were up-regulated and 59 were down-regulated,with nullification of stimulatory/inhibitory effects on most of the genes. Expression of several new genes was noted in this group. CONCLUSION: The commensal bacterial strains used in this study induced the expression of a large number of genes in colonocyte-like cultured cells and changed the expression of several genes involved in important cellular processes such as regulation of transcription,protein biosynthesis,metabolism,cell adhesion,ubiquitination,and apoptosis. Such changes induced by the presence of probiotic bacteria may shape the physiologic and pathologic responses they trigger in the host.

Effect of lactobacillus on the gut microflora and barrier function of the rats with abdominal infection

AIM:To investigate the effect of probiotics supplemented by gut on the tight junctions of epithelial cells, barrier function and the microflora of rats with abdominal infection. METHODS: After the model of cecal ligation and perforation established, SD rats were divided into two groups: parenteral nutrition (PN) group and PIM+probiotics (probiotics) group, PN solution was supplemented by neck vein and probiotics was delivered via the jejunostomy tube for five days. Vena cava blood and the homogenated tissue of liver, lung and mesenteric lymph nodes were cultured to determine the bacterial translocation rate (BTR). The ultra-structure of epithelial tight junctions and microvilli of the gut were observed by electron microscopy; occluding expression was measured by indirect-immune fluorescence method; anaerobic bacterial growth by anaerobic culture and DNA fingerprint of bacterial colonies of the feces by PCR. RESULTS: The quantity of lactobacteria and bifydobacteria in probiotics group was higher than that of PN group. The profiles of DNA fingerprint expression in probiotics group were similar to that in the normal group, a new 16S rDNA sequence appeared in the profile in PN group. The occludin expression, the integrality of the gut epithelial tight junction and microvilli in probiotics group were improved as compared with PN group. The BTR and endotoxin in blood were reduced more significantly in probiotics group as compared with PN group. CONCLUSION: The probiotics could improve the gut microflora disturbance, increase occludin expression, maintain the gut epithelial tight junction and decrease the bacterial translocations rate.

Batch and fed-batch production of butyric acid by Clostridium butyricum ZJUCB

The production of butyric acid by Clostridium butyricum ZJUCB at various pH values was investigated. In order to study the effect of pH on cell growth, butyric acid biosynthesis and reducing sugar consumption, different cultivation pH values ranging from 6.0 to 7.5 were evaluated in 5-L bioreactor. In controlled pH batch fermentation, the optimum pH for cell growth and butyric acid production was 6.5 with a cell yield of 3.65 g/L and butyric acid yield of 12.25 g/L. Based on these results, this study then compared batch and fed-batch fermentation of butyric acid production at pH 6.5. Maximum value (16.74 g/L) of butyric acid concentration was obtained in fed-batch fermentation compared to 12.25 g/L in batch fermentation. It was concluded that culti- vation under fed-batch fermentation mode could enhance butyric acid production significantly (P<0.01) by C. butyricum ZJUCB.

Distinct immune response induced by peptidoglycan derived from Lactobacillus sp

AIM： To analyze the distinct immune responses induced by Lactobacillus peptidoglycan （PG）. METHODS： BALB/c mice were intraperitoneally injected with PG once a day for three consecutive days, Peritoneal macrophage and splenocyte mRNA was extracted and the gene expression profile was studied using high-density oligonudeotide microarrays. Inhibitory effects of Lactobacillus PG on colon tumor tissue were studied in vitro and in vivo, RESULTS： The gene expression profiles revealed that the TLR-NF-kB and Jak-STAT signaling pathways were highly activated. An inflammatory phenotype was induced when peritoneal macrophages were initially exposed to Lactobacillus PG and switched to a more complex phenotype when BALB/c mice were treated with three doses of Lactobacillus PG. A protective physiological inflammatory response was induced after three consecutive days of PG treatment. It was tending toward Thl dominant immune response. Lactobacillus PG also appeared to induce a significant in vivo anti-colon tumor effect. CONCLUSION： Lactobacillus PG is responsible for certain immune responses induced by Lactobacilli. Anti-tumor effects of Lactobacilliare likely to attribute to the activation of macrophages by PG expressed on the bacterial cell surface.

Infant intestinal Enterococcus faecalis down-regulates inflammatory responses in human intestinal cell lines

AIM: To investigate the ability of Lactic acid bacteria (LAB) to modulate inflammatory reaction in human intestinal cell lines (Caco-2, HT-29 and HCT116). Different strains of LAB isolated from new born infants and fermented milk, together with the strains obtained from culture collections were tested.METHODS: LABs were treated with human intestinal cell lines. ELISA was used to detect IL-8 and TGF-β protein secretion. Cytokines and Toll like receptors (TLRs) gene expression were assessed using RT-PCR. Conditional medium, sonicated bacteria and UV killed bacteria were used to find the effecter molecules on the bacteria. Carbohydrate oxidation and protein digestion were applied to figure out the molecules' residues. Adhesion assays were further carried out.RESULTS: It was found that Enterococcus faecalis is the main immune modulator among the LABs by downregulation of IL-8 secretion and upregulation of TGF-β. Strikingly, the effect was only observed in four strains of E. faecalis out of the 27 isolated and tested. This implies strain dependent immunomodulation in the host. In addition, E. faecalis may regulate inflammatory responses through TLR3, TLR4, TLR9 and TRAF6. Carbohydrates on the bacterial cell surface are involved in both its adhesion to intestinal cells and regulation of inflammatory responses in the host. CONCLUSION: These data provide a case for the modulation of intestinal mucosal immunity in which specific strains of E. faecalis have uniquely evolved to maintain colonic homeostasis and regulate inflammatory responses.