<i>In-Vitro</i>Fermentation by Human Fecal Bacteria and Bile Salts Binding Capacity of Physical Modified Defatted Rice Bran Dietary Fiber

Defatted rice bran dietary fiber (DRBDF) was modified by micronization, ultrasound, microwave and extrusion cooking. We investigated the impacts of these physical treatments on the fermentation ability and bile salts binding capacity of DRBDF. In-vitro fermentation by human fecal bacteria of modified fibers showed that the major fermentation products were propionic, acetate and butyrate acid. Fermentation of extruded fiber gave the highest amounts of propionic and acetic acid 135.76 and 25.45 mmol/L respectively, while, the fermented product with microwaved fiber had the highest butyric acid content (10.75 mmol/L). The amount of short-chain fatty acid increased from 12 h to 24 h and propionic acid was the predominant. On the other hand,in-vitrobile salts binding showed that extruded fiber had higher affinity with sodium deoxycholate and sodium chenodeoxycholate (66.14% and 30.25% respectively) while microwaved fiber exhibited the highest affinity with sodium taurocholate (14.38%). In the light of obtained results we can affirmed that these physical treatments significantly improved the fermentation products and bile salts binding capacity of DRBDF. Extrusion compared to the other physical treatment methods used in this study has greatly and positively influenced the fermentation and bile binding capacity of DRBDF.

Mucin and phospholipids determine viscosity of gallbladder bile in patients with gallstones

AIM An increased viscosity of gallbladder bile has been considered an important factor in the pathogenesis of gallstone disease. Besides lipids and proteins, mucin has been suggested to affect the viscosity of bile. To further clarify these issues we compared mucin, protein and the lipid components of hepatic and gallbladder bile and its viscosity in patients with gallstones.METHODS Viscosity of bile ( mpa. s ) wasmeasured using rotation viscosimetry in regard to the non-Newtonian property of bile at law shear rates.RESULTS Biliary viscosity was markedly higher in gallbladder bile of patients with cholesterol (5.00 ± 0.60 mpa. s, mean ± SEM, n --28) and mixed stones (3.50±0.68 mPa. s; n =8) compared to hepatic bile (0.92 ± 0.06 mpa. s,n -6). A positive correlation between mucin and viscosity was found in gallbladder biles (r=0.65; P＜0.001) but not in hepatic biles. The addition of physiologic and supraphysiologic amounts of mucin to gallbladder bile resulted in a dose dependent non linear increase of its viscosity. A positive correlation was determined between phospholipid concentration and viscosity (r = 0.34, P＜0.005) in gallbladder biles. However, no correlation was found between total protein or the other lipid concentrations and viscosity in both gallbladder and hepatic biles.CONCLUSION The viscosity of gallbladder bile is markedly higher than that of hepatic bile in patients with gallstones. The concentration of mucin is the major determinant of biliary viscosity and may contribute by this mechanism to the role of mucin in the pathogenesis of gallstones.

Cloning of Bile Salt Hydrolase Gene and Its Expression in Lactic Acid Bacteria

According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.

Cloning and Expression of Bile Salt Hydrolase Gene from Lactobacillus plantarum M1-UVS29

We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH, pNZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol· min^-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.

Effect of bile salts and bile acids on human gastric mucosal epithelial cells

Objective：To explore the effect of bile salt and bile acid on cultured eternalized human gastric mucosa epithelium GES-1 cells. Methods：Cultured eternalized human gastric mucosa epithelium GES-1 cells were treated with media containing 6 different kinds of bile salts and 3 different kinds of bile acids and their mixture with different concentrations： GCDC（glycochenodeoxychoμte）, GDC （glycodeoxychoμte）, GC（glycochoμte）, TCDC（taurochenodeoxychoμte）, TDC（taurodeoxychoμte）, TC （taurochoμte）, LCA （lithocholicacid）, CA（cholic acid）, DCA（deoxycholic acid）（50 μ mol/L,250 μ mol/L,500 μ mol/L,1000 μ mol/L）, DY（mixture of bile salts） and DS（mixture of bile acids）（250 μ mol/L,500 μ mol/L,1000 μ mol/L,1500 μ mol/L, 2000 μ mol/L）, in comparison with the control group（in normal media without bile salts and bile acids）. Cell proliferation was assessed by MTT（3-[4,5-Dimethylthiaolyl]-2,5- diphenyl-tetrazolium bromide） assay for 72 hours with different concentrations and the apoptotic cells were assayed by flow cytometry （FCM） with Annex V-FITC conjugated with propidium iodide（PI） staining for 24 hours with different concentrations（1500,2000 μt mol/L）. Results：There was no significant difference in morphology and cell proliferation in GC group after 24-72 h. Low concentration（50 μ mol/L） of GCDC, GDC, TCDC, TDC and TC accelerated gastric epithelial cell growth in a dosage-time dependent manner. At middle concentration （250-500 μ mol/L）, it showed positive effect after 24-48 h, while negative effect after 72 h. At high concentration（1000 μ mol/L）, it accelerated gastric epithelial cell growth after 24h and show consistent inhibition even leading to necrosis after 48-72 h. LCA and CA showed a positive effect on the concentration of 50 μ mol/L after 24-72 h, while 250-1000 μ mol/L showed a trend towards apoptosis after 24-72 h. At 50-500 μmol/L, DCA showed proliferation after 24 h and apoptosis after 48-72 h, but showed necrosis after 24-72 h at 1000 μmol/L. DY and DS could facilitate normal gastric mucosa epithelial cell growth at low concentration （250-500 μ mol/L）, however at 1000-2000 μ mol/L the trend shifted from apoptosis to necrosis. FCM with Annexin-V conjugated with PI staining revealed that GCDC, GDC, GC, TCDC, TDC, TC, LCA, CA, DCA, DY and DS induced apoptosis of human gastric mucosal epithelial cells. They were all significantly higher than that of the control（P 〈 0.05）, but there was no significant difference in GC group （P 〉 0.05）. The bile salts induced apoptosis in a time-dose-dependent manner. Conclusion：Our results suggested that bile acid and bile salt is the trigger of injury in human gastric mucosal epithelial cells.

Denaturation studies on bovine serum albumin–bile salt system:Bile salt stabilizes bovine serum albumin through hydrophobicity

Protein denaturation is under intensive research, since it leads to neurological disorders of severe consequences. Avoiding denaturation and stabilizing the proteins in their native state is of great importance,especially when proteins are used as drug molecules or vaccines. It is preferred to add pharmaceutical excipients in protein formulations to avoid denaturation and thereby stabilize them. The present study aimed at using bile salts(BSs), a group of well-known drug delivery systems, for stabilization of proteins.Bovine serum albumin(BSA) was taken as the model protein, whose association with two BSs, namely sodium cholate(Na C) and sodium deoxycholate(Na DC), was studied. Denaturation studies on the preformed BSA-BS systems were carried out under chemical and physical denaturation conditions. Urea was used as the chemical denaturant and BSA-BS systems were subjected to various temperature conditions to understand the thermal(physical) denaturation. With the denaturation conditions prescribed here,the data obtained is informative on the association of BSA-BS systems to be hydrophobic and this effect of hydrophobicity plays an important role in stabilizing the serum albumin in its native state under both chemical and thermal denaturation.

Bile salts inhibit growth and induce apoptosis of human esophageal cancer cell line

AIM: To explore the effect of six bile salts, including glycocholate (GC), glycochenodeoxycholate (GCDC), glycodeoxycholate (GDC), taurocholate (TC), taurochenodeoxycholate (TCDC), taurodeoxycholate (TDC), and two bile acids including cholic acid (CA) and deoxycholic acid (DCA) on esophageal cancer Eca109 cell line.METHODS: Eca109 cells were exposed to six bile salts, two bile acids and the mixed bile salts at different concentrations for 24-72 h. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)assay. Sub-G1 DNA fragmentations and early apoptosis cells were assayed by flow cytometry (FCM) with propidium iodide (PI) staining and annexin V-FITC conjugated with PI staining. Apoptosis DNA ladders on agarose were observed. Activation of caspase-3 was assayed by FCM with FITC-conjugated monoclonal rabbit anti-active caspase3 antibody and expressions of Bcl-2 and Bax proteins were examined immunocytochemically in 500 μmol/L-TC-induced apoptosis cells.RESULTS: Five bile salts except for GC, and two bile acids and the mixed bile salts could initiate growth inhibition of Eca109 cells in a dose- and time-dependent manner.TUNEL, FCM, and DNA ladder assays all demonstrated apoptosis induced by bile salts and bile acids at 500 μmol/L,except for GC. Early apoptosis cell percentages in Eca109 cells treated with GCDC, GDC, TC, TCDC, TDC,CA at 500 μmol/L for 12 h, DCA at 500 μmol/L for 6 h,and mixed bile salts at 1 000 μmol/L for 12 h were 7.5%,8.7%, 14.8%, 8.9%, 7.8%, 9.3%, 22.6% and 12.5%,respectively, all were significantly higher than that in control (1.9%). About 22% of the cell population treated with TC at 500 μmol/L for 24 h had detectable active caspase-3, and were higher than that in the control (1%). Immunocytochemical assay suggested that TC down-regulated Bcl-2 protein level and up-regulated Bax protein level.CONCLUSION: GCDC, GDC, TC, TCDC, TDC, CA and DCA,except for GC, can inhibit growth and induce apoptosis of esophageal cancer Eca109 cells. Activation of caspase-3,decreased Bcl-2 protein and increased Bax protein are involved in TC-induced apoptosis of Eca109 cells.

Bile salts inhibit growth and induce apoptosis of culture human normal esophageal mucosal epithelial cells

AIM: To investigate the effect of six bile salts:glycocholate (GC), glycochenodeoxycholate (GCDC),glycodeoxycholate (GDC), taurocholate (TC),taurochenodeoxycholate (TCDC), taurodeoxycholate (TDC), and their mixture on cultured human normal esophageal mucosal epithelial cells.METHODS: Human normal esophageal mucosal epithelial cells were cultured with serum-free keratinocyte medium. 3-[4,5-Dimethylthiaolyl]-2,5-diphenyl-tetrazolium bromide assay was applied to the detection of cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Sub-G1 DNA fragmentations and early apoptotic cells were assayed by flow cytometry (FCM) with propidium iodide (PI) staining and annexin V-FITC conjugated with PI staining.Apoptotic DNA ladders on agarose gel electrophoresis were observed.RESULTS: Except for GC, GCDC, GDC, TC, TCDC, TDC and their mixture could initiate growth inhibition of esophageal mucosal epithelial cells in a dose- and time-dependent manner. TUNEL and FCM assays demonstrated that the bile salts at 500 μmol/L and their mixture at 1 500 μmol/L induced apoptosis except for GC. The percentage of sub-G1 detected by FCM with PI staining was 83.5% in cells treated with 500μmol/L TC for 2 h, and 19.8%, 20.4%, 25.6%, 13.5%, and 75.8% in cells treated with 500 μmol/L GCDC, TCDC, GDC,TDC, and 1 500 μmol/L mixture for 24 h, respectively,which were higher than that of the control (1.5%). The percentage was 1.4% in cells with 500 μmol/L GC for 24 h.DNA ladders on agarose gel electrophoresis were seen in cells treated with 500 μmol/L TC for 2 h and 1 500 μmol/Lmixture for 24 h.CONCLUSION: All GCDC, GDC, TC, TCDC, TDC and their mixture can inhibit growth and induce apoptosis of cultured human normal esophageal mucosal epithelial cells, but GC is well tolerated by the cells.

Increased susceptibility for intrahepatic cholestasis of pregnancy and contraceptive-induced cholestasis in carriers of the 1331T>C polymorphism in the bile salt export pump

AIM: To study the association of three common ABCB11 and ABCC2 polymorphisms (ABCB11: 1331T>C V444A; ABCC2: 3563T>A V1188E and 4544G>A C1515Y) with intrahepatic cholestasis of pregnancy (ICP) and contraceptive-induced cholestasis (CIC). METHODS: ABCB11 and ABCC2 genotyping data were available from four CIC patients and from 42 and 33 ICP patients, respectively. Allele-frequencies of the studied polymorphisms were compared with those in healthy pregnant controls and Caucasian individuals. Furthermore, serum bile acid levels were correlated with the presence or absence of the 1331 C allele.RESULTS: The ABCB11 1331T>C polymorphism was significantly more frequent in cholestatic patients than in pregnant controls: C allele 76.2% (CI, 58.0-94.4) vs 51.3% (CI 35.8-66.7), respectively (P = 0.0007); and CC allele 57.1% (CI 36.0-78.3) vs 20% (CI 7.6-32.4), respectively (P = 0.0065). All four CIC patients were homozygous carriers of the C allele. In contrast, none of the studied ABCC2 polymorphism was overrepresented in ICP or CIC patients. Higher serum bile acid levels were found in carriers of the 1331CC genotype compared to carriers of the TT genotype.CONCLUSION: Our data support a role for the ABCB11 1331T>C polymorphism as a susceptibility factor for the ←←← development of estrogen-induced cholestasis, whereas no such association was found for ABCC2. Serum bile acid and γ-glutamyl transferase levels might help to distinguish ABCB4- and ABCB11-related forms of ICP and CIC.

Partial external biliary diversion in bile salt export pump deficiency: Association between outcome and mutation

AIM To investigate the relation of two different mutations to the outcome of partial external biliary diversion(PEBD)in severe bile salt export pump(BSEP) deficiency.METHODS Mutations in the gene encoding BSEP leading to severe BSEP deficiency in two unrelated patients were identified by genomic sequencing. Native liver biopsies and transiently transfected human embryonic kidney(HEK) 293 cells expressing either wild-type or mutated BSEP were subjected to immunofluorescence analysis to assess BSEP transporter localization. Bile acid profiles of patient and control bile samples were generated by ultra-performance liquid chromatographytandem mass spectrometry. Wild-type and mutant BSEP transport of [~3H]-labeled taurocholate(TC) and taurochenodeoxycholate(TCDC) was assessed by vesicular transport assays.RESULTS A girl(at 2 mo) presented with pruritus, jaundice and elevated serum bile salts(BS). PEBD stabilized liver function and prevented liver transplantation. She was heterozygous for the BSEP deletion p.T919 del and the nonsense mutation p.R1235 X. At the age of 17 years relative amounts of conjugated BS in her bile were normal, while total BS were less than 3% as compared to controls. An unrelated boy(age 1.5 years) presenting with severe pruritus and elevated serum BS was heterozygous for the same nonsense and another missense mutation, p.G1032 R. PEBD failed to alleviate pruritus, eventually necessitating liver transplantation. BS concentration in bile was about 5% of controls. BS were mainly unconjugated with an unusual low amount of chenodeoxycholate derivatives(< 5%). The patients' native liver biopsies showed canalicular BSEP expression. Both BSEP p.T919 del and p.G1032 R were localized in the plasma membrane in HEK293 cells. In vitro transport assays showed drastic reduction of transport by both mutations. Using purified recombinant BSEP as quantifiable reference, per-molecule transport rates for TC and TCDC were determined to be 3 and 2 BS molecules per wild-type BSEP transporter per minute, respectively.CONCLUSION In summary, our findings suggest that residual function of BSEP as well as substrate specificity influence the therapeutic effectiveness of PEBD in progressive familial intrahepatic cholestasis type 2(PFIC-2).

Bile salt derivatives with novel skeleton from sea lamprey function as putative pheromone

OBJECTIVE The invasive sea lamprey(Petromyzon marinus)has devastated the ecosystem of the Laurentian Great Lakes.Application of pheromones to manipulate adult sea lamprey behavior is among the options considered for alternative sea lamprey control techniques.The male sea lamprey sex pheromone is hypothesized to be possess multiple functions through actions of multiple components,some of which have yet to be characterized.Our objective is to isolate and characterize the bioactive components from water conditioned with sexually mature male sea lamprey.METHODS The water conditioned with sexually mature male sea lamprey was extracted by solid phase extraction and concentrated in vacuo.The compounds were isolated by liquid chromatography and elucidated by spectrometry and spectroscopy.Their biological activities were evaluated by electro-olfactogram recordings and two-choice maze behavioral assays.RESULTS Five novel bile salts,petromyzene A and B and petromyzone A-C,have been characterized.Petromyzene A and B featured either a unique,rearranged side chain or a rare cis-11,12-diol on the steroidal B-ring.Petromyzone A-C represented three novel highly oxidized sulfated bile alcohols possessing different hydroxylation,oxidation,and double bond patterns,which exemplify the chemical diversity of bile salts.These five bile salts were potent odorants that stimulated the adult sea lamprey olfactory epithelium in a concentration dependent manner and showed detection thresholds between 10–13mol·L^(-1) and 10^(–11)mol·L^(-1)(paired t-test,P<0.05).Experiments in the two-choice maze showed that all isolated compounds induced behavioral responses in ovulated females.CONCLUSION The five novel compounds are likely additional components of pheromones released by sexually mature male sea lamprey,and may provide useful behavioral manipulation tools to be implemented with the integrated management of the destructive and invasive sea lamprey in the Laurentian Great Lakes.

深共熔溶剂提取桑黄黄酮及体外降血糖、结合胆酸盐能力分析

从7种深共熔溶剂中筛选最适合用于桑黄黄酮化合物提取的溶剂体系,采用响应面优化深共熔溶剂提取桑黄黄酮的工艺,筛选最佳黄酮回收树脂,并分析其体外降血糖和结合胆酸盐能力。试验结果表明,乙二醇与氯化胆碱组成的深共熔溶剂体系最适合提取桑黄黄酮,最佳提取工艺为深共熔溶剂含水率30%,乙二醇与氯化胆碱的物质的量比为8.21∶1,料液比=50∶1(mg∶m L),提取温度64℃,提取时间43 min,在此条件下,桑黄黄酮得率为(8.85±0.34)%,较体积分数为60%乙醇提取的得率提高了2.02倍。9种大孔树脂中,AB-8型大孔树脂对桑黄黄酮的回收率最高,为85.25%;纯化后桑黄黄酮的纯度提高了6.12倍。桑黄黄酮抑制α-淀粉酶和α-葡萄糖苷酶活性的IC_(50)值分别为1.19 mg/m L和1.28 mg/m L,结合牛磺胆酸钠和甘氨胆酸钠的IC_(50)值分别为0.62 mg/m L和1.19 mg/m L,表明桑黄黄酮具有较强的调节血糖和血脂能力。实验表明,深共熔溶剂结合大孔吸附树脂富集回收可以绿色高效地从桑黄中提取黄酮类化合物。

高产胆盐水解酶乳杆菌的筛选及对新生大鼠黄疸的防治作用

为筛选高产胆盐水解酶的乳杆菌,探究其对新生儿黄疸的防治作用。采用添加了25 U/mL制霉菌素的LBS选择性培养基,从健康新生儿粪便和母乳中筛选乳杆菌并鉴定种类;以鼠李糖乳杆菌LGG为阳性对照,体外评估菌株的益生菌特性;利用盐酸苯肼诱导新生SD大鼠黄疸模型,通过分析血清胆红素水平和肝脏组织的损伤情况,以及肝脏炎症因子、核转录因子的相对表达水平,探究高产胆盐水解酶乳杆菌对新生大鼠黄疸的防治作用及机制。结果表明,来自婴儿粪便的格氏乳杆菌FWJL-5在体外具良好的益生特性,并且产胆盐水解酶能力优于LGG,能够显著缓解新生大鼠胆红素水平升高、肝脏组织肿胀和溶血症状,减少肝脏损伤中肝酶的释放,抑制促炎因子的分泌,促进UGt1A1和上游核转录因子孕烷X受体(pregnane X receptor,pXR)、法尼醇X受体(farnesol X receptor,FXR)的表达。综上所述,婴儿粪便来源的格氏乳杆菌FWJL-5可通过上调核受体FXR/pXR促进UGt1A1表达以调节肝脏胆红素代谢,从而减轻新生大鼠黄疸症状,本研究可为格氏乳杆菌防治新生儿黄疸提供新思路。

乳杆菌在胆盐MRS培养基中的传代稳定性

该实验探究提高乳杆菌传代稳定性的方法以提高乳杆菌在胃肠道的定植能力,为工业生产提供依据。该研究以植物乳杆菌M547、M621、M748、戊型乳杆菌M750为研究对象,研究胆盐环境对菌株生长的影响,利用生长曲线仪确定合适胆盐培养菌株的浓度,对比胆盐培养和MRS培养的菌株在耐胆盐能力、产酸能力、耐酸能力的表现。0.04%的胆盐培养后,菌株在传代过程中对于0.075%胆盐的耐受存活率提升11%~18%左右,0.1%胆盐的耐受存活率提升20%~34%。胆盐培养对于产酸能力,耐酸能力及稳定性的提高没有明显作用,但对菌株的耐胆盐能力及传代稳定性有提高作用,可为研究改善益生菌的益生特性的方法提供理论支持。

冷冻干燥对益生菌传代稳定性的影响

益生菌随着传代的进行,菌株的产酸能力、耐胆盐能力、耐酸能力和抗氧化能力产生变化。冷冻干燥是菌株常用的保藏方法,对菌体转录水平影响较大,能够临时改变菌株的多种特性。文章以植物乳杆菌M547、M621、M748、戊糖乳杆菌M750为研究对象,探究冷冻干燥对菌株传代稳定性的影响。结果表明,冷冻干燥影响菌株的耐胆盐稳定性,对产酸能力、耐酸能力、抗氧化能力无显著影响。

茶多酚对大豆分离蛋白乳液性质及界面蛋白胆盐置换的影响

通过添加不同含量的茶多酚提取物修饰大豆分离蛋白制备水包油乳液,考察大豆分离蛋白乳液的界面张力、蛋白吸附比例、乳液粒径以及Zeta电位等性质变化,探究茶多酚对大豆分离蛋白乳液性质和界面蛋白置换的影响。实验结果表明:添加茶多酚后,大豆分离蛋白的界面张力升高;大豆分离蛋白溶液(1%,质量分数)按照9∶1(质量比)与大豆油混合后,经过高速剪切和超声制备成水包油乳液,添加茶多酚制备的乳液稳定性提高,与空白对照组相比,当茶多酚添加量为0.04%时,乳液平均粒径从1.702μm显著下降至1.203μm(P<0.05),乳液的蛋白吸附比例从9.22%显著上升至20.68%(P<0.05),Zeta电位绝对值从25.7 mV显著上升至27.1 mV(P<0.05);大豆分离蛋白在油-水界面上具有抗胆盐置换的特性,由茶多酚修饰的大豆分离蛋白更加难以被胆盐置换,这可能是添加茶多酚后大豆分离蛋白具有较强的静电相互作用以及较厚的界面层导致的。肠道中的脂质消化是界面过程,探究脂滴界面上蛋白与胆盐的置换反应对研究脂质代谢和食品精准设计具有指导作用。

乳酸乳球菌的单菌包埋技术及在胃肠道环境中的性能评价

由于益生菌对消化系统的强酸和高浓度胆汁盐环境较为敏感,并且在肠道中定殖能力较低,因此开发新颖有效的益生菌包埋体系用于解决上述问题很有必要。本研究使用羧甲基化β-葡聚糖(m GN),通过金属-酚醛网络结构(Fe-TA)的桥联作用,将其黏附在乳酸乳球菌(LL)表面,构建乳酸乳球菌的包埋体系LL@Fe-TA@mGN,并评价LL@Fe-TA@mGN对胃液与胆盐的抗性及其在体内的滞留能力。结果表明:当mGN的质量浓度为0.12 mg/mL时,LL@Fe-TA@mGN体系的粒径和zeta-电位都达到峰值,表明该质量浓度为mGN包埋单个乳酸乳球菌的最佳质量浓度。在MRS液体培养基中,LL@FeTA@mGN的生长曲线与LL的生长曲线没有显著性差异,说明mGN的毒性可以忽略不计。在扫描电镜与透射电镜分析中均可清晰地看到一层“膜”完整地包覆在乳酸乳球菌表面。经2 h模拟胃液与胆盐的孵育,mGN包埋后的LL抗胃液能力与抗胆盐能力相比于未包埋的LL分别提升14.63倍与1.94倍。体内荧光成像实验证实LL@Fe-TA@mGN具有更强的肠道滞留能力。综上,本研究开发的乳酸乳球菌单菌包埋技术能够显著提升乳酸乳球菌的胃肠道抗性与体内滞留能力。研究结果可为益生菌包埋系统的开发与利用提供新的思路和策略。

杜仲叶多糖对植物乳杆菌CICC 20022胆盐耐受性的影响

以植物乳杆菌CICC 20022为研究对象,探讨在胆盐胁迫环境中添加杜仲叶多糖对菌株活菌数、微观结构、细胞膜脂肪酸组成、细胞膜和细胞壁结构完整性的影响。结果表明:在胆盐胁迫环境中添加杜仲叶粗多糖或杜仲叶精多糖并培养24 h时,植物乳杆菌CICC 20022的活菌数显著增加(P<0.05),分别是对照组的1.30倍或1.10倍;与对照组相比,添加杜仲叶粗多糖或杜仲叶精多糖均可较好地保护菌体的微观结构,显著减少因胆盐胁迫造成的菌体损伤,且菌体的PI荧光强度、碱性磷酸酶(AKP)活性和DNA渗漏量均显著降低(P<0.05),细胞膜中不饱和脂肪酸/饱和脂肪酸的比例(U/S)显著增加(P<0.05),达0.37。因此,在胆盐胁迫环境中添加杜仲叶多糖能较好地保持植物乳杆菌CICC 20022细胞结构的完整性,并提高其胆盐耐受性。

一株具有缓解酒精性肝损伤的发酵粘液乳杆菌及其功效评价研究

该研究从新疆奶疙瘩中分离鉴定一株发酵粘液乳杆菌(Limosilactobacillus fermentum)WHH2438,以商业菌株鼠李糖乳酪杆菌(Lacticaseibacillus rhamnosus)GG为对照,从抗氧化特性、改善肠道屏障功能、耐受性和黏附性等方面,进行体外功效评价。之后以C57BL/6 J小鼠为实验对象,使用液体酒精饲料建立慢性酒精性肝损伤模型,验证菌株的缓解酒精性肝损伤功能。结果显示,该发酵粘液乳杆菌清除DPPH自由基和羟自由基能力显著优于LGG,在保护肠道细胞和肠道屏障方面也有优秀的能力。通过酒精性肝损伤功能动物剂量效价实验,发现菌株WHH2438高剂量组和灭活组的血清谷丙转氨酶和谷草转氨酶相比模型组明显下降、肝脏甘油三脂和还原型谷胱甘肽下降且接近对照,总体有较明显的改善效果,说明该菌株降低酒精对肝脏的氧化损伤,延缓酒精性脂肪肝的形成,具有良好的缓解酒精性肝损伤功能。综上,发酵粘液乳杆菌WHH2438具有良好的缓解慢性酒精性肝损伤功能,且菌株特性优良,能顺利到达肠道,并黏附于肠道上皮细胞,发挥益生功效。

Alleviative effects of Bacillus coagulans strains on irritable bowel syndrome-unraveling strain specificity through physiological and genomic analysis

The high intraspecies heterogeneity of Baciillus coagulans leads to significant phenotypic differences among different strains.Thus,6 B.coagulans strains were tested in the present study using an irritable bowel syndrome(IBS)animal model to determine whether the IBS-alleviating effects of B.coagulans strains are strain-specific.The results of this study showed that the ingestion of B.coagulans GBI-30,6086,and B.coagulans CCFM1041 significantly alleviated IBS symptoms in mice.In contrast,other B.coagulans strains showed no or limited alleviating effects on IBS symptoms.According to our experimental results,the two main common features of these strains were as follows:1)The resistance of vegetative cells to bile salts,and 2)ability to synthesize specific lipids and secondary metabolites.Screening strains based on these two indicators may greatly reduce costs and provide a basis for mining new functional B.coagulans strains.Our results also suggest that administration of B.coagulans could significantly regulate microbiota dysbiosis in animal models.Moreover,the close relationships between the gut microbiota,gut microbiota metabolites,and IBS were further confirmed in this study.