双歧杆菌胞外多糖的特性及与宿主关系的研究进展

双歧杆菌是人类肠道菌群中的主要菌群之一,该菌属中的特定菌株能够对人类产生益生作用。双歧杆菌产生的胞外多糖(bifidobacterial exopolysaccharides,B-EPS)和特定菌株的多种益生作用密切相关,对理解菌株与宿主间的共生关系具有十分重要的作用。B-EPS的合成基因和结构在种内/种间存在高度差异性,能够影响双歧杆菌的肠道耐受和定殖能力,特异性调节宿主肠道菌群和免疫应答功能。因此,该文总结了B-EPS的来源、组成及结构特点,并重点分析了B-EPS与宿主关系的研究进展,以期为菌株筛选和B-EPS在功能食品领域中的应用提供理论指导。     

干酪乳杆菌胞外多糖基因簇的调控基因在大肠杆菌中异源表达

为研究干酪乳杆菌胞外多糖的生物合成和转录调控,对其生物合成基因簇中假定的转录调控因子LC2W_2170进行克隆,并在大肠杆菌(Escherichia coli)中异源表达。首先,利用PCR技术从干酪乳杆菌LC2W中扩增出目的基因LC2W_2170,将其插入到大肠杆菌表达载体p ET24a和p ET30a中,构建出LC2W_2170 N端或C端和N端均含有His蛋白标签的重组质粒。含上述质粒的E.coli经异丙基硫代半乳糖苷(IPTG)诱导后,通过聚丙烯酰胺凝胶电泳(SDS-PAGE)分析并未发现LC2W_2170蛋白可溶性表达。对LC2W_2170氨基酸序列利用Phobius服务器分析,发现该蛋白N端存在跨膜区(前27个氨基酸为跨膜序列)。切除此跨膜区域重新构建重组表达质粒后,在E.coli中成功实现LC2W_2170蛋白可溶性表达。研究为进一步纯化该转录调控因子,并研究其功能和对胞外多糖生物合成基因簇的转录调控机制奠定了基础。     

双歧杆菌胞外多糖对酸豆乳品质的影响

为探索益生菌及其胞外多糖对酸豆乳的影响,利用产胞外多糖双歧杆菌及其活性多糖与传统发酵剂共同发酵豆浆,研究其对酸豆乳理化、感官及质构特性的作用。结果表明：添加双歧杆菌（107 CFU/mL）和其活性胞外多糖（质量分数0.3%,下同）均对酸豆乳凝乳速度及后酸化无显著影响（P＞0.05）。添加0.3%胞外多糖能显著提高酸豆乳的黏度、持水力和质构特性（P＜0.05）。添加产胞外多糖双歧杆菌107 CFU/mL,对发酵期间酸豆乳发酵速率及凝胶特性影响不大;在后酵期间产生86 mg/L 胞外多糖,显著提高酸豆乳的黏度和质构特性（P＜0.05）,但对产品持水力的影响并不显著（P＞0.05）。双歧杆菌胞外多糖可有效改善酸豆乳凝胶特性,提高产品品质。     

改性双歧杆菌胞外多糖体外免疫活性研究

对双歧杆菌胞外多糖进行乙酰化、羧甲基化、多羟基化、主链降解修饰,并测定修饰前后多糖对IL-2、IFNγ细胞因子的影响。试验表明,与对照组相比,除主链降解修饰外,其他3种改性产品均能显著提高IL-2、IFNγ细胞因子的浓度,表现出体外免疫活性。其中,羧甲基化和多羟基化改性多糖的体外免疫活性高于未改性多糖。     

五株双歧杆菌胞外多糖功能特性的差异分析

该研究旨在分析比较5株双歧杆菌(假小链双歧杆菌BP-1、长双歧杆菌BL-3、动物双歧杆菌BA-5、动物双歧杆菌BA-6和婴儿双歧杆菌BI-38)胞外多糖(exopolysaccharides, EPS)理化性质的差异。通过水提醇沉法提取了5株双歧杆菌的EPS,分析比较了其抗氧化活性、抑菌特性、表观形态、官能团种类和流变特性等。结果表明,BA-5的产量为(552.69±3.38) mg/L,显著高于其他菌株(P<0.05);体外抗氧化试验结果表明,5种EPS对DPPH自由基和羟自由基均具有较强的清除能力,其中BA-6 EPS对超氧阴离子自由基的清除率达到(67.78±0.68)%,显著高于其他EPS。抑菌试验结果发现,5种EPS对大肠杆菌、金黄色葡萄球菌和单增李斯特菌均有抑菌活性,其中BA-6 EPS对单增李斯特菌抑菌率高达(94.96±1.43)%。扫描电子显微镜(scanning electron microscope, SEM)观察发现5种EPS的表观形态存在差异,傅里叶中远变换红外光谱(Fourier middle far transform infrared spectr...     

双歧杆菌RH胞外多糖提取纯化及体外抗凝血活性评价

为得到高纯度双歧杆菌RH胞外多糖,并探讨其抗凝血功效,采用正交试验优化酶解提取工艺。优化最佳酶解条件为：蛋白酶添加量2 g/L,中性蛋白酶与胰蛋白酶质量比例为1∶2,在pH 7.5的条件下酶解120 min。再经凝胶Sepharose CL-6B柱层析纯化,得到胞外多糖纯品。利用凝胶色谱结合多角度激光散射仪测得该胞外多糖分子质量为5.946×104 D。进一步对其抗凝血活性进行评价,不同质量浓度胞外多糖可以显著延长活化部分凝血活酶时间和凝血酶时间,且具有剂量依赖效应。纯化后胞外多糖纯度可以达到97%以上,纯多糖具有较好的体外抗凝血活性,主要参与内源凝血途径。     

乳酸菌胞外多糖研究新进展

综述了产EPS乳酸菌的筛选方法、已报道的EPS化学结构、分离纯化和结构鉴定的方法、EPS的生理功能、EPS的生物合成及EPS的遗传学和调控方面的研究进展.     

高效阴离子交换色谱检测双歧杆菌胞外多糖的单糖组成

利用离子交换色谱柱(DEAE-Sepharose Fast Flow,2.6×20cm)和凝胶过滤色谱柱(SepharoseCL-6B,1.6×80cm)对双歧杆菌RH所产EPS进行分离纯化,得到EPSa和EPSb两个组分。然后,采用高效阴离子交换-脉冲安培检测色谱法(high-performance anion-exchange chromatography withpulsed amperometric detection,HPAEC-PAD)分别对2个组分的单糖组成进行了定性和定量测定。结果表明,用20%H2SO4于100℃水解2.5h,可将多糖完全水解。HPAEC-PAD检测结果表明EPSa和EPSb均为杂多糖,EPSa含有Rha(1.00%),Ara(3.54%),Gal(18.13%),Glc(42.84%),Man(33.47%)和Fru(1.03%),而EPSb含有Rha(9.41%),Ara(7.88%),Gal(34.82%),Glc(17.82%),Man(27.83%)和Fru(2.24%)。     

双歧杆菌22-5胞外多糖(EPS)的分离、纯化及纯度鉴定

为了得到双歧杆菌22-5胞外多糖(EPS)的纯品,对EPS分离纯化方法进行了摸索,得到EPS产量较高的分离条件:发酵上清液中加入3倍体积95%冷乙醇,-20℃沉淀12h以上,8000r/min离心4次,热水溶解后用Sevag法去蛋白6次,通过SephoroseCL-6B凝胶过滤得到的吸收峰,经紫外光谱扫描和HPLC分析,鉴定为单一多糖组分。     

鼠李糖乳杆菌JAAS8胞外多糖生物合成基因的克隆和序列分析

根据已报道乳杆菌胞外多糖生物合成基因的同源性,利用其保守区设计引物,扩增出鼠李糖乳杆菌JAAS8RmlA基因部分序列,并通过染色体步移法扩增出Rml(ACB)的序列。获得了鼠李糖乳杆菌JAAS8胞外多糖生物合成基因4.1 kb序列片段,编码4个可读框。利用BLAST和ClustalX程序对获得序列进行生物信息学分析,预测其结构和功能。结果表明:该序列与已知鼠李糖乳杆菌胞外多糖生物合成基因具有高度同源性,RmlA、RmlC和RmlB基因与dTDP-L-鼠李糖前体的生物合成密切相关。     

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion, which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and R1 stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS R1 also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10–24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS R1 and with the increases in Bacteroides in cultures with both microbial EPS (R1 and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids.     

变形假单胞菌吡咯喹啉醌合成基因簇的克隆与分析

从变形假单胞菌JUIM01中克隆到吡咯喹啉醌(PQQ)合成基因簇,阐明了其基因组成和生物学信息。根据已报道的假单胞菌的基因组进行简并引物设计,采用LA-PCR技术克隆变形假单胞菌的PQQ合成基因簇,对克隆的基因片段进行测序并使用生物信息学方法进行综合分析。结果表明:克隆到的基因片段全长为11 659 bp,其中包括pqqF、pqqA、pqqB、pqqC、pqqD、pqqE、pqqM、pqqH和pqqI共9个基因,编码PQQ生物合成的前体短肽PqqA和合成途径的相关酶;这些基因与荧光假单胞菌Pf0-1的PQQ合成基因簇的基因组成类似,相应基因的序列一致性达41%～94%。本研究中首次从变形假单胞菌中克隆到PQQ合成基因簇,并对其进行生物信息学分析,为变形假单胞菌的PQQ生物合成途径和胞内再生机制的研究奠定了基础,进而为提高2KGA的生产强度提供了理论支撑。     

母乳源长双歧杆菌的筛选鉴定及耐氧驯化

目的：从母乳中筛选双歧杆菌,并提高双歧杆菌在有氧条件下的耐受性。方法：采用稀释涂布法结合16S rDNA鉴定法从母乳中筛选出双歧杆菌,并采用逐渐增加氧分压和有氧厌氧交替的方法进行驯化。结果：筛选得一株MEFZ-2201菌株,通过16S rDNA测序比对,其与长双歧杆菌模式菌（NCBI登录号：ON631733.1）的同源性达到了100%,鉴定为长双歧杆菌（Bifidobacterium longum）。将长双歧杆菌MEFZ-2201进行驯化后,其有氧培养最高的活菌数为8.9×10⁹ CFU/mL,比未经驯化的菌株活菌数提高了一个数量级;此外,驯化前后菌株的生理生化及形态特征并未发生变异;在短链脂肪酸的产生量上,驯化后菌株在厌氧条件下产酸量也显著高于驯化前的菌株。结论：驯化后的长双歧杆菌MEFZ-2201在有氧条件下的活菌数明显提高,有望成为潜在的益生菌菌株进一步开发利用。     

Phytate degradation by human gut isolated Bifidobacterium pseudocatenulatum ATCC27919 and its probiotic potential

The growing awareness of the relationship between diet and health has led to an increasing demand for food products that support health above and beyond providing basic nutrition. Probiotics are live organisms present in foods, which yield health benefits related to their interactions with the gastrointestinal tract. Phytases are a subgroup of phosphatases that catalyse the desphosphorylation of phytate, which reduces its negative impact on mineral bioavailability, and generates lower inositol phosphates. The aims of this investigation were to (i) study the ability of the probiotic candidate Bifidobacterium pseudocatenulatum to degrade phytate in synthetic medium, to (ii) identify the lower inositol phosphates generated, to (iii) study its survival under conditions mimicking gastrointestinal passage and finally to (iv) assess adhesion of the bacteria to Caco-2 cells. The first steps of InsP₆ degradation by B. pseudocatenulatum phytate-degrading enzyme/s were preferentially initiated at the DL-6-position and 5-position of the myo-inositol ring. It suggests that the main InsP₆ degradation pathway by B. pseudocatenulatum by sequential removal of phosphate groups was D/L-Ins(1,2,3,4,5)P₅ or D/L-Ins(1,2,3,4,6)P₅; D/L-Ins(1,2,3,4)P₄; to finally Ins(1,2,3)P₃ and D/L-Ins(1,2,4)P₃/D/L-Ins(1,3,4)P₃. This human strain also showed a notable tolerance to bile as well as a selective adhesion capacity (adhesion to control surfaces was zero), to human intestinal Caco-2 cells comparable to the commercial probiotic B. lactis. The phytate-degrading activity constitutes a novel metabolic trait which could contribute to the improvement of mineral absorption in the intestine as a nutritional probiotic feature with potential trophic effect in human gut.     

耐盐芽孢杆菌R1高效壳聚糖酶异源表达及特性分析

采用同源克隆获得耐盐芽孢杆菌壳聚糖酶基因,并异源表达制备重组耐盐芽孢杆菌壳聚糖酶（CsnBh46）,通过表征分析获得重组CsnBh46酶学特性。重组CsnBh46全长278个氨基酸,三维结构显示其分为上下两个半球,包含9个α螺旋和2个β折叠。在7 L发酵罐中,重组工程菌bh-5的最高酶活力和总蛋白质质量浓度分别为6 375 U/mL和4.3 g/L。纯化后的重组CsnBh46最适反应pH和温度分别为6.0和60 ℃,金属离子Mn²⁺、Ca²⁺和Mg²⁺对重组CsnBh46具有激活作用。重组CsnBh46最适底物为脱乙酰度95%胶体壳聚糖。重组CsnBh46能够高效水解胶体壳聚糖并且根据反应时间可以定向制备不同聚合度壳寡糖。本研究为CsnBh46产业化应用奠定基础。     

脲酶基因挖掘及其在枯草芽孢杆菌中的重组表达

氨基甲酸乙酯（ethyl carbamate,简称EC）是一种存在于黄酒中的潜在致癌物,酶法降解EC及其前体物质尿素因此备受关注。该研究通过基因挖掘获得了解淀粉芽孢杆菌IT-45的脲酶（Ba-urease）基因并在食品级系统枯草芽孢杆菌中实现了表达与应用。通过密码子优化、核糖体结合位点优化重构脲酶基因簇后,酶活力从6.85 U/mL提升至9.01 U/mL;通过调整基因ureC的位置,脲酶活力进一步提升至10.15 U/mL。研究发现,重组脲酶的最适反应温度为37 ℃;最适反应pH范围为6.5~7.0,为中性脲酶。摇瓶发酵的最佳培养条件为：TB培养基、接种体积分数5%、诱导温度28 ℃、Ni²⁺添加量4 mmol/L。在最佳发酵条件下,酶活力达到12.5 U/mL,在酶法降解黄酒中的尿素具有应用前景。     

链霉菌SG17代谢产物对玉米黄曲霉的抑制作用及其稳定性分析

黄曲霉是玉米贮存过程中主要的污染之一,直接影响食品以及饲料的品质和安全.利用微生物及其代谢产物进行生物防治具有广泛的应用价值.作者以前期筛选获得的SG17菌株为材料,通过形态学观察、生理生化检测及16S rRNA序列分析,初步确定为链霉菌;平板对峙实验显示该菌株能够显著抑制黄曲霉等多种病原真菌的生长,具有一定的广谱性;...     

Fermentation of rice using amylolytic Bifidobacterium

Twenty-two amylolytic bifidobacteria grown in BHI-starch media were compared for the amylase activity of the intra- and extra-cellular enzymes. The activity of the cells grown in the liquid medium differed considerably. Among the strains tested B. adolescentis Int57 and B. adolescentis ZS8 exhibited higher activities than others. In rice medium containing 0.05% -cysteine·HCl and 0.2% yeast extract, the amylolytic Bifidobacterium strains grew considerably better than non-amylolytic Bifidobacterium strains. B. adolescentis Int57, which showed highest growth and amylase activity in the rice medium, was chosen and rice fermentation was investigated. The titratable acidity (TA) reached 2.43 and pH decreased to 4.4 after 24 h fermentation. The relative ratio of acetic acid to lactic acid gradually decreased during fermentation. The concentration of reducing sugar and amylase activity gradually increased and reached 14 mg maltose equivalent/ml and 35 mU/ml min, respectively, in 24 h. The accumulated reducing sugar was mostly maltotriose. The layer-separation of fermented product was stabilized by the addition of 1% gelatin. It was suggested that amylolytic bifidobacteria may be used for the production of fermented rice products.     

芽孢杆菌活性多糖的分离纯化及合成基因研究

选用自行选育的1 株活性多糖高产菌株（Bacillus amyloliquefaciens LPL061）,经醇沉、除蛋白、透析、冷冻干燥从其发酵上清中分离得到活性多糖粗品,并采用色谱纯化技术进一步纯化,得到1 个中性糖组分（EPS1）和1 个酸性糖组分（EPS2）。根据GeneBank中已报道芽孢杆菌产糖相关基因设计引物,经PCR扩增获得解淀粉芽孢杆菌LPL061的5 个EPS生物合成相关基因。对其功能预测分析表明这些基因主要参与EPS合成过程多糖聚合、糖基转移、糖链长度控制以及多糖转运和输出。     

DeoR家族转录因子PsrB调控黏质沙雷氏菌合成灵菌红素

灵菌红素（prodigiosin,PG）,一种由粘质沙雷氏菌产生的次级代谢产物,因其具有抗细菌、抗疟疾、抗肿瘤和免疫抑制等重要功能而备受关注,然而,对黏质沙雷氏菌合成灵菌红素的调控机制的了解依然有限。作者以黏质沙雷氏菌JNB5-1为出发菌株,通过构建Tn5G转座子插入突变文库,发现并解析了DeoR家族转录调控因子BVG90_04085（PsrB）正调控黏质沙雷氏菌合成灵菌红素的分子机制。结果发现,LB培养基中psrB突变株SK8-61和ΔPsrB产灵菌红素能力仅为出发菌株JNB5-1的0.22倍和0.26倍。RT-qPCR分析菌株JNB5-1及ΔPsrB中pig基因簇的表达水平发现,相比于出发菌株JNB5-1,在psrB缺失突变株ΔPsrB中,灵菌红素合成途径关键基因pigABCDEFGHIJKLMN的表达水平下调了5.81~22.93倍。凝胶阻滞EMSA等实验证实转录因子PsrB可直接与pig基因簇启动子区域结合,表明转录因子PsrB正调控黏质沙雷氏菌合成灵菌红素的分子机制可能为：PsrB与灵菌红素合成基因簇启动子区域直接结合,正向调控pig基因簇的转录水平,进而影响黏质沙雷氏菌合成灵菌红素。最后,发酵培养基中psrB过表达菌株JNB5-1/pXW1906可合成8.61 g/L的灵菌红素,为出发菌株JNB5-1（5.53 g/L）的1.56倍。