基于基因组学分析嗜热链球菌KLDS SM的蛋白质水解系统和氨基酸合成途径

为从遗传水平上探究嗜热链球菌KLDS SM蛋白质水解和氨基酸合成的能力,首先基于Illumina Hiseq 2500与Pacbio RSII测序平台对嗜热链球菌KLDS SM进行全基因组测序并绘制基因组图谱;随后从胞外蛋白酶、转运系统、胞内肽酶以及氨基酸合成等方面所涉及的基因进行生物信息学分析;最后对15?株已完成全基因组测序的嗜热链球菌的氨基酸合成能力进行比较基因组学研究。结果表明：菌株KLDS?SM的基因组由一个1?856?787?bp环状染色体组成,GC含量为39.08%,含有1?732?个蛋白质编码基因;菌株KLDS?SM具有完整的蛋白水解系统和8?种氨基酸的合成能力;15?株嗜热链球菌在氨基酸合成方面上相对保守,仅在组氨酸合成途径存在较大的差异。本研究为该菌株氮代谢能力的挖掘提供了理论依据,并在将其开发为发酵剂方面上具有一定指导意义。     

嗜热链球菌KLDSSM胞外多糖生物合成途径的分析

为了从遗传水平上探究嗜热链球菌KLDS SM合成胞外多糖（EPS）的能力,本研究从糖被转运进入胞内、糖核苷酸合成及EPS基因簇三方面所涉及的基因进行了相关分析。结果发现,该菌能够转运葡萄糖、乳糖、蔗糖、海藻糖及甘露糖5种糖,具有代谢葡萄糖、乳糖、果糖、蔗糖、海藻糖与甘露糖形成糖酵解中间代谢产物的酶类,拥有合成EPS前体物质UDP-葡萄糖、UDP-半乳糖、UDP-N-乙酰基葡糖胺、UDP-呋喃半乳糖与d TDP-鼠李糖的潜力。同时序列比对分析发现该菌具有一个与高产EPS的菌株ASCC 1275一致性极高的EPS基因簇,且EPS初步产量测定约为331 mg/m L,表明该菌是一株高产EPS潜力菌株,具有较大的应用潜能。      

嗜热链球菌KLDS3.1012胞外多糖合成途径的基因组学及表型特征分析

在分子水平上挖掘嗜热链球菌KLDS3.1012合成胞外多糖的途径并且通过表型特征进行验证。首先,基于Illumina HiSeq与Illumina MiSeq测序平台对菌株KLDS3.1012进行基因组测序并构建基因组图谱;随后,从糖代谢、糖核苷酸合成、胞外多糖基因簇等方面进行生物信息学分析;最后,利用API 50CH测定菌株KLDS3.1012的糖发酵情况及采用高效离子交换色谱法测定胞外多糖的单糖组成。生物信息学分析结果表明菌株KLDS3.1012具有半乳糖、葡萄糖、果糖、甘露糖、乳糖和蔗糖转运系统和4 种糖核苷酸（UDP-葡萄糖、dTDP-鼠李糖、UDP-半乳糖和UDP-N-乙酰葡糖胺）合成的相关基因及1 个胞外多糖合成基因簇。表型特征分析结果表明菌株KLDS3.1012可以利用以上6 种碳源并能形成由鼠李糖、半乳糖和葡萄糖组成的胞外多糖。本研究为分析该菌株合成胞外多糖的遗传基础与胞外多糖结构的关系提供理论依据,并对将其开发为发酵剂具有一定指导意义。     

嗜热链球菌产胞外多糖的培养条件及分离提取

以高产胞外多糖的西藏雪莲嗜热链球菌为菌株,IRIE培养基为基础培养基,研究了影响嗜热链球菌产胞外多糖培养条件及多糖提取的工艺条件。采用正交实验法分析了温度、时间及接种量对嗜热链球菌胞外多糖产量的影响。结果表明嗜热链球菌培养的最佳条件为:3%接种量,42℃发酵48h,粗多糖含量为382.8mg/L。多糖提取工艺的研究结果表明:Sevage法脱蛋白3次、上清液超滤(滤膜MWCO3ku,压力0.2×105Pa,温度25℃)浓缩4倍,多糖的提取效果最好。     

Short communication: effect of oxygen on symbiosis between Lactobacillus bulgaricus and Streptococcus thermophilus

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus are traditionally used for the manufacture of yogurt. It is said that a symbiotic relationship exists between Strep. thermophilus and L. bulgaricus and this decreases fermentation time. It is well known that L. bulgaricus is stimulated by the formate produced by Strep. thermophilus, and Strep. thermophilus is stimulated by free amino acids and peptides liberated from milk proteins by L. bulgaricus in symbiotic fermentation. We found that acid production by starter culture LB81 composed of L. bulgaricus 2038 and Strep. thermophilus 1131 was greatly accelerated by decreasing dissolved oxygen (DO) to almost 0 mg/kg in the yogurt mix (reduced dissolved oxygen fermentation) and that DO interferes with the symbiotic relationship between L. bulgaricus 2038 and Strep. thermophilus 1131. We attributed the acceleration of acid production of LB81 by reduced dissolved oxygen fermentation mainly to the acceleration of formate production and the suppression of acid production of LB81 by DO mainly to the suppression of formate production.     

Short communication: Molecular epidemiology of Streptococcus agalactiae differs between countries

Group B Streptococcus or Streptococcus agalactiae continue to be challenging for milk quality programs in countries with emerging dairy industries, such as Colombia, where high prevalence has been reported. Molecular typing of isolates is needed to understand the variability and epidemiology of this pathogen and to develop effective control and eradication programs. We characterized the molecular profile of Strep. agalactiae isolated from cows with subclinical mastitis in 21 Colombian dairy herds and measured diversity within and between herds using multilocus sequence typing. Isolates belonged to sequence type 248 [clonal complex (CC) 103; n = 30), ST1 (CC1; n = 6) or ST22 (CC22; n = 4)], whereas members of CC67/61, the dominant type in North America, were not detected. Presence of multiple clonally unrelated sequence type within a herd was common, which contrasts with the situation in European countries and suggests introduction from multiple sources. Our results demonstrate that conclusions from molecular epidemiological studies in 1 region cannot necessarily be extrapolated to other regions, and no single bovine-adapted CC of Strep. agalactiae exists in Colombia. Improvements in internal and external biosecurity will be needed to reduce Strep. agalactiae prevalence in Colombian dairy herds.     

正交试验优化向日葵盘果胶的提取和基本分析

研究向日葵盘果胶(SFHP)的提取工艺。通过单因素试验,比较酸化水、0.75%六偏磷酸钠、0.5%乙二胺四乙酸二钠、0.5%草酸-草酸铵、0.5%草酸和0.5%草酸铵6种提取剂的提取效果,确定较优的提取剂种类。选择pH值、提取温度、提取时间和提取固液比为主要工艺参数,通过正交试验,确定较优的提取参数组合:pH3.50、提取时间45min、提取温度85℃、固液比1:30(g/mL)。在此条件下,向日葵盘果胶得率为20.9%(以半乳糖醛酸计)。采用高效尺寸排阻色谱-多角度激光散射检测器(HPSEC-MALLS)和高效阴离子交换色谱-脉冲安培检测器(HPAEC-PAD)测定了向日葵盘果胶的分子质量和单糖组成。结果表明,向日葵盘果胶的分子质量高于柑橘果胶,半乳糖醛酸为最主要单糖成分,占单糖总量的82.1%,鼠李糖为次要成分,仅占单糖总量的9.1%。     

Short communication: Comparison of growth kinetics at different temperatures of Streptococcus macedonicus and Streptococcus thermophilus strains of dairy origin

Within the genus Streptococcus, S. thermophilus and S. macedonicus are the 2 known species related to foods. Streptococci are widely used as starter cultures to rapidly lower milk pH. As S. macedonicus has been introduced quite recently, much less information is available on its technological potential. Because temperature is an important factor in fermented food production, we compared the growth kinetics over 24 h of 8 S. thermophilus and 7 S. macedonicus strains isolated from various dairy environments in Italy, at 4 temperatures, 30°C, 34°C, 37°C and 42°C. We used the Gompertz model to estimate the 3 main growth parameters; namely, lag phase duration (λ), maximum growth rate (µ_max), and maximum cell number at the stationary phase (N_max). Our results showed significant differences in average growth kinetics between the 2 species. Among the strains tested, 37°C appeared to be the optimal temperature for the growth of both species, particularly for S. macedonicus strains, which showed mean shorter lag phases and higher cell numbers compared with S. thermophilus. Overall, the growth curves of S. macedonicus strains were more similar to each other whereas S. thermophilus strains grew very differently. These results help to better define and compare technological characteristics of the 2 species, in view of the potential use of S. macedonicus in place of S. thermophilus in selected technological applications.     

Detection and quantification of capsular exopolysaccharides from Streptococcus thermophilus using lectin probes

The aim of this work was to use fluorescently labeled lectins to develop a convenient and reliable method to determine the relative abundance of capsular polysaccharides (CPS) at the surface of Streptococcus thermophilus MR-1C cells. Fluorescein isothiocyanate-labeled peanut agglutinin isolated from Arachis hypogaea was found to interact specifically with the CPS of Strep. thermophilus MR-1C. This labeled lectin was then used as an effective probe to detect and quantify CPS. A fluorescence-based lectin-binding assay was successfully applied to follow the accumulation of CPS during the growth of Strep. thermophilus MR-1C in milk and in M17 broth supplemented with lactose. Our results showed that in both media, CPS production by Strep. thermophilus MR-1C began during the exponential phase of growth and continued for several hours after the culture reached the stationary growth phase.     

Short communication: technological and genotypic comparison between Streptococcus macedonicus and Streptococcus thermophilus strains coming from the same dairy environment

The species Streptococcus thermophilus is widely used for the preparation of several dairy products, and its technological contribution is clear. On the other hand, although Streptococcus macedonicus was first described more than 10 yr ago and, despite the scientific interest around this issue, the exact role of Strep. macedonicus in cheese making has yet to be clarified. In this study, 121 strains belonging to both species and isolated from the same dairy environment were genetically characterized by random amplification of polymorphic DNA (RAPD)-PCR and compared for the main biochemical features of technological interest, such as acid production, galactose utilization, citrate metabolism, exopolysaccharide production, and lipolytic, ureolytic, exocellular proteolytic, and decarboxylasic activities. Analysis by RAPD-PCR highlighted a remarkable genotypic heterogeneity among strains in both species, and, at a similarity level of 78%, all the isolates and reference strains of Strep. thermophilus grouped together and were well separated from the strains of Strep. macedonicus, confirming that these 2 species are different microbial entities. Comparison between genetic and phenotypic or biotechnological data did not reveal any relationships.     

Short communication: Genomic prediction using imputed whole-genome sequence variants in Brown Swiss Cattle

The accuracy of genomic prediction determines response to selection. It has been hypothesized that accuracy of genomic breeding values can be increased by a higher density of variants. We used imputed whole-genome sequence data and various single nucleotide polymorphism (SNP) selection criteria to estimate genomic breeding values in Brown Swiss cattle. The extreme scenarios were 50K SNP chip data and whole-genome sequence data with intermediate scenarios using linkage disequilibrium-pruned whole-genome sequence variants, only variants predicted to be missense, or the top 50K variants from genome-wide association studies. We estimated genomic breeding values for 3 traits (somatic cell score, nonreturn rate in heifers, and stature) and found differences in accuracy levels between traits. However, among different SNP sets, accuracy was very similar. In our analyses, sequence data led to a marginal increase in accuracy for 1 trait and was lower than 50K for the other traits. We concluded that the inclusion of imputed whole-genome sequence data does not lead to increased accuracy of genomic prediction with the methods.     

Short communication: Complete genome sequence of Lactobacillus plantarum J26, a probiotic strain with immunomodulatory activity

Lactobacillus plantarum J26, a significant probiotic isolated from Chinese traditional fermented dairy products, exerts a positive immunomodulatory effect by regulating the expression of immune-related genes. We investigated expression of the cytokines IL-1α, IL-1β, IL-6, and tumor necrosis factor-α in the intestinal tract of mice stimulated by L. plantarum J26. In vivo, these cytokines were upregulated, peaked on d 5, and then decreased to the control level, indicating that L. plantarum J26 could induce expression of the genes encoding these proinflammatory cytokines. Teichoic acids produced by L. plantarum are recognized as key immunomodulatory molecules involved in the regulation of the host immune response. To better understand the genetic basis of this immunomodulatory mechanism, we sequenced and analyzed the whole genome of L. plantarum J26. The genome of L. plantarum J26 contains a circular chromosome and 4 circular plasmids. Lactobacillus plantarum J26 was predicted to synthesize ribitol-type backbones of wall teichoic acid. Furthermore, orthologous average nucleotide identity (OrthoANI) values showed that the genome was highly similar (>98.00%) to other L. plantarum strains, especially to L. plantarum ST-III and JDM1. The genomic data of L. plantarum J26 provide a genetic basis to further elucidate its mechanism of immunoregulation and will facilitate its application in the functional dairy food industry.     

Production of the exopolysaccharides by Streptococcus thermophilus: effect of growth conditions on fermentation kinetics and intrinsic viscosity

Production of exopolysaccharides (EPS) by a commercial Streptococcus thermophilus strain was evaluated at different growth conditions [temperature (32-45 degrees C), carbon source and initial nitrogen (N) content]. Lactose from deproteinized whey and sucrose allowed to obtain EPS yields higher than 1200 mg/mM of the consumed carbon source. Intrinsic viscosity of the EPS was significantly reduced by ionic strength indicating a polyelectrolyte behavior. Growth conditions used for the production of the EPS had a significant effect (p<0.05) on the intrinsic viscosity. This was attributed to the effect of growth conditions on the molecular properties of the EPS [stiffness and molecular weight (MW)]. High MW EPS were produced when the bacteria grew at a high specific growth rate; however MW of the EPS and specific growth rate were not linearly associated. In the lactose fermentations carried out at different temperatures specific EPS synthesis rate was positive and linearly associated with the specific lactose consumption rate (R2=0.967) and specific galactose production rate (R2=0.967). Critical coil overlap parameter, [eta]C*, for the EPS produced in the lactose fermentations carried out at 43 and 45 degrees C was determined to be approximately 7.6, and their critical overlap concentrations (C*) were 0.45 and 0.87 g/dL, respectively.     

酶法提取对玉米皮阿拉伯木聚糖组成及分子质量分布的影响

以鲜玉米皮为原料,利用酶法提取玉米皮阿拉伯木聚糖（arabinoxylan,AX）,对提取物分别进行气相色谱分析和高效体积排阻色谱分析,研究Grindmyl Powerbake 950、Pentopan mono BG、Viscozyme L、Amano HC 90四种食品工业中常见的木聚糖酶提取所得提取物的单糖组成和分子质量分布,筛选出提取玉米皮AX的最佳酶制剂,并确定酶的最适添加量。结果表明：采用酶法提取玉米皮AX,其单糖组成主要是阿拉伯糖和木糖。以Amano HC 90提取24 h的玉米皮AX,其纯度为70.51%,提取率为（17.37±2.74）%,重均分子质量为（67.87±1.46）×104 g/mol,数均分子质量为（15.06±0.09）×104 g/mol,相对分子质量分散系数为4.51±0.13。经Amano HC 90提取得到提取物中AX含量较高,提取率适当,分子质量高且分散均匀,更适合食品工业中鲜玉米皮中大分子质量AX的提取,其最适添加量为1 500 U/g。     

嗜热链球菌胞外多糖的分离纯化及理化性质研究

以一株从西藏灵菇中分离的高产胞外多糖嗜热链球菌为研究对象,利用IRIE培养基进行发酵培养,通过离心、除蛋白、醇沉、透析等分离手段获得嗜热链球菌胞外多糖,并通过DEAE纤维素52和DEAE Sepharose CL-6B柱进一步分离纯化。高效液相色谱证实该多糖为纯品。差热扫描量热(DSC)研究显示该多糖有两个熔点分别为120.83℃和212.89℃。热重分析(TGA)表明该多糖第一步热分解温度为264.88℃,第二步热分解温度为441.82℃。红外光谱分析揭示该多糖具有较长分子链,有β-糖苷键构型特征。     

Proteolytic activities of combined fermentation with Lactobacillus helveticus KLDS 1.8701 and Lactobacillus plantarum KLDS 1.0386 reduce antigenic response to cow milk proteins

Cow milk protein is one of the leading food allergens. This study aimed to develop an effective method for reducing milk sensitization by evaluating antigenicity of fermented skim milk protein using Lactobacillus helveticus KLDS 1.8701, Lactobacillus plantarum KLDS 1.0386, and a combination of both strains. The proteolytic systems of strains in terms of genotype and phenotype are characterized by complete genome sequence, and evaluation the antigenicity of skim milk proteins was determined by ELISA and liquid chromatography with tandem mass spectrometry. Our results showed that the genomes encoded a variety of peptidase genes. For fermented skim milk, the degree of hydrolysis of the combined strains was higher than that of individual strain. Electrophoresis showed that the band color density of α-casein (α-CN) by fermentation of the combined strains was reduced when compared with control group. The fermentation process of the combined strains inhibited α-CN, β-lactoglobulin, and α-lactalbumin antigenicity by 69.13, 36.10, and 20.92, respectively. Major allergic epitopes of α-CN and β-lactoglobulin were cleaved by abundant proteases of combined strains. In all, this study showed that the fermentation process involving both L. helveticus and L. plantarum strains could reduce cow milk protein allergenicity through the combination of cell-envelope proteinase and peptidase on α-CN.     

胶质芽孢杆菌胞外多糖单糖组成的研究

目的对胶质芽孢杆菌PM13菌株的胞外多糖的单糖组成进行研究。方法取培养48 h的斜面菌体,用10倍体积的蒸馏水稀释,稀释液6000 r/min离心30 min后除去沉淀,上清液加4倍体积的95%的乙醇进行沉淀,并于6000 r/min离心30 min得沉淀物,沉淀物烘干得到多糖样品。采用苯酚-硫酸法测定多糖样品中含糖量。采用气相色谱法分析其单糖组成,色谱条件为:进样口温度为250℃,分流比为40:1,FID检测器温度为250℃,VF-5HT熔融石英毛细柱,柱温为160℃,8℃/min升至200℃,200℃保持5 min,载气为高纯氮气,1.6mL/min。结果 PM13菌株胞外多糖的含糖量为90.98%,其单糖组成为葡萄糖、半乳糖、甘露糖和木糖,比例为5.57:3.08:4.04:1。结论本文建立了用气相色谱法检测胶质芽孢杆菌PM13菌株胞外多糖的单糖组成的方法,确定了胶质芽孢杆菌胞外多糖的单糖组成及相应比例。