基于iTRAQ蛋白组技术的沙门氏菌抗酸性机理的研究

目的:以鼠伤寒沙门氏菌CGMCC 1.1190为对象,研究其经柠檬酸反复胁迫处理诱导的抗酸性机理。方法:CGMCC 1.1190在经柠檬酸调节到pH为2.5的TSB培养基中胁迫处理并转接12次培养后,获得的抗酸性菌株。采用iTRAQ技术对CGMCC 1.1190抗酸性菌株的蛋白组进行分析,通过GO功能注释和富集对差异蛋白进行聚类分析,以及KEGG注释和富集对差异蛋白参与的通路进行联合分析。结果:iTRAQ技术结果显示CGMCC 1.1190抗酸性菌株共2373个蛋白,其中195个差异蛋白中,95个显著下调蛋白和100个显著上调蛋白。GO分析和KEGG分析结果显示CGMCC 1.1190抗酸性菌株高表达应激蛋白相关基因;高表达细胞膜相关蛋白可促进CGMCC 1.1190菌体细胞膜的完整性;高表达鞭毛相关蛋白可增强CGMCC 1.1190菌体的运动能力并促进保护性生物被膜的形成;高表达双组分系统相关蛋白来响应酸信号,通过调控酸激蛋白的产生增强CGMCC 1.1190菌体的抗酸性;低表达ABC转运系统相关蛋白来降低细胞膜对H+的通透性,达到保护菌体的作用;高表达能量代谢途径相关蛋白为CGMCC 1.1190菌体应对酸胁迫提供能量。结论:CGMCC 1.1190在经pH为2.5的柠檬酸胁迫后,能通过蛋白水平的表达变化启动一系列应答机制并生存下来,通过合成一些蛋白质来增强CGMCC 1.1190的抗酸性。     

酸胁迫对鼠伤寒沙门氏菌抗酸性、细胞膜及膜蛋白的影响

目的：以鼠伤寒沙门氏菌CGMCC 1.1190为对象，研究柠檬酸反复胁迫处理对其抗酸性、细胞膜及其膜蛋白的影响。方法：将CGMCC 1.1190分别在经柠檬酸调节到pH值为3.0、2.7、2.5的胰蛋白胨大豆肉汤培养基中胁迫处理并转接12 次培养后，获得3 株CGMCC 1.1190的抗酸性菌株，测定了这3 株抗酸性菌株的D值、菌落形态、个体形态、膜通透性、膜流动性和膜蛋白的变化。结果：这3 株抗酸性菌株的D值随着酸处理次数的增加不断增大，酸胁迫的pH值越低，D值越大，菌落形态与个体形态变化越明显；与原始对照菌株相比，当这3 株抗酸性菌株置于pH 2.0的强酸环境下时，其碘化丙啶染色区域的死菌比例显著降低（P＜0.05），表明酸胁迫pH值越低，这3 株抗酸性菌株的细胞膜对H＋通透性越低；随着酸胁迫pH值的降低，CGMCC 1.1190抗酸性菌株细胞膜磷脂的熔点升高，表明细胞膜磷脂的相变温度升高，细胞膜流动性降低；十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果表明，这3 株抗酸性菌株分子质量35～180 kDa范围内的细胞膜蛋白表达量增加，在135 kDa和180 kDa处均增加了特异条带。结论：随着酸胁迫pH值的降低，CGMCC 1.1190抗酸性增加，菌落变小，个体形态变长，细胞膜通透性和流动性均降低，部分膜蛋白表达量和表达种类增加，这些变化有利于其适应在酸胁迫环境下生长，提高生存能力。     

诱变酒酒球菌中抗酸候选基因ATPase和FtsH的表达差异分析及功能验证

为研究胁迫条件下具有不同表达趋势的抗酸候选基因ATPase和FtsH是否可以提高菌株的抗酸性,利用实时荧光定量PCR(RT-qPCR)测定诱变酒酒球菌抗酸菌株b1和酸敏菌株b2中ATPase和FtsH在pH 4.8(最适条件)和pH 3.0(胁迫条件)ATB培养基中的相对表达量,分析其表达趋势;从b1中克隆ATPase...     

多重胁迫对嗜盐四联球菌CGMCC 3792存活率及细胞成分的影响

本研究以嗜盐四联球菌(Tetragenococcus halophilus CGMCC 3792)为实验菌株,考察了单因素(酸、热、盐)胁迫和交互保护对其存活的影响。结果表明,T.halophilus CGMCC 3792在p H4.0和45℃预适应处理60 min后细胞对酸致死胁迫(p H2.5,60 min)的存活率分别提高了10.6倍和8.8倍。此外,还考察了酸胁迫对细胞膜不饱和脂肪酸和胞内氨基酸含量的影响,结果表明,酸预适应后再经酸致死胁迫导致细胞膜不饱和脂肪酸18∶1n-9和18∶2n-6,9含量增加,分别是直接经酸致死胁迫的2.3倍和2.0倍;同时,分析胞内氨基酸含量,胞内谷氨酸、天冬氨酸、亮氨酸和异亮氨酸浓度分别是直接经酸致死胁迫的2.5倍、2.4倍、2.2倍和2.7倍。研究结果将有助于进一步解析嗜盐四联球菌环境胁迫抗性的生理机制。     

一株高产淀粉酶乳杆菌的筛选及其安全性评价

为筛选到可高效降解淀粉且安全的乳杆菌菌株,本研究首先对5株供试菌株（植物乳杆菌JYI-3913和AS1.555、食淀粉乳杆菌CGMCC1.3395、嗜淀粉乳杆菌CGMCC1.3394和副干酪乳杆菌CGMCC1.12731）开展了产淀粉酶能力初筛、复筛及产酶曲线与生长曲线测定实验,以挑选出产淀粉酶能力最佳的菌株,随后对该菌进行了详细的安全性评价,包括药敏试验、耐药基因检测、质粒检测、溶血试验、产硝基还原酶、产偶氮还原酶及产生物胺能力检测与小鼠急性经口毒性试验等。结果表明：相同培养条件下,食淀粉乳杆菌CGMCC1.3395的淀粉降解透明圈明显,淀粉降解率可达97.28%,淀粉酶活性大小为36.94 U/mL,且该菌株的生长延滞期短而对数期和稳定期长,在供试菌中其综合产淀粉酶能力最佳。该菌株对22种参试抗生素多表现为敏感或中介,仅对其中氨苄西林、卡那霉素及4种氟喹诺酮类抗生素耐药,不含相关耐药基因,不含质粒,因此不具有转移抗生素耐药性基因风险;无溶血性,不产硝基还原酶、偶氮还原酶、组胺和酪胺等有害代谢物;小鼠在14 d喂养期间,正常生长、体重增加,该菌株为无毒级。综上,食淀粉乳杆菌CGMCC...     

具有高抗胃肠道胁迫嗜酸乳杆菌的筛选及对肠炎小鼠的治疗作用

目的:从三株嗜酸乳杆菌中筛选出具有高抗胃肠道胁迫特性的嗜酸乳杆菌并研究其对溃疡性结肠炎小鼠的治疗效果。方法:人工模拟人体消化道胃液、肠液和胆盐环境,设置不同浓度梯度,研究三株嗜酸乳杆菌进入人体后对在消化道胃液、肠液和胆盐环境中的存活率,从而反映嗜酸乳杆菌对人体消化道胃液、肠液和胆盐环境的抵抗力。然后以筛选出的具有高抗胃肠道胁迫能力的嗜酸乳杆菌为研究对象,让处理组小鼠自由饮用3%葡聚糖硫酸钠盐(DSS)溶液7 d,以人工诱发的溃疡性结肠炎(UC)小鼠为动物模型,观察此株高性能的嗜酸乳杆菌CGMCC10436的抗溃疡性结肠炎潜力。结果表明,嗜酸乳杆菌CGMCC10436在模拟胃酸pH2.5~4.5时,其活菌数大于10~7CFU/mL,并且在3 h时没有下降的趋势;在模拟肠液中2h时,嗜酸乳杆菌CGMCC10436活菌数有所上升;并且嗜酸乳杆菌CGMCC10436在胆盐浓度为4g/kg时,3 h时其活菌数大于1×107CFU/mL。嗜酸乳杆菌CGMCC10436发挥较好的抗溃疡性结肠炎功效的剂量为3×108CFU,能够改善溃疡性结肠炎小鼠表观状况,修复溃疡性结肠炎小鼠肠粘膜损伤,增强溃疡性结肠炎小鼠的免疫调节。     

一株产细菌素细菌CGMCC 6624分离鉴定及其生物学特性

采用牛津杯法,从山西陈醋新鲜醋醅中分离筛选得到1株产广谱、高效抑菌活性物质的细菌CGMCC 6624。采用酸抑菌排除试验、过氧化氢抑菌排除试验、蛋白酶消化试验、研究菌株生长与抑菌物质代谢特性的关系和菌株CGMCC 6624对自产抑菌物质的免疫性,结果表明细菌CGMCC 6624发酵产生的抑菌物质为H2O2和细菌素。基于形态学观察、生理生化特征分析及16S rDNA基因序列系统发育分析的微生物分类鉴定技术,菌株CGMCC 6624 被鉴定为枯草芽孢杆菌。通过对酸碱、温度、乙醇度的耐受性的研究发现,该菌株可在pH 2～11、乙醇体积分数0%～8%、温度25～50 ℃条件下生长,最优培养温度 30 ℃,pH 5.0。本研究结果为该菌株在微生态制剂和细菌素的开发利用及在山西传统酿造食品中的应用奠定基础。     

超高压处理对牡蛎中副溶血性弧菌的消减作用研究

为探索有效的消减副溶血性弧菌(Vp)的超高压处理条件,利用CDC-1(临床分离株,血清型O3:K6)和CGMCC1.1997(标准菌株,血清型O1:K1)两种菌株,研究了不同Vp经超高压处理的消减变化情况;比较了CDC-1菌株接种到牡蛎体内与混合到牡蛎肉中的超高压处理的差异性。结果显示,超过200MPa的压力对两种Vp均有明显的消减作用,增大压力和延长处理时间可以提高超高压处理对Vp的消减作用;多重比较显示两种Vp对超高压的耐受力存在显著差异(p<0.05),CDC-1较CGMCC1.1997有更强的超高压耐受力;超高压处理对两种接种方式Vp的消减效果存在显著差异(p<0.05),接种到牡蛎体内的CDC-1菌株的耐受力高于混合在牡蛎肉中的CDC-1菌株;用300MPa处理10min或350MPa处理5min能有效的消减牡蛎中的Vp。     

高温胁迫对德尔卑沙门氏菌抗逆性的影响

以德尔卑沙门氏菌(Salmonella Derby,SD)为研究对象,通过高温培养驯化获得高温耐受性SD,研究其在环境胁迫条件下的耐受性。经高温驯化后,55℃处理20min,高温耐受菌株的存活率是对照菌株存活率的1.5倍。在pH 3.0,2.4环境下,高温耐受菌株存活率高于对照组,但处于碱胁迫过程中,高温耐受菌株变得更加敏感,pH 12处理30min,对照组存活率为33.83%,高温耐受菌株完全被杀灭。在乙醇、NaClO环境下,高温耐受菌株相较于对照组更加敏感;但在125mmol/L H_O_2条件下,高温耐受菌株存活率高于对照组(30.05%)存活率;对-20℃冷冻处理前期,其耐受性明显高于对照组。SD经高温胁迫后提高对酸、H_2O_2、低温的交叉抗性,同时也增加对碱、乙醇、NaClO等胁迫的敏感性,联合使用有助于提高灭菌效果。     

耐盐乳酸菌和酵母菌强化技术对高盐稀态发酵酱醪品质的影响

分别将耐盐乳酸菌Tetragenococcus halophilus CGMCC 3792、耐盐酵母菌Zygosaccharomyces rouxii CGM-CC 3791和Candida versatilis CGMCC 3790菌悬液接入酱醪中,利用顶空-固相微萃取偶联气质联用技术(HS-SPME/GC-MS)检测了发酵125 d酱醪中挥发性组分的变化。研究结果表明,T.halophilus CGMCC 3792强化的酱醪中酸类、醇类、酮类、呋喃类、吡嗪类和其他组分的相对含量降低,酯类、醛类和酚类组分的相对含量增加,其中苯乙醇、乙酸乙酯、辛酸乙酯、癸酸乙酯和4-乙基愈创木酚等的相对含量显著增加,而Z.rouxii CGMCC 3791、C.versatilis CGMCC 3790共培养强化的酱醪中醇类、酯类、醛类和其他组分的相对含量增加,酸类、酚类、呋喃类和吡嗪类组分的相对含量降低,其中苯乙醇、长链脂肪酸乙酯(月桂酸乙酯、棕榈酸乙酯、亚油酸乙酯等)的相对含量显著增加。此外,本研究中T.halophilus CGMCC 3792强化的酱醪中4-乙基愈创木酚的相对含量大幅增加,且在整个发酵过程中其含量也单调增加,对于T.halophilus CGMCC 3792是否具有分泌4-乙基愈创木酚的能力以及是否冷温工艺赋予该菌株合成4-乙基愈创木酚的能力需进一步探究。     

Behavior of acid-adapted and unadapted Escherichia coli O157:H7 when exposed to reduced pH achieved with various organic acids.

A study was done to determine if various organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted Escherichia coli O157:H7 cells. E. coli O157:H7 strain EO139, isolated from venison jerky, was grown in tryptic soy broth (TSB) and in TSB supplemented with 1% glucose (TSBG) for 18 h at 37 degrees C, then plated on tryptic soy agar (TSA) acidified with malic, citric, lactic, or acetic acid at pH 5.4, 5.1, 4.8, 4.5, 4.2, and 3.9. Regardless of whether cells were grown in TSB or TSBG, visible colonies were not formed when plated on TSA acidified with acetic, lactic, malic, or citric acids at pH values of < or =5.4, < or =4.5, < or =4.2, or < or =4.2, respectively. Cells not adapted to reduced pH did not form colonies on TSA acidified with lactic acid (pH 3.9) or acetic acid (pH 3.9 and 4.2); however, a portion of acid-adapted cells remained viable on TSA containing lactic acid (pH 3.9) or acetic acid (pH 4.2) and could be recovered in TSB. Inactivation of acid-adapted cells was less than that of unadapted cells in TSB acidified at pH 3.9 with citric, lactic, or acetic acid and at pH 3.4 with malic acid. Significantly (P< or =0.05) higher numbers of acid-adapted cells, compared with unadapted cells, were detected 12 h after inoculation of TSB acidified with acetic acid at pH 3.9; in TSB containing lactic acid (pH 3.9), the number of acid-adapted cells was higher than the number of unadapted cells after 5 h. In TSB acidified at pH 3.9 with citric acid or pH 3.4 with malic acid, significantly higher numbers of acid-adapted cells survived. This study shows that organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted E. coli O157:H7 cells, and acid-adapted cells are more tolerant than unadapted cells when subsequently exposed to reduced pH caused by these acids.     

Effect of organic acids and temperature on survival of Shigella flexneri in broth at pH 4

The survival of bacterial pathogens in acidified foods depends not only on the hydrogen ion concentration, but also on the type of acid and the storage temperature. Shigella flexneri is a foodborne pathogen that is acid tolerant. The survival of S. flexneri 5348 in brain heart infusion broth supplemented with 0.04 M acetic, citric, lactic, malic, or tartaric acid and adjusted to pH 4 with HCI or NaOH was studied. The control medium was brain heart infusion broth adjusted to pH 4 with HCI. Stationary-phase cells were inoculated into media at initial populations of 6 to 7 log10 CFU/ml and incubated at 4, 19, 28, and 37 degrees C. A two-phase linear inactivation model was applied to plate count data to derive lag times (tL) and slopes of the curves, from which D-values and time required for a 4-log10 decrease in population (T4D) were calculated. In all cases, survival increased with decreasing temperature. For each acid, tL, the D-value, and T4D increased with decreasing temperature. All acids inhibited S. flexneri to some extent but to differing degrees as follows: lactic acid, acetic acid > citric acid, malic acid, tartaric acid > HCl. The T4D values for the control medium and for media containing acetic, citric, lactic, malic, and tartaric acids were 64, 47, 50, 34, 58, and 52 h, respectively, at 37 degrees C and 2,607, 1,498, 1,905, 1,346, 1,726, and 2,134 h, respectively, at 4 degrees C. The results of this study indicate that organic acids may aid in the inactivation of Shigella. However, these data also suggest that foods stored at or below room temperature containing low levels (< 1%) of acids could cause illness if contaminated with Shigella.     

发酵前后甜石榴汁中有机酸的变化研究

本试验利用反相液相色谱（RP-HPLC）法分离测定发酵前后甜石榴汁中有机酸种类和含量。在甜石榴汁中检测到的有机酸包括草酸、苹果酸、α-酮戊二酸、乳酸、乙酸、柠檬酸与富马酸,其中主要有机酸为乳酸（占总有机酸的51.2%）、草酸（占总有机酸的26.8%）和柠檬酸（占总有机酸的16.8%）。发酵后甜石榴汁中的草酸与乳酸含量下降,乙酸含量增加,新生成酒石酸、丙酮酸与琥珀酸;苹果酸与柠檬酸含量增加。发酵后甜石榴汁的有机酸总量下降了7.8%.     

啤酒中有机酸含量的RP-HPLC检测条件优化及其酿造过程中动态分析

该研究对反相高效液相色谱（RP-HPLC）法测定啤酒中有机酸含量的方法进行优化,最终确定了RP-HPLC法测定啤酒中草酸、乳酸、酒石酸、乙酸、苹果酸、α-酮戊二酸、抗坏血酸、柠檬酸、琥珀酸9种有机酸的检测条件,并结合发酵机理对啤酒酿造过程中的有机酸含量动态变化进行分析。结果表明,RP-HPLC优化条件为：检测波长215 nm、流动相缓冲液0.10 mol/L KH2PO4、pH值3.0、流速0.6 mL/min。有机酸在质量浓度0.2～400.0 mg/L范围内线性关系良好（R2均＞0.99）,加标回收率79.3%～110.0%,精密度试验结果相对标准偏差（RSD）为0.3%～11.5%,表明该方法精密度高、准确性良好。在啤酒酿造过程中有机酸总量及乙酸、琥珀酸、苹果酸、乳酸、柠檬酸、α-酮戊二酸含量呈现先增长后平稳的趋势,草酸、抗坏血酸、酒石酸含量变化相对平稳。     

原汁苹果醋中的有机酸

以苹果汁为原料 ,采用液态酒精发酵和醋酸发酵法制作苹果醋 ,采用反相高效液相色谱法分析苹果醋中有机酸 ,研究了苹果品种、醋酸菌种、醋酸发酵方法及过程对有机酸的影响。认为苹果酸是苹果醋中的主要有机果酸 ,其与液态粮食醋有机酸组成的差别主要体现在苹果酸、酒石酸和乳酸的含量上 ,苹果品种是影响原汁苹果醋中有机酸的种类和含量主要因素 ,醋酸菌在醋酸发酵过程中有代谢各种有机酸的作用 ,发酵方法会影响醋酸菌对有机酸代谢作用的程度 ,试验中所采用的 5种醋酸菌在醋酸发酵过程中通过消耗苹果酸、乳酸、琥珀酸 ,而产生和积累柠檬酸。     

Influence of acetic, citric, and lactic acids on Escherichia coli O157:H7 membrane lipid composition, verotoxin secretion, and acid resistance in simulated gastric fluid

The effect of organic acid (acetic, citric, and lactic acids) adaptation at equivalent initial pH values (6.4 and 5.4) on changes in membrane lipid composition, verotoxin concentration, and acid resistance in simulated gastric fluid (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 ATCC 43895 (HEC) and an rpoS mutant of E. coli O157:H7 ATCC 43895 (RM, FRIK 816-3). For HEC, lactic acid-adapted (pH 5.4) cells had the greatest D-value (32.2 min) and acetic acid-adapted (pH 5.4) cells had the smallest D-value (16.6 min) in simulated gastric fluid. For RM, D-values of citric and acetic acid-adapted cells were similar to those for nonadapted cells grown at pH 7.3, but D-values increased from 13.1 to 27.9 min in lactic acid-adapted cells (from pH 7.3 to pH 5.4). For both strains, the ratio of cis-vaccenic to palmitic acids decreased for citric and lactic acid-adapted cells, but the ratio increased for acetic acid-adapted cells at pH 5.4. Organic acid-adapted cells produced less total verotoxin than did nonadapted cells at approximately 10(8) CFU/ml. Extracellular verotoxin concentration proportionally decreased with decreasing pH for both HEC and RM. Changes in membrane lipid composition, verotoxin concentration, and acid resistance in HEC and RM were dependent on both pH and organic acid. Deletion of the rpoS gene did not affect these changes but did decrease acid resistance in citric acid-adapted cells. Results indicate that decreased membrane fluidity may have caused increased acid resistance and decreased verotoxin secretion.     

Comparison of acids on the induction of an Acid Tolerance Response in Salmonellatyphimurium, consequences for food safety

Salmonellatyphimurium inactivation at pH 3.0 in Brain Heart Infusion (BHI) and Meat Extract (ME) was studied using stationary-phase cells grown in non-acidified BHI (pH 7.4) and ME (pH 6.6) and acidified BHI and ME at pH values of 6.4, 5.4 and 4.5 with acetic, ascorbic, citric, lactic, malic and hydrochloric acids. Cells grown in buffered BHI (pH 7.0) were used as non-acid adapted control cells.     

未除气与除气淡爽型啤酒中9种有机酸的阈值

采用ASTM679标准方法测定了未除气与除气淡爽型啤酒中乙酸、乳酸、柠檬酸、苹果酸、琥珀酸、富马酸、丙酮酸、酒石酸和草酸的个人和小组阈值。结果发现,在未除气啤酒中,9种有机酸的小组阈值大小顺序为苹果酸、富马酸、乳酸、柠檬酸、丙酮酸、琥珀酸、乙酸、草酸、酒石酸。其中品评员对苹果酸、琥珀酸、富马酸、草酸和柠檬酸的口感差异相对较小,而对酒石酸、乙酸、丙酮酸和乳酸的口感差异较大。在除气啤酒中,9种有机酸的小组阈值大小顺序为丙酮酸、乳酸、乙酸、富马酸、琥珀酸、苹果酸、酒石酸、草酸、柠檬酸。品评员对柠檬酸、苹果酸和乳酸的口感差异相对较小,而对丙酮酸、草酸、酒石酸、富马酸、琥珀酸和乙酸的口感差异较大。此外还发现未除气和除气啤酒中有机酸阈值差异很大,其中差异最大的是三元酸柠檬酸,其次为二元酸,包括苹果酸、草酸、酒石酸、琥珀酸和富马酸,差异最小的是一元酸,包括乳酸、乙酸和丙酮酸。