植物乳杆菌(Lactobacillus plantarum)CW5的筛选及细菌素抑菌活性的研究

为了提高抑菌活性,对植物乳杆菌(Lactobacillus plantarum)CW5产细菌素的发酵条件进行了优化,分别研究了培养时间、温度、接种量、培养基起始pH、培养基碳源、氮源等因素对细菌素产生的影响,通过单因素水平实验和正交实验,确定产细菌素的最佳培养基组合和最佳发酵条件为:葡萄糖3%,胰蛋白胨1.5%,蛋白胨1.5%,酵母膏1%,硫酸镁0.058%,吐温80 0.2%,30℃培养24 h,培养基起始pH为6.5,接种量2%。CW5在优化前效价为367.82 IU/mL,优化后效价为1619.85 IU/mL,提高了340.39%。     

植物乳杆菌拮抗致病大肠杆菌机制与应用研究进展

大肠杆菌（Escherichia coli）是最为常见的食源性病原菌之一，随着致病大肠杆菌严峻的耐药性形势出现，防控致病大肠杆菌成为一个难题。植物乳杆菌（Lactiplantibacillus plantarum）是一种具有良好的抑菌效果及提升机体免疫功能作用的益生菌，其可通过多种不同的作用机制拮抗致病大肠杆菌，是一种极具开发潜力的新型抗菌物质。该研究系统综述了植物乳杆菌拮抗致病大肠杆菌的研究进展，从代谢活性物质、增强黏膜屏障、竞争粘附和肠道免疫调节多角度剖析植物乳杆菌在体内外拮抗大肠杆菌的作用机制，进而总结植物乳杆菌在畜禽/水产养殖业、食品加工制造业及临床治疗中的应用模式，以期对益生菌在食品安全防控应用提供参考。     

肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌作用及抑菌机理研究

研究了肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌作用及其抑菌机理。本文采用滤纸片扩散法测定抑菌圈大小,双倍稀释法测定最低抑菌浓度、最低杀菌浓度,以此评价肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌性,通过扫描电镜观察、胞膜通透性、胞膜完整性、膜电位实验,阐述肉桂醛抑菌的机理。结果表明:肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌圈分别为21.75 mm、29.37mm,最低抑菌浓度均为0.25μL/m L,最低杀菌浓度均为0.5μL/m L;肉桂醛破坏了菌体细胞的形态,出现形变;随着肉桂醛浓度的增大,悬液中相对电导率迅速升高,表明肉桂醛影响细菌的膜通透性,而成倍数增长的核酸、蛋白质含量表示胞膜的完整性遭到破坏,肉桂醛降低菌体膜电位,影响其代谢活性,从而抑制细菌生长。肉桂醛主要作用于细胞膜,适宜作为天然防腐剂,抑制食品中腐败菌和致病菌的生长,延长食品货架期。     

植物乳杆菌DMDL 9010胞外多糖合成蛋白cpsB的生物信息学分析

为了探讨植物乳杆菌DMDL 9010胞外多糖合成蛋白cpsB的结构特性,该文通过生物信息学技术研究了胞外多糖合成蛋白cpsB的理化性质、亲/疏水性、跨膜结构、功能位点、磷酸化位点、信号肽、结构域、保守功能域、序列同源性以及空间结构进行预测与分析。结果表明胞外多糖合成蛋白cpsB相对分子质量约为2 920,等电点6.86,含有191个氨基酸;其为疏水蛋白,具有较高的稳定性;经过跨膜结构预测,发现其不存在跨膜结构,为胞内蛋白;胞外多糖合成蛋白cpsB中含有15个磷酸位点;氨基酸序列中不包含信号肽,所编码蛋白均为内分泌型蛋白;保守功能域预测中,基因中仅发现一段PHP结构域,该基因可能参与调节胞外多糖的基因表达。胞外多糖合成蛋白cpsB的二级结构中α-螺旋占51.14%,并不存在β-折叠和β-转角结构,其三级结构比例与二级结构基本相似。该研究对植物乳杆菌DMDL 9010的胞外多糖合成蛋白cpsB功能机制进行深入研究,对利用基因工程技术研究其胞外多糖的生物合成具有重要意义。     

外源诱导及共培养对植物乳杆菌J23合成细菌素Lac-B23的影响研究

目的 研究外源诱导物及共培养对植物乳杆菌J23合成细菌素Lac-B23的影响,提高细菌素产量。方法 利用双层琼脂牛津杯扩散法测定细菌素活性,通过单因素实验、自诱导和共培养验证实验探究外源诱导物及共培养对细菌素J23产量的影响。结果 当初始pH为7、37℃培养12 h,细菌素Lac-B23的产量达到最大值,为2560 AU/mL。甘油和丙酮酸基本不影响细菌素Lac-B23的合成;但酪氨酸、苯丙氨酸、精氨酸、谷氨酰胺和亮氨酸则能促进细菌素Lac-B23的产量。此外,自我诱导研究发现植物乳杆菌J23发酵培养基中含有可以诱导细菌素Lac-B23合成的信号分子。一定浓度范围内,植物乳杆菌J23与单增生李斯特菌或金黄色葡萄球菌共培养时可以提高细菌素Lac-B23的产量。然而,诱导菌的灭活菌体和无细胞上清液并不能提高细菌Lac-B23的产量。结论 环境诱导因素对细菌素的合成有较大影响。初始pH、发酵温度、培养时间、酪氨酸、苯丙氨酸、精氨酸、谷氨酰胺和亮氨酸可以作为诱导因子,提高细菌素Lac-B23的产量。此外,共培养诱导分子没有分泌到培养基中,可能需要活的诱导细菌来诱导细菌素Lac-B22的合成。     

Purification,characterization, and mode of action of a novel bacteriocin BM173 from Lactobacillus crustorum MN047 and its effect on biofilm formation of Escherichia coli and Staphylococcus aureus

There is an increasing demand for dairy products, but the presence of food-spoilage bacteria seriously affects the development of the dairy industry. Bacteriocins are considered to be a potential antibacterial or antibiofilm agent that can be applied as a preservative. In this study, bacteriocin BM173 was successfully expressed in the Escherichia coli expression system and purified by a 2-step method. Furthermore, it exhibited a broad-spectrum antibacterial activity, high thermal stability (121°C, 20 min), and broad pH stability (pH 3–11). Moreover, the minimum inhibitory concentration values of BM173 against E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were 14.8 μg/mL and 29.6 μg/mL, respectively. Growth and time-kill curves showed that BM173 exhibited antibacterial and bactericidal activity. The results of scanning electron microscopy and transmission electron microscopy demonstrated that BM173 increased membrane permeability, facilitated pore formation, and even promoted cell lysis. The disruption of cell membrane integrity was further verified by propidium iodide uptake and lactic dehydrogenase release. In addition, BM173 exhibited high efficiency in inhibiting biofilm formation. Therefore, BM173 has promising potential as a preservative used in the dairy industry.     

Antibacterial activity of dextran-conjugated lysozyme against Escherichia coli and Staphylococcus aureus in cheese curd

The purposes of this research were to glycosylate lysozyme with dextran under Maillard reaction conditions and assess the antimicrobial characteristics of the lysozyme-dextran conjugate in a culture medium and cheese curd. Solutions containing lysozyme and dextran were incubated at 60 degrees C and at 79% relative humidity. Gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to follow the glycosylation process. Under optimum conditions 3.7 mol of dextran were coupled to 1 mol of lysozyme. Lytic activity of the conjugate against the cell wall of Micrococcus luteus was about 62% of that of native lysozyme. Evaluation of the lysozyme-dextran conjugate against test microorganisms (Staphylococcus aureus and Escherichia coli) in culture media indicated a progressive increase in antimicrobial activity, with an increase in enzyme-conjugate concentration. The lysozyme-dextran conjugate was also effective against E. coli in a natural food system, as it reduced the bacterial count by 3 log in cheese curd after 40 days of storage. Unlike E. coli, the antimicrobial action of lysozyme against S. aureus was not improved by conjugation with dextran in both in vitro and in vivo tests. Antimicrobial activity of the lysozyme-dextran conjugate against gram-negative bacteria is probably related to the remaining lytic activity as well as the excellent surfactant properties of the lysozyme-dextran conjugate. These results might increase the applicability of lysozyme as a natural antimicrobial ingredient in different food products.     

乳源抗菌肽BCp12对金黄色葡萄球菌多靶点抑菌机制

为研究新型酪蛋白源抗菌肽BCp12对金黄色葡萄球菌的抑菌机理,本实验通过酶标仪、流式细胞仪、透射电子显微镜分析BCp12对金黄色葡萄球菌的壁膜损伤机制;采用荧光光谱、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳研究BCp12对菌体DNA结合及蛋白质合成的影响;利用赖氨酸乙酰化、琥珀酰化、2-羟基异丁酰化、丙二酰化4 种泛抗体结合免疫印迹分析BCp12对菌体蛋白翻译后修饰的影响。结果表明：BCp12的最小抑菌质量浓度（minimum inhibitory concentration,MIC）为2 mg/mL,经质量浓度超过MIC的BCp12处理后的菌体细胞膜疏水性显著下降（P≤0.001）,通透性增加,菌体形变严重,部分细胞内容物外泄形成空腔;BCp12与溴化乙锭竞争性结合菌体DNA,使核酸合成受到抑制,胞内蛋白质量浓度显著下降（P≤0.001）,特别是分子质量为15～35 kDa的蛋白变化最为明显,说明BCp12可抑制菌体蛋白质的合成;金黄色葡萄球菌蛋白存在大量的赖氨酸乙酰化、琥珀酰化、丙二酰化修饰,经BCp12处理的菌体赖氨酸丙二酰化修饰水平明显下调,而对赖氨酸琥珀酰化和2-羟基异丁酰化修饰的影响不明显。本实验揭示了BCp12对金黄色葡萄球菌的多靶点抑菌机制,对新型乳源抗菌肽的应用和保障食品安全具有一定的意义。     

植物乳杆菌CGMCC.5297产细菌素条件的优化及应用

用响应面法对植物乳杆菌CGMCC.5297生产细菌素的培养基进行了优化。通过Plackett-Burman设计和中心组合试验设计,植物乳杆菌CGMCC.5297代谢产细菌素的最佳培养条件为酵母粉5.09g/L,牛肉膏10.85g/L,葡萄糖55.34g/L。此时的细菌素上清与指示菌单核细胞增多性李斯特菌CVCC1595共培养4h后,指示菌OD600nm为0.001 73,效价为4499 IU/mL,提高了1.4倍。在最优发酵条件下获得的试验结果与模型预测值吻合,说明所建立的模型是切实可行的。将优化后的植物乳杆菌上清加入到自制酸奶中,发现此细菌素对酸奶具有良好的保鲜效果。     

A novel bacteriocin against Staphylococcus aureus from Lactobacillus paracasei isolated from Yunnan traditional fermented yogurt: Purification,antibacterial characterization,and antibiofilm activity

Staphylococcus aureus and its biofilm have emerged as a significant threat to the safety of dairy products. In recent years, lactic acid bacteria (LAB) bacteriocins have been widely acknowledged as the potential natural antibacterial substance in food biopreservation due to their excellent antibacterial effects. However, few LAB bacteriocins with antibacterial and antibiofilm activity against S. aureus have been reported in dairy products. In the present study, a novel bacteriocin LSX01 of Lactobacillus paracasei LS-6 isolated from a traditional fermented yogurt produced in Yunnan, China, was purified and characterized extensively. The LSX01 possessed a molecular weight of 967.49 Da and an AA sequence of LDQAGISYT. The minimum inhibitory concentration of LSX01 against S. aureus_45 was 16.90 μg/mL, which was close to or lower than the previously reported bacteriocins. The LSX01 exhibited an extensive antimicrobial spectrum against both gram-positive and gram-negative bacteria. Moreover, LSX01 exhibited excellent tolerance to heat and acid-base treatments, and sensitivity to the proteolytic enzymes, such as pepsin and proteinase K. Furthermore, the treatment of S. aureus_45 planktonic cells with LSX01 significantly reduced their metabolic activity and disrupted the cell membrane integrity. Scan electron microscopy results demonstrated that LSX01 induced cytoplasmic content leakage and cell deformation. Additionally, biofilm formation of S. aureus_45 was also significantly inhibited by LSX01. Overall, the results suggested that the novel LAB bacteriocin LSX01 possessed antibacterial activity and antibiofilm activity against S. aureus and, hence, could have potential for improving safety of dairy products.     

双歧杆菌细菌素Bifidocin A对金黄色葡萄球菌的抑菌作用及其机制

Bifidocin A是由Bifidobacterium animalis BB04代谢合成的一种新型广谱高效细菌素。以革兰氏阳性的金黄色葡萄球菌为测试敏感菌,分析细菌素Bifidocin A的最低抑菌浓度及不同质量浓度下的抑菌效果,并从敏感菌细胞形态与结构、细胞膜的通透性、细胞膜的完整性以及细胞膜质子移动势的变化4个角度分别探讨该细菌素的抑菌作用机制。结果表明,细菌素Bifidocin A对金黄色葡萄球菌CVCC 26112的最低抑菌浓度为0.058μg/m L,抑菌活性较强且存在浓度依赖性;并初步推测其抑菌作用机制是通过耗散细胞膜质子移动势,增加细胞膜通透性,形成孔洞,进而破坏细胞膜完整性,并最终瓦解细胞。     

金属抗菌肽SIF4对大肠杆菌的抑菌机制

大肠杆菌为低感染剂量肠道致病病原体,可引起严重的食品公共卫生问题。该研究从细胞壁通透性、胞内K⁺和生物大分子泄露、细胞表面疏水性、细胞膜通透性和表面zeta电位等方面系统研究了金属抗菌肽SIF₄对大肠杆菌的抑菌机制。研究发现,SIF₄的最小抑菌质量浓度为0.4 mg/L;SIF₄处理后菌体稳定期与衰亡期提前;细胞壁通透性与SIF₄浓度及温育时间呈正相关,温育1 h后,组别间细胞壁通透性有显著差异（P<0.05）;胞内K⁺泄露随SIF₄浓度升高和温育时间延长呈递增趋势;胞内生物大分子泄露随SIF₄浓度升高与温育时间延长呈增加趋势,温育2 h后,试验组与对照组有显著差异（P<0.05）;表面疏水性随SIF₄浓度增加呈递增趋势,SIF₄对细胞膜损伤可能以“毯式模型”进行;细胞表面zeta电位与SIF₄浓度呈线性递减趋势。研究认为,SIF     

戊糖乳杆菌素pentocin LPEM818的初步纯化及特性研究

对戊糖乳杆菌(Lactobacillus pentosus)LPEM818所产戊糖乳杆菌素(pentocin)LPEM818进行了初步纯化,并对其生物学特性进行了研究。结果表明,采用80%硫酸铵沉淀和Sephadex G-25凝胶层析分离纯化后,细菌素的纯化倍数为11.09倍,回收率为6.4%;该细菌素在pH 2.0～8.0条件下稳定,121℃加热15 min保留84.52%的抑菌活性;对胰蛋白酶和蛋白酶K敏感;该细菌素的作用方式为杀菌;对供试的部分革兰氏阳性菌(G+)和革兰氏阴性菌(G-)具有较强抑制作用,因其抑菌谱较广,对多数致病菌和食品腐败菌有较好抑菌作用,所以具有作为食品生物防腐剂的潜在应用价值。     

姜厚朴水提物对大肠杆菌和金黄色葡萄球菌的抑菌机理研究

为了探讨姜厚朴水提物(GMB)对大肠杆菌和金黄色葡萄球菌的抑菌机理,试验对GMB作用下菌体形态结构、膜系统上离子通道的酶活力和能量代谢等方面进行了研究。结果表明,GMB对大肠杆菌和金黄色葡萄球菌的MIC分别为6.25、12.5 mg/m L。大肠杆菌胞外AKP酶和β-半乳糖苷酶吸光度值分别增加1.78和4.24倍,GMB作用4 h后电导率显著上升,膜上Na+K+-ATP酶活性从0.42增加到1.74 mg prot/m L,且为阴性对照的1.7倍;金黄色葡萄球菌体表出现囊泡状、不规则的突起结构,SDH酶活性、总ATP酶活性和胞内蛋白质含量分别降低40%、23.4%和17.9%,且AKP酶活和电导率均有所增加。由此推测出GMB主要是通过破坏大肠杆菌细胞壁、膜结构,增加其渗透性和通透性,造成胞内物质外流和蛋白质合成量下降等现象,进而抑制菌体生长。而GMB抑制金黄色葡萄球菌的作用机制是增加细胞壁的通透性、降低能量代谢相关酶的活性,干扰其正常的代谢活动。     

Effects of papain,Lactiplantibacillus plantarum 1-24-LJ and their combinations on bacterial community changes and flavour improvement in Suanzhayu,a Chinese traditional fish

Suanzhayu is a traditional Chinese fermented fish that often faces a long fermentation time (about 1 month) and unstable quality. This study evaluated the combined effects of papain and Lactiplantibacillus plantarum 1-24-LJ, a selected starter culture, on the quality of Suanzhayu. The addition of L. plantarum and/or papain increased the content of alcohols, esters, ketones and umami amino acids (UAAs), but decreased the content of bitter amino acids (BAAs). Besides, Lactobacillus might play a role in reducing harmful bacteria and BAAs content and increasing UAAs content, while Lactococcus and Weissella contributed to the characteristic flavour of Suanzhayu. Overall, no significant improvement in sensory quality was observed with the addition of papain alone, while better quality and faster fermentation processes were obtained with the addition of L. plantarum 1-24-LJ alone or in combination with papain. The results contribute to quality control in Suanzhayu production and the selection of starter cultures.     

基于细胞膜损伤的蔗糖月桂酸酯对金黄色葡萄球菌的作用机制

研究蔗糖月桂酸酯对金黄色葡萄球菌的抑菌活性及其对细胞膜的损伤机制。首先采用二倍稀释法考察了蔗糖月桂酸酯对金黄色葡萄球菌的最小抑菌浓度,用平板计数法绘制了时间-杀菌曲线;通过DiSC3(5)探针标记荧光分光光度法考察了蔗糖月桂酸酯对菌体细胞膜电势差的影响,使用荧光显微镜结合流式细胞术研究了菌体细胞膜渗透性的影响,使用紫外分光光度法考察了大分子物质的泄漏情况,使用PBFI钾离子探针测定了胞内钾离子泄漏量,最后通过透射电子显微镜观察了蔗糖月桂酸酯处理前后菌体的超微结构。结果表明：蔗糖月桂酸酯对金黄色葡萄球菌的最小抑菌浓度（minimum inhibitory concentration,MIC）为0.312 5 mg/mL;经蔗糖月桂酸酯作用的金黄色葡萄球菌与对照组相比,DiSC3(5)探针的荧光强度呈现剂量依赖性增大;PI探针对菌体的沾染率增加,MIC处理30 min菌体PI沾染率达到84.7%;260 nm波长处的吸光度随作用时间的延长逐渐增大,但增大量较小;蔗糖月桂酸酯会导致金黄色葡萄球菌细胞钾离子泄漏,且泄漏量与添加的蔗糖月桂酸酯质量浓度呈正相关;经MIC蔗糖月桂酸酯作用1 h后,金黄色葡萄球菌体表面变粗糙,边缘模糊。结论：蔗糖月桂酸酯可以通过破坏细胞膜渗透性消散细胞膜电势,从而导致胞内物质发生轻微泄漏,最终达到抑菌作用,本研究可为多功能性糖酯抑菌产品的开发提供理论依据。