LC-MS/MS测定果汁饮料中乳酸链球菌素

该试验旨在建立LC-MS/MS测定果汁饮料中乳酸链球菌素的分析方法。通过含0.1%甲酸的20%乙腈水溶液超声提取样品中的乳酸链球菌素,C₁₈色谱柱分离,0.1%甲酸水溶液和乙腈进行梯度洗脱,经质谱法检测,外标法定量。结果表明,最佳提取条件为提取溶剂乙腈、超声提取时间15 min。乳酸链球菌素的线性范围在0~100μg/L之间,线性相关系数为0.9996。方法的检出限为20μg/kg,定量限为50μg/kg;在50,100和500μg/kg三个水平下进行加标试验,其加标回收率在79.5%~111.5%之间,相对标准偏差(RSD)<10%,符合GB 27404—2008相关规定。该方法简单、便捷,适用于饮料中乳酸链球菌素含量的测定。     

白酒黄水中提取乳酸链球菌素的研究

对大孔吸附树脂粗提取白酒黄水中乳酸链球菌素的条件进行了研究,结果表明,各个因素对粗提取效果影响大小的顺序为树脂添加量提取温度提取时间转速。乳酸链球菌素最适合粗提取条件为:树脂添加量1.5%,提取温度50℃,提取时间24 h,转速150 r/min,粗提取物效价为572.02 IU/m L。     

有机溶剂沉淀法提取乳酸链球菌素的效果

利用水微溶性有机溶剂二氯甲烷、正丁醇,采用沉淀法从发酵浓缩液中提取乳酸链球菌素。结果表明：在有机溶剂和发酵浓缩液体积比1:1条件下,丁醇提取乳酸链球菌素纯度可达到63.32%,单步沉淀收率24.3%,二氯甲烷提取产品纯度39.96%,沉淀收率36.04%;二氯甲烷和丁醇体积比1:1组成的复合溶剂,提取效果优于单一有机溶剂,产品纯度和单步收率分别为45.94%和59.3%。该方法可克服现有乳酸链球菌素提取工艺产品纯度低、盐含量高的缺陷,具有过程简便、产品纯度较高的优点,具有较好的工业化应用前景。     

高原酸奶中乳酸链球菌产生的乳酸链球菌素纯化鉴定研究

目的:对从高原酸奶中提取的乳酸链球菌产生的乳酸链球菌素进行纯化及鉴定。方法:采用分离、凝胶层析、抑菌实验、高效液相色谱法来纯化乳酸链球菌素,用凝胶电泳来鉴定其纯度,确定其分子量。结果:生产的乳酸链球菌素具有抑菌性,将该抑菌成分与乳酸链球菌素标准品进行SDS-PAGE凝胶电泳,通过比较电泳图谱一致。结论:本实验得到的抑菌成分即为乳酸链球菌素,且纯度高,分子量大约为3300u。     

天然食品防腐剂尼塞的研究

<正>Nisin即乳酸链球菌素(N inhibitory subs-tance),也叫乳酸链球菌肽或尼生素,是从链球菌属(streptococcus)的乳酸链球菌(S.lactis)发酵产物中提取制备的一类多肽化合物。这种     

乳酸链球菌素(Nisin)抑菌作用及其抑菌机理的研究

通过抑菌试验确定了乳酸链球菌素（Nisin）的高产菌株．乳酸乳球菌乳酸亚种T0625菌株的抑菌谱。结果显示,含有乳酸链球菌素的发酵液对革兰氏阳性细菌有抑制作用,但对革兰氏阴性细菌、酵母菌和霉菌无抑制作用。从T0625菌株发酵液中提取乳酸链球菌素加入金黄色葡萄球菌培养液中进行抑菌试验,结果培养液蛋白质含量提高、电导率增加,未出现对数生长期。透射电镜观察发现,加入乳酸链球菌素后,菌体变形,出现质壁分离现象,进而菌体细胞破裂,细胞内容物大量外泄,菌体成为不规则残片。SDS-PAGE凝胶电泳显示,乳酸链球菌素作用使金黄色葡萄球菌菌体内蛋白质减少,培养液中蛋白质增加。推测乳酸链球菌素的作用位点为细胞膜,具有破坏菌体细胞膜完整性的作用。试验结果支持了“孔道形成”理论的观点。     

食品添加剂乳酸链球菌素的摄入量评估研究

目的进行乳酸链球菌素的摄入量评估研究,了解我国居民的乳酸链球菌素摄入情况,为采取恰当的管理措施提供科学依据。方法以2002年全国居民营养调查获得的人群食物摄入量数据和《食品添加剂使用卫生标准》(GB2760—2007)中规定的乳酸链球菌素允许使用的食品类别、最大使用量作为数据来源,利用点评估方法计算我国居民乳酸链球菌素的摄入量。按照不同的年龄、性别、地区组合将人群分为27组,分别计算食物摄入量平均值和第97.5百分位数(P97.5)情况下各组人群乳酸链球菌素的摄入量和各类食物对摄入量的贡献率。结果在食物摄入量为均值的情况下,各组摄入量占每日允许摄入量(ADI)的比例为1.49%～17.16%。在食物摄入量为第97.5百分位数的情况下,各组乳酸链球菌素摄入量为该组ADI的9.35%～77.95%。在食物摄入量为均值和食物摄入量为第97.5百分位数的情况下,乳制品和预制肉制品是乳酸链球菌素摄入量的主要来源。结论无论在食物摄入量为均值情况下还是在食物摄入量为第97.5百分位数的情况下,各组乳酸链球菌素膳食摄入量均未超过其ADI,表明按照目前标准规定使用该添加剂是安全的。     

乳酸链球菌发酵培养基的优化

对乳酸链球菌发酵产生乳酸链球菌素(Nisin)的培养基进行了优化研究,根据代谢发酵控制原理,采用二次回归正交旋转组合设计,考查了乳酸链球菌摇瓶发酵培养基中4种主要成分对乳酸链球菌素(Nisin)产量的影响,通过DPS软件获得最佳发酵培养基配比为蔗糖0.4%,蛋白胨1.2%,酵母浸出膏0.4%,KH2PO4 0.5%。     

蔬菜罐头中的一种有效抗菌剂——乳酸链球菌素

一、绪言乳酸链球菌素是乳酸链球菌属微生物的代谢产物,可用乳酪链球菌(Streptococcu Cremoris)发酵提取而得。在乳酸、酸乳和发酵蔬菜中,有少量天然品存在。五十年代中期,人们发现乳酸链球菌素能有效地防止干酪的芽孢杆菌腐败,自从那时起,人们对它的结构和生化特性逐渐了解,后来,英国的阿普林·巴雷特公司(Aplin & Barrett Ltd)研制生产出浓缩的乳酸链球菌素——商业上称     

乳酸链球菌素对金黄色葡萄球菌抑制条件的研究

研究温度、pH、使用浓度及保存条件等对乳酸链球菌素抑 制金黄色葡萄球菌的影响,结果表明,温度、pH会显著影 响乳酸链球菌素的活性,酸性条件下,乳酸链球菌素对温 度较稳定,随着pH的增加,温度越高,乳酸链球菌素活 性下降越显著。乳酸链球菌素对沙门氏菌无抑制作用,在 温度为10℃、pH5.0的条件下100mg/kg浓度的乳酸链球 菌素在96h内可以抑制培养基中浓度为5.0×106个/mL 的金黄色葡萄球菌的生长。     

DMDC联合Nisin对模拟果汁中肠膜状明串珠菌细胞膜功能的影响

本文研究了二甲基二碳酸盐(DMDC)和Nisin处理对模拟果汁中肠膜状明串珠菌的杀菌效果及其细胞膜功能的影响。结果表明:DMDC联合Nisin作用于该菌时的部分杀菌浓度指数为0.38,小于0.50,两者之间具有很好的协同杀菌作用。扫描电镜分析发现该菌经DMDC和Nisin处理后其细胞形态没有发生明显变化。该菌经DMDC处理后仅有个别菌体细胞的膜通透性出现增加,而该菌经Nisin处理后,约7%菌体细胞的膜通透性增加。Nisin处理虽能改变该菌的细胞膜通透性,增加胞外极性物质的摄入,但并不能明显促进胞内物质的流失,高浓度的DMDC处理能导致该菌溶液的紫外吸收值增加约60%。DMDC和Nisin两者对该菌细胞内物质的流失、细胞膜的通透性的增加和胞内p H的下降没有相互促进作用,但DMDC和Nisin的联合作用能促进该菌细胞内脱氢酶的进一步失活。     

乳酸链球菌素与L-乳酸对嗜水气单胞菌的协同抑杀作用

为探讨乳酸链球菌素(Nisin)与L-乳酸对嗜水气单胞菌的协同抑杀作用,分别采用比浊法、活菌计数法、膜电动势探针等检测Nisin与L-乳酸对嗜水气单胞菌(ATCC 35654、CICC 10500两株菌)生长、存活、生物被膜形成以及菌体膜完整性的影响。结果表明,Nisin与低浓度(3.5 mmol/L)L-乳酸协同,显著抑制了两株嗜水气单胞菌的生长,使生长延滞期延长,最大比生长速率下降,成熟期最高活菌数量降低,且抑制效果随Nisin浓度增加而增强。Nisin和L-乳酸对指示菌有协同致死作用,Nisin与L-乳酸协同处理12 h,可显著(P<0.05)降低嗜水气单胞菌活菌数,造成菌体细胞死亡。机理初探表明:低浓度L-乳酸可以使菌株外膜脂多糖(Lipopolysaccharide,LPS)释放,Nisin进一步使菌体膜电动势消散。研究结果为利用Nisin与L-乳酸或二者的产生菌控制水产品中的嗜水气单胞菌提供了理论依据。     

陶瓷膜在卤制品加工废弃液微滤中的应用

探讨了陶瓷膜用于卤制加工废弃液微滤的工艺条件。研究不同预处理方法、膜孔径、操作温度、操作压力等因素对膜通量的影响,并通过正交实验确定微滤的最佳工艺参数为:预处理网筛300目、陶瓷膜孔径0.22μm、操作温度50℃、操作压力0.075 MPa,通过质量分数0.75%NaOH和0.5%柠檬酸复合清洗后,陶瓷膜的通量恢复率可达到94.1%。     

小孔径陶瓷膜澄清甜叶菊提取液的工艺研究

该文研究了小孔径陶瓷膜澄清甜叶菊提取液的效果。通过比较4、5、8、10 nm孔径的陶瓷膜过滤甜叶菊的甜菊糖提取液时的过滤通量、脱色率和收率的区别,确定较优的陶瓷膜孔径;再优化陶瓷膜操作参数。结果表明,5 nm陶瓷膜较优,在40 ℃时,操作压力5 bar,膜面流速4 m/s,浓缩10倍,加30%原液体积水洗滤效果最佳,陶瓷膜平均过滤通量可达102.6 kg/（m2·h）,甜菊糖收率可达99.2%,陶瓷膜过滤结束后先利用质量分数1%~2%的NaOH清洗1 h,再用0.5%~1%硝酸清洗1 h,陶瓷膜水通量恢复率可以达到99%以上,再生效果比较好,可以重复使用。相比絮凝工艺,陶瓷膜脱色率提高了2.6%,甜菊糖收率提高了6.8%,因此膜法工艺可取代传统絮凝工艺实现对甜叶菊提取液的澄清。     

乳酸链球菌素Q13的纯化及其对保加利亚乳杆菌的抑菌机理

为探讨乳酸链球菌素Q13对保加利亚乳杆菌LMG-1（Lactobacillus bulgaricus LMG-1）的抑菌机理，本研究利用硫酸铵沉淀、超滤和葡聚糖凝胶柱层析等对乳酸乳球菌Q13（Lactococcus lactis Q13）所产细菌素进行分离纯化，并运用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析其纯化效果和分子质量，同时通过研究乳酸链球菌素Q13对保加利亚乳杆菌细胞膜通透性、胞内酶活性及菌体形态结构等的影响，阐明乳酸链球菌素对保加利亚乳杆菌的抑菌作用机制。结果表明，乳酸乳球菌Q13所产细菌素硫酸铵沉淀的最适饱和度为60%，硫酸铵沉淀得到粗提物超滤后仅分子质量大于10 kDa组分具有抑菌效果，将具有抑菌活性的超滤组分利用葡聚糖凝胶柱层析进行纯化后，共收集到2 个具有抑菌活性的洗脱峰，电泳图谱仅呈现出一条分子质量约为17.5 kDa的蛋白条带，分析其可能为乳酸链球菌素的多聚体形式，并将其命名为乳酸链球菌素Q13；抑菌机理实验结果显示，乳酸链球菌素Q13对保加利亚乳杆菌LMG-1的抑菌机制主要是通过在靶细胞细胞膜上形成孔洞、增加细胞膜通透性和破坏细胞膜完整性，导致内容物外泄，胞内Na＋/K＋-ATP酶和碱性磷酸酶等关键酶含量降低，影响细胞正常能量代谢活动，最终诱导菌体细胞死亡。     

乳酸链球菌素(Nisin)与超高压结合对E.coli的协同杀菌效应

本文探究了乳酸链球菌素(Nisin)与超高压(High pressure processing,HPP)结合处理对E.coli O157:H7产生的协同杀菌效果,并分析其协同作用机制。以革兰氏阴性菌E.coli为目标菌,用0.005%或0.01% Nisin结合350 MPa/2 min超高压处理,通过扫描电镜和透射电镜分别观察E.coli细胞外部形态与内部结构、激光共聚焦显微镜与流式细胞仪检测细胞膜通透性。结果表明,与单独超高压处理相比,0.005%或0.01% Nisin结合350 MPa/2 min超高压对E.coli具有明显协同杀菌效果,E.coli菌落数下降6~8个数量级,细胞外形变化不明显,但细胞膜和内部结构受到了更严重的破坏,细胞膜完整的细菌仅占总数的2.13%~5.68%,细胞发生更多内溶物外泄和出现更多的空腔。因此,Nisin和HPP存在协同杀菌机制,推测HPP首先破坏E.coli细胞外膜,Nisin作用于外膜受损的细胞进而破坏细胞膜,细胞内溶物外泄形成空腔,从而导致细胞死亡。     

Transmission electron microscopy study of Listeria monocytogenes treated with nisin in combination with either grape seed or green tea extract

The objective of this study was to use transmission electron microscopy to investigate the morphological changes that occurred in Listeria monocytogenes cells treated with grape seed extract (GSE), green tea extract (GTE), nisin, and combinations of nisin with either GSE or GTE. The test solutions were prepared with (i) 1% GSE, 1% GTE, 6,400 IU of nisin, and the combination of these dilutions with nisin or with (ii) the pure major phenolic constituents of GSE (0.02% epicatechin plus 0.02% catechin) or GTE (0.02% epicatechin plus 0.02% caffeic acid) and their combinations with 6,400 IU of nisin in tryptic soy broth with 0.6% yeast extract (TSBYE). Test solutions were inoculated with L. monocytogenes at approximately 10(6) CFU/ml and incubated for 3 or 24 h at 37 degrees C. After 3 h of incubation, cells were harvested and evaluated under a transmission electron microscope (JEOL-100 CX) operating at 80 kV (50,000X). Microscopic examination revealed an altered cell membrane and condensed cytoplasm when L. monocytogenes cells were exposed to a combination of nisin with either GSE or GTE or to pure compounds of the major phenolic constituents in combination. After 24 h of incubation at 37 degrees C, the combinations of nisin with GSE and nisin with GTE reduced the L. monocytogenes population to undetectable levels and 3.7 log CFU/ml, respectively. These observations indicate that the combination of nisin with either GSE or GTE had a synergistic effect, and the combinations of nisin with the major phenolic constituents were most likely associated with the L. monocytogenes cell damage during inactivation in TSBYE at 37 degrees C.     

Synergetic effects of high-pressure carbon dioxide and nisin on the inactivation of Escherichia coli and Staphylococcus aureus

Synergetic effects of high-pressure carbon dioxide (HPCD) and nisin on Escherichia coli and Staphylococcus aureus were evaluated. Changes in morphology, interior structure, and membrane permeability were analyzed by scanning and transmission electron microscopy, and flow cytometry. Synergetic effects were found, especially in S. aureus. HPCD alone or with nisin led to morphological and intracellular alterations in both bacteria, but nisin alone led to these damages only in S. aureus. A positive correlation between membrane damage and inactivation was found, but ratios of inactivation were higher, probably because of viable but non-culturable state. Mechanisms were proposed for synergism: for E. coli, outer membrane was damaged first by HPCD, and then HPCD and nisin jointly acted on and destroyed the cytoplasmic membrane, leading to further intracellular damage by HPCD; for S. aureus, HPCD and nisin acted on the cytoplasmic membrane together leading to cell death.Industrial RelevanceEscherichia coli and Staphylococcus aureus are two common microorganisms, which exist widely in the environment and easily contaminate food such as vegetables and dairy products, respectively. Considering heat treatment may destroy some heat-sensitive quality of the products, this study evaluated synergetic effects of high-pressure carbon dioxide (HPCD) combined with the bacteriocin nisin. The investigations provided evidence for potentially combined application of HPCD and nisin to help keep food safe in the industry.     

陶瓷膜过滤万古霉素的研究

以预处理后万古霉素发酵液为料液,连续洗滤（CFD）过滤的效果更佳,表现在较高通量和收率。考察了陶瓷膜过滤万古霉素发酵液的分离效果,结果表明,通量与黏度成负相关性,50 nm陶瓷膜较优,且设定操作压力290 kPa,膜面流速5 m/s,温度20～30 ℃,浓缩1.66倍,连续洗滤2.5 BV,CFD过滤的平均通量可达78.9 kg/（h·m2）,收率可达99.1%,与数学模型的理论值相近。采用质量分数为2%～3% NaOH与0.5%～1.0% NaClO混合清洗的方法,清洗后陶瓷膜的水通量重复恢复率可达98%以上,再生性较好。     

THE ACTION OF NISIN ON BEER SPOILAGE LACTIC ACID BACTERIA

Within one minute of adding nisin to suspensions of Lactobacilli clumping of cells was observed. This happened with all the strains assayed, irrespective of their sensitivity or resistance to nisin. There was, however, no evidence of cell lysis. Two different assays for cell viability were used to show that the vast majority of cells of sensitive strains were killed within one minute of contact with nisin. The time needed to kill all the cells depended on the concentrations of both nisin and cells. One strain was more resistant and only 50–60% of the cells died within one minute of nisin addition . Using an ATP-bioluminescence assay it was shown that the addition of nisin to both Lactobacilli and Pediococci caused a rapid fall in intracellular ATP levels, which was reflected in a simultaneous appearance of ATP in the extracellular medium. Actively growing cells lost ATP more rapidly than did those in stationary phase. The amount of intracellular ATP lost was affected both by the concentration of nisin used and the sensitivity of the bacteria. It appears that an initial effect of nisin is to make the cell membrane ‘leaky’ as happens with many antibiotics, yeast killer factors (zymocins), colicins, and other gram-positive bacteriocins .