乳链菌肽的特殊结构与功能

乳链菌肽的特殊结构与功能庄绪亮马桂荣（山东大学微生物技术国家重点实验室,济南２５０１００）关键词乳链菌肽细菌素羊毛硫氨酸乳链菌肽（ｎｉｓｉｎ）是目前研究最多的一种细菌素,它由某些乳酸链球菌（Ｓｔｒｅｐｔｏｃｏｃ－ｃｕｓｌａｃｔｉｓ）所产生,含有３４个...     

ptsH基因过表达对乳酸乳球菌N8自身免疫耐受性及其生理代谢的影响

目的:通过基因工程方法提高HPr蛋白编码基因ptsH在乳链菌肽(nisin)高产野生乳酸乳球菌株N8中的表达,揭示ptsH基因与乳酸乳球菌乳链菌肽耐受性等相关生物学功能的关系。方法:构建ptsH过表达质粒pLEV16-ptsH并转化至N8,使其ptsH基因过量表达,进而对比分析ptsH过表达菌株与野生菌株在生长曲线、乳链菌肽耐受性、效价、Biolog等方面的差异。结果:N8-ptsH过表达菌株与N8菌株在菌落形态、大小、表面湿滑程度及生长曲线等方面没有明显差异;ptsH基因过表达使N8菌株的乳链菌肽耐受性提高了8.3%,2个乳链菌肽耐受性相关基因nisI和nisF的表达量分别提高了15.45倍和近45倍;ptsH基因过表达略微减缓了N8菌株中乳链菌肽的产生,但乳链菌肽的最终产量略有提高;ptsH基因过表达菌株中PTS系统糖苷和磷酸化糖类的利用率比原始菌株显著提高。结论:ptsH基因主要与乳酸乳球菌的乳链菌肽耐受性有关。     

天然食品防腐剂乳链菌肽的性质及其应用

乳链菌肽是一种天然食品防腐剂。本文概述了乳链菌肽的理化性质、抑菌性能、作用机制及在食品防腐中的应用。     

乳链菌肽前体基因（nisZ）在乳酸乳球菌中的克隆和表达

用PCR技术从克隆有完整乳链菌肽生物合成基因簇（来自于乳链菌肽高产菌株L.lactis AL2)的重组噬菌体λHJ-3中扩增了编码乳链菌肽的前体基因,与pMG36e连接得到重组质粒pHJ201,用电击转化法将pHJ201转化到L.lactis NZ9800中,经活性测定和Tricine－SDS－PAGE电泳证实乳链菌肽前体基因获得了功能表达。DNA序列分析表明乳链菌肽高产菌株L.lactis AL2产生的是NisinZ。发现pHJ201d L.lactis NZ9800 中有良好的稳定性。     

乳链菌肽细胞分子传感器的构建与应用

【背景】乳链菌肽主要是由乳酸乳球菌生产的一类多肽,对革兰氏阳性菌有抑菌作用,是目前联合国粮食及农业组织/世界卫生组织唯一批准使用的天然食品防腐剂。但是其产量低、缺乏简便高效的检测方法,限制了其研究和应用。【目的】构建一种可输出肉眼可见红色荧光的细胞分子传感器,以期能简单方便地检测样品中的乳链菌肽,同时应用该传感器筛选乳链菌肽生产菌株。【方法】用Golden-Gate克隆方法构建含乳链菌肽诱导启动子和下游红色荧光蛋白基因(两种)的载体,转入Lactococcus lactis中。用细胞传感器筛选可能的乳链菌肽生产菌株。【结果】构建的两种乳链菌肽细胞分子传感器都能对2-200 ng/mL乳链菌肽有灵敏的响应,可用于定量测定。两种传感器的最大荧光强度和表型也有所不同。利用细胞传感器确定了Lactococcus lactis ATCC 11454乳链菌肽的产生,同时排除了一个能产其他抗菌化合物的菌株。【结论】构建的细胞分子传感器能特异性地响应乳链菌肽,并能简单快速地筛选乳链菌肽菌株。     

一个含有乳链菌肽抗性基因的乳酸乳球菌质粒pTS50的鉴定

在添加乳链菌肽、乳糖及溴甲酚紫的M1 7选择培养基上 ,从 1 97个新鲜牛奶样品中筛选到 3株乳链菌肽抗性菌株 ,PCR扩增证实它们都含有乳链菌肽抗性基因。菌种生理生化特性鉴定及特异性 1 6SrDNAPCR扩增产物的序列测定结果表明这 3株菌都属于乳酸乳球菌乳酸亚种。质粒转化实验发现乳酸乳球菌乳酸亚种TS 1 640中的乳链菌肽抗性基因位于一个约47kb的大质粒pTS50上。BamHI、EcoRI、HindⅢ、NcoI、PstⅠ酶切分析和Southern杂交 ,进一步将乳链菌肽抗性基因定位于pTS50的一个约 1 9kbEcoRI酶切片段中     

乳链菌肽产生菌的定向筛选及发酵产物的鉴定

利用乳链菌肽产生菌中nip^+nis^rsuc^+紧密连锁的原理,在添加乳链菌肽、蔗糖及溴甲酚紫的选择培养基上,从牛奶样品中定向筛选乳链菌肽产生菌。对筛选到的L. lactis1409菌株发酵产物的分析鉴定结果揭示:该产物对多种革兰氏阳性菌有强烈抑制作用,而对革兰氏阴性菌、酵母菌和霉菌无效,在pH值低的条件下对热稳定,对α—胰凝乳蛋白酶敏感,具有生物活性的蛋白质的分子量与乳链菌肽相当,而1409菌株的质粒分布与L. laclisATCC11454和L. lactis 7962不同,说明筛选到的L. laclis 1409菌株确是一株新的乳链菌肽产生菌。     

生物合成乳链菌肽及其螯合物的研究

从酸奶中筛选到一株乳链菌肽产生菌A1-06,研究了各种条件因素对其合成能力的影响。通过发酵培养基的优化,在以质量分数2.0%的蔗糖为唯一碳源、0.25%的酵母膏为唯一氮源条件下,A1-06合成乳链菌肽的产率为0.3 g.L-1,效价为1.018×106U.L-1,比在基础培养基中合成的活性提高17%。Mn2+对A1-06的合成能力有抑制作用,而吐温-80则有促进作用。对乳链菌肽及其Zn2+和Fe2+的螯合物的抑菌效果进行了比较,结果表明,乳链菌肽螯合Fe(Ⅱ)对G-菌有抑制作用,6 h的抑菌率为50.3%,而乳链菌肽、乳链菌肽螯合Zn(Ⅱ)对G-菌无明显的抑制作用。     

乳酸乳酸球菌AL2产生的乳链菌肽的提纯和性质

用NaCl饱和的乳酸乳酸球菌(Lactococcus lactis subsp. Lactis)AL2发酵液经正丙醇提取和CM-Sephadex C-25柱层析,得到聚丙烯酰胺凝胶电泳纯的乳链菌肽组分,比活力从24427IU/mg提高到39865IU/mg,活力回收为41.7％。Α—胰凝乳蛋白酶可使乳链菌肽丧失活性;在低pH条件下,乳链菌肽对热较稳定;对许多革兰氏阳性菌有强烈抑制作用,而对革兰氏阴性菌、酵母菌和霉菌没有作用。     

乳酸乳球菌AL2基因文库的构建及乳链菌肽生物合成基因簇的筛选

用本实验室获得的乳链菌肽高产菌株乳酸乳球菌AL2总DNA为供体,以λ EMBL3为载体,构建了该菌株的基因文库,共获得3900多个噬斑。通过Southern杂交、PCR扩增及DNA序列测定,证实从该文库中筛选到一个含有完整乳链菌肽生物合成基因簇的重组噬菌体λHJ\|3。     

Mechanisms of nisin resistance in Gram-positive bacteria

Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain Gram-positive bacteria. However, the effectiveness of nisin is often affected by environmental factors such as pH, temperature, food composition, structure, as well as food microbiota. The development of nisin resistance has been seen among various Gram-positive bacteria. The mechanisms under the acquisition of nisin resistance are complicated and may differ among strains. This paper presents a brief review of possible mechanisms of the development of resistance to nisin among Gram-positive bacteria.     

Applications of the bacteriocin,nisin

Nisin was first introduced commercially as a food preservative in the UK approximately 30 years ago. First established use was as a preservative in processed cheese products and since then numerous other applications in foods and beverages have been identified. It is currently recognised as a safe food preservative in approximately 50 countries. The established uses of nisin as a preservative in processed cheese, various pasteurised dairy products, and canned vegetables will be briefly reviewed. More recent applications of nisin include its use as a preservative in high moisture, hot baked flour products (crumpets) and pasteurised liquid egg. Renewed interest is evident in the use of nisin in natural cheese production. Considerable research has been carried out on the antilisterial properties of nisin in foods and a number of applications have been proposed. Uses of nisin to control spoilage lactic acid bacteria have been identified in beer, wine, alcohol production and low pH foods such as salad dressings. Further developments of nisin are likely to include synergistic action of nisin with chelators and other bacteriocins, and its use as an adjunct in novel food processing technology such as higher pressure sterilisation and electroporation. Production of highly purified nisin preparations and enhancement by chelators has led to interest in the use of nisin for human ulcer therapy, and mastitis control in cattle.     

Chemical Modification of the Carboxyl Terminal of Nisin A with Biotin does not Abolish Antimicrobial Activity Against the Indicator Organism, Kocuria rhizophila

Nisin is an antimicrobial peptide that is widely used for food preservation. Although it has potent activity against a number of food pathogens, suggesting potential therapeutic applications, its potential for clinical use is limited by proteolytic susceptibility and poor oral bioavailability. Derivatization of nisin could overcome these issues; however, many nisin analogues, prepared by modification at the N-terminal and C-terminal have previously been shown to be inactive. A method for the C-terminal modification was developed using biotinylation as a model derivative. Purification of the modified nisin was carried out using reverse phase chromatography. Confirmation of nisin modification was confirmed by Mass Spectroscopy. The C-terminal modification of nisin resulted in only a twofold reduction in antimicrobial activity of the conjugate against the indicator organism, Kocuria rhizophila. The C-terminal modification could be used to increase the therapeutic potential of nisin by creating more favourable physicochemical characteristics. This is the first study that showed that nisin modification can be carried out successfully without destroying its antimicrobial activity.     

Efficacies of Nisin A and Nisin V Semipurified Preparations Alone and in Combination with Plant Essential Oils for Controlling Listeria monocytogenes

The food-borne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates, reaching up to 25 to 30%. Listeria is a hardy organism, and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semipurified preparations (SPP) containing either nisin A or an enhanced bioengineered derivative, nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol, and trans-cinnamaldehyde, to control Listeria monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing SPP (25 μg/ml) with thymol (0.02%), carvacrol (0.02%), or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil-nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V-carvacrol or nisin V-cinnamaldehyde combinations was twice that of the equivalent nisin A-essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural, and effective means to enhance food safety by inhibiting food-borne pathogens such as L. monocytogenes.     

Effect of Nisin’s Controlled Release on Microbial Growth as Modeled for Micrococcus luteus

The need for safe food products has motivated food scientists and industry to find novel technologies for antimicrobial delivery for improving food safety and quality. Controlled release packaging is a novel technology that uses the package to deliver antimicrobials in a controlled manner and sustain antimicrobial stress on the targeted microorganism over the required shelf life. This work studied the effect of controlled release of nisin to inhibit growth of Micrococcus luteus (a model microorganism) using a computerized syringe pump system to mimic the release of nisin from packaging films which was characterized by an initially fast rate and a slower rate as time progressed. The results show that controlled release of nisin was strikingly more effective than instantly added (“formulated”) nisin. While instant addition experiments achieved microbial inhibition only at the beginning, controlled release experiments achieved complete microbial inhibition for a longer time, even when as little as 15% of the amount of nisin was used as compared to instant addition.     

The impact of nisin on sensitive and resistant mutants of Listeria monocytogenes in cottage cheese

Aims: Listeria monocytogenesΔgadD1 and ΔlisK mutants display enhanced and reduced sensitivity, respectively, to the food preservative nisin in laboratory media. However, the behaviour of these strains in a nisin‐containing food has not been assessed. Here we use cottage cheese as a model food to address this issue. Materials and Results: Antibiotic‐resistant forms of the wild‐type and mutant strains were employed to investigate the behaviour of multiple strains in a single food sample, thereby eliminating the problem of intersample variation. Using this approach, it was established that percentage survival of the ΔlisK mutant was greater than the parent strain in the absence of nisin and that this relative difference became even more dramatic in cottage cheese supplemented with nisin. The numbers of the ΔgadD1 mutant decreased more rapidly than the parent in cottage cheese without nisin, but surprisingly this trend was reversed in nisin‐supplemented cheese. Upon the addition of 10 mmol l^?1 monosodium glutamate, a substrate for the glutamate decarboxylase (GAD) system, the wild‐type LO28 strain regained its relative advantage over ΔgadD1. Conclusions: Care needs to be taken when predicting the behaviour of mutants of L. monocytogenes with altered resistance to nisin in food as experiments in laboratory media are not always a good indicator of how the strains will behave in such food environments. Significance and impact of the Study: This study further emphasizes the importance of utilizing food matrices to confirm observations made using laboratory media.     

Complete nisin A gene cluster from Lactococcus lactis M78 (HM219853) — obtaining the nucleic acid sequence and comparing it to other published nisin sequences

Nisin is an antimicrobial peptide produced by Lactococcus lactis. It has a long history of safe use, mainly in food production. This bacteriocin has been studied from many aspects of genetics, biosynthesis, immunity, regulation and mode of action. The strain Lac. lactis M78 has already been described in previous studies as a good nisin A producer with equally good potential to be used in food production. The main objective of the present study was to determine the complete nucleic acid sequence of the nisin A gene cluster from this strain. This is the first time that all 11 genes that form the nisin A gene cluster were sequenced. The obtained sequence (GenBank: HM219853) was compared to other known nucleic acid sequences of bacteriocin nisin. The results of the comparison showed certain differences in sequences that might influence the structure and function of proteins involved in nisin biosynthesis, immunity and regulation.     

Characterization of Lactic Acid Bacteria Coexisting with a Nisin Z Producer in <Emphasis Type="Italic">Tsuda</Emphasis>-Turnip Pickles

Bacteriocin-producing lactic acid bacteria (LAB) are believed to be associated with many types of fermented food. The present study reports the identification of lactic acid bacterium MS27 producing a bacteriocin isolated from the Tsuda-turnip pickle, which is a Japanese fermented food, and characterization of LAB coexisting with the bacteriocin producers in the Tsuda-turnip pickle. The strain MS27 was identified as Lactococcus lactis subsp. lactis based on a partial 16S rRNA gene sequence and sugar fermentation pattern analyses. Mass spectroscopy and genetic analysis revealed that it produces nisin Z. Microbial population analysis revealed that the LAB community in the Tsuda-turnip pickle comprises nisin Z-sensitive and nisin Z-insensitive LAB (nonbacteriocin producers) and nisin Z producers at population rates of 52.5%, 37.5%, and 10.0%, respectively. This revealed that Leuconostoc spp. (nisin Z insensitive) is the dominant species among LAB microflora and that nisin Z insensitivity of a bacterial strain is proportional to its ability to dominate the population in Tsuda-turnip pickles. Competitive growth assay revealed that Leuconostoc spp. considerably suppressed the bacteriocin production of L. lactis MS27. These results suggested that Leuconostoc spp. contributes to the formation of the LAB community with a wide variety of microorganisms in Tsuda-turnip pickles.     

Suppression of Listeria monocytogenes colonization following adsorption of nisin onto silica surfaces.

Nisin is an antimicrobial peptide proven to be an effective inhibitor of gram-positive bacteria. It is known that nisin can adsorb to various surfaces and still retain much of its original activity (M. A. Daeschel, J. McGuire, and H. Al-Makhlafi, J. Food Prot. 55:731-735, 1992). In this study, nisin films were allowed to form on silanized silica surfaces and then exposed to medium containing Listeria monocytogenes. Representative areas were selected from each surface, and images of resident listeriae were obtained at 4-h intervals for 12 h. During this time, cells on surfaces that had been in contact with a high concentration of nisin (1.0 mg/ml) exhibited no signs of growth and many displayed evidence of cellular deterioration. Surfaces treated with a lower concentration of nisin (0.1 mg/ml) had a smaller degree of inhibition. In contrast, both protein-free surfaces and those with films of heat-inactivated nisin allowed attached L. monocytogenes cells to grow and reproduce. These studies, when repeated with a nisin-resistant strain of L. monocytogenes, resulted in no inhibition of growth on surfaces with adsorbed nisin. The bactericidal effect of adsorbed nisin was also studied with iodonitrotetrazolium violet, a tetrazolium salt, which is reduced to a red formazan crystal by viable bacteria. Crystals were visible in 95% of the cells adhered to control surfaces but were present in less than 20% of the cells on surfaces with adsorbed nisin. These data indicate that adsorbed nisin may have potential for use as a food grade antimicrobial agent on food contact surfaces.     

食品级高效诱导表达系统-NICE系统

乳酸菌NICE系统是在乳链菌肽诱导下由nisA启动子控制目的基因表达的,含nisR和nisK的两组分调节系统的高效诱导表达系统。由于NICE系统的诱导剂、宿主菌和载体都是食品级的,其应用前景十分广阔。