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。     

环境条件对防腐剂抑菌效果影响的研究

研究了环境条件对抑菌效果的影响，着重研究了环境pH、加热处理温度及时间对乳酸链球菌素、山梨酸钾抑菌效果的影响。结果表明：乳酸链球菌素、山梨酸钾抑菌效果，随加热温度及时间的升高和延长而降低，其降低的多少与应用环境的pH、加热温度及时间有直接关系。     

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。     

采用原子力显微镜分析乳酸链球菌素结构的研究

目的:采用原子力显微镜对乳酸链球菌素的分子形貌进行观测,为研究其抑菌机理提供有力的科学依据和可靠、直观的实验方法。方法:从川西高原牧民自然发酵酸奶中分离筛选乳酸链球菌素产生菌,利用其发酵获得产乳酸链球菌素效价高的产生菌C201,用原子力显微镜观察其分子形貌。结果:乳酸链球菌素分子在溶液中呈树状的不规则"峰"状聚集体,带有很多侧链分支,并且肽键之间形成稳定的螺旋结构。结论:原子力显微镜能直观地表征乳酸链球菌素的分子结构,是对研究乳酸链球菌素的抑菌机理在形态学上的完善。     

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。     

乳酸链球菌素对梨汁抑菌作用的研究

利用正交实验来探讨乳酸链球菌素浓度、pH值、杀菌强度和EDTA浓度等因素对乳酸链球菌素在梨汁中抑菌效果的影响。     

肉中多种组分对乳酸链球菌素与香芹酚抑菌活性的影响

目的:在模拟体系中,抑菌剂或其活性成分与菌体直接接触时,可以发挥良好的抑制作用,然而,当其被添加到复杂的食物体系中使用时,相较模拟环境下的抑制效果变差。本研究旨在探索复杂的肉品体系中多组分对乳酸链球菌素和香芹酚的抑菌活性的影响。方法:采用琼脂扩散法测定抑菌圈直径,采用肉汤稀释法测定菌落总数,分别探究肉中多种组分对乳酸链球菌素与香芹酚抑菌活性的影响。结果:水溶性的肌红蛋白和水不溶性的肌原纤维蛋白都对乳酸链球菌素和香芹酚的抑菌活性产生了不利影响;亚油酸对乳酸链球菌素和香芹酚的抑菌活性也产生一定的抑制作用;而多种游离氨基酸和糖原未见明显的影响。结论:较高浓度的蛋白组分和脂肪组分对乳酸链球菌素与香芹酚的抑菌活性都有负面影响,而多种游离氨基酸和糖原基本没有影响。     

冷鲜鸡肉抑菌剂筛选及保鲜效果研究

目的筛选出最优的天然抑菌剂并探究抑菌剂对冷鲜鸡肉的保鲜效果。方法以鸡肉主要腐败菌为研究对象,比较茶多酚、山竹多酚和乳酸链球菌素对鸡肉腐败菌的抑菌活性,筛选出适合的冷鲜鸡抑菌剂。利用筛选的抑菌剂溶液浸泡鸡肉,从肉的pH、出汁率、挥发性盐基氮(TVB-N)和细菌总数等评价其对鸡肉冷藏品质的影响。结果山竹多酚/乳酸链球菌素复配液(山乳复配液)和茶多酚/乳酸链球菌素复配液(茶乳复配液)比单一抑菌活性好,均呈现浓度效应关系。在整个贮藏期间,鸡胸肉的pH值、出汁率、TVB-N值、菌落总数与对照组相比有显著差异,2种复配抑菌液均有一定的保鲜效果,且山乳复配液抑菌的保鲜效果较好。结论0.4%山竹多酚和1.2‰乳酸链球菌素的复配保鲜液对冷鲜肉的保鲜抑菌效果最好。     

响应面法优化山药鱼肉饼的生物抑菌保鲜剂配方

研究了生物保鲜剂对山药鱼肉饼的抑菌效果。在单因素试验的基础上,采用响应面法分析了壳聚糖、乳酸钠、乳酸链球菌素对山药鱼肉饼抑菌保鲜作用的影响,建立了二次回归方程,得到复合生物抑菌保鲜剂的最优配方为:壳聚糖0.35%、乳酸钠4.0%、乳酸链球菌素250 mg/kg,添加此配方保鲜剂的山药鱼饼冷藏10 d测得细菌生长对数值为2.56。     

1株乳酸链球菌素产生菌的筛选鉴定及其抑菌特性的研究

从白菜叶上分离得到21株对乳酸链球菌素(Nisin)有抗性的乳酸菌株,采用杯碟法从中筛选得到抑菌活性较强的菌株T0625,经鉴定该菌株为乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.Lactis)。经测定,发酵液经加热处理,在低pH条件下能够保持较好的抑菌活性,对革兰氏阳性细菌具有明显的抑制作用,但对革兰氏阴性细菌、酵母菌和霉菌不表现抑制作用。研究还证实,T0625的发酵产物的抑菌活性不受胃蛋白酶和胰蛋白酶的影响。经提取纯化,确定抑菌物质为乳酸链球菌素。     

Inactivation of Listeria innocua in skim milk by pulsed electric fields and nisin

Pulsed electric fields (PEF) is an emerging nonthermal processing technology used to inactivate microorganisms in liquid foods such as milk. PEF results in loss of cell membrane functionality that leads to inactivation of the microorganism. There are many processes that aid in the stability and safety of foods. The combination of different preservation factors, such as nisin and PEF, to control microorganisms should be explored. The objective of this research was to study the inactivation of Listeria innocua suspended in skim milk by PEF as well as the sensitization of PEF treated L. innocua to nisin. The selected electric field intensity was 30, 40 and 50 kV/cm and the number of pulses applied was 10.6, 21.3 and 32. The sensitization exhibited by PEF treated L. innocua to nisin was assessed for 10 or 100 IU nisin/ml. A progressive decrease in the population of L. innocua was observed for the selected field intensities, with the greatest reduction being 2 1/2 log cycles (U). The exposure of L. innocua to nisin after PEF had an additive effect on the inactivation of the microorganism as that exhibited by the PEF alone. As the electric field, number of pulses and nisin concentration increased, synergism was observed in the inactivation of L. innocua as a result of exposure to nisin after PEF. The reduction of L. innocua accomplished by exposure to 10 IU nisin/ml after 32 pulsed electric fields was 2, 2.7, and 3.4 U for an electric field intensity of 30, 40, and 50 kV/cm, respectively. Population of L. innocua subjected to 100 IU nisin/ml after PEF was 2.8-3.8 U for the selected electric field intensities and 32 pulses. The designed model for the inactivation of L. innocua as a result of the PEF followed by exposure to nisin proved to be accurate in the prediction of the inactivation of L. innocua in skim milk containing 1.2 or 37 IU nisin/ml. Inactivation of L. innocua in skim milk containing 37 IU nisin/ml resulted in a decrease in population of 3.7 U.     

Thermal resistance of bacterial spores in milk-based beverages supplemented with nisin.

The effect of nisin, added in the form of Nisaplin, on the thermal resistance of bacterial spores and the effects of medium composition, exposure time, and pH on nisin enhancement of heat sensitivity were evaluated. Nisin apparently required specific nutrients to sensitize spores to heat. For example, D130 degrees C values of approximately 10 s were observed in sodium phosphate buffer with and without 6% sucrose with no significant (P> or =0.05) differences detected as a result of increased nisin concentration. In a nutrient-rich chocolate milk model system (CMMS), increasing either the time of exposure to nisin (5, 15, or 24 h) before heating or nisin concentration (0, 2,000, or 4,000 IU/ml) increased the sensitivity of Bacillus stearothermophilus spores to heat. In the CMMS with 10 to 12% fat cocoa powder, increasing nisin concentration (at 5 h of exposure) significantly (P< or =0.05) reduced D130 degrees C values; D130 degrees C values were 21.7, 17.2, and 17.8 s, respectively, for the 0-, 2,000-, and 4,000-IU/ ml nisin treatments. Fifteen and 24 h of exposure further reduced D130 degrees C values in the nisin-containing treatments compared to the control (0 IU of nisin per ml). A lower-fat CMMS (0 to 1% fat cocoa powder) had lower D130 degrees C values (19.3, 15.8, and 14.7 s for the 0-, 2,000-, and 4,000-IU/ml nisin treatments, respectively). Nisin activity was enhanced by lowering pH in the CMMS (10 to 12% fat cocoa powder), with reductions in D130 degrees C values across all pH values (ranging from 18.0% at pH 6.4 to 41.9% at pH 5.0). zD values were 9.6, 9.0, and 8.4 degrees C for the 0-, 2,000-, and 4,000-IU/ml nisin treatments, respectively. Spores of B. licheniformis yielded results similar to those obtained with B. stearothermophilus. For example, decreasing CMMS (10 to 12% fat cocoa powder) pH values from 6.4 to 5.0 produced D100 degrees C values of 3.3, 2.8, and 2.8 min (pH 6.4) and 1.0, 0.8, and 0.8 min (pH 5.0) for the 0-, 2,000-, and 4,000-IU/ml nisin treatments. This study clearly verified that the addition of Nisaplin to dairy-based beverages, such as a chocolate milk drink, or other foods intended to be heated reduces the thermal resistance of selected bacterial spores. Increased spore sensitivity to heat may provide food processors with an opportunity to reduce their thermal processes and expenses while maintaining product quality, functionality, and shelf stability.     

Potential of a nisin-containing bacterial cellulose film to inhibit Listeria monocytogenes on processed meats

A bacterially produced cellulose film containing nisin was developed and used in a proof-of-concept study to control Listeria monocytogenes and total aerobic bacteria on the surface of vacuum-packaged frankfurters. Bacterial cellulose pellicles were produced by Gluconacetobacter xylinus K3 in Corn Steep Liquor-Mannitol Medium and were subsequently purified before nisin was incorporated into them. Investigations into the effect of nisin concentrations and contact times on incorporation of nisin into cellulose films showed that the lowest nisin concentration and shortest time needed for production of an effective antimicrobial cellulose film were 625IUml(-1) and 6h, respectively. The active cellulose films produced under these conditions did not, however, significantly reduce L. monocytogenes populations on frankfurters (P>0.05) during refrigerated storage for 14 days as compared to the controls. Films produced using a higher concentration of nisin (2500IUml(-1)) with the same exposure time (6h) resulted in a significant (P<0.05) decrease in L. monocytogenes counts on frankfurters of approximately 2logCFUg(-1) after 14 days of storage as compared to the control. Both the above-mentioned films showed a similar effectiveness in reducing total aerobic bacterial populations as measured by total aerobic plate counts on frankfurters. For both films, total aerobic bacterial levels were significantly (P>0.05) reduced by approximately 3.3logCFUg(-1) after 14 days of storage as compared to control samples. Bacterial cellulose films were demonstrated in this study to have potential applicability as antimicrobial packaging films or inserts for processed meat products.     

Effective control of Listeria monocytogenes by combination of nisin formulated and slowly released into a broth system

In order to identify conditions for efficient food preservation by nisin, the sensitivity of Listeria monocytogenes to this preservative was studied under the following three model conditions: (1) the instantaneous addition of nisin into broth medium to simulate the formation of nisin in foods, (2) the slow delivery of nisin solution into broth medium using a pump to simulate the slow release of nisin from packaging materials to foods, (3) a combination of the two delivery methods. Based on the following results, we conclude that the antimicrobial effectiveness of nisin strongly depends on its mode of delivery. The instantaneous and slow methods for adding nisin inhibited L. monocytogenes, but over time of exposure, L. monocytogenes developed tolerance to nisin. Our data indicate that cells treated with instantaneously added nisin developed resistance to higher concentrations of nisin (200 IU/ml), compared to cells treated with slowly added nisin at the same total amount of the antimicrobial. Further studies indicated that nisin-tolerant cells recovered from treatments in which 200 IU/ml nisin was added instantaneously were likely to be mutants, which became resistant to the bacteriocin. In contrast, when 200 IU/ml of the antimicrobial was added slowly to the cells, only a temporary tolerance was developed; these cells became nisin-sensitive after passage through nisin-free medium. Due to the development of nisin-resistant cells, excessive amounts of nisin in the model system did not further inhibit L. monocytogenes. These results signify that excess nisin in foods does not necessarily improve the efficiency of controlling L. monocytogenes. Our data suggest that the combination of packaging material containing nisin used in conjunction with nisin-containing foods will provide the most effective means of preventing L. monocytogenes growth.     

Comparison of the sensitivity of commercial strains and infant isolates of bifidobacteria to antibiotics and bacteriocins

Eighteen bifidobacteria, six isolated from infant feces and 12 commercial strains (of which 10 were purchased from the American Type Culture Collection), were tested for sensitivity to 14 antibiotics and four bacteriocins including nisin A, nisin Z, pediocin PA-1 and mutacin B-Ny266. All bacteria were resistant to vancomycin, kanamycin, neomycin and streptomycin. Infant isolates were more sensitive than commercial strains to cloxacillin, ampicillin, chloramphenicol, tetracycline, rifampicin and novobiocin. Sensitivity to bacteriocins determined by a microplate assay varied widely. Generally, nisin A was the most effective bacteriocin followed by nisin Z and mutacin B-Ny266. Pediocin PA-1 appeared to have no inhibition at 70 μg mL⁻¹. Commercial strains showed relatively variable sensitivity to bacteriocins compared to infant isolates, which were inhibited within a narrow range of bacteriocin concentration. Bacteriocin tolerance could be easily gained by prolonging the exposure time. Death–time curves and transmission electron microscopy (TEM) of logarithmic and stationary cells of two strains (Bifidobacterium adolescentis ATCC 15704 and infant isolate Bifidobacterium sp. RBL67) incubated with twice the minimum inhibitory concentration of nisin A and nisin Z for 3 h revealed that log-phase cells were more sensitive than stationary-phase cells. TEM showed that the cell membrane is the most likely site of bactericidal effects of nisin.     

Inhibition of Salmonella on poultry skin using protein- and polysaccharide-based films containing a nisin formulation

The objective of this study was to examine the use of protein- arid polysaccharide-based films containing bacteriocin formulations for inhibiting salmonellae on fresh broiler skin. The lethality of the films containing a nisin-based formulation was determined against Salmonella Typhimurium-contaminated broiler drumstick skin samples coated with the film. In the first study, varying concentrations of nisin (0, 100, 300, and 500 microg/ml) plus 3% citric acid, 5.0 mM EDTA, and 0.5% Tween 80 were incorporated into 0.5% calcium alginate films at a 20% level (wt/wt) and then applied to Salmonella TyphimuriumNAr-contaminated skin samples (log10 5.0) at a 1:2 weight ratio (film:skin). Salmonella TyphimuriumNAr skin population reductions ranged from 1.98 to 3.01 log cycles after a 72-h exposure at 4 degrees C. In comparison to the 0- and 100-microg/ml nisin concentrations, significantly greater population reductions were achieved at nisin concentrations of 300 and 500 microg/ml. In related studies, the 500-degreesg/ml nisin formulation was incorporated into 0.75 and 1.25% agar gels and applied to contaminated broiler drumstick skin samples (log10 7.0). Salmonella TyphimuriumNAr skin population reductions following a 96-h exposure at 4 degrees C were 1.8-(1.25% agar gel) and 4.6-log cycles (0.75% agar gel). These results demonstrated that the inclusion of nisin-based treatments into either calcium alginate or agar gels that were subsequently applied to contaminated broiler drumstick skin yielded significant Salmonella TyphimuriumNAr population reductions ranging between 1.8 to 4.6 log cycles after 72 to 96 h of exposure at 4 degrees C. The level of kill was affected by film type and gel concentration (i.e., gel network formation), exposure time, and nisin concentration.     

Fast induction of nisin resistance in Streptococcus thermophilus INIA 463 during growth in milk

Streptococcus thermophilus INIA 463 became nisin-resistant after exposure in skim milk to subminimal inhibitory concentrations of nisin (1-3 IU/ml) for less than 2 h. Addition of 20 IU/ml caused a 4 log unit decrease in S. thermophilus counts of a culture not exposed previously to nisin, whereas no decrease was observed in the culture exposed to nisin for 2 h. Transfer of immunity genes as responsible for nisin resistance was discarded. The presence of extracellular or intracellular specific nisin-degrading enzymes was not detected in the nisin-resistant variant of S. thermophilus INIA 463. Nisin resistance was caused by the induction of a resistance mechanism. Transmission electron microscopy (TEM) revealed that the nisin-resistant variant of S. thermophilus INIA 463 had a thickened cell wall compared to the wild strain. Resistance to nisin was lost after one transfer (4 h growth) in nisin-free skim milk.     

Effects of combined exposure of micrococcus luteus to nisin and pulsed electric fields

Death and injury following exposure of Micrococcus luteus to nisin and pulsed electric field (PEF) treatment were investigated in phosphate buffer (pH 6.8, σ=4.8 ms/cm at 20°C). Four types of experiment were carried out, a single treatment with nisin (100 IU/ml at 20°C for 2 h), a single PEF treatment, a PEF treatment followed by incubation with nisin (as before) and addition of nisin to the bacterial suspension prior to the PEF treatment. The application of nisin clearly enhanced the lethal effect of PEF treatment. The bactericidal effect of nisin reduced viable counts by 1.4 log₁₀ units. Treatment with PEF (50 pulses at 33 kV/cm) resulted in a reduction of 2.4 log₁₀ units. PEF treatment followed by nisin caused a reduction of 5.2 log₁₀ units in comparison with a 4.9 log₁₀ units reduction obtained with nisin followed by PEF. Injury of surviving cells was investigated using media with different concentrations of salt. Sublethally damaged cells of M. luteus could not be detected by this means, following PEF treatment.     

Effect of combining nisin and/or lysozyme with in-package pasteurization on thermal inactivation of Listeria monocytogenes in ready-to-eat turkey bologna

Achieving a targeted lethality with minimum exposure to heat and preservation of product quality during pasteurization is a challenge. The objective of this study was to evaluate the effect of nisin and/or lysozyme in combination with in-package pasteurization of a ready-to-eat low-fat turkey bologna on the inactivation of Listeria monocytogenes. Sterile bologna samples were initially treated with solutions of nisin (2 mg/ml = 5,000 AU/ml = 31.25 AU/cm2), lysozyme (10 mg/ml = 80 AU/ml = 0.5 AU/cm2), and a mixture of nisin and lysozyme (2 mg/ml nisin + 10 mg/ml lysozyme = 31.75 AU/cm2). Bologna surfaces were uniformly inoculated with a Listeria suspension resulting in a population of approximately 0.5 log CFU/cm2. Samples were vacuum packaged and subjected to heat treatment (60, 62.5, or 65 degrees C). Two nonlinear models (Weibull and log logistic) were used to analyze the data. From the model parameters, the time needed to achieve a 4-log reduction was calculated. The nisin-lysozyme combination and nisin treatments were effective in reducing the time required for 4-log reductions at 62.5 and 65 degrees C but not at 60 degrees C. At 62.5 degrees C, nisin-lysozyme-treated samples required 23% less time than did the control sample to achieve a 4-log reduction and 31% less time at 65 degrees C. Lysozyme alone did not enhance antilisterial activity with heat. Results from this study can be useful to the industry for developing an efficient intervention strategy against contamination of ready-to-eat meat products by L. monocytogenes.     

Nisin Diffusion in Protein Films: Effects of Film Type and Temperature

ABSTRACT: Protein films [cast corn zein (CCZ), heat-pressed corn zein (HPCZ), cast wheat gluten (CWG), and heat-pressed wheat gluten (HPWG)] were studied at different exposure temperatures (5, 25, 35, and 45 °C) to determine nisin diffusion in aqueous model systems. Kinetics of nisin diffusion in protein films followed a Fickian diffusion model. The CCZ had the lowest nisin diffusivity and highest nisin retention. Diffusivities in HPCZ, CWG, and HPWG films were not significantly different. Temperature dependence of nisin diffusion in all films followed an Arrhenius model, which indicated no morphological changes within 5 to 45 °C. The activation energy for nisin diffusion in corn-zein films was higher than that in wheat-gluten films.