饮料中乳酸菌包埋的研究

以玉米为基础原料,采用全酶法糖化工艺进行液化和糖化,接种适宜的乳酸菌进行发酵,然后采用复凝聚法在液相中将乳酸菌包埋,以此延长菌体在液相中的存活时间.乳酸菌被包埋后,在饮料中添加乳酸链球菌素和低聚果糖,对饮料中未被包埋或已被包埋但又释放的菌体的产酸进行控制,同时也保证其在饮料中存活.通过正交试验法找到最佳的微胶囊造粒条件及乳酸链球菌素和低聚果糖的添加量.     

玉米酒糟为主要氮源的Nisin生产菌株的诱变选育

为使乳酸乳球菌适应玉米酒糟作为发酵生产乳酸链球菌素(Nisin)的主要氮源,首先对培养基进行了初步优化,发现在酒糟清液中加入蔗糖、酵母膏和磷酸氢二钾明显促进了乳酸乳球菌的生长.在此基础上,使用等离子体对乳酸乳球菌进行诱变处理,利用24方孔深孔板技术可实现对高产Nisin突变菌株的选择性筛选.结果表明,诱变菌株在只有5.0%存活率时,得到的正突变菌株可达26.2%.借助摇瓶发酵实验对其生产Nisin的发酵水平进行研究,发现有1株诱变选育菌株发酵Nisin的活性高达6 520 U/mL,并且其发酵能力比诱变起始菌株能力更强.     

Optimization of a capillary zone electrophoresis–contactless conductivity detection method for the determination of nisin

Determination of natural preservatives using electrophoretic or chromatographic techniques in fermented milk products is a complex task due to the following reasons: (i) the concentrations of the analytes can be below the detection limits, (ii) complex matrix and comigrating/coeluting compounds in the sample can interfere with the analytes of the interest, (iii) low recovery of the analytes, and (iv) the necessity of complex sample preparation. The aim of this study was to apply capillary zone electrophoresis coupled with contactless conductivity detection for the separation and determination of nisin in fermented milk products. In this work, separation and determination of natural preservative–nisin in fermented milk products is described. Optimized conditions using capillary zone electrophoresis coupled with capacitance‐to‐digital technology based contactless conductivity detector and data conditioning, which filter the noise of the electropherogram adaptively to the peak migration time, allowed precise, accurate, sensitive (limit of quantification: 0.02 μg/mL), and most importantly requiring very minute sample preparation, determination of nisin. Sample preparation includes following steps: (i) extraction/dilution and (ii) centrifugation. This method was applied for the determination of nisin in real samples, i.e. fermented milk products. The values of different nisin forms were ranging from 0.056 ± 0.003 μg/mL to 9.307 ± 0.437 μg/g.     

Separation and determination of closely related lantibiotics by micellar electrokinetic chromatography

A sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of four closely related lantibiotics: gallidermin, cinnamycin, duramycin and nisin. Factors affecting the separation of the lantibiotics such as pH, phosphate buffer concentration, SDS concentration and wavelength for UV detection were investigated. By optimizing these experimental conditions, successful separation was achieved between class 1A lantibiotics (nisin and gallidermin) and class 1B lantibiotics (duramycin and cinnamycin). The four lantibiotics were separated within 12 min in 50 mM phosphate buffer at pH 3.95 ± 0.1 containing 80 mM SDS with UV detection of 214 nm. The LOD (S/N = 3) were 61 ng/mL for gallidermin, 57 ng/mL for cinnamycin, 55 ng/mL for duramycin and 58 ng/mL for nisin. The method was successfully applied to real samples such as fermentation broth, bovine colostrum and predrop beer. This method yielded satisfactory results, with quantitative recoveries of spiked lantibiotics in the three samples ranging from 86.1 to 99.6%.     

Lantibiotics as surface active agents for biomedical applications

The unique physical structure of lantibiotics, (e.g. double bonds, thioethers rings, and unusual amino acid residues), makes these antimicrobial peptides highly reactive, and thus different in mode-of-action from clinical antibiotics. Members of the lantibiotic group have been successfully tested against pathogenic organisms for decades. Some lantibiotics have been studied for use in treating skin infections, and several have been characterized for their anti-viral activity. In addition to their antimicrobial capabilities, lantibiotics possess amphiphilic characteristics, making them potentially valuable as emulsifiers in drug formulations. The small size and surface activity of these peptides also may allow them to enhance the transport of therapeutic compounds across cell membranes. Over 30 lantibiotics are presently known, and more are being discovered each year. With the rising incidence of resistant bacteria, lantibiotics offer considerable potential as safe, antimicrobial barriers for use on synthetic material surfaces, as emulsifiers in formulation of hydrophobic drugs, and as absorption promoters for selected compounds across mucosal membranes. The major hindrance to research in this area is that lantibiotics are difficult to obtain. But with improved methods for production and purification of these unusual antimicrobial agents, the promise of cost-effective application of lantibiotics may eventually be realized.     

Detection of nisin expression by Lactococcus lactis using two susceptible bacteria to associate the effects of nisin with EDTA

Nisin, a bacteriocin produced during the exponential growth phase of Lactococcus lactis ATCC 11454, inhibits the growth of a broad range of Grampositive bacteria. Gram-negative bacteria can also be inhibited by nisin with EDTA. In this study, nisin production was assayed by the agar diffusion method using Lactobacillus sake ATCC 15521 and a recombinant Escherichia coli DH5-α expressing the recombinant green fluorescent protein as the nisin-susceptible test organisms. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/mL of medium) and converted to standard nisin concentration (Nisaplin^®, 25 mg of pure nisin with an activity of 1×10⁶ AU/mL). The expression and release of nisin by L. lactis in skimmed milk (9.09% total solids) with Man Rugosa Shepeer-Bacto Lactobacilli broth (1∶1) was monitored in a 5 L New Brunswick fermentor. Combining EDTA with nisin increased the bactericidal effect of nisin on the bacteria examined. The presence of EDTA was necessary to inhibit E. coli growth with nisin. L. sake was shown to be a good indicator for the evaluation of nisin release in the culture media, including with the addition of EDTA.     

Stress Response Kinetics of Two Nisin Producer Strains of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.     

Production of nisin by Lactococcus lactis in media with skimmed milk

Nisin is a bacteriocin that inhibits the germination and growth of Gram-positive bacteria. With nisin expression related to growth conditions of Lactococcus lactis subsp. lactis, the effects of growth parameters, media components, and incubation time were studied to optimize expression. L. lactis ATCC 11454 was grown (100 rpm at 30°C for 36 h) in both M17 and MRS standard broth media (pH 6.0–7.0) supplemented with sucrose (1.0–12.5 g/L), potassium phosphate (0.13 g/L), asparagine (0.5 g/L), and sucrose (0.24 g/L), and diluted 1:1 with liquid nonfat milk. Liquid nonfat milk, undiluted, was also used as another medium (9% total solids, pH 6.5). Nisin production was assayed by agar diffusion using Lactobacillus sake ATCC 15521 (30°C for 24 h) as the sensitive test organism. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/L of medium) and converted into known concentrations of “standard nisin” (Nisaplin®, g/L). The detection of nisin activity was <0.01 AU/L in M17 and MRS broths, and 7.5 AU/L in M17 with 0.14% sucrose or 0.13% other supplements, and the activity increased to 142.5 AU/L in M17 diluted with liquid nonfat milk (1:1). The 25% milk added to either 25% M17 or 25% MRS provided the highest levels of nisin assayed.     

Improvement of microbiological safety of sous-vide meals by gamma radiation

Experimental batches of smoked-cured pork in stewed beans sauce were inoculated with spores of psychrotrophic Bacillus cereus, more heat and radiation resistant than spores of non-proteolytic C. botulinum. After vacuum packaging, the meals were treated with combinations of pasteurizing heat treatments and gamma irradiation of 5 kGy. Prior and after treatments, and periodically during storage at 10°C, total aerobic and total anerobic viable cell counts, and selectively, the viable cell counts of B. cereus and sulphite-reducing clostridia have been determined. The effects of the treatment order as well as addition of nisin to enhance the preservative efficiency of the physical treatments were also studied. Heat-sensitization of bacterial spores surviving irradiation occurred. The quality-friendly sous-vide cooking in combination with this medium dose gamma irradiation and/or nisin addition increased considerably the microbiological safety and the keeping quality of the meals studied. However, approx. 40% loss of thiamin content occurred as an effect of combination treatments, and adverse sensorial effects may also limit the feasible radiation doses or the usable concentrations of nisin.     

鲜湿米粉中两种乳酸链球菌素的HPLC-MS/MS检测方法

建立了高效液相色谱-串联质谱(HPLC-MS/MS)同时测定鲜湿米粉中乳酸链球菌素A(Nisin A)和乳酸链球菌素Z(Nisin Z)的分析方法。样品经p H 3.0的水(甲酸调节p H值)提取,以Hilic Plus色谱柱分离待测物,在电喷雾正离子化模式下,采用多反应监测(MRM)模式检测。结果表明,Nisin A和Nisin Z在100~2 000μg/L范围内线性关系良好,相关系数均大于0.999,方法检出限分别为0.01、0.02 mg/kg,回收率为95.6%~107.0%,RSD均小于2.0%。该方法准确、可靠,适用于鲜湿米粉中Nisin A和Nisin Z的含量检测。