High nisin-Z production during repeated-cycle batch cultures in supplemented whey permeate using immobilized Lactococcus lactis UL719

Nisin-Z production was studied during repeated-cycle pH-controlled batch (RCB) cultures using Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 immobilized in κ-carrageenan/locust bean gum gel beads in supplemented whey permeate. After an initial colonization of gel beads during the first two cycles, nisin-Z production in bulk medium and gel beads was very similar for 1-h and 2-h cycle RCB cultures. A very high nisin-Z production (8200 IU mL⁻¹) was measured in the broth after the 1-h cycles, with a corresponding volumetric productivity of 5730 IU mL⁻¹ h⁻¹. This productivity is much higher than maximum nisin productivities reported in literature or maximum productivities obtained previously for free-cell batch cultures (850 IU mL⁻¹ h⁻¹), and free-cell (460 IU mL⁻¹ h⁻¹) or immobilized-cell (1760 IU mL⁻¹ h⁻¹) continuous cultures, using the same strain and fermentation conditions. The stability of RCB cultures was demonstrated for 24 and 36 1-h cycles carried out over 3 and 6-day periods, respectively. Changing environmental conditions during batch cultures resulted high nisin production.     

乳酸片球菌R-4细菌素PA-1原核表达及其理化特性

为实现原核表达产出细菌素并检测其理化特性,作者将乳酸片球菌 R-4细菌素pedA基因进行扩增回收,与pMD19-T载体连接后转入E.coli DH5α感受态细胞进行克隆。提取克隆后的pedA基因与表达载体pET-32a（+）连接,形成重组质粒pET-32a-pedA并转入E.coli BL21(DE3)感受态细胞,经异丙基硫代半乳糖苷诱导,乳酸片球菌R-4细菌素PA-1在大肠杆菌细胞进行表达。表达蛋白质经Ni-NTA柱纯化后,以金黄色葡萄球菌为指示菌检测其理化特性。结果表明,在E.coli BL21(DE3)细胞中成功表达相对分子质量为26 000的乳酸片球菌R-4细菌素PA-1并完成纯化。纯化后的乳酸片球菌R-4细菌素PA-1在40~121 ℃作用20 min、在pH 2~12、紫外线照射0~10 h、过氧化氢酶作用2 h后,其抑菌范围分别为14.7~15.6 mm、14.0~16.5 mm、15.1~15.8 mm和14.9 mm,而分别经胃蛋白酶和胰蛋白酶作用2 h均失去抑菌作用。这表明乳酸片球菌R-4细菌素PA-1对高温、强酸强碱、紫外线和过氧化氢酶均具有较好的稳定性,而胃蛋白酶和胰蛋白酶会使其失活。     

乳酸片球菌素PA-1在大肠杆菌中的表达与纯化

为了实现该细菌素的外源表达,本实验首先利用聚合酶链式反应从乳酸片球菌PAF中扩增出乳酸片球菌素PA-1的结构和免疫基因,然后克隆到表达载体pGEX-6p-1,构建了N端含有GST-His-DDDDK标签的重组质粒pGEX/his-pedAB,然后转化进入大肠杆菌Rosetta（DE3）感受态细胞,经异丙基硫代半乳糖苷诱导,重组乳酸片球菌素PA-1在大肠杆菌胞内成功表达。表达的融合蛋白先经过镍亲合层析柱纯化,然后注入谷胱甘肽S-转移酶亲和色谱柱用肠激酶处理,释放出成熟的乳酸片球菌素PA-1。利用高效液相色谱和质谱技术检测乳酸片球菌素PA-1纯度。以单核细胞增生李斯特氏菌CMCC54004为指示菌,利用琼脂扩散法检验乳酸片球菌素PA-1活性。结果表明,携带GST-His-DDDDK标签的融合蛋白无活性,标签切除后其抑菌活性恢复,且其纯度达90%以上。     

Ethanol production by repeated batch culture using yeast cells immobilized within porous cellulose carriers

Ethanol was efficiently produced for 20 d by repeated batch culture using yeast cells immobilized in porous cellulose carriers. The physical structure of the carrier beads, which had small pores distributed on the outer surface and relatively large pores distributed in the interior, was found to be effective for cell immobilization. The effects of chemical modifications to the basic cellulose carrier on cell immobilization and ethanol production were also examined in repeated batch cultures. The chemical modifications had little effect on either cell immobilization or ethanol production.     

Pediocin PA-1的结构、生物合成与作用机制

乳酸菌素是乳酸菌通过核糖体合成机制产生的一类具有生物活性的蛋白质、多肽或前体多肽。最近将乳酸菌素分为三大类,其中Ⅱa类类片球菌素是研究的热点,它的典型代表是Pediocin PA-1。Pediocin PA-1具有较强的抗单增李斯特菌特性和热稳定性,是一种具有广阔应用前景的食品添加剂。本文综述了Pediocin PA-1的结构、生物合成和作用机制,概述了国内外对Pediocin PA-1的研究进展和发展方向,以期对细菌素的深入研究和广泛应用起到抛砖引玉的作用。      

足球菌ISK—1的发酵产物足球菌素ISK—1的生产最适化

研究了足球菌ＩＳＫ－１发酵中温度，ｐＨ值以及无机阳离子对足球菌素ＩＳＫ－１活性的影响，３７℃，ｐＨ值为５．５～６．０是产生足球菌素ＩＳＫ－１的较适宜条件，Ｍｎ＾２＋和Ｍｇ＾２＋对足球菌素ＩＳＫ－１的产生是必需要，而Ｎａ＾＋，Ｃａ＾２＋，ＮＨ４等阳离子均能提高足球菌素ＩＳＫ－１的活性，其中以Ｎａ＾＋的效果最显著。     

NaCl对足球菌ISK—1的发酵产物足球菌素ISK—1活性的影响

研究了足球菌ＩＳＫ－１发酵中ＮａＣｌ对足球菌素ＩＳＫ－１活性的影响。在ＭＲＳ培养基中添加８％的ＮａＣｌ，发酵２１￣２４ｈ，足球菌素ＩＳＫ－１活性达到最高，比无ＮａＣｌ时提高２５％。表明ＮａＣｌ具有提高足球菌素ＩＳＫ－１活性的作用。     

Growth inhibition of Listeria monocytogenes by a bacteriocin of Pediococcus acidilactici M during fermentation of kimchi

Four lactic acid bacteria known to produce bacteriocins were evaluated for the inhibitory effect against Listeria monocytogenes strain Scott A. Lactococcus lactis subsp. lactis biovar diacetylactis 26-2 was the least antilisterial, whereas Pediococcus acidilactici M resulted in the largest zone of inhibition on an agar medium. Leuconostoc paramesenteroides OX and Lactobacillus sake Lb 706 were intermediate. Aqueous solutions of crude bacteriocin powders derived from supernatant fluids of L. sake Lb 706 and P. acidilactici M. cultures inhibited the growth of Lactobacillus plantarum UGA8 in MRS agar. The addition of 10 mg of crude bacteriocin powder from L. sake to 150 g of kimchi fermented at 14 ± 1°C for 16 days had no effect on the growth of L. monocytogenes. The same amount of crude bacteriocin derived from P. acidilactici, however, had an initial lethal effect on L. monocytogenes and controlled growth of the pathogen throughout the 16-day fermentation. The successful application of bacteriocins to control the growth of L. monocytogenes in lightly fermented foods such as kimchi would appear to be achievable.     

Evaluation of Chinese hamster ovary cell stability during repeated batch culture for large-scale antibody production

Pharmaceutical manufacturing plants can be operated continuously for several months. It is therefore important to use cells with long-term stability for the production of active ingredients. We investigated the reliability and long-term stability of an antibody-producing cell line. A recombinant Chinese hamster ovary (CHO) cell line was cultivated in spinner flasks and reactors, including a practical production-scale reactor (1600 L), for 109 days to produce monoclonal antibodies against the HM1.24 antigen. During cultivation, the cells remained stable and there was an increase in the rate of cell proliferation, yielding viable cells at high density. A decrease in cell-specific productivity was associated with this increase in the rate of cell proliferation. The cells were genetically stable and other measures of cellular function remained consistent throughout the cultivation period.     

Use of autochthonous Pediococcus acidilactici and Staphylococcus vitulus starter cultures in the production of "chorizo" in 2 different traditional industries

The present study determined how the different ripening conditions affected the growth and development of 3 autochthonous starter cultures, and the physico-chemical and sensory characteristics of chorizo. Each of 3 strains of Pediococcus acidilactici (MC184, MS198, and MS200) and one of Staphylococcus vitulus (RS34) were associated to prepare the starter cultures, P184S34, P198S34, and P200S34. Then, chorizo was prepared following 2 manufacturing procedures. The autochthonous starter cultures were able to compete and colonize the sausages in both ripening procedures. The use of the starter cultures showed evident differences by the texture analysis, with the control batches being generally tougher than the starter culture batches. Also, the highest biogenic amine (BA) levels were found in control batches and the lowest in P200S34 batches. While the use of these starter cultures does not change the sensory characteristics of these traditional fermented sausages, it improves their homogeneity and safety, except for P184S34 batch in which more BAs are detected in industry 2. PRACTICAL APPLICATION: The 3 autochthonous starter cultures selected could be used in traditional industries because they are able to compete well and colonize the dry fermented sausages "chorizo." The use of these starter cultures improves the texture and homogeneity of traditional fermented sausages. Biogenic amines decreased in the starter cultures batches improving the safety.     

外源信号分子AI-2对发酵乳杆菌TG4-1-1和乳酸片球菌11-3产胞外多糖的影响

研究群体感应信号分子AI-2对发酵乳杆菌TG4-1-1和乳酸片球菌11-3胞外多糖产量的影响,探讨信号分子AI-2对乳酸菌分泌胞外多糖的调控机制。采用苯酚-硫酸法和哈维氏弧菌BB170生物发光法测定菌株不同培养时间胞外多糖产量和AI-2活性,筛选添加最佳浓度的外源信号分子AI-2,测定菌株的生长量、胞外多糖产量、AI-2活性,扫描电镜观察菌体形态及冷冻干燥后多糖形态。结果表明:发酵乳杆菌TG4-1-1和乳酸片球菌11-3均在10 h时AI-2活性最强,22 h时胞外多糖产量最高,分别达到(195.863±1.643)mg/L和(125.179±1.458)mg/L。100μmol/L外源AI-2为菌株TG4-1-1和11-3的最佳添加浓度,而该浓度对两株菌各阶段生长量均无显著影响(P>0.05),对菌株TG4-1-1在16～22 h和菌株11-3在13～22 h的胞外多糖产量有显著促进作用(P<0.05)。同时,显著增强试验菌株在10 h时AI-2的活性(P<0.05)。添加100μmol/L外源AI-2培养13 h后菌体表面光滑,形状规则,形态饱满,其对胞外多糖形态...     

Comparative study of batch and continuous multi-stage fixed-bed tower (MFBT) bioreactor during wine-making using freeze-dried immobilized cells

A freeze-dried immobilized biocatalyst produced by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on gluten pellets and subsequent freeze-drying was used in a multistage fixed-bed tower (MFBT) bioreactor for batch and continuous wine-making. The MFBT bioreactor resulted in higher alcohol productivity compared to fermentations carried out in a packed bed (PB) bioreactor and showed an important operational stability and no decrease in activity, even at low fermentation temperature (5 °C) and after storage for 6 months at 4 °C. The production of amyl alcohols proved to be temperature dependent and was significantly reduced at low temperatures. Re-activation of the freeze-dried immobilized cells after storage for 6 months resulted in further decreased content of amyl alcohols. The SPME GC/MS analysis of volatile compounds revealed no significant differences in the wines produced by MFBT and PB bioreactors, while the preliminary sensory evaluation ascertained the overall improved quality of the produced wines. Potential industrial application of MFBT bioreactor is also assessed and discussed.     

固定化细胞生产乳酸的研究

以海藻酸钠为材料固定鼠李糖乳杆菌ATCC10863．6^#,研究不同条件对L-乳酸的影响。结果表明,利用2％海藻酸钠得到的固定化细胞颗粒稳定性好,可维持150h无破裂,同时L-乳酸产量也相对较高。发酵培养基中酵母粉的最适浓度为3．0g／L,最适发酵温度为42℃,加入的最适接种细胞量为15％,最佳凝胶珠直径为2．5mm-3．0mm。通过4批次的重复发酵培养,第1批次发酵时间为48h,L-乳酸产量为40．1g／L。后3批次发酵时间均缩短为36h,L-乳酸产量约为45g/L。     

Optimal conditions for the production of exopolysaccharide by Pediococcus acidilactici and impact of the bacterium,its EPS and dextran on the quality of Kariesh cheese

The effect of some growth conditions on the production of exopolysaccharide (EPS) by Pediococcus acidilactici was studied. Furthermore, the impact of utilisation of the isolated EPS, dextran and P. acidilactici on some properties of Kariesh cheese was investigated. The maximum EPS production by P. acidilactici was obtained after 10 h of incubation at 37°C in MRS medium with an initial pH of 7. Kariesh cheese manufactured with dextran or P. acidilactici exhibited the highest (P ≤ 0.05) moisture content and the lowest hardness values. Protein, fat, ash, total bacterial and yeast and mould counts were not significantly affected by the applied treatments. However, the body and texture of Kariesh cheese were significantly improved.     

Immunity gene pedB enhances production of pediocin PA-1 in naturally resistant Lactococcus lactis strains

Heterologous production of the antilisterial bacteriocin pediocin PA-1 in lactococci is an attractive objective to increase the safety of dairy products. In a previous paper, we developed a system for the heterologous production of the bacteriocin pediocin PA-1 in pediocin-resistant lactococcal hosts through a leader exchange strategy. The system was based on 3 genes, 1 encoding the fusion between the lactococcin A leader and propediocin PA-1, and the other 2 encoding the lactococcin A secretion machinery. In this study, we investigated whether the addition of the pediocin PA-1 immunity gene (pedB) to this system has any effect on pediocin production. Introduction of the plasmid(s) carrying the genes described above into nisinproducing and non-nisinproducing lactococcal hosts led to a significant increase in the production of pediocin compared with the equivalent pedB-devoid systems. In addition, we obtained a nisin-producing strain with the ability to secrete pediocin PA-1 at a level equivalent to that of the parental strain Pediococcus acidilactici 347, which represents a notable improvement over our previous systems.     

Effect of aeration and dilution rate on nisin Z production during continuous fermentation with free and immobilized Lactococcus lactis UL719 in supplemented whey permeate

The influence of dilution rate (D) and aeration on soluble and cell-bound nisin Z production was investigated during continuous free (FC) and immobilized cell (IC) cultures with Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in supplemented whey permeate. Maximum total bacteriocin titres during non-aerated continuous FC and IC cultures were obtained for low D, with 1490 and 1090 IU mL⁻¹ for 0.15 h⁻¹ or 0.25 and 0.5 h⁻¹, respectively. For both systems, aeration increased nisin total production with maximum titres of 2560 and 2430 IU mL⁻¹ for low D, respectively, as well as specific production. Volumetric productivity was the highest for an intermediate D of 0.4 h⁻¹ during FC cultures (460 IU mL⁻¹ h⁻¹ for both aerated and non-aerated cultures), while it increased continuously with D during IC cultures, reaching high values of 1090 and 1760 IU mL⁻¹ h⁻¹ at 2.0 h⁻¹ without and with aeration, respectively. In comparison with previous data for FC batch cultures, data from this study may indicate that during continuous fermentations at steady state, some steps in nisin biosynthesis are limiting. In these conditions, nisin production by immobilized cells is reduced.     

Continuous production of pectinase by immobilized yeast cells on spent grains

A yeast strain secreting endopolygalacturonase was used in this work to study the possibility of continuous production of this enzyme. It is a feasible and interesting alternative to fungal batch production essentially due to the specificity of the type of pectinase excreted by Kluyveromyces marxianus CCT 3172, to the lower broth viscosity and to the easier downstream operations. In order to increase the reactors' productivity, a cellulosic carrier obtained from barley spent grains was tested as an immobilization support. Two types of reactors were studied for pectinase production using glucose as a carbon and energy source--a continuous stirred tank reactor (CSTR) and a packed bed reactor (PBR) with recycled flow. The highest value for pectinase volumetric productivity (P(V)=0.98 U ml(-1) h(-1)) was achieved in the PBR for D=0.40 h(-1), a glucose concentration on the inlet of S(in)=20 g l(-1), and a biomass load in the support of X(i)=0.225 g g(-1). The results demonstrate the attractiveness of the packed bed system for pectinase production.     

Chimeras of mature pediocin PA-1 fused to the signal peptide of enterocin P permits the cloning, production, and expression of pediocin PA-1 in Lactococcus lactis

Chimeras of pediocin PA-1 (PedA-1), a bacteriocin produced by Pediococcus acidilactici PLBH9, fused to the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, permitted the production of PedA-1 in Lactococcus lactis. Chimeric genes encoding the EntP signal peptide (SP(entP)) fused to mature PedA-1 (pedA), with or without its immunity gene (pedB), were cloned into the expression vector pMG36c to generate the recombinant plasmids pMPP9 (SP(entP):pedA) and pMPP14i (SP(entP):pedA + pedB). Transformation of competent L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. lactis DPC5598 with the recombinant plasmids has permitted the detection and quantitation of PedA-1 and the coproduction of nisin A and PedA-1 in supernatants of producer cells with specific anti-PedA-1 antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay. Recombinant L. lactis hosts carrying pMPP9 or pMPP14i displayed antimicrobial activity, suggesting that mature PedA-1 fused to SP(EntP) is the minimum requirement for the synthesis, processing, and secretion of biologically active PedA-1 in L. lactis. However, the production and antimicrobial activity of the PedA-1 produced by L. lactis was lower than that produced by the P. acidilactici control strains.