乳酸链球菌素的研究和应用

综述了一种新型天然食品防腐、保鲜剂-乳酸链球菌素的研究开发进展,主要从乳酸链球菌素的性质、作用机理、使用的安全性、活力单位的测定、作为食品添加剂的优缺点、在食品方面的用途及工业化秤现状等方面进行了探讨,以便我国更好地研究开发和利用这一生物工程产品造福食品加工业。     

变形链球菌致龋抗原基因在乳酸乳球菌的表达

目的 克隆变形链球菌葡聚糖结合蛋白B(GbpB)功能区的基因片段,并在乳酸乳球菌中表达.方法 在实验中利用了分子克隆技术构建携带GbpB基因的重组原核表达质粒pNI1,将重组质粒转化乳酸乳球菌YF02株,筛选鉴定阳性菌落,诱导表达的GbpB蛋白用SDS-PAGE进行鉴定.结果 成功克隆了GbpB功能区的基因片段,并在乳酸乳球菌中得到其融合蛋白的表达.结论 利用分子生物学技术能够成功克隆GbpB功能区基因并获得乳酸乳球菌融合蛋白的表达,为后续研究奠定了基础.     

产抑菌素菌株SM-A的分离和鉴定

自市售酸乳酪中分离到一株乳球菌SM-A菌株。该菌株产生的抑菌素能抑制或杀死芽孢杆菌、葡萄球菌、微球菌、链球菌、棒杆菌和梭菌等革兰氏阳性细菌,但对革兰氏阴性细菌、霉菌和酵母无效。SM-A菌株多为链球状,也有成对存在。革兰氏染色阳性,抗酸染色阴性,兼性厌氧生长,最适生长温度32℃,不形成芽孢,无荚膜和鞭毛,不运动;可从多种糖类产酸,但不产气;接触酶、苯丙氨酸脱氨酶和酪氨酸脱羧酶均为阴性,精氨酸双水解酶阳性;不液化明胶,还原石蕊牛奶并胨化,生长温度范围10～43℃,DNA中G+Cmol为36.4％。经鉴定,SM-A菌株为乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.lactis)。     

乳酸链球菌素对瘤胃体外发酵、甲烷生成及功能菌群数量的影响北大核心CSCD

【目的】旨在通过体外静态模拟瘤胃发酵法研究乳酸链球菌素(NI)对瘤胃发酵、甲烷生成及功能菌群数量的影响。【方法】以不添加任何添加剂处理做阴性对照(NC),以莫能菌素(MON,5μmol/L)做阳性对照,试验组NI添加水平分别为3(NI-3)、9(NI-9)和27 mg/100 m L(NI-27),每个处理4个重复,分别于培养后的0、3、6、9、12、24 h测定产气量和甲烷产量。培养24 h后,采集发酵液样品,用于发酵参数和菌群数量的测定。【结果】与NC组相比,添加NI和MON均能显著降低产气量和甲烷产量(P<0.05);添加NI对pH值、干物质消失率(DMD)和有机物消失率(OMD)无显著影响(P>0.05);NI-9处理组与NC组相比氨态氮浓度显著降低(P<0.05),而NI-3和NI-27组氨氮浓度没有显著变化(P>0.05);相比而言,MON处理组DMD、OMD和氨氮浓度与NC组相比均显著降低(P<0.05),而pH值与其他各处理组相比没有差异(P>0.05);与NC组相比,NI各处理组和MON组乙酸浓度及乙丙比均显著降低(P<0.05),丙酸浓度显著提高(P<0.05)。功能菌方面,qPCR结果显示添加NI和MON对总菌和拟杆菌门数量均无显著影响(P>0.05);与NC相比,添加NI对原虫、甲烷菌、真菌和厚壁菌门数量均无显著影响(P>0.05),而MON组原虫、甲烷菌、真菌和厚壁菌门数量显著降低(P<0.05);NI和MON处理均显著提高了硫还原菌和C.aminophilum数量(P<0.05),但C.sticklandii数量不受影响(P>0.05)。【结论】添加适宜浓度的NI可降低瘤胃甲烷与氨的生成,但并不影响饲料消化,这种发酵模式的改变可能与瘤胃功能菌群数量与多样性的变化密切相关。     

pH及流加葡萄糖对酵母分批发酵生产谷胱甘肽的影响

在5 L的发酵罐中研究了pH及流加葡萄糖对酵母分批发酵生产谷胱甘肽(GSH)的影响。实验考察了不同浓度的流加葡萄糖和不同的恒pH值的分批发酵对于酵母生产GSH产量的变化。实验结果表明,当pH值控制为5.0,流加葡萄糖流速为5g.L-1.h-1,连续流加30 h,可使GSH产量最高,与之前未流加葡萄糖和控制pH相比,其产量提高了6倍。     

产抑菌素菌株SM—A的分离和鉴定

自市售酸乳酪中分离到一株乳球菌SM-A菌株。该菌株产生的抑菌素能抑制或杀死芽孢杆菌、葡萄球菌、微球菌、链球菌、棒杆菌和梭菌等革兰氏阳性细菌,但对革兰氏阴性细菌、霉菌和酵母无效。SM-A菌株多为链球状,也有成对存在。革兰氏染色阳性,抗酸染色阴性,兼性厌氧生长,最适生长温度32℃,不形成芽孢,无荚膜和鞭毛,不运动;可从多种糖类产酸,但不产气;接触酶、苯丙氨酸脱氨酶和酪氨酸脱羧酶均为阴性,精氨酸双水解酶阳性;不液化明胶,还原石蕊牛奶并胨化,生长温度范围10～43℃,DNA中G Cmol为36.4％。经鉴定,SM-A菌株为乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.lactis)。     

pH控制下绿色木霉（Trichoderma viride）流加发酵生产纤维素酶

绿色木霉（Trichodermaviride）在pH控制发酵条件下,采用流加葡萄糖发酵策略,可显著提高综合滤纸酶活力（FPA）和内切酶（endo—β—1,4-glucanase,EG）、外切酶exo—β-1,4-glucanase,CBH）、纤维二糖酶（cellobiase,CB）酶活。在5L发酵罐中采用pH控制和流加葡萄糖工艺,可提高CB酶含量,改变酶组分之间的比例,使得FPA、EG、CB和CBH酶活分别达到50．0U／mL,210．0U／mL,4．0U／mL和2．5U／mL,比摇瓶发酵分别提高了6．7．4．2、19、2．5倍。     

Pfk基因过表达对乳酸乳球菌N8产nisin速率的影响

【目的】通过基因工程手段增加糖酵解途径中编码限速酶6-磷酸果糖激酶基因Pfk在乳酸链球菌素(nisin)产生菌Lactococcus lactis N8中的表达,增快nisin的产生,从而提高单位时间内nisin的产量,缩短发酵周期。【方法】将pfk基因及编码以c AMP为依赖的蛋白激酶催化亚基基因pka C克隆到表达质粒p MG36e上,将共表达重组质粒转入L.lactis N8中,使Pfk-pka C基因过量表达,得到重组菌株L.lactis N8-p MG36epfk-pka C,并比较该重组菌株与野生菌的生长曲线、胞内6-磷酸果糖激酶活性、发酵上清液的抑菌活性及效价,并从转录水平分析两株菌nis A及pfk-pka C的转录差异,比较野生菌与重组菌在不同葡萄糖含量下培养产nisin的变化。【结果】Pfk基因与pka C基因的过表达对重组菌的生长速度没有明显的影响,却能提高重组菌产nisin的速度,在发酵10 h时nisin的产量比野生菌提高了20%,使得发酵周期缩短近2 h。野生菌及重组菌在不同葡萄糖含量下培养发酵上清液的nisin效价没有明显的变化。【结论】糖酵解途径中6-磷酸果糖激酶基因Pfk的过表达可以加快乳酸乳球菌N8产nisin的速率,缩短发酵周期。     

不同甲醇流加策略对重组毕赤酵母高密度发酵生产水蛭素的影响

考察了不同甲醇流加策略对毕赤酵母高密度发酵生产水蛭素的影响。溶氧控制法不能有效地防止甲醇的过量流加。气相色谱离线检测法虽然防止了甲醇流加过量,但甲醇浓度的波动较大。利用甲醇传感器在线检测控制甲醇的流加可维持较恒定的甲醇浓度。在流加甲醇的同时,以限制性速率流加甘油可以增加表达期间的能量供应,提高产物的表达量。经优化后,采用甲醇甘油混合流加时细胞干重达到162g/L,水蛭素活性达到24×10⁴ATU/mL,即1.7 g/L。      

不同甲醇流加策略对重组毕赤醇母高密度发酵生产水蛭素的影响

考察了不同甲醇流加策略对毕赤酵母高密度发酵生产水蛭素的影响。溶氧控制法不能有效地防止甲醇的过量流加。气相色谱离线检测法虽然防止了甲醇流加过量 ,但甲醇浓度的波动较大。利用甲醇传感器在线检测控制甲醇的流加可维持较恒定的甲醇浓度。在流加甲醇的同时 ,以限制性速率流加甘油可以增加表达期间的能量供应 ,提高产物的表达量。经优化后 ,采用甲醇甘油混合流加时细胞干重达到 16 2g L ,水蛭素活性达到 2 4×10 4ATU mL ,即 1 7g L。     

乳酸链球菌肽发酵动力学的研究

提出了在恒定不同pH的发酵条件下,乳酸链球菌SM526的菌体生长、底物消耗、乳酸及Nisin产生的动力学模型。菌体生长、乳酸及Nisin产生用逻辑方程描述,而底物消耗是菌体生长和乳酸产生速率的函数。模型表明,乳酸链球菌SM526菌体生长和乳酸产生的最佳pH为7．0,而Nisin产生的最佳pH却为6．5。     

Nisin A前体基因nisA的过量表达对Nisin A产量的影响

Nisin是一种广泛应用于食品工业的抗菌素。通过基因工程手段分别构建了Nisin A前体结构基因nisA的穿梭表达载体pMG36ek-nisA和整合型载体pDG780-nisA,并转入Nisin A产生菌乳酸乳球菌Lactococcus lactis ATCC 11454中,得到两株基因工程菌FMM1和FMM2。对比工程菌和原始产生菌的生长状态及Nisin A产量,结果表明FMM1的生长速度、生物量以及发酵液的酸碱度没有显著变化,而Nisin A产量提高了31%;相反,FMM2的生物量较原始菌株显著降低,但Nisin A产量也有一定程度的提高。通过RT-PCR检测工程菌与原始产生菌Nisin A生物合成基因簇中11个基因的转录水平,结果显示FMM1和FMM2的11个基因的转录水平均有提高,其中FMM1提高更为显著。因此推测,nisA是Nisin A高产的关键因素之一,其游离型过量表达能显著提高Nisin A的产量。该研究为采用基因工程手段提高Nisin A的产量提供了新的思路,并对NisinA的大规模工业生产有指导意义,同时,也为其他抗菌肽产生菌的基因工程改造提供了参考。     

乳链菌肽的分离纯化和部分生物学性质

用乳酸链球菌ＳＭ５２６进行乳链菌肽的发酵生产,产量为４０～５０ｍｇ／ｌ．经中空纤维超滤器超滤,非极性大孔吸附树脂ＸＡＤ－２层析,ＣＭ－ＳｅｐｈａｄｅｘＣ－２５层析和ＳｅｐｈａｄｅｘＧ－５０层析纯化了该肽。ＳＤＳ－ＰＡＧＥ表明达均一,ＲＰ－ＨＰＬＣ表明其纯度不低于９５％。ＳＤＳ－ＰＡＧＥ测其Ｍｒ约为３６００,用ＩＥＦ测其等电点为９．５．酸性条件下稳定且抗热;对胰蛋白酶、胃蛋白酶和木瓜蛋白酶不敏感,但对α－胰凝乳酶和蛋白酶Ｋ敏感。乳链菌肽对多种革兰氏阳性菌有强烈的抑制作用;以枯草杆菌和金黄色葡萄球菌为指示菌,其作用方式是杀菌。     

High nisin Z production by Lactococcus lactis UL719 in whey permeate with aeration

The influence of controlled pH (5.0–6.5) and initial dissolved oxygen level (0–90% air saturation) on nisin Z production in a yeast extract/Tween 80-supplemented whey permeate (SWP) was examined during batch fermentations with citrate positive Lactococcus lactis subsp. lactis UL719. The total activity corresponding to the sum of soluble and cell-bound activities, as measured by a critical dilution method, was more than 50% lower at pH 5.0 than in the range 5.5–6.5, although the specific production decreased as pH increased. A maximum nisin Z activity of 8200 AU/ml (4100IU/ml) was observed in the supernatant after 8h of culture for pH ranging from 5.5 to 6.5. Prolonging the culture beyond 12h decreased this activity at pH 6.0 and 6.5 but not at pH 5.5 or 5.0. A corresponding increase in cell-bound activity was probably due to adsorption of soluble bacteriocin to the cell wall. Aeration increased cell-bound and total activity to maximum values of 32800 and 41000 AU/ml (16400 and 20500IU/ml), respectively, with an initial level of 60% air saturation after 24h of incubation at pH 6.0. The specific production at 60% or 90% initial air saturation was eight-fold higher than at 0%.     

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.     

Synergistic effects of nisin and thymol on antimicrobial activities in Listeria monocytogenes and Bacillus subtilis

Nisin Z and thymol were tested, alone and in combination, for antibacterial activity against Listeria monocytogenes ATCC 7644 and Bacillus subtilis ATCC 33712. The antibacterial effect of nisin Z, produced by Lactococcus lactis KE3 isolated from the traditional Moroccan fermented milk, was greatly potentiated by sub-inhibitory concentrations of thymol in both bacterial strains. Our data showed that the concentration of nisin required for effective control of food-borne pathogenic bacteria could be considerably lowered by the use of thymol in combination. The use of low concentrations of nisin could lead to a less favourable condition for the occurrence of nisin-resistant bacterial sub-populations.     

sacA and nisA genes are not always linked in Lactococcus lactis subsp. lactis strains

Sixty-seven lactococcal strains arising from dairy habitat were screened for the presence of the sucrose 6-phosphate hydrolase gene by polymerase chain reaction. Of the strains tested, 35.8% were able to ferment sucrose as well as to harbour the sucrose-6-phosphate hydrolase gene, even though they were unable to produce nisin as well as to show the nisin structural gene. After pulsed-field gel electrophoresis and hybridisation all Suc⁺Nis⁻ strains exhibited physical linkage between sacA gene and the left end of lactococcal transposons (Tn5276 or Tn5301) without linkage to nisin genes. However, we were unable to transfer the sacA gene as well as to detect Suc⁻ derivatives from Suc⁺Nis⁻ strains after conjugation and curing experiments.     

Evidence for a role of NisT in transport of the lantibiotic nisin produced by Lactococcus lactis N8

Abstract The biosynthesis, immunity and regulation of nisin, a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis , is encoded by two gene clusters, nisAIZBTCIPRK and nisFEG . The mutant strain LAC46 with a deletion in the translocator gene nisT could not secrete nisin but nisin activity was detected from cell lysates. The nisT mutation was complemented by a NisT-expression plasmid resulting in restored capacity to secrete nisin. These results demonstrate that NisT is the transport protein dedicated to translocate nisin and that dehydration and lanthionine formation in nisin maturation can occur independently of transport.     

pH值对厚颌鲂幼鱼的耗氧率及窒息点的影响

在密封式流水呼吸室内,对厚颌鲂Megalobrama pellegrini幼鱼在pH值分别为5.5、6.0、6.5、7.0、7.5、8.0和8.5试验用水条件下的耗氧率进行了测定.结果 显示:厚颌鲂幼鱼耗氧率存在明显的昼夜变化,白天高于晚上,下午高于上午,夜间变化不大.不同pH值的平均耗氧率为0.7135 mg/g · h,平均耗氧率最高是在pH7.5时,为0.8665 mg/g · h,平均耗氧率最低是在pH5.5时,为0.5397 mg/g · h.在密封式呼吸室内测定pH值在5.5、6.0、6.5、7.0、7.5、8.0和8.5试验用水条件下的厚颌鲂幼鱼的窒息点,分别为1.3920、1.3067、1.1547、1.0683、1.0933、1.1733和1.2331 mg/L,平均窒息点为1.2008 mg/L.