乳酸菌代谢途径的基因工程调控

随着乳酸菌基因工程技术的发展，对乳酸菌的代谢途径进行基因工程调控，可以使其产生除乳酸之外的其它具有重要工业用途的成分。通过对乳酸菌丙酮酸代谢途径中某些关键酶基因进行敲除或过量表达，可以使乳酸菌大量地产生双乙酰或L-丙氨酸；在乳球菌中过量地表达叶酸或核黄素生物合成的关键酶，可以促进这两种B族维生素的生物合成：对乳酸菌胞外多糖生物合成途径中的限制性步骤进行调控。可以有效地提高胞外多糖的产量。利用这种基因工程改造的乳酸菌可以开发新型功能性发酵食品。     

响应面法优化乳酸乳球菌KLDS4.0325产叶酸的培养基成分及发酵条件

探究并提高实验室保藏的分离自新疆酸马奶的乳酸乳球菌KLDS4.0325的产叶酸能力。首先基于实验室前期对KLDS4.0325的全基因组测序结果,对其在基因水平表现出的产叶酸能力进一步运用高效液相色谱法进行测定。然后,以叶酸产量为响应值,通过响应面法对发酵条件进行优化,确定最优发酵条件。利用单因素试验,确定最佳氮源为酵母浸粉,最佳碳源为葡萄糖,主要影响因素为初始pH值、对氨基苯甲酸(p-aminobenzoic acid,p ABA)添加量、谷氨酸添加量与温度。通过Box-Behnken响应面试验,确定最优发酵条件为初始p H 5.40、p ABA添加量42.0 mg/L、谷氨酸添加量6.30 g/L、温度35.40℃,在此条件下叶酸产量为0.814 μg/m L,是优化前(0.260 μg/m L)的3.13倍。通过对乳酸乳球菌KLDS4.0325培养基成分及发酵条件的优化,得出该菌株产叶酸的最优培养基成分及发酵条件,为天然叶酸的产业化生产与应用提供了理论支持。     

产细菌素乳酸菌的筛选及相关基因分析

以Escherichia coli为指示菌,采用双层平板法从7株乳酸菌中筛选出一株高产细菌素的优良菌株KLDS 4.0325;通过菌体形态观察及16S r RNA序列分析鉴定菌株KLDS 4.0325为乳酸乳球菌,通过分析该菌株全基因组序列,预测其细菌素合成基因簇有AOI_1和AOI_2,并分别做了简要介绍,在细菌素数据库中比对,均属于二类细菌素中的Lactococcin_B_(LCN-B)型细菌素;最后通过向指示菌菌液中添加无细胞发酵上清液进一步证明了该菌株所产细菌素对指示菌的作用方式是杀菌。     

基于基因水平研究乳酸乳球菌的有氧呼吸代谢

本研究旨在对能和不能有氧呼吸代谢的乳酸乳球菌呼吸链基因同源性和细胞色素氧化酶活力进行分析,为确定乳酸乳球菌的有氧呼吸必要条件奠定基础。首先,分别测定已筛选到的菌株KLDS4.0325和KLDS4.1102在发酵和有氧呼吸代谢时的生长曲线;其次,提取KLDS4.1102的基因组DNA,随后利用PCR扩增其呼吸链编码基因并进行DNA测序;最后,从Gen Bank数据库下载KLDS4.0325的呼吸链编码基因序列,利用DNAMAN软件比较这两株菌呼吸链编码基因的同源性,并测定这两株菌的细胞色素氧化酶活性。结果表明在有氧呼吸条件下KLDS4.0325的生物量和p H都显著增大,而KLDS4.1102在两种条件下呈现一致的生长趋势,呼吸链编码基因的同源性分析显示KLDS4.1102具备完整的呼吸链,而酶活测定结果却显示KLDS4.1102无细胞色素氧化酶活性,这表明KLDS4.1102不能进行有氧呼吸代谢不是由于编码基因缺陷造成的,可能是某些编码基因不能正常表达,导致其细胞色素氧化酶不能正常发挥作用。     

产B族维生素植物乳杆菌的来源及其在发酵食品中的应用

整理了与合成核黄素、叶酸以及钴胺素相关的基因,分析总结了国内外在产B族维生素植物乳杆菌的筛选、表达优化、合成机制、调控技术、食品加工中应用及植物乳杆菌调节肠道菌群改善宿主维生素缺乏效果等方面的研究进展,为开发富含B族维生素发酵食品,防治人体B族维生素缺乏提供参考。     

乳酸乳球菌KLDS4.0325产细菌素最佳培养条件的研究

以分离自新疆牧民家庭自制酸马奶中的乳酸乳球菌KLDS4.0325为研究对象,以大肠杆菌为指示菌,以抑菌圈直径为考察指标,考察了不同温度、p H、转速及培养时间等培养条件对该菌株细菌素产量的影响,经单因素实验优化得出乳酸乳球菌KLDS4.0325在培养温度33℃,培养基初始p H7.0,120 r/min摇床培养20 h的条件下,无细胞发酵上清液对大肠杆菌的抑菌圈直径最大,抑菌活性最强,细菌素产量最高。在此条件下,抑菌圈直径为25.26 mm,细菌素效价可达5383.07 IU/m L。     

切达干酪发酵菌种的特性测定及发酵干酪的评价

对实验室研究较为成熟的2株乳酸乳球菌进行发酵产酸、产黏测定,同时对其蛋白水解能力进行评价;并对单菌株、不同比例组合菌株制备的切达干酪进行质构特性评价、感官分析和风味物质测定。测定结果表明:乳酸乳球菌乳酸亚种KLDS4.0424是主要的产酸菌种;与商业发酵剂相比,实验室菌株的蛋白水解能力和产黏能力相对较弱;乳酸乳球菌乳酸亚种KLDS4.0424与乳酸乳球菌乳脂亚种KLDS4.0326按1∶1接种制作的干酪具有良好的成熟度、质地和风味,具备开发成干酪发酵剂的潜力。     

乳酸菌发酵代谢合成叶酸的影响因素

对嗜酸乳杆菌以及乳酸乳球菌发酵合成叶酸的影响因素进行了研究。结果表明,乳酸菌代谢合成叶酸的产率为17~100μg/L,菌种、培养时间、pH值、对氨基苯甲酸(PABA)质量浓度会影响乳酸菌合成叶酸的产量。与乳酸乳球菌乳酸亚种相比,嗜酸乳杆菌CH-2生成的叶酸产量要高。不同菌株生成叶酸的能力与pH值有关,嗜酸乳杆菌在pH值为4.2叶酸产率明显下降,乳酸乳球菌乳酸亚种产叶酸的能力则不受pH值影响。添加PABA可以显著提高乳酸菌的叶酸产率。选择适宜的乳酸菌菌株,优化发酵工艺参数可以提高乳及相关食品中叶酸的质量浓度,达到生物方式强化叶酸的效果。     

切达干酪发酵剂用菌种的筛选研究

概述了切达干酪的特点,以实验室现有的3株优良乳酸乳球菌为出发菌株,对3种单一菌株和3种复合菌株进行产酸、产黏、产香、蛋白水解及自溶度等工艺性能的测定,旨在开发适合切达干酪生产的优良发酵剂菌种。结果表明,复合菌株L4(乳酸乳球菌乳酸亚种KLDS4.0424和乳酸乳球菌乳脂亚种KLDS41326)适合用作切达干酪发酵剂,以用于进一步研究。     

高产纯L-乳酸的乳酸乳球菌的发酵条件探究

为了研究一株分离自新疆牧民家庭自制酸马奶中的乳酸乳球菌KLDS4.0325产L-乳酸的能力,以L-/D-乳酸试剂盒验证该菌种在发酵过程中L-乳酸的光学纯度为100%。在已优化的培养基上,进行产酸条件的探究,研究发现该菌株在最适发酵温度为33℃,最适p H 8.0,最佳发酵转速为120 r/min,在24 h产酸量达到最大值。     

一株仅产L-乳酸的乳酸乳球菌发酵培养基的优化

为了研究一株分离自新疆牧民家庭自制酸马奶中的乳酸乳球菌KLDS 4.0325产L-乳酸的能力,以L-/D-乳酸试剂盒验证该菌种在发酵过程中所产L-乳酸的光学纯度为100%。利用Plackett-Burman设计法对影响该菌株发酵的培养基主要组分进行筛选,确定影响L-乳酸产量的主要因素为蔗糖、酵母粉、K2HPO4。在此基础上,采用响应面法优化发酵培养基的组成,结果表明：当蔗糖添加量为102.9 g/L、酵母粉添加量为2.5 g/L、K2HPO4添加量为7.9 g/L时,L-乳酸产量最大,可达86.6 g/L,在最优发酵条件下获得的实测值与模型预测值（86.3 g/L）吻合,说明所建立的模型是切实可行的。     

乳酸乳球菌有氧呼吸能力的优化研究

为了提高乳酸乳球菌KLDS4.0316在血红素存在状态下的有氧呼吸能力,实现高密度培养,分析比较了阶段性和持续有氧呼吸的培养方法。结合不同碳源及浓度、血红素添加量和摇床转速等单因素实验结果,设计正交实验优化获得的最佳有氧呼吸培养条件为:培养物以1/1000接菌量接种于添加20g/L葡萄糖和2μg/mL血红素的M17培养基中,摇床转速250r/min,30℃培养。增菌培养终止时间为8h。研究发现,优化培养的KLDS4.0316菌株消耗更多的葡萄糖,这可能与显著增加的生物量有关。     

Growth stimulation of a proteinase positive Lactococcus lactis strain by a proteinase negative Lactococcus lactis strain

Lactococcus lactis AMP15/pAMP31(D471R) is a proteinase negative, lactose negative strain with a modified oligopeptide transport system, and potential as a debittering agent due to its efficient utilization of hydrophobic peptides. Five wild L. lactis strains of dairy origin, which produced cheeses of high flavour quality, were cocultured with L. lactis AMP15/pAMP31(D471R) in an attempt to select adequate combinations of strains for use as defined cheese starters with potential debittering ability. Four of these strains, L. lactis B6, K16, M21 and P21, inhibited growth of L. lactis AMP15/pAMP31(D471R) at a level of 10(6) to 10(7) cfu mL(-1) after 24 h of incubation, even though production of bacteriocin-like compounds could only be proven for L. lactis M21. When L. lactis AMP15/pAMP31(D471R) was cocultured with the fifth strain, L. lactis N22, its growth was significantly (P<0.001) inhibited whereas growth of L. lactis N22 was significantly stimulated. The nature of the interaction was studied and it was established that L. lactis N22 is auxotrophic for folate, a compound produced and excreted by L. lactis AMP15/pAMP31(D471R).     

不同发酵剂对切达干酪成熟期间品质的影响

应用乳酸乳球菌乳酸亚种LA、乳酸乳球菌乳脂亚种LC以及不同比例混合菌(LA∶LC=1∶1,LA∶LC=1∶2,LA∶LC=2∶1)制作切达干酪,研究这5种发酵剂在干酪成熟过程中对其质构、感官、风味物质形成及蛋白水解程度等方面的影响。结果表明:3种不同比例组合菌株发酵剂的蛋白水解能力适中,生产出的干酪口味清淡,其中按1∶1接种制作的干酪具有良好的成熟度、质地和风味,具有一定的商业应用价值,可将其用于切达干酪的生产。     

Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish

We isolated lactic acid bacteria from the intestinal tract of the pufferfish Takifugu niphobles caught in Shimoda, Shizuoka, Japan by using MRS broth prepared with 50% seawater. Additional screening was carried out using phenotypic tests such as Gram staining, cell morphology, catalase, oxidase and fermentation of glucose. Subsequently 227 isolates screened by the phenotypic tests were subjected to species-specific PCR for Lactococcus lactis, resulting in four positive isolates. The 16S rRNA gene sequences from three isolates were highly similar to that of L. lactis subsp. lactis (DNA database accession number M58837), while that of one isolate was identical to that of Leuconostoc mesenteroides (AB023246). These isolates were characterized by API 50 CH for carbohydrate fermentation and other phenotypic criteria for salt tolerance, and the characteristics were compared with those of L. lactis subsp. lactis from a cheese starter culture. The carbohydrate fermentation profiles of these isolates were characteristic of L. lactis subsp. lactis strains, whereas the tolerance of these isolates to salt was higher than that of L. lactis subsp. lactis from the cheese starter culture: the new L. lactis isolates showed high salt tolerance in MRS-agar plates containing 200% seawater or 6% sodium chloride. This is the first report of the isolation of halotolerant strains of L. lactis subsp. lactis from a marine environment.     

Antimicrobial potential of immobilized Lactococcus lactis subsp. lactis ATCC 11454 against selected bacteria

Immobilization of living cells of lactic acid bacteria could be an alternative or complementary method of immobilizing organic acids and bacteriocins and inhibit undesirable bacteria in foods. This study evaluated the inhibition potential of immobilized Lactococcus lactis subsp. lactis ATCC 11454 on selected bacteria by a modified method of the agar spot test. L. lactis was immobilized in calcium alginate (1 to 2%)-whey protein concentrate (0 and 1%) beads. The antimicrobial potential of immobilized L. lactis was evaluated in microbiological media against pathogenic bacteria (Escherichia coli, Salmonella, and Staphylococcus aureus) or Pseudomonas putida, a natural meat contaminant, and against seven gram-positive bacteria used as indicator strains. Results obtained in this study indicated that immobilized L. lactis inhibited the growth of S. aureus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus curvatus, Lactobacillus sakei, Kocuria varians, and Pediococcus acidilactici. Only 4 h of incubation at 35 degrees C resulted in a clear inhibition zone around the beads that increased with time. With the addition of 10 mM of a chelating agent (EDTA) to the media, results showed growth inhibition of E. coli; however, P. putida and Salmonella Typhi were unaffected by this treatment. These results indicate that immobilized lactic acid bacteria strains can be successfully used to produce nisin and inhibit bacterial growth in semisolid synthetic media.     

Cross-inhibition among wild strains of Lactococcus lactis isolated from the same ecological niche.

The cross-inhibition between 23 Lactococcus lactis subsp. lactis strains and 9 L. lactis subsp. cremoris strains with different randomly amplified polymorphic DNA patterns, all isolated from the same ecological niche--cheese made in the spring at a single factory from raw milk without added lactic starter cultures-was investigated. Cross-inhibition, as determined by the agar well diffusion assay, was recorded in 130 cases (12.7%) out of 1.024 total cases, with 109 cases due to supernatants of L. lactis subsp. lactis strains and 21 cases due to supernatants of L. lactis subsp. cremoris strains. L. lactis strains isolated in April, May, and June showed differences in their inhibitory activities, with cross-inhibition against each other in 34.7, 14.1, and 6.1% of the cases, respectively. Polymerase chain reaction techniques using specific primers for nisin, lacticin 481, and lactococcin A only revealed the presence of the structural gene of lacticin 481 in two L. lactis subsp. lactis strains.     

酸应激对1株乳酸乳球菌质膜F_1F_0-ATPase活性的影响

评价10株乳酸乳球菌的酸应激能力,选用应激能力较高的菌株,确定其最佳酸应激条件。将4 h、10 h、20 h培养的该菌菌体在此条件下应激,以硫酸亚铁钼蓝比色法测定应激前后的质膜F_1 F_0-ATPase活性。结果表明,菌株KLDS 4.0312具有较高应激能力,应激后耐酸性可提高7542倍,其最佳酸应激条件为pH4.5,30 min。4 h、10 h、20 h培养的该菌菌体应激后,质膜F_1F_0-ATPase活性分别提高56.68%、11.05%、5.16%。     

与特定乳酸菌共培养对植物乳杆菌KLDS1.0391细菌素合成的诱导作用

植物乳杆菌KLDS1.0391与76株乳酸菌分别共培养后,测定培养物的抑菌活性和活菌数,判断与乳酸菌共培养对植物乳杆菌细菌素合成的影响及细菌素合成与菌体密度的关系。结果表明：植物乳杆菌KLDS1.0706、KLDS4.0315、KLDS4.0351、罗伊氏乳杆菌KLDS1.0736这4株菌与植物乳杆菌KLDS1.0391共培养后,抑菌活性显著增加(P＜0.01)。与罗伊氏乳杆菌共培养过程中细菌素的抑菌活性与植物乳杆菌KLDS1.0391的细胞密度呈现明显的正相关性,并且发现只有活的诱导菌与植物乳杆菌KLDS1.0391共培养才能够诱导细菌素的合成。