乳酸乳球菌乳酸亚种IMAU11823全基因组测序及胞外多糖基因簇分析

目的:乳酸乳球菌乳酸亚种IMAU11823是从内蒙古锡林郭勒盟自然发酵乳制品嚼口中分离得到的1株具有优良发酵特性且高产胞外多糖的乳酸菌。为探究其产胞外多糖的作用机制,对其进行全基因组测序并解析基因组序列信息。方法:使用PacBio SMRT三代测序技术对该菌株进行全基因组测序,采用生物信息学方法对其进行序列组装、基因预测和功能注释,并深入挖掘可能参与胞外多糖生物合成相关的基因信息。结果:乳酸乳球菌乳酸亚种IMAU11823包含1条染色体DNA(基因组大小为2 458 520 bp,GC含量为35.2%)和3个环状质粒(pIMAU11823A、pIMAU11823B、pIMAU11823C,基因组大小和GC含量分别为127 965,24 554,7 734 bp和35.7%,39.5%,32.7%),同时发现该菌株基因组内具有乳糖、纤维二糖、蔗糖、果糖、甘露糖、半乳糖转运系统和7种糖核苷酸合成相关基因以及1个胞外多糖合成基因簇epsRXCDBEF(GTF1)H(GTF2)JKM。结论:本研究揭示了乳酸乳球菌乳酸亚种IMAU11823的基因组特征,并预测了其胞外多糖的合成机制,为研究其产生的胞外多糖结构特征提供了理论依据,同时为该菌株在工业生产中的应用奠定理论基础。     

嗜热链球菌m6A甲基化差异研究

嗜热链球菌是乳制品工业生产中最为重要的发酵剂之一，具有良好的发酵特性。DNA甲基化是DNA化学修饰的一种形式，可在不改变DNA序列的前提下影响基因表达。本文选取2株产酸表型具有较大差异的嗜热链球菌为研究对象，通过甲基化组学探究m6A甲基化与遗传表型的关系。利用PacBio SMRT 测序平台对嗜热链球菌 IMAU80278与嗜热链球菌IMAU20416进行基因组测序及m6A甲基化测定，比较2株嗜热链球菌功能基因及m6A甲基化差异。结果表明，嗜热链球菌IMAU80278与嗜热链球菌 IMAU20416基因组相似，且功能基因均以碳水化合物代谢及氨基酸代谢为主。嗜热链球菌IMAU80278与嗜热链球菌 IMAU20416 限制修饰系统，m6A甲基化位点和m6A基序均存在显著差异，特别是在碳水化合物代谢及氨基酸代谢相关功能基因的m6A甲基化存在明显差异。推测m6A甲基化的差异可能导致嗜热链球菌IMAU80278与嗜热链球菌IMAU20416产酸表型的不同。此外，从表观遗传学角度探究嗜热链球菌DNA甲基化对发酵特性的影响，为优良发酵剂的筛选及利用奠定理论基础。     

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。     

开菲尔(Kefir)酸牛乳酒中乳酸菌分离及发酵性能测定

对自制的开菲尔酸牛乳酒进行分离得到16株乳酸菌,通过显微镜观察及生理生化特性研究,结果为肠膜明串珠菌乳脂亚种两株,乳酸乳球菌乳酸亚种2株,乳酸乳球菌乳脂亚种2株,粪肠球菌1株,瑞士乳杆菌3株,德氏乳杆菌保加利亚亚种2株,嗜酸乳杆菌4株;经发酵性能测定,筛选出2株乳酸球菌LC2、LC6和3株乳酸杆菌LB3、LB4、LB8发酵活力较高、发酵乳组织状态及风味较好,可作为Kefir酸牛乳酒纯培养发酵剂乳酸菌的备选菌株.     

开菲尔粒中一株乳酸乳球菌的分离及性能研究

为了筛选益生乳酸菌,从传统发酵乳制品之发酵剂开菲尔粒中分离到一株优势菌株,命名为HUCM 201,经形态学特征、16S rDNA基因序列及生理生化特性分析,将其鉴定为乳酸乳球菌乳亚种(Lactococcus lactis subsp.lactis)。该菌株对酸和胆汁均表现出了一定的耐受能力,在模拟人工胃液的环境中培养3 h后,活菌数接近于105 CFU/mL水平;而经1%牛胆汁作用4 h后,活菌数仅出现了微弱的减少。同时,以Caco-2细胞为模型模拟肠道上皮细胞检测细菌的黏附能力,菌株HUCM 201对Caco-2细胞表现出了一定的黏附性,黏附率为1.93%。通过最低抑制浓度法(MICs)评价了其对抗生素的敏感性,发现其对氨苄青霉素、万古霉素、红霉素、氯霉素、青霉素5种常见抗生素均敏感。乳酸乳球菌乳亚种HUCM 201可作为一株潜在的优良候选菌株进行深入研究。     

产胞外多糖乳酸球菌的分离筛选及在Mozzarella干酪中的应用

从东北传统的发酵制品大酱、糖蒜和辣酱中分离出116株乳酸菌,其中LJ35和LJ51两株菌胞外多糖产量相对较高且稳定,分别为乳酸乳球菌乳酸亚种(L.lactis subsp.lactis)和乳酸乳球菌乳脂亚种(L.lactis subsp.cremoris),胞外多糖的产量分别为62.19 mg/L和74.24 mg/L.以菌株LJ35和LJ51制作Mozzarella千酪的研究表明,添加2%LJ35或2%LJ51作发酵剂制作的Mozzarellla干酪保水性分别提高了2.1%和3.2%;同时干酪的融化性得到改善,干酪硬度降低,变得柔软.     

不同保加利亚乳杆菌与嗜热链球菌S10复配对发酵乳特性的影响

嗜热链球菌S10分离自青海地区自然发酵酸牛乳，具有良好的弱后酸化能力和发酵特性。本研究将分离自不同国家传统发酵乳及酸马奶的4株保加利亚乳杆菌IMAU20450、IMAU95110、IMAU62091和IMAU62161，分别与嗜热链球菌S10进行复配组成发酵剂(A、B、C和D)，以商业发酵剂(E)为对照，采用多频扩散波谱法研究发酵过程中的微流变学特性，并对贮藏稳定性和后酸化进行综合评价。结果表明:发酵过程中A组发酵乳的固液平衡值最低，而黏性因子、弹性因子高于B、C、D组发酵乳，表明A组发酵乳形成具有较高强度的乳凝胶结构。在4 ℃贮藏21 d期间，A组和C组硬度与对照组无差异，均显著大于其它试验组(P<0.05)，各试验组发酵乳的黏度、持水与对照组无显著性差异(P>0.05)。25 ℃贮藏14 d后酸化评价表明，B组和D组发酵乳pH值和滴定酸度均分别小于和大于其它各组(P<0.05)，而A组和C组的发酵乳与对照组无显著性差异，说明A组和C组发酵剂具有弱后酸特性。同时A、C组感官评价得分最高，发酵乳酸甜比恰当，组织均匀，质地细腻丝滑。综上，保加利亚乳杆菌IMAU20450、IMAU62091分别与嗜热链球菌S10复配时酸乳发酵和贮藏特性较好，具有进一步研究价值和较好应用前景，为乳酸菌发酵剂开发提供物质基础和数据参考。     

分离自传统发酵乳制品中具有优良发酵特性的保加利亚乳杆菌的筛选

以分离自传统发酵乳制品中的11株德氏乳杆菌保加利亚亚种(Lactobacillus delbeueckii subsp.bulgaricus)为研究对象,通过全脂乳单菌发酵,对其发酵乳的发酵时间、pH值、滴定酸度、乙醛和双乙酰含量等指标进行分析,最终筛选出1株具有优良发酵特性的菌株IMAU20775。该菌株发酵时间较短,后酸化能力弱,产乙醛能力较好,产双乙酰能力较稳定,适于进一步作为发酵剂菌种的筛选和应用研究。     

新疆酸马奶中乳酸菌的多态性分析及发酵性能研究

以新疆牧民传统家庭自制酸马奶样品为研究对象,考察乳酸菌的多态性及筛选优良性状的乳酸菌菌株。通过16S r RNA基因序列分析和生理生化试验等方法对分离出的菌株进行鉴定,并进行发酵性能测试。结果表明,本实验共分离出19株菌株,包括乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.lactis)(8株)、粪肠球菌(Enterococcus faecalis)(2株)、屎肠球菌(Enterococcus faecium)(2株)、嗜热链球菌(Streptococcus thermophilus)(1株)、干酪乳杆菌(Lactobacillus casei)(3株)、植物乳杆菌(Lactobacillus plantarum)(2株)和徳氏乳杆菌乳酸亚种(Lactobacillus delbrueckii subsp.Lactis)(1株)。其中,干酪乳杆菌JDB1.1505是一株产酸和产黏性能都比较突出的乳酸菌,发酵脱脂乳的滴定酸度达到了130.1°T,黏度为1420.1m Pa·s,具有良好的乳品发酵应用潜能。     

乳酸菌在大豆黄浆水中发酵条件的优化

以乳酸乳球菌乳亚种为发酵菌株,大豆黄浆水为培养基质,产酸量、pH值为考察指标,探讨不同发酵温度、发酵时间、种子液接种量、葡萄糖添加量对乳酸乳球菌乳亚种在黄浆水中发酵产酸的影响。在单因素试验的基础上,采用均匀设计法对其发酵条件进行优化,并对产酸量进行二次多项式逐步回归分析。结果表明,乳酸乳球菌乳亚种在黄浆水中最适发酵条件为发酵温度35℃、种子液接种量9%(V/V)、发酵时间68h、葡萄糖添加量5%(m/V)、初始pH 6.2,该条件下产酸量达0.791 3g/100mL,pH为3.40。     

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.     

Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates

In this study, bacteriocins from two Lactococcus lactis subsp. lactis isolates from raw milk samples in Turkey designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after alpha-amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of the purified bacteriocins were determined by SDS-PAGE. PCR amplification was carried out with specific primers for the detection of their structural genes. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Examination of plasmid contents of the isolates and the results of plasmid curing and conjugation experiments showed that in L. lactis subsp. lactis OC1 strain the 39.7-kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity, whereas the 16.0-kb plasmid is responsible for lacticin 481 production and lactose fermentation in L. lactis subsp. lactis OC2 strain.     

乳酸乳球菌KLDS4.0325的B族维生素生物合成途径分析

为了探究乳酸乳球菌KLDS4.0325的B族维生素合成潜力,利用各类B族维生素生物合成途径的相关蛋白序列针对该菌株的氨基酸序列进行同源性搜索,并与其他9 株乳酸乳球菌的叶酸生物合成途径进行比较分析。结果表明：与参考菌株相比,乳酸乳球菌KLDS4.0325具有较为完整的叶酸和核黄素合成途径编码基因,在基因水平上可以有效合成叶酸和核黄素,具有相当大的工业潜能。     

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).     

Bacteriophage resistance in Lactococcus lactis ssp. lactis using antisense ribonucleic acid.

Antisense RNA against a conserved bacteriophage gene when expressed in a Lactococcus lactis ssp. lactis strain renders it resistant to bacteriophage infection. Two open reading frames have been identified in a L. lactis ssp. lactis bacteriophage that are conserved in a majority of isolates. They code for an 18-kDa (designated GP18C) protein and a 24-kDa (GP24C) protein, respectively, which are arranged along with previously identified open reading frames in a tandem motif similar to other bacteriophages. The presence of gp18C and gp24C in a number of bacteriophage isolates was confirmed by polymerase chain reaction using primers specific for these regions. Plasmids bearing various fragments of gp18C, gp24C, or both were constructed such that the respective open reading frames were positioned in the antisense direction relative to the Lactococcus lactis ssp. cremoris Wg2 promoter, p59. These antisense RNA-producing vectors inhibited the efficiency of plaquing of L. lactis ssp. lactis bacteriophage phi 7-9 up to 50%; the resulting plaques were extremely small and irregular in shape. The replication of the bacteriophage was severely inhibited, and the total number decreased over the first 3 h during infection in strains expressing antisense RNA compared with the host strain alone, in which the bacteriophage number increased 10(4)-fold.     

Is thermotolerance correlated to heat-shock protein synthesis in Lactococcus lactis subsp. lactis?

Exposure of Lactococcus lactis subsp. lactis cells to a heat shock at 40 degrees C for 30 min induces thermotolerance, the increased ability of bacterial cells to survive exposure to lethal temperature (52 degrees C for 25 min). This transient state of thermal resistance is accompanied, as in Escherichia coli, by the synthesis of a new set of specific proteins termed heat-shock proteins (Hsps). Pre-treatment of the bacterial cells by antibiotics (streptomycin, spiramycin, kanamycin and erythromycin) known to act on translation, induces the major Hsps synthesis but no thermal protection; conversely, puromycin and amino acid analogues treatments, known to produce abnormal and incomplete peptides, triggers the thermotolerance state without inducing significant Hsps synthesis. These results demonstrate that heat-shock response and induced thermotolerance are not tightly correlated phenomena in L. lactis subsp. lactis.     

镉胁迫下泡菜乳酸乳球菌的形态变化

通过扫描电镜和透射电镜分别观察不同质量浓度水平的Cd2+对泡菜乳酸乳球菌（Lactococcus lactis subsp.lactis）细胞的影响,扫描电镜结果显示：Cd2+质量浓度在0、10 mg/L时,泡菜乳酸乳球菌呈椭圆形、表面光滑、菌体生长繁殖旺盛,随着Cd2+质量浓度的增加菌体细胞表面产生白色颗粒状物质、菌体细胞存活数量大幅下降（OD600 nm值由1.336下降到0.515）。当添加200 mg/L Cd2+时,几乎没有见到明显的菌体、显示有少量棱形晶状物。透射电镜结果显示：当Cd2+质量浓度为0～50 mg/L时泡菜乳酸乳球菌结构完整、细胞内容物分布均、菌体生长较为正常,当菌体暴露于100、200 mg/L Cd2+时菌体细胞出现异常现象,如细胞破裂、内容物从薄膜穿孔中释放、质壁分离等。两类电镜结果均表明：在低质量浓度Cd2+（≤50 mg/L）胁迫下,对泡菜乳酸乳球菌的生长几乎不产生影响,添加Cd2+质量浓度上升到100、200 mg/L时泡菜乳酸乳球菌正常生长受到抑制。     

Growth characteristics of Candida kefyr and two strains of Lactococcus lactis subsp. lactis isolated from Zimbabwean naturally fermented milk.

Lactic acid bacteria (LAB) and yeasts constitute part of the microflora in Zimbabwean traditional fermented cows' milk, amasi. The present study was carried out to investigate the growth characteristics of Candida kefyr 23, Lactococcus lactis subsp. lactis biovar. diacetylactis C1 and L. lactis subsp. lactis Lc261, previously isolated from amasi, in ultrahigh temperature (UHT)-treated cows' milk. The strains were inoculated into the UHT milk as both single and yeast     

Incidence of nisin Z production in Lactococcus lactis subsp. lactis TFF 221 isolated from Thai fermented foods

Lactic acid bacteria isolated from various Thai fermented foods were screened for the presence of nisin gene by using PCR with primers specific to nisin A structural gene. Only one strain, Lactococcus lactis subsp. lactis TFF 221, isolated from kung jom, a traditional shrimp paste, was found to carry a nisin gene. The TFF 221 nisin had antimicrobial activity against not only closely related lactic acid bacteria but also some foodborne pathogens. It was heat stable and inactivated by alpha-chymotrypsin and proteinase K. Some characteristics of TFF 221 nisin were found to be very similar to those of nisin A produced by Lactococcus lactis subsp. lactis NCDO 2111. Both of them had the same antimicrobial spectrum and MICs against all indicator bacteria. However, when assayed with indicator organisms, in all cases the TFF 221 nisin produced larger zones of inhibition in agar diffusion assays than the nisin A did. Sequencing of the TFF 221 nisin gene showed that it was the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The nisin determinant in strain TFF 221 was found to be located on a conjugative transposon residing in the chromosome. The ability of the nisin produced by L. lactis subsp. lactis TFF 221 to inhibit a wide range of foodborne pathogens may be useful in improving the food safety of the fermented product, especially in the Thai environment, which suffers from perennial problems of poor food hygiene.     

乳酸乳球菌胞外多糖磷酸化的工艺优化

采用单因素和正交试验,对乳酸乳球菌胞外多糖的磷酸化工艺进行研究,探讨磷酸盐用量、反应温度、反应时间、反应pH值对乳酸乳球菌胞外多糖最终PO43-接枝量的影响。所得的乳酸乳菌球菌胞外多糖磷酸化的工艺优化条件为：胞外多糖与磷酸盐质量比为6:1、温度90℃、时间4h、pH6.0,此条件下所得PO43-的接枝量为1.639mg/g。