A protein which masks galactose receptor mediated phage susceptibility in Lactococcus lactis subsp. lactis MPL56

A 28.5-kb plasmid, isolated from Lactococcus lactis subsp. lactis MPL56, causes complete inhibition of four lactococcal phages. Cell wall characteristics of wild-type strain MPL56 were compared with its 28.5 kb plasmid-cured, phage-sensitive derivative MPL56-22. After proteolytic enzyme treatments, adsorption of phages occurred at high levels, an example is 94.6–98.5% in MPL56 cells. Analysis of cell wall extracts of MPL56-22 by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) indicated that the only difference between strains was the 55.4 kDa band in protein patterns of MPL56. Adsorption of the four phages was completely inhibited when MPL56-22 cells were subjected to SDS, Triton-X-100, HCl and NaOH treatments. Lectins that were specific for glucose/mannose and N-acetylglucosamine did not prevent adsorption of phages in cell wall extracts of MPL 56-22. However a lectin specific for galactose (MCA; Momordica charantia ) completely inhibited adsorption of these phages in cell wall extracts of MPL56-22. HPLC patterns of cell wall carbohydrates of MPL56-22 and its HCl treated preparations showed that the most prevalent difference was the galactose on untreated MPL56-22 cell wall chromatograms.     

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.     

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。     

乳酸乳球菌细胞壁蛋白酶的分离纯化

本研究确定了分离纯化乳酸乳球菌细胞壁蛋白酶(CEP)的最佳技术路线.用裂解液(50mmol/L Tris-HCI,2mmol/L EDTA-Na2,100mmol/L NaCl,0.5%Tritonx-100,1mg/ml溶菌酶,pH8.5)悬浮菌体(20ml/g),37℃下保温3h,离心后取上清液即为粗酶液.粗酶液通过45%硫酸铵沉淀,DEAE-Sephadcx A-25和Sephacryl-S-300 HR两步层析,可以得到纯化的细胞壁蛋白酶.蛋白酶提纯倍数达到74.048%,最后回收率为14.865%,PAGE电泳检测为一条带,SDS-PAGE检测蛋白酶为单体结构,分子量大约为53kD.用纯化后CEP酶解乳清蛋白,酶解液ACE抑制率为45%.     

Citrate metabolism in Lactococcus lactis subsp. lactis var. diacetylactis strains

The formation of diacetyl, acetoin, 2,3-butylene glycol, acetaldehyde, ethanol and lactic acid during 24 h of cultivation in milk with 0.19 and 0.5 % of citrate has been studied. Depending on the strain, bacteria produced 1.5 - 1.9 mg of diacetyl, 212 - 311 mg of acetoin and 137 - 156 mg of butylene glycol in 1 1 milk. An increase of the citrate concentration in milk to 0.5 % resulted in an increase in the production of diacetyl from 58 to 74 % and of acetoin by 2.8 - 3.7 times. The strains of distinct activity of acetoin reductase produced in these conditions 2.3 - 2.7 times as much as 2,3-butylene glycol. The recovery of citrate in the from of C₄-compounds ranged from 76 to 98 %, yet barely 0.18 - 0.44 % in the from of diacetyl. Increased concentration of citrate in milk stimulated the production of diacetyl and acetaldehyde to the similar extent, thereby it did not result in the deterioration of organoleptic qualities of starters and milk products. Within the doses used citrate did not significantly affect growth and acidifying activity of the bacteria.     

乳酸乳球菌乳酸亚种产丁二酮的优化研究

分别研究了10种不同因素对乳酸乳球菌乳酸亚种产生丁二酮含量的影响。实验结果表明,各因素产生丁二酮最高量的条件分别为:培养温度为37℃,凝乳后0h,后熟12h,培养基pH调节为5.6,培养基中添加柠檬酸的量为0.15%;培养基中添加Vc的量为0.01%,培养基中添加金属离子的量分别为Mg2+0.03%、Cu2+0.02%、Mn2+0%,培养基中添加甘氨酸的量为0.2%,培养基中添加碳水化合物的量分别为葡萄糖为0%、蔗糖为0%;随着基质浓度增加丁二酮产量也随之增加。     

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。     

Autolysis of Lactococcus lactis

During cheese making, autolysis of Lactococcus lactis starter bacteria affects cheese flavour development through release of intracellular enzymes. The gene for the major autolysin in L. lactis, N-acetyl muramidase (AcmA), has been cloned and sequenced. The activity of AcmA alone, however, does not explain the huge variation in the extent of autolysis found in commercial L. lactis starter strains. Many such strains have multiple cell wall hydrolases that can be seen as different sized clearance bands in zymograms. In addition, the recently completed L. lactis subsp. lactis IL1403 genome sequence shows the presence of several open reading frames that putatively encode cell wall hydrolases having up to 42% predicted amino acid identity to AcmA. These enzymes could have roles in the autolysis of L. lactis. In this paper, we review the literature on autolysis of L. lactis and provide experimental evidence, based on Western blot and zymogram analysis, that commercial L. lactis starter strains express varying levels of AcmA and contain other cell wall hydrolases.     

High-level coproduction of the bacteriocins nisin A and lactococcin A by Lactococcus lactis

In this study, a two-plasmid system for enhanced and consistent biosynthesis of the model lactococcal bacteriocin lactococcin A in non-producing Lactococcus lactis hosts was developed. The system comprised a plasmid carrying the genes lcnA and lciA under the control of the nisin-inducible nisA promoter, and a second plasmid harbouring the lcnC and lcnD genes. The introduction of both plasmids into two strains containing the nisRK genes required for nisin-controlled expression, Lc. lactis FI5876 (a nisin A-producer strain) and FI7847, resulted in production of extracellular lactococcin A at a higher level than that for the parental strain, Lc. lactis WM4. In addition, transformation of the nisin-producing host with both plasmids led to a high-level production of both lactococcal bacteriocins, which may provide a means to exploit their complementary properties in cheese ripening.     

Spray Drying of Lactococcus lactis ssp. lactis C2 and Cellular Injury

Starter cultures were spray-dried at five outlet-air temperatures using four concentrations of cells in the feed solution. Powders made using the lowest outlet-air temperature and the highest cell concentration had the highest viability. Storage at 4°C for 3 mo caused a 34–86% loss of viability. Cellular injury resulted from dehydration, and exposure to high temperatures in the atomizer and during droplet drying. Lactic acid production was similar for frozen, freeze-dried and spray-dried cultures made from a single cell paste. The lag time before lactic acid production was apparently an inherent characteristic of each specific cell paste.     

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.     

Hydroxypropyl β-cyclodextrin improving multiple stresses tolerance of Lactococcus lactis subsp. lactis

L. lactis is known as industrial starter in the fermentation of dairy and meat products, and it plays an important role in human health as an edible probiotic. During industrial production, L. lactis often experiences different stresses that delay the growth and decrease the survival in some serious conditions. In this study, the protective effects of hydroxypropyl β-cyclodextrin (HP β-CD) on L. lactis under multiple stresses were investigated. The microbial cells were treated with different stresses including heat, NaCl, cold, and H₂O₂ stresses, and the results were showed by measuring the OD₆₀₀ or spot plating method. The growth and tolerance were improved when HP β-CD was added during different stress conditions, better than that of trehalose. Besides, the scanning electron microscopic and fluorescence spectrum studies showed that HP β-CD could combine with L. lactis to protect the cell structure, suggesting that HP β-CD may act as a protective agent of L. lactis. Therefore, HP β-CD could be considered as a potential protective agent to be applied in food industry, and its protective mechanism on L. lactis still needs further investigation.     

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。     

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.     

Xylooligosaccharide fermentation with Leuconostoc lactis

Strains of Leuconostoc lactis SHO-47 and Le. lactis SHO-54, producing the clinically useful enzyme NAD-specific 6-phosphoglucanate dehydrogenase, were cultivated with a hydrolyzed birch wood xylan as the unique carbon source to produce D-lactic acid for poly(D-lactic acid). In addition to the strains SHO-47 and SHO-54, Lactococcus lactis IO-1, well known as a good xylose-utilizing lactic acid bacterium, was used as a control to confirm the extent of hemicellulose hydrolysis. The fermentation time for lactic acid of strains SHO-47 and SHO-54 was 12 h, and produced respectively 2.3 and 2.2 g/l lactic acid from 8.5 g/l hydrolyzed xylan, whereas the fermentation time of strain IO-1 was 21 h, and produced 1.3 g/l lactic acid. Xylooligosaccharides from xylobiose to xylohexose were utilized more rapidly than xylose in the cultures of strains SHO-47 and SHO-54. However, xylose concentration increased temporarily and then decreased in the culture of strain IO-1. On the other hand, xylooligosaccharides larger than xyloheptaose were not utilized by these three strains. The xylosidase activities of SHO-47, SHO-54, and IO-1 were induced by xylose or a mixture of xylobiose and xylotriose. The xylosidases of these three strains were localized in their cytoplasm.