Production of pediocin PA-1, and coproduction of nisin A and pediocin PA-1, by wild Lactococcus lactis strains of dairy origin

Heterologous production of pediocin PA-1 in nisin and non-nisin-producing Lactococcus lactis strains, which had been previously selected because of their technological properties for cheese making, was investigated. Plasmid pFI2160, which contains a hybrid gene (L-pedA) encoding the fusion between the lactococcin A leader and propediocin PA-1, and also the genes lcnC and lcnD, that encode the lactococcin A secretion apparatus, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis⁺). The pediocin production level of their respective transformants, L. lactis CL1 and L. lactis CL2 (Nis⁺), was approximately 600 and 400 ng mL⁻¹, respectively, which represents a 30% and a 20% of the quantity produced by the natural pediocin PA-1 producer Pediococcus acidilactici 347. Transformation of L. lactis ESI 515 with pFI2160 did not affect its ability to produce nisin. Pediocin bioassays showed the stability of pFI2160 in both heterologous hosts under selective and non-selective conditions.     

Mode of action of lactococcin R produced by Lactococcus lactis R

We investigated the mode of action and factors affecting adsorption of lactoccocin R produced by Lactococcus lactis R. It was found that lactococcin R adsorbed to all Gram-positive but not to the Gram-negative bacteria tested and its adsorption was dependent on pH. It was observed that the binding of lactococcin R was prevented by anions of several salts (Cl-, PO4(-3)) and lipoteichoic acid. Pretreatments of sensitive cells and cell walls with detergents, organic solvents or enzymes did not reduce subsequent binding of lactococcin R. However, treatment of cell wall preparations with methanol:chloroform and hot 20% trichloroacetic acid (TCA) caused such walls to lose their ability to adsorb lactococcin R. Sensitive cells treated with lactococcin R lost high amounts of intracellular K+ ions, UV-absorbing materials and became more permeable to o-nitrophenol-beta-D-glactopyranoside (ONPG). In addition, different lactococcin R concentrations (0-2560 AU/mL) decreased the colony counts of Listeria monocytogenes by 99% and also a reduction in the absorbance values. These results show that the mode of action of lactococcin R is bactericidal rather than bacteriostatic.     

Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp. lactis IPLA 729, a nisin Z producer

Lactococcus lactis ssp. lactis IPLA 729 is a nisin Z producer isolated from raw milk cheese able to grow and produce nisin Z in milk. The ability of this strain to inhibit the growth of Clostridium tyrobutyricum CECT 4011, a late blowing agent, in Vidiago cheese, a semi-hard farmhouse variety, manufactured in Asturias, Northern Spain, was investigated. For control purposes, cheeses were manufactured with the mesophilic mixed starter IPLA-001. In experimental cheeses, the nisin-producing strain L. lactis IPLA 729 was combined with this starter. Nisin Z activity reached a concentration of 1600 AU/ml in 1-day cheeses and this level was maintained until 15 days of ripening. Furthermore, to compare the inhibitory activity of the nisin-producing strain to nitrate, cheeses were also manufactured with a commercial starter culture and potassium nitrate as anti-blowing agent was added in accordance with Vidiago's cheesemakers. The control, experimental and commercial cheeses were contaminated with C. tyrobutyricum CECT 4011. The composition of the three different cheeses showed only slight differences with respect to total solids, protein and fat, although control and experimental cheeses showed a richer flavour-compound profile than commercial cheeses. The level of the spoilage strain C. tyrobutyricum CECT 4011 decreased from 1.2x10(6) to 1.3x10(3) cfu/g during ripening in presence of the nisin Z producer, while it increased to 1.99x10(9) cfu/g in control cheeses and to 3.5x10(7) cfu/g in commercial cheeses.     

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.     

Inhibition of a methicillin-resistant Staphylococcus aureus strain in Afuega'l Pitu cheese by the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729

Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently. This article focuses on the inhibitory effect of the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain. S. aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain. A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period. In milk, viable counts of S. aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 x 10(4) and 7.2 x 10(6) CFU/ml). The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.     

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).     

Robustness of cascade pH and dissolved oxygen control in symbiotic nisin production process system of Lactococcus lactis and Kluyveromyces marxianus

In symbiotic processes, different organisms coexist stably and interact by sharing the same metabolites and environmental conditions. The robustness of a symbiotic nisin production process system composed of the lactic acid bacterium Lactococcus lactis subsp. lactis (ATCC11454) and dairy yeast Kluyveromyces marxianus (MS1) was studied. It was found that this symbiotic process system was robust to the initial disturbance in the inoculum sizes of both microorganisms and pH.     

Preliminary characterization of bacteriocins from Lactococcus lactis,Enterococcus faecium and Enterococcus mundtii strains isolated from turbot (Psetta maxima)

The aim of this work was the characterization of new strains of lactic acid bacteria (LAB) from farmed fish and with potential application as biopreservatives against both Listeria monocytogenes and Staphylococcus aureus. Twenty-five strains of LAB isolated from the muscle of farmed turbot were investigated. Genetic identification of the bacteriocin-producing LAB strains was performed by means of a PCR method using novel BAL1/BAL2 16S ribosomal-RNA-targeted primers. Maximum bacteriocin production by Lactococcus lactis ssp. lactis USC-39, Enterococcus faecium USC-46 and Enterococcus mundtii USC-51 was detected in the stationary phase of growth. Both acidification and the production of hydrogen peroxide by LAB were ruled out as the source of the inhibition. In contrast, the antimicrobial activity of all three LAB strains was inactivated by the addition of proteinase K, thus confirming the proteinaceous nature of the inhibition. The activity against L. monocytogenes was maintained in the 3.5–5.5 or 3.5–6.5 pH range, depending on the LAB strain. Likewise, inhibition of S. aureus strains was observed in the 3.5–4.5 and in the 3.5–5.5 pH ranges, depending on the LAB strain and on the S. aureus strain tested. Bacteriocin activity was stable in all three strains after heating the cell-free extract for 60 min at 100 °C, or even for 15 min at 121 °C, in all the three LAB strains. The acidic and heat-resistant bacteriocins produced by the three LAB strains isolated from turbot, able to inhibit the growth of both L. monocytogenes and S. aureus may find application as biopreservatives in fermented and/or heated food products.     

乳酸乳球菌与酵母菌混合培养时菌间的相互影响

研究了干酪常用发酵剂——乳酸乳球菌与干酪中两株常见酵母菌——耶罗解脂酵母及汉逊德巴利酵母混合培养时的相互影响.在营养肉汤中对三株菌单独及混合培养,比浊法测定其生长浓度,同时在脱脂乳培养基中做相同培养,然后检测活菌数、球菌产酸及菌株自溶情况.结果表明:乳酸乳球菌、耶罗解脂酵母及汉逊德巴利酵母三株菌混合培养时其菌数及自溶度,均有显著增加,表明混合培养加快了菌株生长代谢的速率.这对研究干酪促熟及风味调整具有重要意义.     

Amino acid catabolism by Lactococcus lactis during milk fermentation

Two wild-type Lactococcus lactis strains isolated from naturally Tunisian fermented milk (Leben), and one laboratory strain, were used to investigate the ability of L. lactis to transform amino acids into aroma compounds during milk fermentation. The α-ketoacid acceptor used for leucine transamination, the first step of catabolism, was identified by gas chromatography/mass spectrometry analysis of the ¹⁵N-labelled amino acids that formed from ¹⁵N-labelled leucine in fermented milk. Furthermore, the amino acids produced or catabolized by the laboratory strain via transamination were identified by comparing the free amino acids in milk fermented with the wild-type strain and the double mutant for aromatic and branched-chain aminotransferases, which cannot transaminate amino acids. The three L. lactis strains strongly catabolized leucine and valine during milk fermentation. The principal amino acid formed via leucine and valine transamination was glutamate indicating that α-ketoglutarate was the principal α-ketoacid acceptor and was generated during milk fermentation.     

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%.     

Fluorescence assessment of Lactococcus lactis viability

The reproduction and activity of lactic acid bacteria (LAB) are essential in their applications in the dairy industry and other fermentations. Traditionally used methods like plate counting and acidification tests require long incubation times and provide limited information. Fluorescence techniques provide possibilities for rapid assessment of cell physiology. We used traditional and fluorescence assays to assess the physiological condition of L. lactis subsp. lactis ML3 cultures that were exposed to various stress conditions. After exposure to some of the stress conditions, carboxyfluorescein (cF) labelling did not agree with plate counts. Therefore, a two-step method was developed in which cF labelling was followed by a lactose-energized efflux assay. The combined assay proved to be a good and rapid indicator for reproduction and acidification capacity of stressed L. lactis. This novel assay has potential for physiological research and dairy applications related to LAB.     

Improved viability of bifidobacteria in fermented milk by cocultivation with Lactococcus lactis subspecies lactis

The poor survival of probiotic bacteria in commercial yogurts may limit their potential to exert health benefits in humans. The objective was to improve the survival of bifidobacteria in fermented milk. Cocultivation with some strains of Lactococcus lactis ssp. lactis improved the survival of bifidobacteria in fermented milk during refrigerated storage. Studies on one strain, Lc. lactis ssp. lactis MCC866, showed that the concentrations of dissolved oxygen were kept lower in the cocultivated fermented milk during storage compared with monocultured Bifidobacterium longum BB536 or samples cocultured with another noneffective Lc. lactis ssp. lactis strain. Degradation of genomic DNA was suppressed in the cocultivating system with Lc. lactis ssp. lactis MCC866. Several genes that participated in protection from active oxygen species (e.g., genes coding for alkyl hydroperoxide reductase and Fe²⁺ transport system) were expressed at higher levels during refrigerated storage in Lc. lactis ssp. lactis MCC 866 compared with another noneffective Lc. lactis ssp. lactis strain. Concentration of free iron ion was also lower in supernatants of fermented milk cocultivated with B. longum BB536 and Lc. lactis ssp. lactis MCC866. These results suggest that Lc. lactis ssp. lactis MCC 866 is potentially superior in reducing oxygen damage and consequently improves the survival of bifidobacteria in the cocultivating system. This cocultivation system is of industrial interest for producing fermented milk containing viable bifidobacteria with long shelf life.     

产胞外多糖乳球菌的优化培养

以苯酚硫酸法测定乳酸乳球菌乳酸亚种WH-C1在不同培养基中的胞外多糖合成量,试验以WHEY作为基础培养基,比较了添加不同碳源、氮源对EPS合成的影响,并采用正交实验L9（3^4）对碳源、氮源的添加量及培养基初始pH进行了优化,确定最佳组合为：葡萄糖添加量15g／L,胰蛋白胨添加量5g／L,培养基初始pH值为6．5。研究了不同培养温度和接种量条件下该菌株的EPS合成曲线,结果表明温度对发酵液中EPS总积累量的影响较大,通过本研究确定了乳酸乳球菌乳酸亚种WH-C1最佳培养条件为：培养温度25℃,接种量为2％（体积分数）,培养时间32h,EPs合成量为325．8mg／L。     

乳酸乳球菌高效电转化的方法

以质粒pMG36e和pNZ8148为材料,对乳酸乳球ATCC19435的电转化条件进行了优化研究.实验结果表明,乳酸乳球菌的最佳电转化条件为对数生长中后期的细胞,电场强度12 kV/cm,质粒浓度0.1～1 mg/L,复苏时间2 h,此时质粒pMG36e和pNZ8148时供试菌株的电转化效率分别为1.95x105μg-1和2.18x105μg-1,比优化前电转化效率均提高10倍以上.本研究为外源基因电转化乳酸乳球菌提供了可靠的试验参数依据.     

PCR Identification of Lysogenic Lactococcus lactis Strains

Lactococcus lactis plays an essential role as a starter in the dairy industry. Unfortunately this species is susceptible to bacteriophage infections that result in fermentation failures and subsequent economic losses. This paper reports a PCR screening method that detects prophages in the genomes of wild lactococcal strains isolated from Cabrales cheese, a traditional, Spanish, blue-veined cheese, which is not inoculated. Approximately, one sixth of the samples tested carried a prophage in the genome. PCR positive strains were treated with mitomycin C to induce the lytic cycle and to determine the viability of the detected prophages. 66.6% of the PCR-positive strains lysed after induction, showing the threat these viruses pose to the dairy industry. The results of this work show that PCR could be routinely used as a rapid screening method for the detection of lysogenic Lactococcus strains.

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Zusammenfassung (Redaktion). Für die Milch-verarbeitende Industrie ist Lactococcus lactis als Starter-Kultur von besonderer Bedeutung. Da L. lactis von Bacteriophagen infiziert werden kann, ist es m?glich, dass dann die gewünschte Fermentation der Milch-Produkte ausbleibt und somit ein wirtschaftlicher Schaden eintritt. Daher wurde ein PCR-Screening-Verfahren entwickelt, um das Vorhandensein von Prophagen im Genom von L. lactis frühzeitig nachweisen zu k?nnen. Mit diesem Verfahren wurden Wildst?mme von L. lactis untersucht, die aus „ „Cabrales“-K?se isoliert worden waren. Bei etwa einem Fünftel der Wildst?mme wurden Prophagen im Genom von L. lactis nachgewiesen; 66,6% dieser Wildst?mme konnten zur Zell-Lyse induziert werden.

     

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.     

高原酸奶中乳酸链球菌产生的乳酸链球菌素纯化鉴定研究

目的:对从高原酸奶中提取的乳酸链球菌产生的乳酸链球菌素进行纯化及鉴定。方法:采用分离、凝胶层析、抑菌实验、高效液相色谱法来纯化乳酸链球菌素,用凝胶电泳来鉴定其纯度,确定其分子量。结果:生产的乳酸链球菌素具有抑菌性,将该抑菌成分与乳酸链球菌素标准品进行SDS-PAGE凝胶电泳,通过比较电泳图谱一致。结论:本实验得到的抑菌成分即为乳酸链球菌素,且纯度高,分子量大约为3300u。     

Fluorescent labelling negatively affects the physiology of Lactococcus lactis

Culturability on plates, growth behaviour in liquid media and acidification activity of three Lactococcus lactis strains was negatively affected by carboxyfluorescein diacetate (cFDA), the corresponding succinimidyl ester cFDA-SE, propidium iodide (PI) and TOTO-1 iodide. Single staining with the vitality dye cFDA-SE decreased the reproductive capability and acidification activity of L. lactis cells more than cFDA. Since TOTO-1 proved to have a higher toxicity than PI, the application of PI was favoured over TOTO-1 as counterstain for cells labelled with one of the vitality dyes. The overall extent to which double staining with cFDA/cFDA-SE and PI impaired bacterial physiology was determined by the dye with the greater influence on L. lactis cells during single staining. The observed strain-dependent differences in sensitivity highlight the importance of studying the impact of fluorescent labelling on cell physiology before using the dyes in combination with flow cytometric cell sorting for physiological characterisation of subpopulations.     

The effect of lactose, NaCl and an aero/anaerobic environment on the tyrosine decarboxylase activity of Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis

The aim of this work was to study, under model conditions, combined effects of the concentration of lactose (0-1% w/v), NaCl (0-2% w/v) and aero/anaerobiosis on the growth and tyramine production in 3 strains of Lactococcus lactis subsp. lactis and 2 strains of L. lactis subsp. cremoris. The levels of the factors tested were chosen with respect to the conditions which can occur during the real process of natural cheese production, including the culture temperature (10 ± 1 °C). In all strains tested, tyrosine decarboxylation was most influenced by NaCl concentration; the highest production of tyramine was obtained within the culture with the highest (2% w/v) salt concentration applied. Two of the strains L. lactis subsp. lactis produced tyramine only in broth with the highest NaCl concentration tested. In the remaining 3 strains of L. lactis, tyramine was detected under all conditions applied. The tested concentration of lactose and aero/anaerobiosis had a less significant effect on tyramine decarboxylation. However, it was also found that at the same concentrations of NaCl and lactose, a higher amount of tyramine was detected under anaerobic conditions. In all strains tested, tyramine decarboxylation started during the active growth phase of the cells.