Biodiversity of bacteriophages infecting Lactococcus lactis starter cultures

In the current study, we characterized 137 Lactococcus lactis bacteriophages that had been isolated between 1997 and 2012 from whey samples obtained from industrial facilities located in 16 countries. Multiplex PCR grouping of these 137 phage isolates revealed that the majority (61.31%) belonged to the 936 group, with the remainder belonging to the P335 and c2 groups (23.36 and 15.33%, respectively). Restriction profile analysis of phage genomic DNA indicated a high degree of genetic diversity within this phage collection. Furthermore, based on a host-range survey of the phage collection using 113 dairy starter strains, we showed that the c2-group isolates exhibited a broader host range than isolates of the 936 and P335 groups.     

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.     

Screening for natural defence mechanisms of Lactococcus lactis strains isolated from traditional starter cultures

Three different bacterial defence mechanisms were identified in the seventeen Lactococcus lactis isolates from starter cultures in three Slovenian dairy plants. Isolates MB18, KR7, PT4, PT13 and PT19 inhibited phage adsorption by means of exopolysaccharides production. The most extensive polysaccharides production was detected in PT19 isolate, which was susceptible only to phage ΦPT19. Eight isolates exhibited nuclease activity, and seven of them were susceptible up to four phages out of thirteen from our collection. Eight isolates possessed the abiB gene, fourteen isolates abiH, two isolates abiJ and one isolate abiQ. Isolates PT27 and PT28 possessed AbiB, AbiH and AbiJ mechanisms as well as inhibition of phage adsorption. Isolate MB18, which was susceptible to one phage only, possessed the abiQ gene, nuclease activity and ability to prevent adsorption of most phages. Isolates PT67 and PT70, possessing only AbiH mechanism, were susceptible to only two phages.     

Kinetics of the thermal inactivation of the Lactococcus lactis bacteriophage P008

The thermal resistance of the lactococcal bacteriophage P008 was investigated between 55 and 80 degrees C. Inactivation kinetics revealed an order of reaction above 1 and could be determined by a non-1st-order regression model. Phage inactivation was influenced by the medium (milk and Ca-M17-broth). Within the investigated temperature range, milk had a protective effect on phage P008. This was reflected in the rate constant and in the activation energy. Thermal phage inactivation studies reported in literature were re-analysed using non-1st-order regression. The obtained kinetic parameters showed that phage P008 belongs to the most heat resistant lactococcal phages investigated so far.     

Effectiveness of thermal treatments and biocides in the inactivation of Argentinian Lactococcus lactis phages

The thermal and chemical resistance levels of four autochthonal bacteriophages of Lactococcus lactis subsp. lactis, isolated from cheese processes, was investigated. The times required to obtain 99% inactivation of phages (T99) at 63 and 72 degrees C in three suspension media (M17 broth, reconstituted commercial nonfat skim milk, and Tris magnesium gelatin buffer) were determined. Thermal resistance was dependent on the phage studied, and the results of this study demonstrate that pasteurization treatments used in dairy industries may leave viable viral particles in milk. It was possible to determine that M17 broth was generally the least protective medium, while phosphate buffer was the most protective one. Peracetic acid (0.15%, vol/vol) was the most effective viricidal agent, with exposures of 5 min being sufficient to inactivate high-titer phage suspensions (>10(6) PFU/ml). To achieve total inactivation (<10 PFU/ml) of viral suspensions, sodium hypochlorite was effective at 100 ppm for only two phages, while the other two phages needed concentrations of 200 and 300 ppm. Ethanol at concentrations of 100 and 75% proved to be very efficient in inactivating phages, but isopropanol was not effective against them.     

具有溶栓活性的重组乳酸乳球菌的构建

利用PCR方法从纳豆芽孢杆菌(Bacillus subtilis subsp.natto)基因组DNA中扩增纳豆激酶原基因,构建了纳豆激酶原基因的表达载体pFY002,转化Lactococcus lactis NZ9000,得到活性表达纳豆激酶的重组菌Lactococcus lactis NZ9000/pFY002。利用纤维蛋白平板法检测其溶栓活性,并对诱导剂乳链菌肽(NisinA)的浓度、温度、诱导时间对重组菌产酶的影响进行探究。     

DNA sequence analysis of three Lactococcus lactis plasmids encoding phage resistance mechanisms

The three Lactococcus lactis plasmids pSRQ700, pSRQ800, and pSRQ900 encode the previously described anti-phage resistance mechanisms LlaDCHI, AbiK, and AbiQ, respectively. Since these plasmids are likely to be introduced into industrial Lactococcus lactis strains used to manufacture commercial fermented dairy products, their complete DNA sequences were determined and analyzed. The plasmids pSRQ700 (7784 bp), pSRQ800 (7858 bp), and pSRQ900 (10,836 bp) showed a similar genetic organization including a common lactococcal theta-type replicon. A second replication module showing features of the pMV158 family of rolling circle replicons was also found on pSRQ700. The theta replication regions of the three plasmids were associated with two additional coding regions, one of which encodes for HsdS, the specificity subunit of the type I restriction/modification system. When introduced into L. lactis IL1403, the HsdS of pSRQ800 and pSRQ900 conferred a weak resistance against phage P008 (936 species). These results indicated that both HsdS subunits can complement the chromosomally encoded type I restriction/modification system in IL1403. The genes involved in the phage resistance systems LlaDCHI, AbiK, and AbiQ were found in close proximity to and downstream of the replication modules. In pSRQ800 and pSRQ900, transfer origins and putative tyrosine recombinases were found upstream of the theta replicons. Genes encoding recombination proteins were also found on pSRQ700. Finally, open reading frames associated with bacteriocin production were found on pSRQ900, but no anti-lactococcal activity was detected. Based on our current knowledge, these three plasmids are safe and suitable for food-grade applications.     

Fermented soymilk with a monoculture of Lactococcus lactis

Lactococcus lactis strain (LL3) isolated from mothers' milk was used to produce fermented soymilk. The strain survived at levels of over 7 log cfu/ml for 3 weeks in the fermented soymilk. A consumer survey was carried out to compare the acceptability of the fermented product with a similar product made with L. lactis ATCC11545 originally isolated from cow's milk. Blind samples produced by fermentation with the two strains were rated equally attractive, whereas information on the origin of the strains significantly enhanced the pleasantness of the fermented soymilk.     

Purification and characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis H-559 isolated from kimchi

Lactic acid bacteria were isolated from kimchi and screened for bacteriocin production. Strain H-559, identified as Lactococcus lactis subsp. lactis, exhibited the strongest antibacterial activity among them and was active against pathogenic bacteria such as Listeria monocytogenes and Staphylococcus aureus as well as many lactic acid bacteria. The antimicrobial substance produced by L. lactis subsp. lactis H-559 was inactivated by alpha-chymotrypsin, and protease type IX and XIV and was confirmed to be a bacteriocin. The bacteriocin activity was stable from pH 2.0-11.0 and up to 10 min heating at 100 degrees C. The bacteriocin was sequentially purified by ammonium sulfate precipitation, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography (HPLC). Its molecular weight was determined to be 3343.7 Da by MALDI-mass spectrometry. Isoleucine was detected as the first N-terminal amino acid residue but the remaining amino acid sequence could not be determined by the Edman degradation method. It was different from other bacteriocins in terms of pH stability, molecular weight, amino acid composition, and the partial amino acid sequences of peptides obtained by acid hydrolysis.     

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.     

Identification and characterization of two bacteriocin-producing strains of Lactococcus lactis isolated from vegetables.

Isolated from mixed salad and fermented carrots, 123 strains of lactic acid bacteria were screened for bacteriocin production. Two strains, D53 and 23, identified as Lactococcus lactis by DNA-DNA hybridizations, produced heat stable bacteriocins which were resistant to trypsin and pepsin, but were inactivated by alpha-chymotrypsin and proteinase K. The bacteriocins were active from pH 2 to 9 and inhibited species of Listeria, Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Carnobacterium, Bacillus and Staphylococcus. Strain D53 produced bacteriocin at pH values of 4.5-8.0 and from 10 to 37 degrees C.     

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.     

Effects of high pressure treatment on glycolytic enzymes of Lactococcus lactis subsp. lactis,Streptococcus thermophilus and Lactobacillus acidophilus

High pressure processing (HPP) reduces the glycolytic activity of lactic acid bacteria (LAB) and provides a means to control further production of acidic metabolites in fermented dairy products during storage. However, there is limited information on the effects of HPP on specific enzymes of dairy starter bacteria responsible for the metabolism of lactose. The aim of this study was to determine pressure-induced inactivation of glycolytic enzymes in Lactococcus lactis subsp. lactis C10, Streptococcus thermophilus TS1 and Lactobacillus acidophilus 2400. Cultures were grown for 16 h in M17 or MRS broth containing 5% (w/v) lactose at pH 6.5 (maintained by addition of 10 M NaOH). The cells were harvested by centrifugation, washed and resuspended in 100 mM phosphate buffer (pH 6.5) and pressure-treated at 300 and 600 MPa (≤ 22 °C, 5 min). The ability of pressure-treated resting cells of Lactococcus, incubated with 5% (w/v) lactose at 30 °C, to ferment lactose was evaluated by determining titratable acidity (TA) during incubation. The activities of phospho-β-galactosidase (P-β-gal), β-galactosidase (β-gal) and lactate dehydrogenase (LDH) were determined in cell-free extracts of untreated and pressure-treated cells. Resting cells of Lactococcus treated at 600 MPa had a substantially lower rate of acidification than the controls and those treated at 300 MPa. Both P-β-gal and β-gal were significantly inactivated (p < 0.01) in the starter cultures treated at 300 or 600 MPa. The LDH in Lactococcus and Lactobacillus was highly resistant to pressure treatment at 300 MPa. In contrast, the LDH in Streptococcus was almost completely inactivated at ≥ 300 MPa.Industrial relevanceContinuing production of acidic metabolites in fermented dairy products during storage can be a technological challenge that adversely affects product quality. The current study demonstrates that high pressure processing (HPP) offers the potential of controlling this problem by inactivation of glycolytic enzymes in various mesophilic and thermophilic starter cultures. The findings of this research will assist in establishing optimised operating parameters for HPP treatment of cultured products to extend shelf-life, by reducing acid production during storage.     

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%.     

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).     

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.     

Biological characteristics of luminescent Lactococcus lactis transformed with lux genes

Until now there has been no effective method to measure the adhesion of probiotics to the intestinal tract because it is very difficult to determine the ability of cells to attach to intestinal epithelium in vivo. Methods for construction of luminescent Lactococcus sp. transformed with lux genes and the biological characteristics of these transformants were investigated so as to lay a foundation for in vivo studies of the distribution and survival of probiotics, and their mechanisms to benefit human health. The results showed that the plasmid pMG36e is a suitable vector for expressing lux genes derived from a gram negative bacterium in the gram positive bacterium, Lactococcus lactis. Experiments revealed that luxAB genes in pMG36e can be inherited stably in Lactococcus even without selective pressure. The introduction of lux genes into Lactococcus did not affect its growth or acid production. Luminescence was affected by all factors tested, including lux gene size, media components and pH.     

Subspecies-Specific Nested PCR Assay for Detection of Lactococcus lactis spp. lactis and spp. cremoris

A nested PCR-based assay composed of Lactococcus lactis species-specific primers for the nest 1 amplification and subspecies-specific primers for the nest 2 amplification was validated with the identified strains of L. lactis isolated from dairy and nondairy sources and positive and negative control strains. Forward and reverse primer set was designed for nest 1 amplification targeting the conserved housekeeping gene yueF encoding nonproteolytic protein from peptidase family M16 of L. lactis. Amplicons of 447 bp of yueF were subjected for nest 2 amplification producing amplicons of 372 bp. The designed outer primer set for nest 1 amplification was observed to be specific to L. lactis because the DNA from other bacteria could not be amplified and the inner primer set for nest 2 amplification was found to be specific for the detection of ssp. lactis and cremoris of L. lactis.     

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。