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.     

Characterization of four new Lactococcus lactis bacteriophages isolated from dahi whey

Four lytic phages of Lactococcus lactis ssp. diacetylactis isolated from indigenous dahi whey were examined for their stability, growth characteristics and morphology. All these phages were partially inactivated by CHCl₃ , remained stable at 40°C and were partially inactivated at pH 3. There was a marked difference among these phages with respect to latent period, rise period and burst size. All phages belonged to Bradley's group B.     

Lactococcus lactis phages from the perspective of their diversity,thermal and biocidal resistance

Lactic acid bacteria (LAB), particularly Lactococcus lactis, are of great significance in dairy fermentations. Many LAB strains are susceptible to attack by phages that affect their technological, biochemical and physiological functions. Phages of L. lactis are a serious concern because of the economic importance of this bacterium in the dairy industry. Members of L. lactis phages belonging to the P335, 936 and c2 groups are more problematic for the dairy industry. Many phages of the 936 group are resistant to various thermal and biocidal treatments commonly used in the dairy industry. This article reviews the diversity of L. lactis bacteriophages of the P335, 936 and c2 groups and discusses their interaction with their bacterial hosts. In addition, this review provides an overview of the resistance of L. lactis phages to thermal treatments and chemical biocides, and highlights some novel strategies to destroy these phages.     

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.     

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.     

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。     

Immunomodulatory action of Lactococcus lactis

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高产丁二酮乳球菌的选育及发酵条件优化

从收集到的6株乳酸菌中筛选得到一株高产丁二酮菌株DX。经过16S rDNA测序和构建系统发育树,结果表明菌株DX的16S rDNA基因序列同Lactococcus lactis的同源性为99%,综合系统发育树结果表明该菌株为乳酸乳球菌。通过单因素试验和中心组合试验确定菌株DX的最佳发酵条件为：脱脂乳质量浓度10g/100mL、接种量2%、发酵培养基初始pH6.5、培养温度37℃、静置培养,丁二酮产量达到22.383mg/L。     

Data mining and fuzzy modelling of high pressure inactivation pathways of Lactococcus lactis

A mathematical model to predict lethal and sublethal pressure effects on Lactococcus lactic was established through combined use of Principal Component Analysis (PCA) and Fuzzy Logic. The model was based on data comprising pressure inactivation kinetics with 64 combinations of the parameters pressure, temperature, pH, and buffer composition, samples were analysed with respect to 5 physiological states describing lethal or sublethal injury of L. lactis, i.e. viable cell counts (CFU), undamaged cell counts (CFUsub), membrane integrity (MI), metabolic activity (MA) and the activity of the membrane bound enzyme LmrP (LmrP).Correlations found by PCA were used to generate a bi-layer fuzzy model using clustering methods and rule oriented statistical analysis as well as the physiological states CFU and LmrP as autonomous output variables. The result of these variables is used to accurately predict dependent output variables MA, CFUsub, and MI taking into account the combined effects of the inactivation process.Industrial relevanceThe study provides a predictive model for the inactivation of an industrially relevant micro-organism. Predictive models are useful for process design on the industrial scale. Further, our model provides information about intermediate steps of high pressure inactivation.     

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.     

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.     

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.     

Use of waste materials for Lactococcus lactis development

BACKGROUND: Lactococcus lactis is an interesting microorganism with several industrial applications, particularly in the food industry. As well as being a probiotic species, L. lactis produces several metabolites with interesting properties, such as lactic acid (LA) and biosurfactants. Nevertheless, L. lactis is an especially demanding species since it has strong nutritional requirements, implying the use of complex and expensive culture media. RESULTS: The results showed the potential of L. lactis CECT‐4434 as a LA and biosurfactant producer. The economical cost of L. lactis cultures can be reduced by replacing the MRS medium by the use of two waste materials: trimming vine shoots as C source, and 20 g L^?1 distilled wine lees (vinasses) as N, P and micronutrient sources. From the hemicellulosic fraction, 14.3 g L^?1 LA and 1.7 mg L^?1 surfactin equivalent were achieved after 74 h (surface tension reduction of 14.4 mN m^?1); meanwhile, a simultaneous saccharification and fermentation process allowed the generation of 10.8 g L^?1 LA and 1.5 mg L^?1 surfactin equivalent after 72 h, reducing the surface tension by 12.1 units at the end of fermentation. CONCLUSIONS: Trimming vine shoots and vinasses can be used as alternative economical media for LA and cell‐bound biosurfactant production. Copyright © 2010 Society of Chemical Industry     

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。