Immunity gene pedB enhances production of pediocin PA-1 in naturally resistant Lactococcus lactis strains

Heterologous production of the antilisterial bacteriocin pediocin PA-1 in lactococci is an attractive objective to increase the safety of dairy products. In a previous paper, we developed a system for the heterologous production of the bacteriocin pediocin PA-1 in pediocin-resistant lactococcal hosts through a leader exchange strategy. The system was based on 3 genes, 1 encoding the fusion between the lactococcin A leader and propediocin PA-1, and the other 2 encoding the lactococcin A secretion machinery. In this study, we investigated whether the addition of the pediocin PA-1 immunity gene (pedB) to this system has any effect on pediocin production. Introduction of the plasmid(s) carrying the genes described above into nisinproducing and non-nisinproducing lactococcal hosts led to a significant increase in the production of pediocin compared with the equivalent pedB-devoid systems. In addition, we obtained a nisin-producing strain with the ability to secrete pediocin PA-1 at a level equivalent to that of the parental strain Pediococcus acidilactici 347, which represents a notable improvement over our previous systems.     

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.     

Enhanced production of pediocin PA-1 in wild nisin- and non-nisin-producing Lactococcus lactis strains of dairy origin

In this work, heterologous production of pediocin PA-1 in Lactococcus lactis ESI 153 and ESI 515 (Nis⁺), two strains selected because of their technological properties for cheesemaking, was achieved after transformation with plasmids pMC117, pRK119 and pCNC1, which contain the complete pediocin operon under the control of the strong P32 promoter. The pediocin production of the L. lactis ESI 153 derivatives containing pRK119 or pCNC1 was higher (approximately 165%) than that achieved by the natural pediocin PA-1 producer Pediococcus acidilactici 347. In the case of the L. lactis ESI 515 derivatives, those containing pRK119 or pCNC1 showed a pediocin production level similar (95–100%) to that of P. acidilactici 347.     

A food-grade system for production of pediocin PA-1 in nisin-producing and non-nisin-producing Lactococcus lactis strains: application to inhibit Listeria growth in a cheese model system

Food-grade heterologous production of pediocin PA-1 in nisin-producing and non-nisin-producing Lactococcus lactis strains, previously selected because of their technological properties for cheese making, was achieved. Plasmid pGA1, which contains the complete pediocin operon under the control of the strong P32 promoter and is devoid of any antibiotic marker, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis+). Transformation of L. lactis ESI 515 with pGA1 did not affect its ability to produce nisin. The antimicrobial activity of the pediocin-producing transformants on the survival of Listeria innocua SA1 during cheese ripening was also investigated. Cheeses were manufactured from milk inoculated with 1% of the lactic culture and with or without approximately 4 log CFU/ml of the Listeria strain. L. lactis ESI 153, L. lactis ESI 515, and their transformants (L. lactis GA1 and GA2, respectively) were used as starter cultures. At the end of the ripening period, counts of L. innocua in cheeses made with the bacteriocin-producing lactococcal strains were below 50 CFU/g in the L. lactis GA1 cheeses and below 25 CFU/g in the L. lactis GA2 ones, compared with 3.7 million CFU/g for the controls without nisin or pediocin production.     

Expression and purification of a fusion-typed pediocin PA-1 in Escherichia coli and recovery of biologically active pediocin PA-1

Pediocin PA-1 is a representative class IIa bacteriocin which is small and heat-stable and has a consensus motif, -YGNGV-. The plasmid pQE40PED, encoding pediocin PA-1 fused with His-tagged mouse dihydrofolate reductase (DHFR), was constructed and introduced into Escherichia coli M15 strain. The fusion protein was overexpressed in the strain after induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. For the recovery of biologically active pediocin PA-1, the purified fusion protein was cleaved by Factor Xa protease and the liberated pediocin PA-1 was finally purified by ultrafiltration with a 75% yield. The molecular mass of the purified recombinant pediocin PA-1 was the same as that of native pediocin PA-1 on an electrophoresis gel.     

Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria

The present work compares, under the same stated experimental conditions, the antimicrobial activity of crude and purified enterocin L50, pediocin PA-1, nisin A and lactocin S, produced by lactic acid bacteria (LAB) isolated from Spanish dry-fermented sausages. The bacteriocins were purified to homogeneity by ammonium sulphate precipitation, gel filtration (for lactocin S), and cation-exchange, hydrophobic-interaction, and reverse-phase-chromatography; high yields of pure bacteriocins were obtained. Minimal inhibitory concentration (MIC) of pure enterocin L50, pediocin PA-1, nisin A and lactocin S was determined against a broad spectrum of Gram-positive bacteria, including spoilage and foodborne pathogenic bacteria. The purified bacteriocins showed a broad antimicrobial spectrum similar to that exerted by crude bacteriocins. Enterocin L50 and pediocin PA-1 were very active againstListeria monocytogenes, which was quite resistant to nisin A and lactocin S. Enterocin L50 also displayed antimicrobial activity againstStaphylococcus aureus,Clostridium perfringensandClostridium botulinum. However, these pathogens were weakly inhibited, or not at all, by the other pure bacteriocins.     

重组抗冻肽在乳酸乳球菌中表达及其抗冻活性研究

为了优化抗冻蛋白SF-P的重组表达,利用乳酸链球菌素Nisin对已构建的重组乳酸乳球菌进行表达。通过对诱导时间、诱导pH、诱导温度和诱导剂Nisin浓度等诱导表达条件进行优化,利用SDS-PAGE和Western blot确定最佳的表达条件;通过比较冷冻胁迫前后菌体的生长状况、发酵活力和细胞内钠钾离子含量的变化,研究重组菌在冷冻胁迫作用下的生理功能特性。结果表明:优化后的最佳表达条件为pH 7.0,诱导剂Nisin浓度15ng/mL,温度25℃,诱导时间6h;SF-P2诱导重组菌株可以显著改善乳酸菌由于经受冷冻胁迫导致的对数生长延滞期和稳定生长延滞期的增加,表现出较强的酸化活力,并且可以有效降低冷冻胁迫过程对乳酸菌细胞膜通透性的影响,起到保护细胞生理功能的作用,表明SF-P2诱导重组菌具有显著的抗冷冻胁迫保护作用。     

Proteolytic activities,peptide utilization and oligopeptide transport systems of wild Lactococcus lactis strains

The increasing demand for tasty dairy products has raised the interest for strains of lactic acid bacteria with novel properties. In this study we have explored the proteolytic system of 24 wild Lactococcus lactis strains isolated previously from Spanish raw milk cheeses, to select proteolytic strains with high peptidase activity and efficient peptide transport capability. Large variations in overall proteolysis, proteolytic activities, as well as in peptide utilization, were recorded among strains. Peptide utilization correlated well with the presence of oligopeptide transport systems (Opp and Dpp) in wild L. lactis strains. Eighteen of the 24 wild L. lactis strains possessed both Opp and Dpp systems, what supports the theory that the presence of both oligopeptide transport systems could confer an advantage to the strain. Differences in the nucleotide sequence of genes coding for the oligopeptide binding proteins were shown by means of restriction endonuclease analyses. Based on the characteristics determined in this work, L. lactis strains ESI197, M21 and P21 seem to be promising candidates for use as components of starter cultures.     

Heterologous extracellular production of enterocin P in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by a food-grade expression system

In order to develop an entirely food-grade enterocin P expression system for the food industry, the enterocin P structural gene (entP) with or without the enterocin P immunity gene (entiP) was cloned in plasmid pLEB590 under control of the lactococcal constitutive promoter P₄₅. Introduction of the recombinant vectors in L. lactis MG1614 resulted in production of biologically active enterocin P in the supernatants of recombinant L. lactis MG1614. Moreover, coexpression of the entP and entiP genes could increase the production of enterocin P in all L. lactis MG1614 hosts. Recombinant enterocin P from L. lactis MG1614 (pLEB590-entP2) was purified by a three-step procedure involving ammonium sulfate precipitation, SP-Sepharose Fast Flow cation exchange, and hydrophobic adsorption chromatography. The purified bacteriocin protein concentration from recombinant L. lactis MG1614 (pLEB590-entP2) was 3.9-fold greater than that of E. faecium LM-2, and the final recovery of enterocin P activity from the supernatant of L. lactis MG1614 (40.2%) was dramatically improved compared with that of the native host strain (19.9%). Bacteriocin activity and Tricine-SDS–PAGE analysis revealed that purified recombinant enterocin P is biologically active and has a molecular mass corresponding to the native enterocin P from E. faecium LM-2, suggesting that the synthesis, process, and secretion of enterocin P progresses efficiently in recombinant L. lactis MG1614 hosts. The enterocin P was expressed successfully in this food-grade system.     

Thermal inactivation of the heat-resistant Lactococcus lactis bacteriophage P680 in modern cheese processing

The thermal inactivation of the highly thermo-resistant test phage (P680), was investigated in whey, whey protein concentrate (WPC) and whey cream. After a heat treatment at 90 °C for 15 min, only a 6-log reduction was obtained and the phage was still detectable in each medium. Kinetic parameters for the inactivation of the phage were calculated for temperatures ranging from 70 to 90 °C using a non-linear model. With the help of the parameters obtained, the lines of equal effects showing a 9-log inactivation of the phage were calculated. High temperature short time pasteurization was not sufficient for 9-log inactivation of phage P680 in skim milk, whey, WPC or whey cream. Temperature and time combinations ranging from 100 °C for 20 min to 140 °C for 2 s are necessary for a 9-log inactivation of P680.     

Arginine metabolism in sugar deprived Lactococcus lactis enhances survival and cellular activity, while supporting flavour production

Flavour development in cheese is affected by the integrity of Lactococcus lactis cells. Disintegrated cells enhance for instance the enzymatic degradation of casein to free amino acids, while integer cells are needed to produce specific flavour compounds from amino acids. The impact of the cellular activity of these integer cells on flavour production remains to be elucidated. In this study we investigated whether lactose-deprived L. lactis cells that use arginine as an alternative energy source can extend cellular activity and produce more specific flavours. In cheese experiments we demonstrated that arginine metabolising cells survived about 3 times longer than non-arginine metabolising cells, which suggests prolonged cellular activity. Cellular activity and flavour production of L. lactis was further studied in vitro to enable controlled arginine supplementation. Comparable with the results found in cheese, the survival rates of in vitro incubated cells improved when arginine was metabolised. Furthermore, elongated cellular activity was reflected in 3-4-fold increased activity of flavour generating enzymes. The observed prolonged cellular activity resulted in about 2-fold higher concentrations of typical Gouda cheese flavours. These findings provide new leads for composing starter cultures that will produce specific flavour compounds.     

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.     

In vitro evaluation of the probiotic attributes of two pediococci strains producing pediocin PA-1 with selective potency as compared to nisin

Eighteen dairy starter cultures, spoilage and food-borne pathogenic strains were analyzed for susceptibility to antimicrobial peptides pediocin PA-1 (PedPA-1) and nisin, through the individual 50 % inhibitory concentrations (IC₅₀) determination. The IC₅₀ of purified PedPA-1 was found to be more potent than nisin against five spoilage and food-borne pathogenic strains, i.e., Bacillus cereus NCDC 240, Enterococcus faecalis NCDC 114, Enterococcus faecium NCDC 124, Streptococcus agalactiae NCDC 208 and Staphylococcus aureus NCDC 110. The IC₅₀ of PedPA-1 and nisin ranged from 6.58 to 0.29 µM and 18.91 to 0.03 µM, respectively. Further, PedPA-1 producing Pediococcus pentosaceus NCDC 273 and Pediococcus acidilactici NCDC 252 strains were evaluated for potential probiotic attributes by in vitro studies. Both pediococci strains were able to survive at low pH and 2 % bile with a good bile salt hydrolase activity, cell surface hydrophobicity and β-galactosidase activity that makes them potentially good candidates for probiotics. These strains with proven promising probiotic attributes are good candidates for further investigation through in vivo studies to elucidate their potential health benefits.     

Effect of Lactococcus garvieae, Lactococcus lactis and Enterococcus faecalis on the behaviour of Staphylococcus aureus in microfiltered milk

The effect of four strains of Lactococcus garvieae, three strains of Lactococcus lactis and one strain of Enterococcus faecalis on Staphylococcus aureus SA15 growth in microfiltered milk was evaluated. Lactococcus and Enterococcus strains were co-cultured with S. aureus in microfiltered milk and in medium buffered at pH 6.8. All Lactococcus and Enterococcus strains were able to inhibit S. aureus growth after 6h of incubation. Inhibition by L. lactis and E. faecalis strains could be partially attributed to the decrease in pH below 6.0 as it has been observed in medium buffered at pH 6.8. L. garvieae strains were the most effective to inhibit S. aureus growth without acidification. Inhibition of S. aureus could not be attributed neither to production of lactate, acetate or nor to antistaphylococcal substance. Amino acids competition was not involved in the inhibition by L. garvieae as addition of valine, isoleucine, threonine, methionine and phenylalanine did not suppress the inhibition of S. aureus.     

乳酸乳球菌胞外多糖的分离及其体外抗氧化活性研究

本实验对乳酸乳球菌胞外多糖(EPS)的离心除菌、提取、粗胞外多糖中蛋白质的去除进行研究,同时探究胞外多糖的体外抗氧化活性。结果表明：6000r/min 离心15min;温度15℃;旋转蒸发浓缩到原体积的1/4;醇沉最佳条件为３倍糖溶液体积的90% 乙醇沉淀12h;选择TCA 沉淀蛋白,TCA 的最佳浓度为10%;胞外多糖有显著的体外抗氧化性。     

Diversity Analysis,Batch Fermentation and Characterization of Nisin in Identified Strains of Lactococcus lactis spp. lactis

Thirty-one strains of Lactococcus lactis spp. lactis were identified out of 89 isolates of lactic acid bacteria (LAB) from dairy and nondairy sources. Of the 31 strains, 24 (46.1%) were obtained from dairy and seven (18.9%) from non-dairy sources. The cluster analysis of rep-PCR showed that (GTG)₅-PCR followed by ERIC-PCR exhibited more discriminating potential than BOX-PCR. The obtained banding patterns characterized the polymorphism among strains. The strain level polymorphism was also obtained by the combined cluster analysis of (GTG)_5, ERIC and BOX-PCR which exhibited a level of heterogeneity among strains but not with the sources of isolation. Among 31 strains, 17 strains were able to produce zones of inhibition against Lactobacillus acidophilus NCDC 015 and therefore considered as nisin producers. Nisin production by strains was further confirmed by PCR amplification of nisA/Z of 174 bp size. The nisin activity and cell growth observed to be higher in pH controlled batch fermentation than in uncontrolled fermentation. The nisin activity, cell concentration, and acidity were high in immobilized cell system than free cell batch fermentation. The hot acid and chloroform extraction method was found to be the efficient way for the partial purification of nisin from fermented broth.     

The conjugal plasmid pLL10236 encodes lactose fermentation ability,restriction/modification activity,bacteriocin production and immunity in Lactococcus lactis subsp. lactis LL102

A 36 kb plasmid, designed as pLL 10236, was determined in Lactococcus lactis subsp.Lactis LL102. This plasmid mediates lactose fermentation ability (Lac⁺), bacteriocin production (Bac⁺) and immunity (Imm⁺), and restriction/modification (R⁺M⁺) activity against bacteriophage Ø1120 in strain LL102. The conjugal ability of pLL 10236 and expression of pLL 10236 encoding traits were determined by using a Lac⁻, Bac⁻, Imm⁻and (R⁻M⁻) recipient strain L. lactis subsp. lactis P81-1.