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.     

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.     

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.     

Chimeras of mature pediocin PA-1 fused to the signal peptide of enterocin P permits the cloning, production, and expression of pediocin PA-1 in Lactococcus lactis

Chimeras of pediocin PA-1 (PedA-1), a bacteriocin produced by Pediococcus acidilactici PLBH9, fused to the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, permitted the production of PedA-1 in Lactococcus lactis. Chimeric genes encoding the EntP signal peptide (SP(entP)) fused to mature PedA-1 (pedA), with or without its immunity gene (pedB), were cloned into the expression vector pMG36c to generate the recombinant plasmids pMPP9 (SP(entP):pedA) and pMPP14i (SP(entP):pedA + pedB). Transformation of competent L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. lactis DPC5598 with the recombinant plasmids has permitted the detection and quantitation of PedA-1 and the coproduction of nisin A and PedA-1 in supernatants of producer cells with specific anti-PedA-1 antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay. Recombinant L. lactis hosts carrying pMPP9 or pMPP14i displayed antimicrobial activity, suggesting that mature PedA-1 fused to SP(EntP) is the minimum requirement for the synthesis, processing, and secretion of biologically active PedA-1 in L. lactis. However, the production and antimicrobial activity of the PedA-1 produced by L. lactis was lower than that produced by the P. acidilactici control strains.     

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.     

Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection

The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in starter formulations. The 33 out of 100 isolates of L. lactis, originated from farmhouse cheeses, were found to be resistant to a collection of 34 phages belonging to the c2 and 936 groups. Six of the isolates were discarded as potential starters because they were lysogenic and other five because they produced tyramine. Plasmid and chromosomal profiles of the 22 remaining isolates allowed their classification into 16 different strains. All of these were good lactic acid producers from lactose, moderately proteolytic and, in eight cases, diacetyl production from citrate was observed. The mechanism(s) leading to the phenotype of phage resistance was identified for all the strains used in this study. Inhibition of adsorption was the most frequent one, although genetic determinants for some abortive infection systems were also detected (abiB, abiG and abiI). Frequently, more than one mechanism was present in the same strain. One of the strains, L. lactis IPLA542, was selected as a model starter for pilot fermentations. It clotted milk normally both in the absence and in the presence of phage at concentrations that completely abolished the process when promoted by a phage-susceptible strain.     

Technological performance of several Lactococcus and Enterococcus strains of dairy origin in milk

Thirty-nine strains (29 Lactococcus strains and 10 Enterococcus strains) isolated from five different artisanal cheeses were subjected to technological characterization. Several strains of lactococci and enterococci produced lactic acid at a rate and final concentration suitable for large-scale cheesemaking. However, extensive phenotypic differences between strains were encountered. Proteolytic activity correlated quite well with acidification for all strains, with the more proteolytic strains being the best acidifiers. The strains were also assayed for the production of organic acids and volatile components in milk. With few exceptions, enterococcus isolates produced more formic acid and acetic acid than did lactococcus isolates. The volatile-compound profiles obtained were rather simple. The main volatile component produced by most strains was ethanol. Since the inclusion of enterococcus strains in food systems is controversial, tests were also performed to detect recognized determinants of virulence (namely, aggregation, gelatinase and hemolysin production, and antibiotic resistance). Aggregation in both liquid and solid media was observed only for two Enterococcus durans isolates. None of the strains studied produced gelatinase under the conditions of the assay. Beta-hemolysin activity was clearly detected in two Enterococcus faecalis strains, which also produced the biogenic amine tyramine from tyrosine in a laboratory medium. In general, the enterococcus strains were more resistant to the antibiotics assayed than were the lactococcus strains. Both the minimum inhibitory concentration (MIC) modes and the highest MIC values were consistently higher for the enterococci. Nevertheless, particular strains of lactococci were resistant to antibiotics such as bacitracin, cephalothin, clindamycin, streptomycin, and tetracycline.     

Angiotensin-converting enzyme inhibitory activity of milk fermented by wild and industrial Lactococcus lactis strains

Angiotensin I-converting enzyme inhibitory (ACEI) activity was evaluated and compared in <3 KDa water-soluble extracts (WSE) isolated from milk fermented by wild and commercial starter culture Lactococcus lactis strains after 48 h of incubation. The highest ACEI activities were found in WSE from milk inoculated with wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures. On the other hand, the lowest ACEI activities were found in WSE from milk inoculated with wild strains isolated from vegetables. Moreover, the IC₅₀ values (concentration that inhibits 50% activity) of WSE from artisanal dairy products were the lowest, indicating that these fractions were the most effective in inhibiting 50% of ACE activity. In fact, a strain isolated from artisanal cheese presented the lowest IC₅₀ (13 μg/mL). Thus, it appears that wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures showed good potential for the production of fermented dairy products with ACEI properties.     

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.     

Cross-inhibition among wild strains of Lactococcus lactis isolated from the same ecological niche.

The cross-inhibition between 23 Lactococcus lactis subsp. lactis strains and 9 L. lactis subsp. cremoris strains with different randomly amplified polymorphic DNA patterns, all isolated from the same ecological niche--cheese made in the spring at a single factory from raw milk without added lactic starter cultures-was investigated. Cross-inhibition, as determined by the agar well diffusion assay, was recorded in 130 cases (12.7%) out of 1.024 total cases, with 109 cases due to supernatants of L. lactis subsp. lactis strains and 21 cases due to supernatants of L. lactis subsp. cremoris strains. L. lactis strains isolated in April, May, and June showed differences in their inhibitory activities, with cross-inhibition against each other in 34.7, 14.1, and 6.1% of the cases, respectively. Polymerase chain reaction techniques using specific primers for nisin, lacticin 481, and lactococcin A only revealed the presence of the structural gene of lacticin 481 in two L. lactis subsp. lactis strains.     

Genotypic and phenotypic diversity of Lactobacillus delbrueckii subsp. lactis strains of dairy origin

Eighty-nine strains of Lactobacillus delbrueckii subsp. lactis isolated from Italian hard and semi-hard cheeses and artisan starter cultures were characterised by phenotypic and genotypic methods. Phenotypic diversity was evaluated by studying biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR and pulsed field gel electrophoresis (PFGE). Phenotypic characterisation indicated a wide variability of the acidifying activity within Lact. delbrueckii subsp. lactis. Although the data was variable, it allowed us to evidence groups of strains with different acidifying properties, especially in terms of acidification intensity. Concerning peptidase activity, Lact. delbrueckii subsp. lactis showed a homogeneously high x-prolil-dipeptidil-aminopeptidase activity and a considerable but more heterogeneous lysil-aminopeptidase activity. The increased resolution obtained by the use of two molecular typing techniques, i.e. RAPD-PCR and PFGE, allowed to widen the level of strain heterogeneity. Technological and ecological pressures are determinant in selecting Lact. delbrueckii subsp. lactis sub-populations which are more functional to the different cheese technologies.     

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.     

Antimicrobial activity of pediocin PA-1 against Oenococcus oeni and other wine bacteria

Pediocin PA-1 is an antimicrobial peptide produced by lactic acid bacteria (LAB) that has been sufficiently well characterised to be used in food industry as a biopreservative. Sulphur dioxide is the traditional antimicrobial agent used during the winemaking process to control bacterial growth and wine spoilage. In this study, we describe the effect of pediocin PA-1 alone and in combination with sulphur dioxide and ethanol on the growth of a collection of 53 oenological LAB, 18 acetic acid bacteria and 16 yeast strains; in addition, production of pediocin PA-1 by Pediococcus acidilactici J347-29 in presence of ethanol and grape must is also reported. Inhibitory concentrations (IC) and minimal bactericide concentrations of pediocin PA-1 were determined against LAB, and revealed a bacteriostatic effect. Oenococcus oeni resulted more sensitive to pediocin PA-1 (IC₅₀ = 19 ng/ml) than the other LAB species (IC₅₀ = 312 ng/ml). Cooperative inhibitory effects of pediocin PA-1 and either sulphur dioxide or ethanol were observed on LAB growth. Moreover, the pediocin PA-1 producing P. acidilactici strain J347-29 was able to grow and produce the bacteriocin in presence of ethanol (up to 4% ethanol in the fermentation broth) and grape must (up to 80%), which indicated that pediocin PA-1 can be considered as a potential biopreservative in winemaking.     

Detection of pediocin PA-1-producing pediococci by rapid molecular biology techniques

Five bacteriocinogenic lactic acid bacteria strains (347, X13, Z102, A172 and P20) independently isolated from fermented sausages were identified asPediococcus acidilacticiby carbohydrate fermentation patterns and other biochemical characteristics. This fact, together with their activity againstListeria monocytogenes, suggested that they could be pediocin PA-1 producers. Rapid molecular biology techniques were used to detect the pediocin PA-1 operon in these strains. PCR, dot-blot and Southern hybridization, and DNA sequencing results confirmed that all of them had the genetic determinants for pediocin PA-1 biosynthesis encoded in a 9.4 kb plasmid. The bacteriocin produced byP. acidilactici347 has been purified by a procedure that included ammonium sulphate precipitation and cation exchange, hydrophobic-interaction and reverse-phase chromatography. The amino acid sequence of this bacteriocin was identical to pediocin PA-1, confirming the expression of the pediocin PA-1 genes.     

Effect of wild strains of Lactococcus lactis on the volatile profile and the sensory characteristics of ewes' raw milk cheese

The production of volatile compounds by wild strains of Lactococcus lactis used as starter cultures and their effect on the sensory characteristics of ewes' raw milk cheese were investigated. Sixteen vats of cheese were manufactured and ripened for 120 d in two experiments, each of them duplicated. In the first experiment, milk was inoculated with different ratios of four wild Lactococcus lactis strains, two producing and two not producing branched-chain volatile compounds, and in the second experiment with different ratios of a commercial starter culture and the two strains producing branched-chain volatile compounds. Cheese pH, proteolysis, and aminopeptidase activity increased when the strains producing branched-chain volatile compounds were inoculated at a higher rate. Fifty volatile compounds were identified in cheeses using a purge and trap system coupled to a gas chromatography-mass spectrometry apparatus. The relative abundances of 30 volatile compounds (8 alcohols, 5 aldehydes, 3 ketones, 12 esters, 1 sulfur compound, and 1 benzenic compound) were influenced by starter culture composition. 2-Methylpropanol, 3-methylbutanol, isobutyl acetate, isoamyl acetate, ethyl butyrate, isobutyl butyrate, and isoamyl butyrate were always more abundant in the cheeses made with a higher level of L. lactis strains producing branched-chain volatile compounds. Flavor intensity was enhanced by a high level of L. lactis strains producing branched-chain volatile compounds in the first experiment, in which four wild L. lactis strains were used as starter culture, but not in the second experiment, in which a combination of two wild L. lactis strains and the commercial starter culture were used. Flavor quality, as judged by trained panelists, was impaired in both experiments by a high level of L. lactis strains producing branched-chain volatile compounds.     

Comparison of growth and survival of single strains of Lactococcus lactis and Lactococcus cremoris during Cheddar cheese manufacture

Traditionally, starter cultures for Cheddar cheese are combinations of Lactococcus lactis and Lactococcus cremoris. Our goal was to compare growth and survival of individual strains during cheesemaking, and after salting and pressing. Cultures used were 2 strains of L. lactis (SSM 7605, SSM 7436) and 2 strains of L. cremoris (SSM 7136, SSM 7661). A standardized Cheddar cheese make procedure was used that included a 38°C cook temperature and salting levels of 2.0, 2.4, 2.8, 3.2, and 3.6% from which were selected cheeses with salt-in-moisture levels of 3.5, 4.5, and 5.5%. Vats of cheese were made using each strain on its own as biological duplicates on different days. Starter culture numbers were enumerated by plate counting during cheesemaking and after 6 d storage at 6°C. Flow cytometry with fluorescent staining by SYBR Green and propidium iodide was used to determine the number of live and dead cells in cheese at the different salt levels. Differences in cheese make times were strain dependent rather than species dependent. Even with correction for average culture chain length, cheeses made using L. lactis strains contained ～4 times (～0.6 log) more bacterial cells than those made using L. cremoris strains. Growth of the strains used in this study was not influenced by the amount of salt added to the curd. The higher pH of cheeses with higher salting levels was attributed to those cheeses having a lower moisture content. Based on flow cytometry, ～5% of the total starter culture cells in the cheese were dead after 6 d of storage. Another 3 to 19% of the cells were designated as being live, but semipermeable, with L. cremoris strains having the higher number of semipermeable cells.     

Interactions between Lactococcus lactis and Streptococcus thermophilus strains in Cheddar cheese processing conditions

The growth of pure and mixed cultures of Lactococcus lactis and Streptococcus thermophilus under simulated Cheddar cheese manufacture was examined. Cell-free wheys (CFW) of the cultures were prepared for analysis by automated spectrophotometry (AS). The maximal growth rate of the lactococci in S. thermophilus R0083 CFW was 13% higher than that noted in their own CFW and three lactococci also gave higher biomass levels (OD_max). During simulated Cheddar cheese fermentations with four paired cultures, one L. lactis strain grew 20% less when paired with S. thermophilus R0083, and an increase in colony forming units (cfu) was found with one other lactococcal strain. Viable counts of S. thermophilus in mixed cultures varied by less than 0.1 log cfu mL^?1. The AS data on OD_max in CFW were useful in predicting the evolution of cfu in the fermented mixed cultures. As a function of strain, the presence of S. thermophilus in a Cheddar fermentation process can enable extended growth of the lactococci.     

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.