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.     

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.     

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.     

Technological characterization of bacteriocin producing Lactococcus lactis strains employed to control Listeria monocytogenes in cottage cheese

In recent years, there has been a particular focus on the application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives to control the growth of spoilage and pathogenic bacteria in food. Bacteriocins are antimicrobial peptides which can be added to foods in concentrated forms as food preservatives, e.g. additives, or they can be produced in situ by starters or protective cultures. In this study, twenty Lactococcus lactis bacteriocin producers previously isolated from Italian fermented foods were subjected to a variety of physical and biochemical tests in order to identify those with the greatest potential as starter cultures in cheese production. Of these, four strains isolated from cheese (one nisin Z producer, one nisin A producer and two lacticin 481 producers) which fulfilled the desired technological criteria were assessed for their ability to control Listeria monocytogenes. The subsequent application of these bacteriocinogenic strains as starter cultures in Cottage cheese established that the nisin A producing Lact. lactis 40FEL3, and to a lesser extent the lacticin 481 producers 32FL1 and 32FL3, successfully controlled the growth of the pathogen. This is the first study to directly compare the ability of nisin A, nisin Z and lacticin 481 producing strains to control listerial growth during the manufacture and storage of Cottage cheese.     

Inhibition of toxicogenic Bacillus licheniformis 553/1 in Nigerian Wara soft cheese by nisin-producing Lactococcus lactis LAC309

Wara soft cheese is a traditionally produced cheese in Nigeria. The production of this cheese includes a heating step for killing vegetative cells. Therefore, mainly spore-forming bacteria surviving the process may function as the first spoilers resulting in lowered shelf-life and safety of the product. In this study, we investigated if the addition of a nisin-producing Lactococcus lactis LAC309 starter after the high temperature treatment could result in nisin production and inhibition of the toxicogenic Bacillus licheniformis 553/1 strain spiked into the cheese. The results showed that L. lactis LAC309 could produce nisin in Wara cheese and that the strain inhibited (3 log reduction) B. licheniformis 553/1 in Wara cheese. Food technologists developing industrialised Wara cheese should therefore consider including a nisin-producing starter strain in the cheese-making process.     

Validation of a model for growth of Lactococcus lactis and Listeria innocua in a structured gel system: effect of monopotassium phosphate

The effect of monopotassium phosphate (KH(2)PO(4)) on the chemical environment and on growth of Listeria innocua and Lactococcus lactis in coculture were investigated in a liquid and in a gelled microbiological medium at 12 degrees C and an initial pH of 6.2. As expected, addition of KH(2)PO(4) to both the liquid and gelled media resulted in an increase in buffering capacity. This effect on buffering capacity changed the profiles of lactic acid dissociation and pH evolution. At all gelatin concentrations studied, addition of KH(2)PO(4) increased the growth rate and the stationary cell concentration of L. lactis. In addition, the growth rate of L. innocua slightly increased but, in contrast, the stationary cell concentration remained unchanged. A new class of predictive models developed previously in our research team to quantify the effect of food model gel structure on microbial growth [Antwi, M., Bernaerts, K., Van Impe, J. F., Geeraerd, A. H., 2007. Modelling the combined effect of food model system and lactic acid on L. innocua and L. lactis growth in mono- and coculture. International Journal of Food Microbiology 120, 71-84] was applied. Our analysis indicate that KH(2)PO(4) influenced the parameters of the chemical and microbiological subprocesses of the model. Nonetheless, the growth model satisfactorily predicted the stationary cell concentration when (i) the undissociated lactic acid concentrations at which L. innocua and L. lactis growth cease were chosen as previously reported, and (ii) all other parameters of the chemical and microbiological subprocesses were computed for each medium. This confirms that the undissociated lactic acid concentrations at which growth ceases is a unique property of a bacterium and does not, within our case study, depend on growth medium. The study indicates that microbial growth depends on the interplay between the individual food components which affect the physicochemical properties of the food, such as the buffering capacity. Towards future research, it can be concluded that mathematical models which embody the effect of buffering capacity are needed for accurate predictions of microbial growth in food systems.     

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.     

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.     

Fate of enterotoxigenic Staphylococcus aureus in the presence of nisin-producing Lactococcus lactis strain during manufacture of Jben,a Moroccan traditional fresh cheese

The inhibitory effect of nisin-producing Lactococcus lactis subsp. lactis UL730 on the growth of enterotoxigenic Staphylococcus aureus J10 during manufacture of Jben, a Moroccan traditional fresh cheese prepared from recombined milk, was investigated.With an inoculum level of 10³ cfu mL⁻¹, S. aureus was absent in Jben four days after inoculation when the nisin-producing lactococcus was used as lactic starter. In contrast, it survived after that period, when the starter was non-nisin-producing. No staphylococcal thermonuclease was detected in all Jben samples made from milk inoculated with S. aureus at the level of 10³ cfu mL⁻¹.With a higher inoculum of 10⁵ cfu mL⁻¹, S. aureus was still present in Jben after manufacture and persisted during the storage of the product for 3 days in the laboratory, even when the starter used was nisin-producing. Staphylococcal thermonuclease and type C enterotoxin were detected in all Jben samples made from milk inoculated with 10⁵ cfu mL⁻¹. Thermonuclease and enterotoxin were already produced in the coagulum, at 24 h after milk inoculation with S. aureus.     

Phenotypic and genetic characterization of a selected set of Lactococcus lactis strains isolated from a starter-free farmhouse cheese

Eleven strains of lactococci isolated from a farmhouse starter-free cheese manufactured from raw cow's milk were analysed in detail for some technologically-related properties. Large phenotypic differences were encountered between the isolates, some of which could be of practical relevance. Several strains produced lactic acid at a rate and at a final concentration suitable for large-scale cheesemaking. The enzymatic capabilities assayed with the API ZYM system showed that all strains possess similar profiles with weak proteinase and moderate leucine-arylamidase and esterase-lipase activities. Interestingly, two related strains presented a strong β -galactosidase activity. Plasmid and chromosomal analyses indicated a high degree of diversity among wild strains and showed low homology with some well-known Lactococcus lactis strains. Under highly stringent conditions, only one plasmid from a single strain gave a clear positive hybridization signal with lactococcal-derived probes for the β -phospho- galactosidase and the proteinase genes. In general, wild strains produced more odorous compounds and in higher amounts than the reference strains.     

Biocontrol of Listeria monocytogenes in a model cultured milk (lben) by in situ bacteriocin production from Lactococcus lactis ssp. lactis

Bacteriocin‐producing (Bac⁺) Lactococcus lactis ssp. lactis CCMM/IAV/BK1 isolated from traditional lben was used in the preparation of lben from pasteurized milk to assess its potential inhibitory activity against Listeria monocytogenes ATCC 7644. Production of bacteriocin (arbitrary units, AU) in MRS broth fortified with yeast extract (MRSY) in a fermentor under controlled and uncontrolled pH conditions was also investigated. This Bac⁺ strain yielded about 35 times more bacteriocin when the pH was maintained constant at 6.5 than under varying pH conditions. To test the effect of in situ bacteriocin production against L. monocytogenes, lben was made from cow's milk artificially contaminated with approximately 10⁷ cfu/mL and fermented with a mixed mesophilic starter culture consisting of the lactococcal Bac⁺ organism and Lc. lactis ssp. lactis biovar diacetylactis 66, a diacetyl‐producing strain, in a ratio of 1 : 1. Numbers of L. monocytogenes were monitored during fermentation and storage of lben at refrigeration temperature (c. 7°C) for up to 6 days. Performances of the Bac⁺ starter were compared to those of an isogenic Bac^? derivative strain obtained from the Bac⁺ starter by curing with ethidium bromide. The results showed that the amount of L. monocytogenes decreased to below the detectable level in a 1‐mL sample within 24 h of storage at 7°C in lben fermented with the Bac⁺ starter culture. On the contrary, L. monocytogenes survived for 6 days of storage at 7°C in lben made with the Bac^? starter. The Bac⁺ wild strain of the starter studied could be adequately used to produce lben or similar indigenous fermented milks of improved hygienic quality on an industrial scale. Alternatively, it could be used as an adjunct in minimally processed products or in products obtained from raw milk to add a safety factor.     

Impact of ropy and capsular exopolysaccharide-producing strains of Lactococcus lactis subsp. cremoris on reduced-fat Cheddar cheese production and whey composition

Cheddar cheese mixed starter cultures containing exopolysaccharide (EPS)-producing strains of Lactococcus lactis subsp. cremoris (Lac. cremoris) were characterized and used for the production of reduced-fat Cheddar cheese (15% fat). The effects of ropy and capsular strains and their combination on cheese production and physical characteristics as well as composition of the resultant whey samples were investigated and compared with the impact of adding 0.2% (w/v) of lecithin, as a thickening agent, to cheese milk. Control cheese was made using EPS-non-producing Lac. cremoris. Cheeses made with capsular or ropy strains or their combination retained 3.6–4.8% more moisture and resulted in 0.29–1.19 kg/100 kg higher yield than control cheese. Lecithin also increased the moisture retention and cheese yield by 1.4% and 0.37%, respectively, over the control cheese. Lecithin addition also substantially increased viscosity, total solid content and concentrating time by ultra-filtration (UF) of the whey produced. Compared with lecithin addition, the application of EPS-producing strains increased the viscosity of the resultant whey slightly, while decreasing whey total solids, and prolonging the time required to concentrate whey samples by UF. The amount of EPS expelled in whey ranged from 31 to 53 mg L⁻¹. Retention of EPS-producing strains in cheese curd was remarkably higher than that of non-producing strains. These results indicate the capacity of EPS-producing Lac. cremoris for enhanced moisture retention in reduced-fat Cheddar cheese; these strains would be a promising alternative to commercial stabilizers.     

Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama

Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.     

The growth and aflatoxin production of Aspergillus flavus strains on a cheese model system are influenced by physicochemical factors

Traditional cheeses may be contaminated by aflatoxin-producing Aspergillus flavus during the ripening process, which has not been sufficiently taken into account. The objectives of this study were to evaluate the influence of water activity (a_w), pH, and temperature on the lag phases, growth, and aflatoxin production of 3 A. flavus strains (CQ7, CQ8, and CG103) on a cheese-based medium. The results showed that the behavior of A. flavus strains was influenced by pH, a_w, and temperature conditions. The CQ7 strain showed the maximum growth at pH 5.5, 0.99 a_w, and 25°C, whereas for CQ8 and CQ103 strains, no differences were obtained at pH 5.5 and 6.0. In general, low pH, a_w, and temperature values increased the latency times and decreased the growth rate and colony diameter, although a_w and temperature were the most limiting factors. Maximum aflatoxin production on the cheese-based medium occurred at pH 5.0, 0.95 a_w, and 25 or 30°C, depending on the strain. This study shows the effect of pH, a_w, and temperature factors on growth and aflatoxin production of 3 aflatoxigenic A. flavus strains on a cheese-based medium. The findings may help to design control strategies during the cheesemaking process and storage, to prevent and avoid aflatoxin contamination by aflatoxigenic molds.     

Antimicrobial activity of pediocin-producing Lactococcus lactis on Listeria monocytogenes,Staphylococcus aureus and Escherichia coli O157:H7 in cheese

The antimicrobial activity of two pediocin-producing transformants obtained from wild strains of Lactococcus lactis on the survival of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 during cheese ripening was investigated. Cheeses were manufactured from milk inoculated with the three pathogens, each at approximately 6 log cfu mL⁻¹. Pediococcus acidilactici 347 (Ped⁺), Lc. lactis ESI 153, Lc. lactis ESI 515 (Nis⁺) and their respective pediocin-producing transformants Lc. lactis CL1 (Ped⁺) and Lc. lactis CL2 (Nis⁺, Ped⁺) were added at 1% as adjuncts to the starter culture. After 30 d, L. monocytogenes, S. aureus and E. coli O157:H7 counts were 5.30, 5.16 and 4.14 log cfu g⁻¹ in control cheese made without adjunct culture. On day 30, pediocin-producing derivatives Lc. lactis CL1 and Lc. lactis CL2 lowered L. monocytogenes counts by 2.97 and 1.64 log units, S. aureus by 0.98 and 0.40 log units, and E. coli O157:H7 by 0.84 and 1.69 log units with respect to control cheese. All cheeses made with nisin-producing LAB exhibited bacteriocin activity throughout ripening. Pediocin activity was only detected throughout the whole ripening period in cheese with Lc. lactis CL1. Because of the antimicrobial activity of pediocin PA-1, its production in situ by strains of LAB growing efficiently in milk would extend the application of this bacteriocin in cheese manufacture.     

Controlled permeabilization of Lactococcus lactis cells as a means to study and influence cheese ripening processes

Cells of Lactococcus lactis were permeabilized by perturbing the membrane structure through the delipidating action of n-butanol to an extent that allowed normally excluded peptidase substrates to enter the cells and be accessible to intracellular enzymes. The degree of permeabilization of cells depended on the concentration of the solvent used, the duration of the treatment, the density of the cell suspension, pH and temperature. This was indicated by the optimum or maximum activities of the intracellular peptidases aminopeptidase N and aminopeptidase X. The possible usefulness of permeabilized cells for cheese-ripening studies was demonstrated with cells that were treated with 5 ml L⁻¹ n-butanol at pH 6.5 and 25 °C using a mixture of chymosin-generated primary cheese peptides as substrate. Production of amino acids could be correlated with conversion of peptides that had entered the permeabilized cells and reflected the course of amino acid production in cheese.     

Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product

Screening for bacteriocin production of 500 strains of lactic acid bacteria (LAB) from various African fermented foods resulted in the detection of a bacteriocin producing Lactococcus lactis (BFE 1500) isolated from a dairy product called wara. The bacteriocin inhibited not only the closely related LAB, but also strains of Listeria monocytogenes, Listeria innocua, Clostridium butyricum, Clostridium perfringens, Bacillis cereus and Staphylococcus aureus. It was heat stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10), but highest activity was observed in the lower pH range. The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K, but not by other proteases. Growth kinetic assay indicated stronger growth inhibition by the bacteriocin produced by Lc. lactis BFE 1500 on L. monocytogenes WS 2250 and B. cereus DSM 2301 than with the nisin A producing strain DSM 20729. Polymerase chain reaction indicated the presence of the nisin operon in strain BFE 1500 and sequencing of its structural gene showed that Lc. lactis BFE 1500 produced the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The genetic determinants for bacteriocin production in strain BFE 1500 are located on a conjugative transposon. The ability of the bacteriocin produced by Lc. lactis BFE 1500 to inhibit a wide range of food-borne pathogens is of special interest for food safety, especially in the African environment with perennial problems of poor food hygiene.