Diacetylactis UL719 Research Articles

Growth, acid production and bacteriocin production by probiotic candidates under simulated colonic conditions

The aim of this study is to evaluate the capacity of three bacteriocin producers, namely Lactococcus lactis subsp. lactis biovar diacetylactis UL719 (nisin Z producer), L. lactis ATCC 11454 (nisin A producer) and Pediococcus acidilactici UL5 (pediocin PA-1 producer), and to grow and produce their active bacteriocins in Macfarlane broth, which mimics the nutrient composition encountered in the human large intestine. The three bacteriocin-producing strains were grown in Macfarlane broth and in De Man-Rogosa-Sharpe (MRS) broth. For each strain, the bacterial count, pH drop and production of organic acids and bacteriocins were measured for different period of time. The ability of the probiotic candidates to inhibit Listeria ivanovii HPB 28 in co-culture in Macfarlane broth was also examined. Lactococcus lactis subsp. lactis biovar diacetylactis UL719, L. lactis ATCC 11454 and Ped. acidilactici UL5 were able to grow and produce their bacteriocins in MRS broth and in Macfarlane broth. Each of the three candidates inhibited L. ivanovii HPB 28, and this inhibition activity was correlated with bacteriocin production. The role of bacteriocin production in the inhibition of L. ivanovii in Macfarlane broth was confirmed for Ped. acidilactici UL5 using a pediocin nonproducer mutant. The data provide some evidence that these bacteria can produce bacteriocins in a complex medium with carbon source similar to those found in the colon. This study demonstrates the capacity of lactic acid bacteria to produce their bacteriocins in a medium simulating the nutrient composition of the large intestine.

Enhancement of Functional Characteristics of Mixed Lactic Culture Producing Nisin Z and Exopolysaccharides During Continuous Prefermentation of Milk with Immobilized Cells

Antagonistic phenomena between strains often occur in mixed cultures containing a bacteriocinogenic strain. A nisin Z producer (Lactococcus lactis ssp. lactis biovar. diacetylactis UL719) and 2 nisin-sensitive strains for acidification (Lactococcus lactis ssp. cremoris ATCC19257) and exopolysaccharide (EPS) production (Lactobacillus rhamnosus RW-9595M) were immobilized separately in gel beads and used to continuously preferment milk at different temperatures, with pH controlled at 6.0 by fresh milk addition. The process showed high volumetric productivity, with an increase from 8.0 to 12.5L of prefermented milk per liter of reactor volume and hour as the temperature was increased from 27 to 35°C. Lactococcus lactis ssp. lactis biovar. diacetylactis UL719 counts in prefermented and fermented (22-h batch fermentation) milks were stable during 3 wk of continuous fermentation (8.1±0.1 and 8.9±0.2 log cfu/mL, respectively). The L. lactis ssp. cremoris population (estimated with real-time quantitative PCR) decreased rapidly during the first week of continuous culture to approximately 4.5 log cfu/mL and remained constant afterward. Lactobacillus rhamnosus counts in prefermented and fermented milks significantly increased with prefermentation time, with no temperature effect. Nisin Z reached high titers in fermented milks (from 177 to 363 IU/mL), with EPS concentration in the range from 43 to 178mg/L. Immobilization and continuous culture led to important physiological changes, with Lb. rhamnosus becoming much more tolerant to nisin Z, and Lb. rhamnosus and L. lactis ssp. lactis biovar. diacetylactis UL719 exhibiting large increases in milk acidification capacity. Our data showed that continuous milk prefermentation with immobilized cells can stimulate the acidification activity of low-acidifying strains and produce fermented milks with improved and controlled functional properties.

Milk fermentation by functional mixed culture producing nisin Z and exopolysaccharides in a fresh cheese model

A nisin Z-producing strain, Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 and two nisin-sensitive cultures, Lactobacillus rhamnosus RW-9595 M producing exopolysaccharide (EPS), and Lc. lactis subsp. cremoris for acidification, were tested in pure and mixed cultures during milk fermentation. The mixed culture of the three strains showed a higher acidifying capacity at 34°C and 38°C, even though populations of Lc. cremoris were largely reduced compared with pure cultures. Bacteriocin production was 3.1–4.6-fold higher in mixed cultures than for pure cultures of Lc. diacetylactis UL719. These data can be explained by commensalism behavior relying on high proteolytic activity of Lc. cremoris and autolysis and nisin Z-induced lysis. In mixed culture, EPS production was 3-fold lower than for Lb. rhamnosus RW-9595 M pure culture. Our data showed that this strain combination, with nisin-producing and sensitive strains, can be used in mixed cultures for manufacture of fresh cheese with improved functional properties.

Quantification by real-time PCR of Lactococcus lactis subsp. cremoris in milk fermented by a mixed culture

During cheese making, interactions between different strains of lactic acid bacteria play an important role. However, few methods are available to specifically determine each bacterial population in mixed cultures, in particular for strains of the same species. The aim of this study was to develop a real-time PCR quantification method to monitor the population of Lactococcus cremoris ATCC 19257 in mixed culture with Lactobacillus rhamnosus RW-9595M and the bacteriocin-producing microorganism Lc. diacetylactis UL719. The specificity of the two primers 68FCa33 and 16SR308 used to amplify a 240-bp fragment of DNA from Lc. cremoris was demonstrated by conventional PCR. Using these primers for real-time PCR, the detection limit was 2 cfu/reaction or 200 cfu of Lc. cremoris ATCC 19257 per millilitre of mixed culture in milk. In pure culture batch fermentation, good correlation was obtained between real-time PCR and the conventional plating method for monitoring Lc. cremoris growth. In mixed culture batch fermentation, Lb. rhamnosus and Lc. cremoris decreased due to nisin Z production by Lc. diacetylactis. The decrease of the Lc. cremoris cell population detected by real-time PCR was not possible to observe by the plate count method in the presence of a Lc. diacetylactis population that was 1 log higher.

Impact of autolytic, proteolytic, and nisin-producing adjunct cultures on biochemical and textural properties of cheddar cheese.

The effect of incorporating a highly autolytic strain (Lactobacillus delbrueckii subsp. bulgaricus UL12) a proteolytic strain (Lactobacillus casei subsp. casei L2A), or a nisin Z-producing strain (Lactococcus lactis, subsp. lactis biovar diacetylactis UL719) into Cheddar cheese starter culture (Lactococcus lactis KB and Lactococcus cremoris KB) on physicochemical and rheological properties of the resultant cheeses was examined. Cheeses were ripened at 7 degrees C and analyzed over a 6-mo period for viable lactococcal and lactobacilli counts, pH, titratable acidity (TA), lipolysis, proteolysis, and textural characteristics. The combination of the nisin-producing strain and autolytic adjuncts significantly increased the production of water-soluble nitrogen, free amino acids, and free fatty acids. The effect of Lc. diacetylactis UL719 alone or of Lb. casei L2A on water-soluble nitrogen and free amino acid contents were also significant, whereas their effect on free fatty acids was not. Viable counts of Lb. bulgaricus UL12 were significantly reduced in the presence of Lc. diacetylactis UL719. Lactobacilli-containing cheeses showed significantly lower values for hardness, fracturability, and springiness. It could be concluded that the addition of Lb. bulgaricus UL12 together with a nisin-producing strain produces a greater increase in cheese proteolysis and an improvement in Cheddar cheese texture.

Impact of Autolytic and Proteolytic Lactobacilli and Nisin‐Producing Culture on Proteolysis and Sensory Characteristics in Cheddar Cheese

ABSTRACT: Cheddar cheeses were made using a nisin‐tolerant starter culture with either Lactobacillus delbrueckii subsp. bulgaricus UL12 (autolytic strain), Lactobacillus casei subsp. casei L2A (proteolytic strain), Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 (nisin producer), or of Lb. bulgaricus UL12 and Lc. diacetylactis UL719. Lb. bulgaricus UL12 produced more trichloroacetic acid‐soluble nitrogen than did Lb. casei L2A, which produced more phosphotungstic acid‐soluble nitrogen than did Lc. diacetylactis UL719. High‐performance liquid chromatography analyses showed that either lactobacilli or Lc. diacetylactis UL719 increased the hydrophilic and hydrophobic peptide contents. Cheeses containing both Lb. bulgaricus UL12 and Lc. diacetylactis UL719 had the most intense old Cheddar cheese flavor after 6 mo of ripening.

Impact of Nisin Producing Culture and Liposome-encapsulated Nisin on Ripening of Lactobacillus added-Cheddar Cheese

This study aimed to evaluate the effects of incorporating liposome-encapsulated nisin Z, nisin Z producing Lactococcus lactis ssp. lactis biovar. diacetylactis UL719, or Lactobacillus casei-casei L2A adjunct culture into cheese milk on textural, physicochemical and sensory attributes during ripening of Cheddar cheese. For this purpose, cheeses were made using a selected nisin tolerant cheese starter culture. Proteolysis, free fatty acid production, rheological parameters and hydrophilic/hydrophobic peptides evolution were monitored over 6 mo ripening. Sensory quality of cheeses was evaluated after 6 mo. Incorporating the nisin-producing strain into cheese starter culture increased proteolysis and lipolysis but did not significantly affect cheese rheology. Liposome-encapsulated nisin did not appear to affect cheese proteolysis, rheology and sensory characteristics. The nisinogenic strain increased the formation of both hydrophilic and hydrophobic peptides present in the cheese water extract. Sensory assessment indicated that acidic and bitter tastes were enhanced in the nisinogenic strain-containing cheese compared to control cheese. Incorporating Lb. casei and the nisinogenic culture into cheese produced a debittering effect and improved cheese flavor quality. Cheeses with added Lb. casei and liposome-encapsulated nisin Z exhibited the highest flavor intensity and were ranked first for sensory characteristics.

Production and characterization of anti-nisin Z monoclonal antibodies: suitability for distinguishing active from inactive forms through a competitive enzyme immunoassay.

As a pre-requisite to monoclonal antibody development, an efficient purification strategy was devised that yielded 72 mg of nisin Z from 14.5 1 of Lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 (L. diacetylactis UL719) culture in supplemented whey permeate. Specific monoclonal antibodies (mAbs) were produced in mice against the purified nisin Z using keyhole limpet hemocyanin as a carrier protein. These antibodies did not recognize nisin A, suggesting that the asparagine residue at position 27 is involved in antibody recognition to nisin Z. However, the high reactivity of mAbs against biologically inactive nisin Z degradation products, produced during storage of freeze-dried pure nisin Z at -70 degrees C, indicated that the dehydroalanine residue at position 5 (Dha5), required for biological activity, is not necessary in nisin Z recognition by the mAb. A competitive enzyme immunoassay (cEIA) using the specific anti-nisin Z mAb was developed and used for rapid and sensitive detection and quantification of nisin Z in fresh culture supernatant, milk and whey. Detection limits of 78 ng/ml in phosphate-buffered saline, 87 ng/ml in culture supernatant, 106 ng/ml in milk and 90.5 ng/ml in whey were obtained for this assay. The cEIA using specific mAbs can be used to quantify nisin Z in food products.

Effect of aeration and dilution rate on nisin Z production during continuous fermentation with free and immobilized Lactococcus lactis UL719 in supplemented whey permeate

The influence of dilution rate (D) and aeration on soluble and cell-bound nisin Z production was investigated during continuous free (FC) and immobilized cell (IC) cultures with Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in supplemented whey permeate. Maximum total bacteriocin titres during non-aerated continuous FC and IC cultures were obtained for low D, with 1490 and 1090 IU mL−1 for 0.15 h−1 or 0.25 and 0.5 h−1, respectively. For both systems, aeration increased nisin total production with maximum titres of 2560 and 2430 IU mL−1 for low D, respectively, as well as specific production. Volumetric productivity was the highest for an intermediate D of 0.4 h−1 during FC cultures (460 IU mL−1 h−1 for both aerated and non-aerated cultures), while it increased continuously with D during IC cultures, reaching high values of 1090 and 1760 IU mL−1 h−1 at 2.0 h−1 without and with aeration, respectively. In comparison with previous data for FC batch cultures, data from this study may indicate that during continuous fermentations at steady state, some steps in nisin biosynthesis are limiting. In these conditions, nisin production by immobilized cells is reduced.

High nisin-Z production during repeated-cycle batch cultures in supplemented whey permeate using immobilized Lactococcus lactis UL719

Abstract Nisin-Z production was studied during repeated-cycle pH-controlled batch (RCB) cultures using Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 immobilized in κ -carrageenan/locust bean gum gel beads in supplemented whey permeate. After an initial colonization of gel beads during the first two cycles, nisin-Z production in bulk medium and gel beads was very similar for 1-h and 2-h cycle RCB cultures. A very high nisin-Z production (8200 IU mL −1 ) was measured in the broth after the 1-h cycles, with a corresponding volumetric productivity of 5730 IU mL −1 h −1 . This productivity is much higher than maximum nisin productivities reported in literature or maximum productivities obtained previously for free-cell batch cultures (850 IU mL −1 h −1 ), and free-cell (460 IU mL −1 h −1 ) or immobilized-cell (1760 IU mL −1 h −1 ) continuous cultures, using the same strain and fermentation conditions. The stability of RCB cultures was demonstrated for 24 and 36 1-h cycles carried out over 3 and 6-day periods, respectively. Changing environmental conditions during batch cultures resulted high nisin production.

Rapid purification of nisin Z using specific monoclonal antibody-coated magnetic beads

Abstract A rapid method is described for the isolation and purification of nisin Z from Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 culture supernatant using magnetic beads. Anti-nisin Z monoclonal antibodies were covalently bound to EDC-activated magnetic beads and the complex was incubated overnight at 4°C with the culture supernatant. Bound nisin Z was then eluted with urea 6 m formic acid 0.1 m mixture. Using this procedure, a high yield of 61.5% was obtained with a nisin Z specific activity of 2.4×10 4 IU mg −1 . Magnetic beads were re-used for two other purification cycles during which a significant decrease of purification yield from 61.5 to 26.7% was observed. The immunomagnetic purification strategy developed in this work was shown to be efficient and offers an alternative rapid procedure for production of high-purity food-grade nisin Z.

Production of a nisin Z/pediocin mixture by pH-controlled mixed-strain batch cultures in supplemented whey permeate

The conditions for high production of nisin Z and pediocin during pH-controlled, mixed-strain batch cultures in a supplemented whey permeate medium with Lactococcus lactis subsp. lactis biovar. diacetylactis UL719, a nisin Z producer strain, and variant T5 of Pediococcus acidilactici UL5, a pediocin-producing strain resistant to high concentrations of nisin, were studied. Mixed cultures were performed at 37 °C and pH 5·5 by first inoculating with variant T5 and then with L. diacetylactis UL719 after 8 h incubation, and were compared with single-strain batch cultures. High productions of both nisin Z and pediocin were obtained after 18 or 16 h incubation during mixed cultures, with titres of 3000 and 730 AU ml−1, or 1060 and 1360 AU ml−1, respectively, corresponding to approximately 75 and 55, or 25 and 100 mg l−1 of pure nisin Z and pediocin, respectively. In pure cultures, nisin Z and pediocin productions were higher than in mixed cultures, and maximum activities were obtained after 10 h incubation, with approximately 10 000 AU ml−1 (250 mg l−1 pure nisin Z) and 2500 AU ml−1 (190 mg l−1 pure pediocin). During mixed cultures, significant pediocin degradation was observed in the culture supernatant fluid after 16 h incubation, together with a sharp drop in Ped. acidilactici UL5 cell viability. In the test conditions, the feasibility of producing a nisin/pediocin mixture by mixed-strain fermentation was demonstrated. The bacteriocin mixture produced in a supplemented whey permeate medium could be used as a natural food-grade biopreservative with a broad activity spectrum.

Characterization of diacetin B, a bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis UL720.

Fourteen Lactococcus lactis strains showing inhibitory activity against Listeria innocua SICC 4202 were isolated from different French raw milks and raw milk cheeses and screened for bacteriocin production by the triple layer method under conditions that eliminate the effects of lactic acid and hydrogen peroxide. Three bacteriocinogenic strains (two Lactococcus lactis subsp. lactis bv. diacetylactis UL719 and UL720 and one Lactococcus lactis subsp. lactis UL730) were selected for their high capacity to inhibit the growth of various food pathogens, including Listeria monocytogenes, Staphylococcus aureus, and clostridial strains. The inhibitory compounds from these three strains are inactivated by selected proteases, indicating their protein nature. They retained their antibacterial activity after heat treatments of 100 degrees C for 60 min and 121 degrees C for 20 min, and in the pH range from 2 to 11. The bacteriocin diacetin B produced by strain UL720 has been purified by a pH-dependent adsorption-desorption procedure, followed by reverse-phase high performance liquid chromatography, with a yield of 1.25% of the original activity. Mass spectrometry analysis indicates that the pure peptide has a molecular mass of 4292.32 or 4490.28 Da, while amino acid sequencing allowed the identification of the primary structure of the bacteriocin composed of 37 amino acid residues. The structure of the peptide did not show similarity with other known bacteriocins from lactic acid bacteria.