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 12.5 L 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 178 mg/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.     

Fresh-cheesemilk formulation fermented by a combination of freeze-dried citrate-positive cultures and exopolysaccharide-producing lactobacilli with liquid lactococcal starters

Milk formulation (4% fat and 5% protein) prepared to simulate fresh cheese production was inoculated with: (1) 10⁷ cfu mL⁻¹ of fresh liquid starters of Lactococcus lactis ssp. lactis T1 and Lc. lactis ssp. cremoris T2, (2) a freeze-dried exopolysaccharide-producing (EPS) strain of Lactobacillus rhamnosus RW-9595M, and (3) freeze-dried Leuconostoc cremoris LM057 or Lc. lactis ssp. lactis var. diacetylactis MD089 strains. The effect of inoculation rate of the freeze-dried starters (between 10⁶ and 10⁷ cfu mL⁻¹) and incubation temperature (between 23.5 and 36.5 °C) on evolution of pH and the various populations during fermentation was examined. Texture (apparent viscosity, syneresis potential) and chemical composition (diacetyl, acetaldehyde) of the fermented milks were also determined. Milk was incubated until a pH of 4.6 was obtained, which required between 6 and 10 h depending on temperature.In the range of inoculation levels used, there was no significant effect of the presence of lactobacilli, Ln. cremoris or Lc. lactis ssp. lactis var. diacetilactis on the growth of the lactococci. There was a direct correlation between the inoculation rates of the freeze-dried cultures and their final populations in the fermented milks. The growth of the cultures were also affected by temperature, Ln. cremoris growing less as incubation temperature increased, while the opposite was noted with Lb. rhamnosus. The apparent viscosity of the fermented milk was significantly affected by incubation temperature, but there was no correlation between apparent viscosity and the final population in lactobacilli. Of the three variables studied, the highest correlation with diacetyl content was obtained with the inoculation level of the Leuconostoc strain.     

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

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 mformic acid 0.1 m mixture. Using this procedure, a high yield of 61.5% was obtained with a nisin Z specific activity of 2.4×10⁴ 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.     

Effect of commercial adjunct cultures on proteolysis in low-fat Kefalograviera-type cheese

The effect of two commercially available adjunct cultures, LBC 80 (Lactobacillus casei subsp. rhamnosus) and CR-213 (containing Lactococcus lactis subsp. cremoris and Lc. lactis subsp. lactis) on the proteolysis in low-fat hard ewes’ milk cheese of Kefalograviera-type was investigated. Two controls, a full-fat cheese (306 g kg⁻¹ fat, 378 g kg⁻¹ moisture) and a low-fat cheese (97 g kg⁻¹ fat, 486 g kg⁻¹ moisture, made using a modified procedure), were also prepared. The effect of adjunct culture on proteolysis, as examined by polyacrylamide gel electrophoresis of cheese and water soluble cheese extracts, was marginal. The reverse-phase HPLC peptide profiles of the water soluble extracts from low-fat cheeses were similar although some quantitative differences were observed between low-fat control cheese and experimental cheeses. The fat content as reflected by the differences in peptide profiles affected the pattern of proteolysis. Proteolysis, as measured by the percentage of total nitrogen soluble in water or in 120 g L⁻¹ trichloroacetic acid, was significantly (P<0.05) affected by the addition of adjunct cultures. Furthermore, the adjunct cultures enhanced the production of low molecular mass nitrogenous compounds; the levels of total nitrogen, soluble in 50 g L⁻¹ phosphotungstic acid, and of free amino acids were significantly (P<0.05) higher in the low-fat experimental cheeses than in the low-fat control cheese.     

Comparison of the functionality of exopolysaccharides produced in situ or added as bioingredients on yogurt properties

The effect on yogurt properties of in situ production of exopolysaccharides (EPS) and addition of 2 EPS powders (crude and purified EPS from Lactobacillus rhamnosus RW-9595M fermentation in whey-based medium) at different concentrations was studied. No effect of purified powder addition for EPS concentrations up to 500 mg/L was observed on acidification rate to the difference of milks supplemented with crude EPS, which exhibited longer acidification times. The addition of EPS from 125 to 500 mg/L or the use of EPS-producing cultures resulted in yogurts with lower yield stress and viscoelastic moduli compared with control yogurts without EPS, with no apparent effect of EPS concentration. However, the consistency index was higher for yogurts produced with the commercial EPS-producing culture, and to a lesser extent with the mixed culture containing Lb. rhamnosus RW-9595M, compared with yogurts supplemented with EPS powders, which were not different from that for control yogurts. Our study showed that the mode of EPS incorporation in yogurts has a major effect on the rheological properties of the final product.     

Production of volatile aroma compounds by kefir starter cultures

Production of carbonyl compounds by single-strain cultures, kefir starter (Lactobacillus delbrueckii subsp. bulgaricus HP1+Lb. helveticus MP12+Lactococcus lactis subsp. lactis C15+Streptococcus thermophilus T15+Saccharomyces cerevisiae A13) and kefir grains during fermentation and storage of kefir was studied. The content of carbonyl compounds produced by kefir starter was greater than that produced by kefir grains. The maximum acetaldehyde concentration (18.3 μg g⁻¹) in kefir with starter culture was mainly due to the metabolic activity of Lb. delbrueckii subsp. bulgaricus HP1 isolated from kefir grains. The highest diacetyl production activity was recorded in the starter culture (1.87 μg g⁻¹) and the single-strain culture St. thermophilus T15 (1.62 μg g⁻¹), followed by Lb. helveticus MP12 (0.85 μg g⁻¹) and Lc. lactis subsp. lactis C15 (0.42 μg g⁻¹). The lactobacilli Lb. delbrueckii subsp. bulgaricus HP1 and Lb. helveticus MP12 produced acetone, which was not found in the cocci cultures. The presence of 2-butanone was related to the production ability of Lb. helveticus MP12. In comparison, Lc. lactis subsp. lactis C15 synthesized ethyl acetate more actively than the other single-strain cultures included in the starter. S. cerevisiae A13 produced ethanol and CO₂ in amounts (3975 μg g⁻¹; 1.80 g L⁻¹) that lent cultured kefir distinctive flavour and aroma characteristic of authentic kefir.     

Stability of autolytic lactococci during starter production and storage in commercial whey-based media

Lactococcus lactis ssp. cremoris strains with different autolytic activities were grown in milk and in a commercial whey-based medium. Fermentation under pH control substantially improved total and viable counts of strain NM33-7; highest populations were obtained on the commercial whey-based medium and the cultures had the highest content of viable cells (80%). Cultures grown in milk without pH control gave only 46% viable cells. Once fermentation was completed, cooling prevented a significant decrease in viable counts. Losses in viability of NM33-7 upon extended incubation at 30 °C or storage at 4 °C were higher when the cultures were prepared on whey-based medium than when prepared on milk without pH control. In pH-controlled fermentations, the autolytic activity of NM33-7 was higher when grown on whey-based medium than when grown on milk. A negative correlation was obtained between the autolytic activity of the Lc. lactis ssp. cremoris strains and their subsequent stability during storage.     

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.     

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.     

Effect of exopolysaccharides and proteolytic activity of Lactococcus lactis subsp. cremoris strains on the viscosity and structure of fermented milks

Two exopolysaccharide (EPS)-producing Lactococcus lactis subsp. cremoris strains (B35 and B891) were used to study the effect of the kinetics of EPS production and bacterial proteolytic activity on the structure of milk gels and the viscosity of stirred milk gels. Strains were grown at 20 °C in milk containing either yeast extract or casitone and at 30 °C in either milk alone or milk containing casitone. Lactococcal counts, pH decrease and production and molecular characteristics (molar mass and radius of gyration) of both EPSs were followed during milk fermentation. The level of proteolysis in the fermented milks was determined after 24 h of incubation. The results obtained showed that the yield of EPS and the timing of EPS production during milk-gel formation were the most important factors that influenced the structure of the milk gels and the viscosity of the stirred product. The proteolytic activity of the strains did not seem to play any significant role.     

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.     

Identification and technological properties of lactic acid bacteria isolated from raw goat milk of four Algerian races

The characteristics of 725 lactic acid bacteria isolated from raw goat milk of four Algerian races were studied. They were phenotypically classified as Lactobacillus (31.6%), Lactococcus (28.4%), Leuconostoc (22.2%), Streptococcus (13.7%) and Pediococcus (4.1%). No major differences were observed as to the distribution of genus isolates in the milk of three races (Makatia, Makatia-Chamia and Kabyle). However, the number of isolates of both Leuconostoc and Lactococcus predominated in Arabia milk while the samples collected from the other races mostly contained lactobacilli isolates.The phenotypic and biochemical analyses gave a diversity of species (28 presumptive species). The most abundant species were Streptococcus thermophilus (46 isolates), Lactobacillus helveticus (45 isolates), Lactobacillus plantarum (30 isolates), Lactobacillus delbruecki subsp. bulgaricus (26 isolates), Lactobacillus brevis (25 isolates) and Lactococcus lactis subsp. lactis (25 isolates).The technological properties of the isolates were as follows: 38.6% of isolates showed a fast acidifying rate (more than 60°D during 18 h of incubation); 25.9% of isolates had a high proteolytic capacity (more than 6 ppm leucine/72 h) and 14.1% of isolates had a high diacetyl production (more than 0.8 ppm diacetyl/16 h).Nine isolates were selected to prepare yoghurt and cheese. Sensory tests performed by a trained panel revealed a pleasant yoghurt prepared with mixed culture St. thermophilus 16 TMC+L. helveticus 20TMC. Cheese prepared with isolates L. lactis subsp. lactis biovar diacetylactis 19 MCA and L. lactis subsp. lactis 10 MCM had a very good sensory quality and a pleasant taste.     

Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro

Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out.     

Identification of cultivable lactic acid bacteria isolated from Algerian raw goat's milk and evaluation of their technological properties

One hundred and fifty-eight strains of lactic acid bacteria isolated from Algerian raw goat's milk were identified and technologically characterized. Five genera were found: Lactobacillus (50.63%), Lactococcus (25.94%), Streptococcus (14.56%), Leuconostoc (7.59%) and Pediococcus (1.26%). The predominant species were Lactococcus lactis (32 strains), Streptococcus thermophilus (23 strains), Lactobacillus bulgaricus (19 strains), Lb. helveticus (16 strains) and Lb. plantarum (14 strains).Approximately 39% of the lactic acid bacteria isolated produced more than 0.6% lactic acid (w/v) after 18 h of incubation, and belonged to the Lactococcus and Lactobacillus genera. The highest proteolytic activity was approximately 3 mg tyrosine l⁻¹ for mesophilic strains and nearly 5 mg tyrosine l⁻¹ for thermophilic lactobacilli after 72 h. High aromatic activity (more than 0.8 mg diacetyl l⁻¹ after 16 h) was detected in 14% of the strains.Nine strains were used to make dairy products (a yoghurt-like product and Edam-type cheese) on a pilot scale in the laboratory. The best-liked organoleptic characteristics were noted in a yoghurt produced with a mixed culture made up of S. thermophilus (strain 16TMC+) and Lb. helveticus (strain 20TMC) and in a cheese made with a starter composed of Lc. lactis subsp. lactis (strain 10MCM) and L. lactis subsp. lactis (V.P. +) (strain 19MCM).     

Selection of probiotic bacteria for the fermentation of a soy beverage in combination with Streptococcus thermophilus

Lactobacillus delbrueckii subsp. lactis R0187, Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011 and Bifidobacterium longum R0175 were examined for their ability to grow in combination with Streptococcus thermophilus cultures in milk and a laboratory soy beverage (LSB; both standardized to 4.5% protein and 2.3% fat). Strains R0011 and R0187 did not rapidly acidify the soy beverage despite good growth rates on soy carbohydrates. The S. thermophilus populations in the LSB were similar to that of milk even though milk had 30% more buffering capacity. In milk but not in soy, symbiosis with respect to acidification rate was observed between S. thermophilus and L. helveticus or B. longum. The populations of L. helveticus in the fermented products were similar in pure cultures or in the presence of the streptococci. However B. longum did not compete well in the mixed culture. Fermentation conditions varied as a function of the ability of S. thermophilus strains to acidify media to a pH of 4.65 (between 8 and 24 h). The probiotic populations in the mixed culture were influenced by the S. thermophilus strain and by the time of fermentation. Variations in growth rates of the bacteria did not appear to be linked to differences in initial redox or α-amino nitrogen levels. Strain selection enabled the preparation of a mixed starter, probiotic-fermented soy beverage containing 1.1 × 10⁸ CFU/mL of L. helveticus R0052, which represented approximately 13% of the total final population.     

Influence of proteolytic Lactococcus lactis subsp. cremoris on ripening of reduced-fat Cheddar cheese made with camel chymosin

This study investigated proteolysis in reduced-fat Cheddar cheese produced with camel chymosin and Lactococcus lactis subsp. cremoris with the ability to cleave the N-terminus of α_S1-casein. The aim was to match the activity of bovine chymosin, which leads to softer cheese structure than camel chymosin. Cheeses were analysed for gross composition, casein and peptide breakdown, release of free amino acids, structure parameters and sensory characteristics. Selected Lc. lactis subsp. cremoris increased the amount of peptides and, to a limited extent, the total amount of free amino acids in the cheeses. One group of experimental cheeses was found to have a significantly firmer structure, higher stress at fracture and modulus of deformability than the reference cheeses. The addition of the selected proteolytic dairy strains of Lc. lactis subsp. cremoris to the cheeses did not result in extended breakdown of α_S1-casein or a softer cheese structure.     

Effects of mixed starter composition on nisin Z production by lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 during production and ripening of Gouda cheese

A starter culture system that produced both acid and nisin at acceptable rates in milk for manufacture of Gouda cheese was developed using nisin Z-producing L. lactis subsp. lactis biovar. diacetylactis UL 719 (UL 719) and a commercial Flora Danica (FD) starter culture. Different compositions of mixed cultures (0, 0.2, 0.4, 0.6 or 0.8% UL 719 with 1.4% FD) were tested for acidification and nisin Z production in milk after 12 h incubation at 30 degrees C. The 0.6/1.4% combination, selected as the optimal mixture of starter cultures, acidified milk to a suitable pH and produced nisin Z at a high concentration of 512 IU/ml. With this optimal combination, FD numbers of citrate-fermenting and non-fermenting bacteria did not change compared with the control (1.4% FD). However, with 0.8% of L. lactis strain UL 719 and 1.4% of the FD starter culture, the numbers of citrate-fermenting and non-fermenting bacteria in fermented milk decreased compared with those obtained when milk was inoculated with 0.2, 0.4 or 0.6% of UL 719 added to 1.4% FD or control cultures (1.4% FD). Mixed starter culture ratios 0.6/1.4%, 0.4/1.4% and 0.5/1.4% (UL 719/FD) were used to manufacture nisin Z containing Gouda cheese which was ripened up to 45 weeks. The composition of control cheeses made with 1.4% FD, and nisin Z-containing Gouda cheeses were similar with respect to percent moisture, fat, salt and protein. During the ripening period, the cell counts observed were approximately two logs higher in cheese made with the 0.6/1.4% mixed starter culture than in control cheese. In experimental cheese produced with 0.6/1.4% (UL 719/FD) mixed starter culture, nisin activity increased from 256 IU/g at the end of manufacture to a maximum of 512 IU/g after 6 weeks of ripening; the levels then decreased to 128 and 32 IU/g after 27 and 45 weeks of ripening, respectively. In contrast, nisin Z was not detected in experimental cheeses made with 0.4/1.4% or 0.5/1.4% (UL 719/FD) mixed starters. Using an affinity purified anti-nisin polyclonal antibody, anti-rabbit gold-conjugate and transmission electron microscopy, nisin Z was found to be localized in the cheese matrix, in fat globules, in the casein phase and concentrated at the fat-casein interface. After 27 weeks of ripening, nisin Z was detected preferentially in the fat globules of the experimental cheese.     

Application of ruthenium red and colloidal gold-labeled lectin for the visualization of bacterial exopolysaccharides in Cheddar cheese matrix using transmission electron microscopy

The objective of the present study was to develop a methodology for direct observation of capsular and ropy strains and their exopolysaccharides (EPS) in a Cheddar cheese matrix. Cheddar cheeses with 50% reduced fat were made from milk containing 1.7% fat using mixed starter culture containing either capsule-forming Lactococcus lactis subsp. cremoris (SMQ-461) or ropy L. lactis subsp. cremoris (JRF-1) strains. Control cheese was made using the EPS-negative L. lactis subsp. cremoris (RBL132) strain. Following cheese pressing, samples were taken from each cheese treatment and examined by transmission electron microscopy (TEM). Samples were divided into two series: the first was prepared following the conventional methods (involving fixation, post fixation, dehydration and embedding in resin) and the second with added ruthenium red at 0.15% (w/v) during the fixation, post fixation and washing procedures. Gold-labeled lectin was also used for the visualization and localization of EPS in cheese matrix. Electron micrographs showed that ruthenium red makes it possible to visualize and enhance the resolution of the EPS in a Cheddar matrix compared with the conventional method. The EPS layer of the capsular strain appeared regular and evenly distributed around the cell, whereas the cell-associated EPS layer produced by the ropy strain was longer, more irregular (having a filamentous structure) and unevenly surrounded the cell. EPS released from the ropy strain appeared to form a network-like structure located principally in whey pockets and appeared to interact with the casein matrix and fat globule membrane. Labeling EPS by lectin conjugated to colloidal gold could only be performed with conventional preparation of cheese samples and appeared to react only with the cell surface rather than with liberated EPS. Besides their ability to bind water and increase cheese yield, capsular and ropy strains used in this study appear to have potential autolytic characteristics, which may have an impact on cheese proteolysis, texture and flavor quality.     

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

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.     

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.