C. Lacroix

Immobilized Cell Technologies for the Dairy Industry

The potential applications of immobilized cell technology (ICT) to the dairy industry are examined. Immobilization modifies the physiology of cells, and the consequences of ICT on lactose as well as citrate metabolism are reviewed. Immobilization also affects the sensitivity of lactic acid bacteria (LAB) to salt and penicillin. ICT can be used to produce starters for the dairy industry, and aspects of biomass production in beads, continuous cell release from beads, and continuous fermentations with filtration cell recycle are examined. Potential applications of ICT to the dairy industry include acidification of raw milk prior to ultrafiltration, inhibition of psychrotrophic bacteria in raw milk, yogurt production, cheese manufacture, and cream fermentations. Impacts of yeast, bacterial, or bacteriophage contaminations in ICT processes as well as their control are discussed.

IMMOBILIZED GROWING LACTIC ACID BACTERIA WITH ?-CARRAGEENAN-LOCUST BEAN GUM GEL

SummaryA cell entrapment process using κ-carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.

Kinetic study of continuous whey permeate fermentation by immobilized Lactobacillus helveticus for lactic acid production

A pH-controlled continuous lactic acid fermentation of whey permeate supplemented with yeast extract was carried out with Lactobacillus helveticus entrapped in κ-carrageenan-locust bean gum gel beads at various dilution rates and pH set points. A kinetic study of the immobilized cell continuous fermentation in a 1-l stirred-tank reactor containing 33% (v/v) gel beads was carried out with an original experimental device, consisting of a 110-ml second-stage free-cell fermenter. This second stage was assumed to reflect the physiological behavior of the free-cell phase in the immobilized-cell bioreactor. A maximum lactic acid production rate of 28.5 g l−1 h−1 was measured at pH 5.5 and dilution rate 1.21 h−1 for the immobilized-cell bioreactor. The overall specific growth rate and the specific lactic acid production rate of entrapped cells were 12–18% those of free cells in the reactor. This low activity may result from the presence of high inhibition in the central part of the beads as a result of substrate, product, and pH gradients. However, entrapped cells were responsible for 75–85% of the lactic acid and biomass productions within the immobilized cell fermenter at pH 4.7–6.3 and close to 90% at pH 4.3.

Influence of growth conditions on production and activity of mesenterocin 5 by a strain of Leuconostoc mesenteroides

The effects of various parameters on production and activity of mesenterocin 5, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides UL5, were investigated. Titres of bacteriocin and minimum inhibitory concentration values were determined by a critical dilution micromethod, using a sensitive strain of Listeria ivanovii as an indicator. Production of the antimicrobial compound was optimal at 37 and 40°C after 9 h of incubation, and was maximized in an aerobic fermentor maintained at pH 5.0. Tween 80 was a major factor in increasing mesenteroxin 5 production and specific production. Large quantities of bacteriocin could be obtained in whey and in whey permeate supplemented with yeast extract in the presence of the surfactant (0.1%). Most of the Listeria strains tested including L. monocytogenes were highly sensitive to the bacteriocin in the pH range 5.5 to 6.0 and at a temperature of 20 to 25°C.

Lactobacillus helveticus growth and lactic acid production during pH-controlled batch cultures in whey permeate/yeast extract medium. Part I. Multiple factor kinetic analysis

Abstract Twenty pH-controlled batch cultures with Lactobacillus helveticus were carried out in whey permeate-yeast extract medium according to a composite design with three factors: pH setpoint, and yeast extract and initial whey permeate concentrations. Growth and production parameters were estimated from experimental data with the Richards and Luedeking and Piret models, respectively, and analyzed statistically with response surfaces. The maximum specific growth rate, μ max , depended primarily on pH, with an optimum of 0.7 h −1 near pH 5.5. Whey permeate concentration had no direct effect on maximum specific growth rate, but showed a strong interaction with pH. Yeast extract addition had a strong positive effect on maximum specific growth rate. During nitrogen-limited batch cultures X max depended on yeast extract concentration and pH, as well as on interactions of these variables with whey permeate concentration. The growth-associated lactic acid production parameter a was constant at 4.5 g lactic acid (g biomass) −1 , while the nongrowth-associated production parameter b, estimated during growth and early stationary phase, was a linear function of pH. This study allowed for the first detailed multifactor kinetic analysis of Lb. helveticus growth and lactic acid production, with a limited number of experiments, and demonstrated the importance of interactions among tested culture conditions.

Purification and structure of mutacin B-Ny266: a new lantibiotic produced by Streptococcus mutans

Mutacins are bactericidal substances of proteinaceous nature produced by Streptococcus mutans. Lantibiotics are antibacterial substances containing post‐translationally modified amino acids such as lanthionine. Mutacin B‐Ny266 was purified from the cell pellet of S. mutans strain Ny266 by ethanol extraction at pH 2.0 followed by reversed‐phase chromatography (Sep‐Pak® cartridge) and by HPLC on a C18 column. The mean purification factor was 3240±81 and the mean yield was 1.0±0.1%. Molecular mass of mutacin B‐Ny266 as determined by mass spectroscopy is 2270.29±0.21 Da. The amino acid sequence of the purified active fraction was obtained by Edman degradation after treatment with alkaline ethanethiol. Twenty‐one amino acids were detected in this analysis. Mutacin B‐Ny266 belongs to the type A lantibiotics. The proposed sequence is: F–K– –W–U–F– – –P–G– –A–K–O–G– –F–N– –Y– . The molecule differs from that of epidermin/staphylococcin 1580 and gallidermin at positions 1, 2, 4, 5 and 6.

Batch fermentation with entrapped growing cells of Lactobacillus casei : optimization of the rheological properties of the entrapment gel matrix

SummaryGrowing cells ofLactobacillus casei were entrapped inκ-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%κ-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K+ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 1010 cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 1011 cells/ml and bead integrity was maintained throughout fermentation.Growing cells ofLactobacillus casei were entrapped inκ-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%κ-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K+ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 1010 cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 1011 cells/ml and bead integrity was maintained throughout fermentation.

Effect of agitation rate on cell release rate and metabolism during continuous fermentation with entrapped growing

The effects of agitation rate (50 to 150 rpm) on cell metabolism and cell release rates were studied during continuous fermentation of de Man, Rogosa and Sharpe medium (MRS) by immobilizedLactobacillus casei subsp.casei in κ-carrageenan/locust bean gum gel beads. Biomass concentration in the outflow was significantly higher at high agitation rates. Shear forces promoted cell loss from the beads by disrupting cell-filled cavities in the gel, near the particle surface. Moreover, high agitation rates enhanced fluid-to-particle mass transfer.

Note: Genetic and biochemical characterization of nisin Z produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719

The bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719 was purified and characterized. Two peaks exhibiting antimicrobial activity were obtained after purification. Primary structure of the peptide of major peak 2 was identical to that of nisin Z when determined by Edman degradation and confirmed by DNA sequence analysis. The molecular mass as determined by mass spectrometry was 3346·39 ± 0·40 Da for peak 1 and 3330·39 ± 0·27 Da for peak 2, which suggests that peak 1 may correspond to an oxidized form of nisin Z. The two purified peaks exhibiting xrantimicrobial activity appear to correspond with the oxidized and native forms of nisin Z.

Comparison of simple neural networks and nonlinear regression models for descriptive modeling of Lactobacillus helveticus growth in pH-controlled batch cultures

A set of 20 Lactobacillus helveticus growth curves was obtained from pH-controlled batch cultures with different pH setpoints, whey permeate and yeast extract concentrations. To find the best descriptive model of the biomass concentration versus time (y = X(t)) growth curve, fitting results of a large number of models were compared with statistical and approximate methods. Models studied included simple neural networks, reparameterized Logistic, Gompertz, Richards, Schnute, Weibull, and Morgan-Mercier-Flodin models, Amrane-Prigent model, and four new models based on autonomous growth functions. Simple neural networks with only four weights were good descriptive models of the growth curves and fitting qualities were similar to those of the best existing four-parameter models, such as the Logistic model. However, meaningful parameters had to be calculated numerically and use of simple neural networks yielded no distinctive advantages over other models. A new five-parameter model, based on an autonomous growth function, yielded the best fitting results, even when the number of model parameters was accounted for in the comparisons. However, the maximum specific growth rate was not always well estimated. Therefore the five-parameter Richards model was chosen as the best descriptive model of the growth curve.