Céline Paquin

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.

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.

Batch fermentations with a mixed culture of lactic acid bacteria immobilized separately in κ-carrageenan locust bean gum gel beads

SummaryStreptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus were immobilized separately in κ-carrageenan-locust bean gum gel beads. The beads were prepared by a dispersion process in a two-phase system (water in oil) and two ranges of bead diameter selected by sieving (0.5–1.0 mm and 1.0–2.0 mm). Fermentations with the two strains were conducted in bench bioreactors in a supplemented whey permeate medium. Free and entrapped cells (two ranges of bead diameter and two levels of initial bead cell load) were grown in mixed culture, and carbohydrate utilization, acid production and cell growth or cell release rate measured. Fermentation rates were influenced by bead diameter and initial cell load of the beads. Beads with high initial cell density increased fermentation rates compared to low cell density beads or free cells. Smaller diameter beads (0.5–1.0 mm) showed a stable tendency (not statistically significant pa > 0.05) towards higher cell release rates, lactose utilization, galactose accumulation and lactic acid production than did larger diameter beads (1.0–2.0 mm). Immobilization of S. salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus in separate beads did not seem to affect protocooperation during batch fermentation, and allowed for high cell release rates into the medium.

Continuous fermentation of a supplemented whey permeate medium with immobilized Streptococcus salivarius subsp. thermophilus

Abstract Streptococcus salivarius subsp. thermophilus was immobilized in k -carrageenan/locust bean gum mixed gel beads (0·5–2·0 mm diameter), using a dispersion process in a two-phase system. Inoculated beads were used to ferment a supplemented whey permeate medium continuously in a 0·75-liter bioreactor equipped with external pH control and mechanical stirring. The pH-stat fermentations were conducted at optimal pH of 5·8, inoculation level of 20% (v/v) and various dilution rates (D = 0·5, 1·0, 1·5, 2·0 and 3·0 h−1). Cell production, carbohydrate utilization and acid production were measured at a steady state. High cell release rates from the gel beads into the broth medium, and growth of free cells in the bioreactor allowed for a steady and efficient inoculation and fermentation of the feed, with cell counts in the outflow varying from 1 × 109 to 3·0 × 108 CFU/ml for D from 0·5 to 3·0 h−1. Lactic acid concentration in the effluent was 17·3 and 4·3 g/liter for D = 0·5 and 3·0 h−1, respectively. Biomass yield showed a slight but not significant tendency to increase with increasing dilution rates. Gel bead integrity was only slightly affected after 72 h continuous fermentation, particularly for bead diameters in the range 1·3–1·7 mm, even though the selected level of broth supplementation with KCl was deliberately suboptimal for the mechanical properties of the gel beads. This process could be used for efficient continuous inoculation and fermentation of dairy fluids, or for continuous single or mixed strain starter production.

Effect of medium and temperature of storage on viability of lactic acid bacteria immobilized in κ-carrageenan-locust bean gum gel beads

The influence of various storage solutions and temperature (4°C and 25°C) on viability ofStreptococcus salivarius subsp.thermophilus andLactobacillusdelbrueckii subsp.bulgaricus entrapped in κ-carrageenan-locust bean gum mixed gel beads was studied. The immobilized strains could be stored at 4°C in all storage solutions studied for at least 14 and 11 days respectively before counts decreased to 105c.f.u./mL, which was considered to be the practical limit for their use as inoculum in a fermentation process. The most effective storage solutions for preserving cell viability at 4°C were NaCl, glycerol and sorbitol solutions forS. thermophilus, and PO4 buffer and sorbitol solutions forL. bulgaricus. At 25°C,S. thermophilus could be stored for over 14 days in all solutions except glycerol, andL. bulgaricus for 4 days in 10% sorbitol.

Production of concentrated freeze-dried cultures of Bifidobacterium longum in kappa-carrageenan-locust bean gum gel

Bifidobacterium longum was immobilized in k-carrageenan/locust bean gum gel beads, and cultured in a medium containing Lactobacillus MRS broth and whey-permeate. The same beads were incubated for 5 successive batch fermentations and freeze-dried following mixing with a protective solution. Viable population in the beads increased from 8 3 10 7 to 4.7 3 10 10 cfu/g after three batch fermentations, but no further increase in viable cell population could be achieved in the last two fermentations. The freeze-dried culture contained 3 3 10 10 cfu/g with a survival rate of approximately 10%. Survival to freeze-drying of immobilized cells was as good as that of classical free-cell cultures. Stability of freeze-dried cultures during storage at minus 17, 4 and 20°C was not influenced by immobilization.

Direct measurement of pH profiles in gel beads immobilizing Lactobacillus helveticus using a pH sensitive microelectrode

A H+-selective liquid membrane microelectrode was prepared and used to measure the pH profile evolution during colonization of gel beads immobilizing Lactobacillus helveticus in a whey permeate medium. A large pH gradient was observed in a highly active periferal layer thickness that decreased from 500 to 300 μm for an immobilized cell population that increased from 5.8 × 109 to 3.1 × 1010 CFU/g. The flat pH profile (pH 4.4) in the central part of the bead was attributed to a high concentration of the inhibitory undissociated form of lactic acid.