固定化乳酸乳球菌连续生产Nisin的研究

以海藻酸钙为材料 ,固定乳酸乳球菌 (Lactococcuslactissubsp .lactis)SM5 2 6 ,研究不同条件对Nisin合成的影响。结果表明 ,利用 2 %海藻酸钠在 1 0mmol LCaCl2 条件下 ,得到的固定化细胞颗粒稳定性较好 ,可维持 90h无破裂 ;在发酵过程中SYS3培养基中的无机盐成分尤其磷酸盐对固定化颗粒有破坏作用 ;用mSYS3培养基代替SYS3 ,通过 72h三批次循环的半连续培养 ,Nisin活性为 85 0IU mL ,无明显的细胞渗漏现象。连续化生产 70h ,Nisin活性达 1 1 5 0IU mL ,相当于游离细胞的发酵水平。     

Production of a particulate hepatitis C vaccine candidate by an engineered Lactococcus lactis strain

Vaccine delivery systems based on display of antigens on bioengineered bacterial polyester inclusions can stimulate cellular immune responses. The food-grade Gram-positive bacterium Lactococcus lactis was engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which abundantly displayed the hepatitis C virus core (HCc) antigen. In mice, the immune response induced by this antigen delivery system was compared to that induced by vaccination with HCc antigen displayed on PHB beads produced in Escherichia coli, to PHB beads without antigen produced in L. lactis or E. coli, or directly to the recombinant HCc protein. Vaccination site lesions were minimal in all mice vaccinated with HCc PHB beads or recombinant protein, all mixed in the oil-in-water adjuvant Emulsigen, while vaccination with the recombinant protein in complete Freund's adjuvant produced a marked inflammatory reaction at the vaccination site. Vaccination with the PHB beads produced in L. lactis and displaying HCc antigen produced antigen-specific cellular immune responses with significant release of gamma interferon (IFN-γ) and interleukin-17A (IL-17A) from splenocyte cultures and no significant antigen-specific serum antibody, while the PHB beads displaying HCc but produced in E. coli released IFN-γ and IL-17A as well as the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 and low levels of IgG2c antibody. In contrast, recombinant HCc antigen in Emulsigen produced a diverse cytokine response and a strong IgG1 antibody response. Overall it was shown that L. lactis can be used to produce immunogenic PHB beads displaying viral antigens, making the beads suitable for vaccination against viral infections.     

Sugar Utilization and Acid Production by Free and Entrapped Cells of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactococcus lactis subsp. lactis in a Whey Permeate Medium

Cells of Streptococcus salivarius subsp. thermophilus and Lactococcus lactis subsp. lactis entrapped in k-carrageenan-locust bean gum gel performed similarly to free cells in the conversion of lactose to lactic acid. Bead diameter influenced the fermentation rate. Cells entrapped in smaller beads (0.5 to 1.0 mm) showed higher release rates, higher lactose, glucose, and formic acid utilization, higher galactose accumulation, and higher lactic acid production than did cells entrapped in larger beads (1.0 to 2.0 mm). Values for smaller beads were comparable with those for free cells. Immobilization affected the fermentation rate of lactic acid bacteria, especially Lactobacillus delbrueckii subsp. bulgaricus. Entrapped cells of L. delbrueckii subsp. bulgaricus demonstrated a lower lactic acid production than did free cells in batch fermentation. The kinetics of the production of formic and pyruvic acids by L. lactis subsp. lactis and S. salivarius subsp. thermophilus are presented.     

Continuous production of mixed lactic starters containing probiotics using immobilized cell technology

The production of a mixed lactic culture containing Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707 was studied during a 17-day continuous immobilized-cell culture at different temperatures between 32 and 37 degrees C. The two-stage fermentation system was composed of a first reactor (R1) containing cells of the two strains separately immobilized in kappa-carrageenan/locust bean gum gel beads and a second reactor (R2) operated with free cells released from the first reactor. The system allowed continuous production of a concentrated mixed culture with a strain ratio whose composition depended on temperature and fermentation time. A stable mixed culture (with a 22:1 ratio of L. diacetylactis and B. longum) was produced at 35 degrees C in the effluent of R2, whereas the mixed culture was rapidly unbalanced in favor of B. longum at a higher temperature (37 degrees C) or L. diacetylactis at a lower temperature (32 degrees C). Strain redistribution in beads originally immobilizing pure cultures of L. diacetylactis or B. longum was observed. At the end of culture, the strain ratio (7:1 L. diacetylactis/B. longum) in bulk bead samples was similar to that of individual beads. The determination of the spatial distribution of the two strains in gel beads by immunofluorescence and confocal laser-scanning microscopy showed that bead cross-contamination was limited to a 100 microm peripheral layer. Data from this study validate a previous model for population dynamics and cell release in gel beads during mixed immobilized-cell cultures.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads.

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

Continuous production of lacticin 3147 and nisin using cells immobilized in calcium alginate

Bacteriocinogenic strains, Lactococcus lactis subsp. lactis DPC 3147 and L. lactis DPC 496, producing lacticin 3147 and nisin, respectively, were immobilized in double-layered calcium alginate beads. These beads were inoculated into MRS broth at a ratio of 1:4 and continuously fermented for 180 h. Free cells were used to compare the effect of immobilization on bacteriocin production. After equilibrium was reached, a flow rate of 580 ml h(-1) was used in the immobilized cell (IC), and 240 ml h(-1) in free-cell (FC) bioreactors. Outgrowth from beads was observed after 18 h. Bacteriocin production peaked at 5120 AU ml(-1) in both IC and FC bioreactors. However, FC production declined after 80 h to 160 AU ml(-1) at the end of the fermentation. Results of this study indicate that immobilization offers the possibility of a more stable and long-term means of producing lacticin 3147 in laboratory media than with free cells.     

Quantitative determination of the spatial distribution of pure- and mixed-strain immobilized cells in gel beads by immunofluorescence

A new method was developed to detect and quantify two strains, Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707, immobilized separately and co-immobilized in gel beads, using specific polyclonal antibodies and confocal laser-scanning microscopy. The establishment of biomass concentration profiles for each strain was measured during colonization of beads using successive pH-controlled batch fermentations. Growth occurred preferentially in 200- and 300-microm peripheral layers of the beads for L. diacetylactis and B. longum, respectively. Repeated-batch cultures with immobilized cells permitted the production of a mixed culture containing a non-competitive strain of bifidobacteria, as a result of immobilized-cell growth and high cell-release activity from the beads. During co-immobilized fermentations, there were no apparent interactions between the strains.     

Nisin formation by immobilized cells of the lactic acid bacterium, Streptococcus lactis]

The problem of microbial cell immobilization at present attracts the ever increasing attention of the scientists, since such organisms may be the source of various enzymes. Production of nizin by the immobilized cells of Str. lactis was studied. It was found that the cells of Str. lactis incorporated into polyacrylamide gel produced nizit on definite media. Still, the amount of the antibiotic was 2-3 times lower than in case of using free cells. The effect of a number of factors on the process of immobilization was studied and the influence of some factors, such as temperature, pH, aeration on nizin synthesis by the immobilized cells of the streptococcus was elucidated. Optimal conditions for nizin biosynthesis by the immobilized cells of Str. lactis were developed.     

Continuous mixed strain mesophilic lactic starter production in supplemented whey permeate medium using immobilized cell technology

The aim of this work was to study a new process for the continuous production of mixed-strain lactic acid bacteria starters using immobilized cells. Three strains of Lactococcus (two Lactococcus lactis subsp. lactis: KB and KBP, and one Lactococcus lactis subsp. lactis biovar diacetylactis: MD) were immobilized separately in kappa-carrageenan-locust bean gum gel beads. Continuous fermentations were carried out in a 1 L pH-controlled stirred tank reactor with a 30% (v/v) bead inoculum (strain ratio 1:1:1), continuously fed with a whey UF permeate medium, supplemented with 1.5% yeast extract and 0.1M KCl. The effects of three parameters-pH, temperature (T), dilution rate (D), and their interactions on the composition and activity of the culture in the effluent at pseudosteady state were studied according to a rotatable central composite design, during a 53-day fermentation. The process showed a high biological stability and no strain became dominant, or was eliminated from the bioreactor. The statistical analysis showed that the three strains were differently affected by the studied parameters, and that a large range of effluent starter composition can be achieved by varying D, pH, and T. However, the acidifying characteristics were not affected by the culture conditions. A cross-contamination from other strains of the mixed culture was observed in gel beads entrapping a pure culture at the fermentation onset, and led to a biomass redistribution within the beads. However, the strain ratio (KB:KBP:MD) observed after the 53-day experiment (1:2:2) was close to the initial bead ratio (1:1:1). The beads demonstrated a high mechanical stability throughout the 53-day continuous fermentation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 502-516, 1997.     

Components of fermentation medium regulate bacteriocin synthesis by the recombinant strain Lactococcus lactis subsp. lactis F-116

The regulation of the synthesis of bacteriocin produced by the recombinant strain Lactococcus lactis subsp. lactis F-116 has been studied. The synthesis is regulated by the components of the fermentation medium, the content of inorganic phosphate (KH2PO4), yeast autolysate (source of amine nitrogen), and changes in carbohydrates and amino acids. The strain was obtained by fusion of protoplasts derived from two related L. lactis subsp. lactis strains, both exhibiting a weak ability to synthesize the bacteriocin nisin. Decreasing the content of KH2PO4 from 2.0 to 1.0 or 0.5% caused bacteriocin production to go down from 4100 to 2800 or 1150 IU/ml, respectively; the base fermentation medium contained 1.0% glucose, 0.2% NaCl, 0.02% MgSO4, and yeast autolysate (an amount corresponding to 35 mg % ammonium nitrogen). The substitution of sucrose for glucose (as the source of carbon) increased the antibiotic activity by 26%, and the addition of isoleucine, by 28.5%. Elevation of the concentration of yeast autolysate in the low-phosphate fermentation medium stimulated both the growth of the lactococci and the synthesis of bacteriocin. Introduction of 1% KH2PO4, yeast autolysate (in an amount corresponding to 70 mg % ammonium nitrogen), 2.0% sucrose, and 0.1% isoleucine increased the bacteriocin-producing activity of the strain by 2.4 times.     

Increased stress tolerance of Bifidobacterium longum and Lactococcus lactis produced during continuous mixed-strain immobilized-cell fermentation

AIMS: The effect of immobilization and long-term continuous culture was studied on probiotic and technological characteristics of lactic acid and probiotic bacteria. METHODS AND RESULTS: A continuous culture in a two-stage system was carried out for 17 days at different temperatures ranging from 32 to 37 degrees C, with a first reactor containing Bifidobacterium longum ATCC 15707 and Lactococcus lactis subsp. lactis biovar. diacetylactis MD immobilized separately in gel beads, and a second reactor operated with free cells released from the first reactor. The tolerance of free cells from both strains produced in the effluent medium of both reactors to hydrogen peroxide, simulated gastric and intestinal juices, antibiotics and nisin, and freeze-drying markedly increased with culture time and was generally higher after 6 days than that of stationary-phase cells produced during free-cell batch fermentations. The reversibility of the acquired tolerance of B. longum, but not L. diacetylactis, to antibiotics was shown during successive free-cell batch cultures. CONCLUSIONS: Free cells produced from continuous immobilized-cell culture exhibited altered physiology and increased tolerance to various chemical and physico-chemical stresses. SIGNIFICANCE AND IMPACT OF THE STUDY: Continuous culture with immobilized cells could be used to produce probiotic and lactic acid bacteria with enhanced technological and probiotic characteristics.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

pH-controlled cell release and biomass distribution of alginate-immobilized Lactococcus lactis subsp. lactis

AIMS: To investigate the growth and release of Lactococcus lactis subsp. lactis in gel beads and to affect rates of cell release by changing the growth conditions. METHODS AND RESULTS: The rate of release and the distribution of immobilized L. lactis subsp. lactis in alginate beads were studied in continuous fermentations for 48 h. A change in operating pH from 6.5 to 9.25 initially reduced the ratio of the rates of cell release to lactate production by almost a factor of 105. Compared with fermentations at pH 6.5, growth at pH 9.25 also increased the final internal bead biomass concentration by a factor of 5 and increased the final rate of lactate production by 25%. After 48 h, the ratio of the rates of cell release to lactate production was still 10 times lower than in fermentations at pH 6.5. CONCLUSIONS: A change in the operating pH from 6.5 to 9.25 reduced rates of cell release throughout 48 h of fermentation and increased the final rates of lactate production and internal bead biomass concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These data illustrate that diffusional limitations and corresponding pH gradients can be exploited in affecting the distribution of immobilized growing cells and their concomitant release.     

Adsorption of the antibiotic, nisin, to Streptococcus lactis cells

Nizin is produced by Str. lactis, strain MSU. During biosynthesis it is excreted into the fermentation broth and gradually adsorbed on the organism cells. This was confirmed by experiments with an inactive variant of Str. lactis IIa. The cells of this culture adsorbed nizin from "active" fermentation broth. Adsorption of nizin depended on pH of the medium; at pH 2,3 the cells did not adsorbe the antibiotic and at pH 6.6 the amount of the antibiotic adsorbed by the cells was maximum.     

Mass transfer analysis for immobilized cells of Lactococcus lactis sp. using both simulations and in-situ pH measurements

Mathematical modeling and in-situ pH measurements were used to characterize the effects of the microenvironment on alginate gel beads immobilized cells of Lactococcus lactis. Mass transfer limitations led to a progressive pH acidification within gel beads which determined both the cell distribution and the cellular activity of entrapped cells. The dynamics of the system is discussed in relation to the overall activity of the immobilized cell reactor.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.