Optimization of Cultivation Conditions for the Production of γ-Cyclodextrin Glucanotransferase by Bacillus macorous

Abstract

The production of γ-cyclodextrin glucanotransferase (γ-CGTase) from Bacillus macorous WSH02-06 was optimized in shake flasks using conventional sequential techniques and statistical experimental design. Effects of nutrients including carbon and nitrogen sources, cation ions, initial pH, and temperature on γ-CGTase production were investigated. Corn starch, peptone, Mn²⁺, and Zn²⁺ were found to be essential for obtaining high γ-CGTase activity and biomass. The promoting effect of manganese and zinc on enzyme production has not been reported previously. According to the results of orthogonal array experiment, the optimal culture medium for high γ-CGTase activity was determined. Maximal γ-CGTase activity obtained in the optimized culture broth was about 250?U/mL which was 10-fold higher than that obtained in the basal medium. Time course of cell growth and γ-CGTase production in a 7 l fermenter showed that enzyme production was growth associated, and that maximum γ-CGTase activity reached 277?U/mL in 17?h.     

Extracellular α-Amylase Production by Bacillus brevis MTCC 7521

Alpha amylases have various applications in food processing industries, for example, baking, brewing and distillery industries. Studies of the Ca²⁺ independent α-amylase production were carried out by a strain of Bacillus brevis MTCC 7521 isolated from a brick kiln soil. The optimum temperature, pH and incubation period for amylase production were 50°C, 6.0 and 36 h, respectively. The enzyme secretion was at par in the presence of any of the carbon sources (soluble starch, cassava starch and cassava flour). B. brevis produced more amylase in presence of beef extract as nitrogen source in comparison to other organic nitrogen sources (peptone, yeast extract and casein) and asparagine, potassium nitrate, ammonium sulphate, ammonium nitrate and urea reduced the enzyme activity. The addition of Ca²⁺ (10–40 mM) or surfactants (Tween 20, Tween 40, Tween 60, Tween 80, and sodium lauryl sulphate at 0.02% concentration) in culture medium did not result in further improvement in the enzyme production. The purified enzyme had a molecular mass of 205 kDa in native SDS-PAGE.     

Extracellular Production of a Novel Ice Structuring Protein by Bacillus subtilis—a Case of Recombinant Food Peptide Additive Production

Bacillus subtilis is an ideal host for the production of extracellular heterologous proteins. Antifreeze protein (ice structuring protein, ISP) is a potent additive in cryogenic preservation of food products. In this investigation, the first production of a recombinant ISP was achieved by using a high-efficient artificial promoter and a signal peptide from subtilisin YaB. The functional ISP was expressed extracellularly at a high level by a protease deficient host, B. subtilis WB800. This study provides an efficient tool to produce recombinant food peptide additive at a high level and overcomes the problem of natural resources limitation as well.     

Enhanced antiviral activity of soybean β-conglycinin-derived peptides by acylation with saturated fatty acids

Abstract: Peptide mixtures prepared from soybean β‐conglycinin (7S‐peptides) were acylated with saturated fatty acids of different chain length (6C‐18C) in order to improve their antiviral activity against Feline calicivirus (FCV) strain F9 which is a typical norovirus surrogate. Among the fatty acids varieties, it was revealed that 7S‐peptides acylated with myristic and palmitic acids potently inhibited FCV replication. Myristorylation and palmitoylation of 7S‐peptides kept host cells viability at 91.51% and 98.90%, respectively. The infectivity of FCV on Crandell–Reese feline kidney cells was further determined after exposure of initial titer of 10^6.47 TCID₅₀/mL. Myristoylated and palmitoylated 7S‐peptides significantly (P < 0.006) reduced FCV infectivity as compared to native 7S‐peptides. Native 7S‐peptides showed 25% FCV inhibitory activity while myristoylated and palmitoylated 7S‐peptides exhibited 98.59% and 99.98% reduction in FCV infectivity, respectively. Myristoylated and palmitoylated 7S‐peptides demonstrated higher anti‐FCV activity in a wide range of concentration with complete reduction at 25 μg/mL. Surface hydrophobicity was significantly (P < 0.05) increased after attachment of long hydrocarbon fatty acids to 7S‐peptides as supported by changes in fluorescence intensity. Enzymatic hydrolysis together with acylation will give an insight into surface and physiological functional lipopeptides derived from soy β‐conglycinin.     

Production of γ-aminobutyric acid during fermentation of Gastrodia elata Bl. by co-culture of Lactobacillus brevis GABA 100 with Bifidobacterium bifidum BGN4

Gastrodia elata Bl. (GE) is a traditional herbal medicine used for the prevention of cerebrovascular disease and for the regulation of one’s blood pressure. γ-Aminobutyric acid (GABA) is known to be beneficial for preventing neurological disorders and hypertension. The objective of this study was to develop fermented GE products containing high levels of GABA. The optimal medium conditions for the production of GABA during fermentation were as follows: an initial pH of 6.5, 3% (w/v) l-monosodium glutamate, 10% (w/v) freeze-dried GE powder, and 0.5% (w/v) yeast extract. The production of GABA was further enhanced by the co-culture of Lactobacillus brevis GABA 100 with Bifidobacterium bifidum BGN4. During fermentation, high amounts of organic acids and GABA were produced, while gastrodin, the main polyphenol compound of GE, was completely converted to 4-hydroxybenzylalcohol. The fermented GE product with GABA was successfully produced by optimizing the fermentation conditions.     

Synthesis of oligosaccharides with lactose and N-acetylglucosamine as substrates by using β-d-galactosidase from Bacillus circulans

In the present study, β-d-galactosidase from Bacillus circulans was proved to be a suitable biocatalyst for the production of N-acetyl-oligosaccharides with lactose and N-acetylglucosamine (GlcNAc) as biocatalyst. During the hydrolysis of lactose, apart from no ultraviolet absorption oligosaccharides such as β-d-Galp-(1 → 6)-d-Glcp (6′-allolactose) and β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-Glcp (4′-galactosyl-lactose), the formation of four N-acetyl-oligosaccharides was followed by high-performance liquid chromatography with a diode-array detector. The four N-acetyl-oligosaccharides were isolated from the reaction mixture and identified to be as β-d-Galp-(1 → 4)-d-GlcpNAc (LacNAc, I), β-d-Galp-(1 → 6)-d-GlcpNAc (allo-LacNAc, II), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (III), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (IV) by authentic standards and the spike technique or high-resolution mass spectrometry with an electrospray ionization source and nuclear magnetic resonance spectroscopy. Furthermore, the effects of synthetic conditions including reaction temperature, concentration of substrate, molar ratio of donor/acceptor and enzyme concentration on the formation of N-acetyl-oligosaccharides were examined. We found that the optimal synthetic conditions were different for production of oligosaccharides with β-(1 → 4) linkages and β-(1 → 6) linkage. The optimal reaction conditions for I, III and IV were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 1.0 U/mL of enzyme. Under such conditions, the N-acetyl-oligosaccharides formed were composed of 28.75% of I, 2.29% of II, 9.47% of III and 5.67% of IV. On the other hand, suitable reaction conditions found for II were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 2.0 U/mL of enzyme.     

Production of fructooligosaccharides and β-fructofuranosidase by batch and repeated batch fermentation with immobilized cells of Penicillium expansum

The production of fructooligosaccharides (FOS) and ??-fructofuranosidase (FFase) by immobilized cells of Penicillium expansum was evaluated. In an initial stage, different low-cost materials including synthetic fiber, polyurethane foam, stainless steel sponge, loofah sponge, and cork oak were tested as carrier for the fungus immobilization. Additionally, the influence of the inoculum age (1 or 3?weeks) on cells immobilization, FOS and FFase production was also verified. Synthetic fiber and polyurethane foam were the best materials for P. expansum immobilization (2.21 and 1.98?g/g carrier, respectively) and FOS production (120.3 and 104.8?g/l), and gave also high results of FFase activity (23.01 and 32.42?U/ml). Then, the production of FOS and FFase by repeated batch fermentation with P. expansum immobilized on synthetic fiber was studied, aiming to improve the batch fermentation results. The results obtained in this stage were very promising with FOS yields of 87, 72, and 44?%, in the 3 initial cycles (60?h), respectively; the FFase activity was constant throughout the process (6 cycles, 96?h). Repeated batch fermentation with immobilized cells of P. expansum was found as being a technology with great potential for FOS and FFase production on industrial scale.     

Role of cysteine in the improvement of γ-aminobutyric acid production by nonproteolytic Levilactobacillus brevis in coculture with Streptococcus thermophilus

Previous research has showed that nonproteolytic Levilactobacillus brevis 145 (L) in coculture with Streptococcus thermophilus 1275 (S), not Lactobacillus delbrueckii ssp. bulgaricus (Lbu), was able to produce γ-aminobutyric acid (GABA) during milk fermentation in the presence of monosodium glutamate (MSG). It was assumed that differences of casein hydrolysis patterns between Strep. thermophilus 1275 and L. bulgaricus caused the phenomenon. Moreover, the GABA content was low and residual MSG was high in SL-fermented milk. In our research, comparison of peptide profiles determined by liquid chromatography/tandem mass spectrometry showed that α_S2-casein, β-casein, and κ-casein degradation by L. bulgaricus and Strep. thermophilus varied. Importantly, the peptide number in the L and Lbu coculture group increased compared with the Lbu monoculture group, whereas the peptide number in the SL coculture group decreased in comparison with S monoculture group, suggesting that L. bulgaricus was not able to provide peptides for the growth of Lb. brevis 145. Furthermore, we found that after supplementation with cysteine (50 mg/L) during milk fermentation by SL, 10 g/L MSG was converted into 4.8 g/L GABA with a minimum level of residual MSG, viable cell counts of Lb. brevis and lactic acid production were increased, and the casein hydrolysis pattern was not influenced. Moreover, sulfhydryl group-containing chemicals including cystine, reduced glutathione, and oxidized glutathione showed effects similar to that of cysteine in improving GABA production. Finally, when L. bulgaricus YIB2 was combined with SL, supplementation of cysteine was also able to significantly improve GABA production.