| ?-ploy-glutamicacid(?-PGA) is a anionic polypeptide polymerizing by homopolymerization with microbial fermentation. Its structure decides the unique physical, chemical and biological characteristics. y-PGA has several special properties such as water-solubility, moisturizing, membranous, thickening and edible. These properties make y-PGA attractive for many potential applications in many fields such as food, cosmetic, medicine, pharmacology, environmental protection. In this study, pgsB which is a key gene of y-PGA synthetise with Bacillus subtilis HCUL-B-115(CGMCC2283) was cloned and bioinformatics analysed. Optimized components of the culture medium producing y-PGA, and studied the frost resistance of ?-PGA in terms of freeze-dried protection of Bifidobacterium longum and quick-frozen dumpling two application.With gene in Bacillus subtilis HCUL-B-115 as template, the PgsB synthase gene was amplified by PCR.Constructed the recombinant plasmid pET-22b-pgsB, and transformed into E.coli TransI. After preliminary identification by PCR, the positive clones were sequenced. And analyzed the biological information of the protein with the corresponding online software. The results of sequencing and Blast were compared, and the results showed that the consistency of the sequence with subtilis Bacillus was 99%. PgsB gene contained 1182 nucleotides, encoding PgsB synthase protein of 393 amino acids with 44KDa of relative molecular weight and predicted pI value of 5.31. Sequence analysis showed that there was a signal peptide between the fifteenth and sixteenth amino acid residues of PgsB protein and a transmembrane helical region in the 1-17 amino acid at the N end. There may be a N-glycosylation site at the location of the 213rd amino acid residues. PgsB was a kind of hydrophilic unstable protein which is secreted to the extracellular and needs to be modified by the endoplasmic reticulum. PgsB had a ATP/GTP binding site, indicating its catalytic role requires ATP to provide energy. PgsB protein was mainly composed of alpha helix and irregular coil, in which the alpha helix is 44.53%, and the rule is 39.19%. The prediction of 3D spatial structure is based on the template le8c.1.A, the similarity of the sequence is 17.33%, and the model quality parameter Z value is 0.49.The culture medium of prouducing y-PGA with Bacillus subtilis HCUL-B-115 was optimized by one factor test, design of PB and response surface method. The optimum medium composition was:saccharification liquid from corn (Brix 200g/L) 400.00mL/L.20.10 g/L C6H5O7 (NH4)3,37.70 g/L C5H8NO4Na,8.00 g/L KH2PO4,12.00 g/L NaCl,0.20 g/L CaCl2, 2.00 g/L MgSO4 and 4.00 g/L MnSO4. Under the optimal culture conditions, the production of y-PGA was 83.64g/L.With Bifidobacterium longum as experimental strain, in polymery-PGA as main protective agent, freeze-drying experiment was carried out. The optimal formulation of protective agent was studied by survival rate, the ability of producing acid, the morphology of the protective agent and the cell morphology that was repeated freezing and thawing after 3 times. The results showed that using 10% dried skim milk as a protectant cell survival rate was 47.3%, with 2.5%y-PGA as a protectant cell survival rate was 54.8%. The survival rate of compound that 1.5%?-PGA mixed with 10% Skim Milk Power was up to 79.8%, which was about 25.0% higher than using the single dried skim milk protectant at10%. Electron microscope results show that, with composite protective agent bacteria surface form a dense structure. After repeat freezing and thawing 3 times, there was no serious damage on bacteria. With strains that freeze-dried bacteria powder watered 2h for fermentation, the fermentation acidity was 65.5 °T after 18 h. The survival rate stored 90 d at room temperature could reach 48.7%.Experiment of antifreeze applied in quick-frozen dumplings with polymer y-PGA and trehalose was carried out. The effect of y-PGA on the quality of frozen dumplings was investigated by analyzing the pasting properties, texture analysis, freezing mark rate, damage rate, sensory evaluation and microscopic electron microscope image. The results showed that adding 10.0% trehalose as antifreeze in frozen dumplings cohesion of structure analysis and taste of sensory evaluation was better than adding y-PGA frozen dumplings, but effect in pasting properties, damage rate of quick-frozen dumpling skin is counterproductive. Frozen dumpling skin adding y-PGA not only had a positive effect in the gelatinization properties, structural analysis, frost crack and breakage structure, but also increases sensory quality. And the electron microscope observation showed that not only the fresh dough with y-PGA was more compact than that of the blank dough and the addition of 10% trehalose, but also had a positive effect on the microstructure of the frozen dumpling skin. Found that holes of the dough adding 0.2%?-PGA was less and small, and most starch granule surface still had the gluten in the package although a small amount of gluten in the fracture; while gluten network structure of the dough adding 10% trehalose occurred in large area of fracture, most of the starch granules exposed from wrapping structure. |
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