Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase fromBacillus mojavensisSY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed intoPichia pastorisX33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

Pigment proteins play a vital role in the red colour change of the red swamp crayfish (Procambarus clarkii) shell after cooking. In this study, two red-change-related pigment proteins with molecular weights of approximately 170 and 43 kDa—denoted as F1 and F2, respectively—were purified by ammonium sulphate salting-out and size exclusion chromatography. F1 and F2 entirely comprised homomultimeric protein complexes composed of 21 kDa subunits. LC-MS/MS analysis showed that the 21 kDa protein subunit belonged to the crustacyanin family, named P. clarkii crustacyanin A2 (PcCRA2). The full-length cDNA of PcCRA2 was cloned, which encoded 190 amino acid residues and was highly homologous (91.58%) with Cherax quadricarinatus crustacyanin A. The predicted 3D structure showed that PcCRA2 had a β-barrel structure for pigment encapsulation. The colour change of F1 was first detected at 40 °C, and the red change occurred upon heating above 60 °C. Additionally, with increasing temperature, its β-sheet content increased, and its α-helix content reduced. Correlation analysis showed that the redness value of F1 was significantly related to the heating temperature and the β-sheet content.

To enhance and stabilise the edible quality of coated tofu, this study explored the changes in the microstructure and intermolecular forces of coated tofu gel and sol under different heat treatments. It elucidated the phase transformation mechanism of coated tofu gel and sol under heat treatment. The results showed that the protein structure unfolded, the fluorescence intensity decreased, and the protein solubility, surface hydrophobicity, and free sulfhydryl group content increased as the coated tofu gel transformed to sol. Disulfide bonding and hydrophobic interactions were the primary intermolecular forces in the heat-induced gel–sol transition. FTIR showed that the content of β-sheets decreased significantly during gel–sol transformation, while the content of β-turns, α-helices and random coils increased significantly. Most remained relatively stable during the gel–sol transformation process, with only the A and B subunits of the 11S protein decreasing slightly. Their reduction became significant when the temperature reached 200 °C. Additionally, the high-temperature heat treatment promoted the gel–sol transition of the coated tofu, with its cross-section gradually transforming from a porous network structure to a more uniform and smooth texture during heat treatment process. The findings of this study provide a theoretical basis for improving the quality of coated tofu by optimising heat treatment parameters, laying the groundwork for future advancements in the development of pre-heat-treated coated tofu.

Leisure dried tofu (LD-tofu) was prepared using two different marinating processes: the repeated heating method (RHM) and the vacuum pulse method (VPM). The quality characteristics and bacterial community succession of LD-tofu and the marinade were evaluated. The results showed that the nutrients in LD-tofu were easily dissolved into the marinade during the marinating process, while the protein and moisture content of RHM LD-tofu changed most dramatically. With the increase in marinade recycling times, the springiness, chewiness and hardness of VPM LD-tofu increased significantly. The total viable count (TVC) of the VPM LD-tofu decreased from the initial value of 4.41 lg cfu/g to 2.51–2.67 lg cfu/g as a result of the marinating process, which had a significant inhibitory effect. Additionally, 26, 167 and 356 communities in the LD-tofu and marinade were detected at the phylum, family and genus levels, respectively. Pearson correlation analysis showed that Pseudomonadaceae, Thermaceae and Lactobacillaceae were closely related to the quality characteristics of LD-tofu, whereas Caulobacteriaceae, Bacillaceae and Enterobacteriae were closely related to the marinade. The present work provides a theoretical basis for the screening of functional strains and quality control in LD-tofu and marinade.

Coated tofu is prone to spoilage and degradation during processing, storage, and transportation. As the material basis for gel of coated tofu, proteins determine coated tofu’s unique qualities, such as its colour, flavour, and texture. This study aimed to investigate the changes in the quality of coated tofu and the physicochemical properties of its proteins during cold storage (4 °C and 10 °C, 14 days), as well as the intrinsic correlations between these variables. Quality deterioration and protein structural changes were significantly slower at 4 °C than at 10 °C, with lower temperature effectively delaying quality loss. The results indicated that as storage time increased, the freshness of coated tofu declined, its textural properties significantly deteriorated, and the protein gel network structure became impaired. Meanwhile, the proteins underwent significant oxidative denaturation, characterized by a decrease in the free thiol group content and an increase in surface hydrophobicity. The tertiary structure exhibited unfolding and disruption, while the secondary structure transitioned from an ordered to a disordered state. Specifically, the contents of α-helixes and β-sheets decreased significantly, reaching 34.96% and 8.68%, respectively, after 14 days of storage at 4 °C. In contrast, the contents of β-turns and random coils increased to 30.11% and 26.25%, respectively, under the same storage conditions. The subunit bands of the 11S and 7S proteins gradually weakened, and the protein structure tended to loosen. Correlation analysis revealed that the oxidative denaturation, structural depolymerization, and reaggregation of proteins were highly significantly correlated with the textural breakdown and colour deterioration of coated tofu, which together contributed to the quality degradation of coated tofu during cold storage. The findings of this study provide fundamental data and technical support for the development of cold storage methods for coated tofu.