A novel aspartic protease (AnproA1) was cloned from Aspergillus niger in this study. Multiple amino acid sequence alignments revealed that the protease belonged to the pepsin-like aspartic protease A1 family and shared the highest amino acid sequence identity of 42.6% with the aspartic protease from Penicillium rubens. The high-level secretory expression of AnproA1 in Komagataella phaffii was successfully achieved using multiple strategies. After high-cell density fermentation in a 5 L fermenter, the fermentation supernatant showed a protease activity of 15250.0 U/mL and a protein concentration of 14.0 mg/mL. The optimum pH and temperature for the purified AnproA1 were 2.5 and 55 ℃, respectively. It was stable within the pH range of 2.5–5.5 and up to 50 ℃. AnproA1 displayed broad substrate specificity and the highest hydrolysis ability towards κ-casein followed by hemoglobin. Furthermore, AnproA1 could hydrolyze duck hemoglobin and plasma proteins into angiotensin-converting enzyme (ACE) inhibitory peptides with half maximal inhibitory concentration (IC₅₀) values of 0.084 and 0.042 mg/mL, respectively. This study offers valuable theoretical insights for the high-level expression of aspartic proteases in K. phaffii and the high-value bioconversion of duck blood proteins.

The de novo synthesis pathway of 2'-fucosyllactose (2'-FL) was established in Escherichia coli BL21 Star (DE3) in this study. The β-galactosidase gene lacZ M15 and the uridine diphosphate (UDP)-glucose lipid carrier transferase gene wcaJ were knocked out using the CRISPR/Cas9 gene editing system. The effects of three different pathway configurations, viz., operon, pseudo-operon, and monocistronic on the synthesis of 2'-FL were explored. The results showed that the concentration of 2'-FL produced in shake flasks was 0.34 g/L after overexpression of the de novo synthesis pathway related genes in E. coli BL21 Star (DE3). The concentration of 2'-FL was increased to 1.26 g/L by deleting the lacZ M15 and wcaJ genes. The highest concentration of 2'-FL of 1.92 g/L was observed in strain BS-7 when regulated by the operon expression. Fed-batch fermentation of strain BS-7 accumulated 14.04 g/L 2'-FL with a productivity of 0.59 g/(L·h) and a lactose conversion rate of 63%, respectively. This study suggested that lower gene expression levels not only increased 2'-FL production, but also could improve the conversion efficiency of substrate in engineered E. coli.

A pathway in Escherichia coli BL21 (DE3) was constructed to produce D-allulose from D-glucose via a phosphorylation-dephosphorylation strategy. The genes related to competitive pathways were deleted using the CRISPR/Cas9 system, and allulose-6-phosphate phosphatases from different sources were selected to evaluate their effects on the synthesis of D-allulose. The expression levels of D-allulose synthetic pathway-related genes were regulated and the fermentation conditions for the engineered strain were optimized. The results showed that the titer of D-allulose increased to 0.95 g/L after the deletion of the genes encoding phosphofructokinase A (pfkA), glucose-6-phosphate dehydrogenase (zwf), allose-6-phosphate isomerase (rpiB), and mannose-6-phosphate isomerase (manA). The allulose-6-phosphate phosphatase BbA6PP from Bacteroides bouchesdurhonensis showed the best performance in D-allulose synthesis, and its use increased the titer of D-allulose to 1.21 g/L in shake flasks. The recombinant strain BE-14, obtained by regulating the expression levels of D-allulose synthetic pathway-related genes through the optimization of plasmid copy number, achieved a maximum D-allulose yield of 2.06 g/L. After optimizing the fermentation conditions, the titer of D-allulose was improved to 2.72 g/L in shake flasks. BE-14 produced the highest titer of D-allulose of 18.4 g/L in a 5 L fermenter after 46 h fermentation. Only trace amounts of glucose (0.7 g/L) and fructose (0.1 g/L) were present in the fermentation broth, which was beneficial for subsequent separation and purification. This study provides a research basis for the efficient production of D-allulose by E. coli, which is of great significance for promoting the industrial production of D-allulose.

To study the effects of Polygonatum kingianum polysaccharides (PKP) on lipid metabolism disorders and brain function impairments in obese mice, the obese mice were treated with PKP, prepared by enzyme-assisted extraction, for 12 weeks. Changes in body mass and blood lipids, as well as pathological changes in liver and adipose tissue were observed. Behavioral changes were detected to assess the autonomic activity and anxiety-like behavior of mice, and inflammation levels in liver, adipose tissue, serum, and brain as well as pathomorphological changes in brain were detected. The results showed that PKP (1500 mg/kg) reduced body mass gain by 29.6% in obese mice, significantly alleviated dyslipidemia and pathological changes in liver and adipose tissue, and promoted the expression of peroxisome proliferator-activated receptors α (PPARα) in liver (46.0%) and the expression of uncoupling protein 1 in adipose tissue (59.0%). PKP significantly increased autonomic activity and suppressed anxiety-like behavior in obese mice. Furthermore, PKP reduced the expression of F4/80 in liver and adipose tissue by 55.0% and 67.2%, respectively, and the serum levels of pro-inflammatory cytokines in including interleukin 1β by 22.9%, interleukin 6 by 44.6%, and tumor necrosis factor α by 7.8%, but increased the serum level of anti-inflammatory cytokine interleukin 10 by 27.1%. Furthermore, PKP reduced the expression levels of ionized calcium binding adaptor molecule 1 in the hippocampus and cerebral cortex by 16.0% and 28.6%, respectively, and inhibited structural damages of the hippocampus and cerebral cortex. PKP mitigate obesity-induced lipid disorders and brain damages, indicating their potential as a functional food ingredient for obesity and suppressing obesity-induced brain damage.

Acid proteases are important enzymes in the food industry. In this study, an acid protease (AopepA) from Aspergillus oryzae was high-level expressed extracellularly in Aspergillus niger using different expression strategies. The highest protease activity of 407.6 U/mL was yielded in a shake flask by the engineered A. niger strain, which was constructed using a mixed expression strategy of constitutive and induction types. High protease activity of 1153.6 U/mL with a protein concentration of 3.2 mg/mL was produced by fed-batch fermentation in a 5 L fermenter. AopepA displayed optimal activity at pH 2.5 and 50 ℃, respectively. It was stable within pH 2.0-7.0 and up to 45 ℃. AopepA had a broad substrate specificity and the highest specific activity of 920.8 U/mg towards hemoglobin. The addition of AopepA (1000 U/kg) during the bread making improved the specific volume by 16.6% and decreased the hardness by 26.9%. Moreover, AopepA was applied to modify soy protein, resulting in a 2.2-fold increase in foaming capacity and a 1.2-fold increase in foaming stability. The emulsifying activity and emulsion stability indexes were enhanced by 1.3-fold and 2.8-fold, respectively. This study provides a valuable way for the efficient expression of proteases in A. niger and broadens the application of acid proteases in the food industry.

Atherosclerosis is driven both by hyperlipidemia and inflammation. Chitin oligosaccharides (NACOS) have shown pharmacological effects on multiple diseases via hypolipidemic and/or anti-inflammatory activities. The present study aims to evaluate whether NACOS treatment can prevent atherosclerosis induced by a high-fat-diet (HFD) in Apolipoprotein E-knockout (ApoE^-/-) mice. Results showed that 300 and 900 mg/kg b.w./day NACOS supplementation for 14 weeks significantly decreased atherosclerotic lesions up to 45% and 67% in compared with the HFD (P < 0.05), as measured in the valve area of the aortic root. Further, NACOS supplementation significantly reduced the serum hyperlipidemia and circulating proinflammatory cytokines including interleukin-1β, interleukin-6, monocyte chemoattractant protein-1 and tumor necrosis factor-α. NACOS decreased the hepatic Hmgcr to reduce cholesterol synthesis, activated the genes involved in reverse cholesterol transport to enhance cholesterol efflux and excretion, and reduced the intestinal Npc1L1 to lower cholesterol absorption. Additionally, NACOS enhanced cecum short chain fatty acids production and intestinal integrity. Thus, NACOS supplementation ameliorated atherosclerosis via altering lipid metabolism and reducing inflammation. These findings indicate that NACOS may be a potential functional food material for attenuating atherosclerosis development.