Xingfei Li

Digestibility is a key determinant of the nutritional quality of food proteins, while disulfide bonds (SSs) restrict enzymatic hydrolysis. This study isolated and characterized endogenous small thiol compounds (ESTCs) from defatted soy meal and investigated their effects on SS cleavage and soy proteins' in vitro digestibility. The results revealed ESTCs are a <500 Da complex mixture of glutathione, cysteine-cysteine dipeptides, and cysteine-rich peptides. ESTC-induced SS cleavage was profoundly influenced by protein predenaturation, temperature, and pH, while excessive heat or high pH treatments impaired SS cleavage, but urea denaturation enabled the highest degree of SS cleavage (DSC) of 44.5%. Reaction kinetics revealed activation energies of 35.3 and 13.6 kJ/mol for undenatured and urea-denatured proteins, respectively. Compared to samples with 0% DSC, SS-cleaved proteins with DSCs of 7.2%, 22.1%, and 44.5% showed significantly increased hydrolysis degrees. This study proposed a green strategy through molecular modification to improve the nutritional quality of soy proteins.

Agarases are enzymes that serve as valuable catalysts in producing bioactive agar oligosaccharides. The thermostability of agarase is crucial for its industrial application since the sol-gel transition of the agar substrate is temperature-dependent. An exo-β-agarase, Aga50D, from Saccharophagus degradans 2-40 was re-engineered based on a single mutation near the interface. The A86D mutant exhibited a melting temperature increase of 23.3 °C with more than a 7-fold improvement in catalytic performance for agarose hydrolysis at 50 °C for 1 h. Small-angle X-ray scattering (SAXS), differential fluorimetry scanning (DFS), and size exclusion chromatography (SEC) analyses provided convincing evidence that the mutation significantly promoted the dimerization. Molecular dynamics simulations of the enzyme further supported our findings, demonstrating that the single mutation near the interface induced the rearrangement of the intrachain salt bridges between the loops and strengthened the interchain salt-bridge interaction at the interface, which were identified as critical mechanisms for improving its thermostability. Meanwhile, the stable interface interaction may play an important role in the dimerization of A86D. These results highlight the design of intersubunit interactions to obtain high-stability enzymes via targeted mutations near the subunit interface. The robust A86D mutant shows great potential in industrial agar oligosaccharide production.

Whole component soy beverage achieve the highest values of in vitro DIAAS (96/80) and digestible protein (29.40/24.41 g per 100 g of soybeans).

Recently, the orientational immobilization of enzymes has attracted extensive attention. In this study, we report the development of a strategy combined with rational design to achieve precise site-specific covalent immobilization of beta-agarase. We first rationally screened six surface sites that can be mutated to cysteine by combining molecular dynamics simulation and energy calculation. Site-specific immobilization was successfully achieved by Michael addition reaction of mutant enzymes and maleimide-modified magnetic nanoparticles (MAL-MNPs). The enzyme activity retention rate of R66C-MAL-MNPs and K588C-MAL-MNPs was greater than 96%. The thermal deactivation kinetics study revealed that the site-specific immobilization strategy significantly improved the thermal stability of Aga50D, resulting in a substantial increase in its antidenaturation activity at elevated temperatures, and the highest t(1/2) of the immobilized mutant enzymes was increased by an impressive 21.25-fold at 40 degrees C. The immobilized mutant enzymes also showed significantly enhanced tolerance to metal ions and organic reagents. For instance, all of the immobilized enzymes maintained over 90% of their enzymatic activity in the 50% (v/v) acetone/water solution. The present work may pave the way for the design of precisely immobilized enzymes, which can help promote green manufacturing.

Natural ginger protease effectively cleaves wheat gluten to produce DPP-4 inhibitory peptides including PPFS with an IC₅₀ value of 56 μM and better in vitro digestion stability.

Hexanal and (E)-2-hexenal in soymilk mainly form during the soaking and grinding of soybeans. In this study, freshly dehulled soybeans were soaked or ground in the presence or absence of different enzyme inhibitors. The results showed that (1) 1-palmitoyl-2-linoleoyl-sn-3-phosphatidylcholine, 1-stearoyl-2-linoleoyl-sn-3-phosphatidylcholine, 1-palmitoyl-2-linolenoyl-sn-3-phosphatidylcholine, and 1-stearoyl-2-linolenoyl-sn-3-phosphatidylcholine were preferentially acted upon by lipoxygenases (LOXs) and made predominant contributions to hexanal/(E)-2-hexenal formation. Phospholipase A₂ (PLA₂) is one of the key enzymes for hexanal/(E)-2-hexenal formation. (2) The ratio of net increase in hexanal/(E)-2-hexenal and net decrease in linoleic acid/linolenic acid was close to 100% during soaking, but it was only 60% during grinding. Only 13-hydroperoxy octadecad(tr)ienoic acid (13-HPOD/T) was formed for the membrane LOX, but both 13- and 9-hydroperoxy octadecad(tr)ienoic acid (9-HPOD/T) were produced for the cytoplasm LOX. Thus, only the membrane LOX was involved during soaking, while both membrane- and cytoplasm-bound LOXs worked during grinding. (3) Hydroperoxides and hexanal/(E)-2-hexenal during soybean grinding were studied. PC hydroperoxides formed almost instantly and reached a maximum in 10 s, while fatty acid hydroperoxides and hexanal/(E)-2-hexenal formed relatively slowly and reached a maximum in 50 s. The experimental data were fitted to the integrated form of the Michaelis–Menten equation, and K_m, V_max, and k_cat for the LOX, PLA₂, and hydroperoxide lyase were obtained, respectively.

Flavor is an essential quality characteristic of soymilk. (E)-2-Heptenal has a fatty and fruity flavor with the sensory threshold value of 13 μg/L in water. This study demonstrated that the formation of (E)-2-heptenal was independent of the lipoxygenase (LOX) and hydroperoxide lyase (HPL) activity as well as oxygen concentration but was related to the presence/absence of Fe²⁺ and chelators. In a dry matter base, soybean hypocotyls generated a much higher amount of (E)-2-heptenal than cotyledons. A phospholipid hydroperoxide was purified from the chloroform/methanol extract of soybean hypocotyls and was identified as 1-palmitoyl-2-(12-hydroperoxyoctadecadienoyl)-sn-glycerol-3-phosphatidylethanol-amine (12-PEOOH). The decomposition of 12-PEOOH in the presence of ferrous ions to form (E)-2-heptenal was studied in a model system. The rate of decomposition decreased sharply at pH values higher than 6, but the molar conversion of 12-PEOOH to (E)-2-heptenal increased with an increase of pH. At a constant pH of 5.8, the decomposition rate of 12-PEOOH was positively linearly related to the Fe²⁺ concentration, while the molar conversion to (E)-2-heptenal was 74% and independent of the Fe²⁺ concentration. The formation of radicals LOO^• and R^• showed similar pH and Fe²⁺ concentration dependence with those of (E)-2-heptenal. (E)-2-Heptenal displayed an enhancement of bean aroma and fruity flavor of soymilk at low concentrations, but a fatty flavor was noticed at high concentrations.

In the study, antibacterial peptides were separated and identified from cottonseed protein hydrolysates and the interactions between antibacterial peptides and Escherichia coli were further investigated. Firstly, by using a combined strategy of Amberlite CG-50 ion exchange chromatography and reversed-phase high-performance liquid chromatography, three peptides with antibacterial activity were purified and identified, including HHRRFSLY, KFMPT, and RRLFSDY. Interestingly, HHRRFSLY and RRLFSDY exhibited higher inhibition activity with the IC₅₀ value of 0.26 mg mL⁻¹ and 0.58 mg mL⁻¹ (p < 0.05), respectively. Flow cytometry results showed that the incubation of antibacterial peptides with E. coli could cause damage to the integrity of the E. coli cell membrane. Transmission electron microscopy and scanning electron microscopy results revealed the damage caused to the bacterial cell surface and the leakage of cytoplasmic content by the antibacterial peptides. Molecular docking studies indicated that HHRRFSLY, KFMPT, and RRLFSDY have a good binding affinity to the active sites of the surface protein (OmpF) mainly through a hydrogen bond and salt bridge. The results here showed that the antibacterial peptides derived from cottonseed protein could be used as a good choice for functional foods or related drugs, and also shed light on further studies of antibacterial mechanism.