Extraction, Investigation, and Potential Application of Baijiu Jiuzao Glutelin

Jiuzao, the primary solid by-product from baijiu (a traditional Chinese spirit) distillation, contains a high protein content due to the high boiling point of these proteins. To enhance the added value of Jiuzao, utilizing its proteins presents an ideal solution. Although various methods have been developed for protein extraction, higher yields still need to be explored. Pulsed electric field (PEF) is a novel non-thermal extraction method known for its high extraction efficiency. In this study, a PEF-assisted extraction method was used to improve protein extraction efficiency from Jiuzao. Jiuzao glutelin (JG) was fractionally extracted, and this study is the first to explore the species type, secondary structure, and functional characteristics of JG. Based on JG, bioactive peptides with antioxidant activity were prepared under protease hydrolysis conditions. An AAPH-induced oxidative damage model using Spragure Dawley (SD) rat was constructed to evaluate the in vivo antioxidant activity of these peptides. For the first time, quercetin (QUE), resveratrol (RES), rhein (RH), and riboflavin were (RIB) carriers were constructed using JG-conjugated polysaccharide (Pullulan, Dextran, Carboxymethyl chitosan, Pectin, and Arabic gum) Maillard products. The incorporation of JG enhances the added value of Jiuzao and mitigates the environmental burden caused by its large-scale production. PEF was used as a supplementary technique for the fractional extraction of Jiuzao proteins (albumin, globulin, gliadin, and glutelin). The extraction efficiency was enhanced by 13.81% compared to the ultrasound-assisted method (0.92 mg/mL) using with 83 pulses, a field strength of 3.26 kV/cm, and a Jiuzao/distilled water ratio of 3:20. Subsequently, it was found that 59.16% of JG was derived from sorghum. Extracted JG showed excellent foaming and foam stability, as well as water and oil holding characteristics. Additionally, in vitro antioxidant assays showed that JG exhibited ABTS, DPPH, hydroxyl radical scavenging capacity, ferrous chelating, and oxygen radical absorbance capacity (ORAC). Furthermore, extracted JG exhibited favourable cytocompatibility in Caco-2 and CCD 841 CON cells. The optimal conditions for JG hydrolysis were explored using various proteases, including alkaline protease, neutral protease, papain, pepsin, trypsin, flavor, and complex proteases, to generate bioactive peptides. The findings indicated that the peak hydrolysis rate of JG attained 80.7% under the following parameters: a pepsin-to-JG ratio of 0.378, a hydrolysis temperature at 41°C, a pH of 1.40, and a hydrolysis duration of 300 minutes. The in vivo antioxidant activity of JG hydrolyzed peptides, including Asp-Arg-Glu-Leu (DREL), Ala-Tyr-Ile (AYI), and Val-Asn-Pro (VNP), was measured using an AAPH-primed SD rat model. All three peptides activated the Nrf2/Keap1-p38/PI3K-MafK antioxidant pathway and the downstream antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and hemoxyrubinase-1 (HO-1), were examined at the gene and protein levels to enhance antioxidant activity in rats. Polysaccharides such as Pullulan, Dextran (Dex), Carboxymethyl chitosan (CTS), Pectin (Pec), and Gum Arabic (GA) were used to modify JG through Maillard reaction. The optimal reaction conditions were as follows: for CTS-JG, the ratio was 2:1, the reaction time was 180 minutes, and the pH was 7 (CTS-JGI-2); for Pullulan-JG, the ratio was 2:1, the reaction time was 180 minutes, and the pH was 11 (PJC-2); for Dex-JG, the ratio was 1:1, the reaction time was 120 minutes, and the pH was 7; for Pec-JG, the ratio was 4:1, the reaction time was 180 minutes, and the pH was 7; and for GA-JG, the ratio was 2:1, the reaction time was 120 minutes, and the pH was 11. These conjugates showed significant improvements in solubility, foaming, foam stability, viscosity, and thermal stability. CTS-JGI-2 was used to construct oil-in-water nanoemulsion for delivering RES and quercetin (QUE) because of its ideal stability, properties, and activities. The results showed that the CTS-JGI-2 stabilized oil-in-water nanoemulsion improved the encapsulation efficiency of RES and QUE (RES was 80.96%, QUE was 93.13%) and enhanced stability during the simulated digestion process (RES was 73.23%, QUE was 77.94%) through hydrogen bonding, anion, sigma, and donor compared to native JG. Furthermore, PJC-2 was combined with enteric-coated materials (polymethacrylic acid, hydroxypropyl methylcellulose phthalates, cellulose acetate phthalates, and D-mannitol) to construct a microencapsulated delivery system for rhein (RH). The encapsulation efficiency of RH in the four enteric-PJC-2 bilayer microcapsules (70.03±3.24%~91.08±4.78%) was significantly higher than that of PJC-2 microcapsules (61.84±0.47%). The antioxidant activity and stability of RH in microcapsules were increased (ABTS, 49.7% -113.93%; DPPH，40.85%-101.82%; ferrous reducing power, 62.32%-126.42%; ferrous chelate, 70.58%-147.20%) compared to free RH under in vitro simulated digestion and extreme environmental conditions. This study successfully created a method to achieve the maximum extraction of JG from Jiuzao (with 83 pulses, a field strength of 3.26 kV/cm, and a Jiuzao/distilled water ratio of 3:20). The utilization of JG encompassed two successful approaches which are the hydrolysis for the generation of functional peptides and conjugation for the delivery of functional components. The incorporation of JG significantly elevated the added value of Jiuzao, preventing potential wastage.