Protease Inhibitors and Hydrolysate from Duck Egg Albumen:Characteristics and Applications in Surimi Gels

Abstract

Duck egg albumen (DEA) was monitored for its physicochemical properties, trypsin inhibitory activity, and gelling properties as affected by store conditions (4 °C and room temperature) and salting time (30 days). Haugh unit, moisture content, and trypsin inhibitory activity decreased, while the pH value increased as the storage time increased (P<0.05). The rate of changes was lower at 4 °C. During salting, moisture content and trypsin inhibitory activity were continuously decreased, whereas the salt content and surface hydrophobicity increased as the salting time increased (P<0.05). However, there was no difference in protein patterns during the storage and salting. Protein with MW of 44 kDa acted as trypsin inhibitor in DEA. Based on texture profile analysis, the highest hardness, gumminess, and chewiness were found at day 3 for room temperature and at day 6 for 4 °C. Gel became weaker and had lowered whiteness with increasing storage time, particularly at room temperature. Additionally, as salting time increased, the lowered gel strength with higher whiteness was recorded. Thus, storage conditions and salting directly affected the quality of DEA. Trypsin inhibitor (TI) from DEA was isolated and purified using ammonium sulfate precipitation at 20%-40% saturation and affinity chromatography using trypsin- CNBr-activated Sepharose 4B column with high purity. Molecular weight of purified TI was 43 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. TI was stable within the temperature range of 40-60°C and pH of 7-9. The concentration of salt higher than 5% led to the decreases in inhibitory activity of TI. The inhibition mode of TI was classified as uncompetitive type with low Ki value (508 nM). When the effects of duck and hen egg albumens at different levels (1-4%, w/w) on proteolysis and gel properties of sardine (Sardinella albella) surimi were comparatively investigated, the addition of both albumens could inhibit autolysis of surimi proteins in a dose dependent manner as indicated by the lower TCA-peptide content, more retained myosin heavy chain and actin, and increased gel strength of surimi gel (P<0.05). Duck albumen showed higher efficiency in enhancing the gelling properties of sardine surimi than hen albumen. Thus, DEA was considered to replace hen egg albumen as protein additive in surimi gel. To prepare DEA powder, desugarization for DEA was optimized as follows: glucose oxidase/catalase concentration of 31.24/781 units/mL albumen and incubation time of 6.55 h. Drying methods also affected properties and TI activity. Higher TI activity was found in freeze-dried albumen powder than spray-dried counterpart (P<0.05). However, no marked differences in protein patterns were observed in all the powders, regardless of desugarization and drying methods. The whiteness and foaming property of powder were improved, while the solubility decreased after desugarization (P<0.05). Furthermore, prior desugarization could markedly improve the whiteness and gelling properties of powder. Prior desugarization could lower browning and increased foaming and gelling properties of DEA, particularly when spray dried with inlet temperature of 160 °C. To widen the exploitation of salted DEA, salted DEA powder (SDEAP) was used as salt replacer at various levels (0.5-2.5%). Autolysis and gelling properties of sardine surimi were investigated. SDEAP had high salt (33.67%) and protein contents (64.52%) with TI activity of 5,975 units/g solid. Sardine surimi gels added with SDEAP had the lowered proteolysis, in which MHC was more retained. The addition of SDEAP could improve the gel strength and sensorial characteristics of surimi gel (P<0.05). SDEAP could be used as a promising ingredient to replace salt used in surimi gel preparation. Protein hydrolysate from DEA was also prepared and characterized. Influences of different ultrasound treatments combined with heat pre-treatment on enzymatic hydrolysis, emulsifying properties and antioxidant activities of duck albumen hydrolysate (DAH) were studied. Heat treatment of DEA at 95 °C for 30 min with subsequent ultrasound pretreatment at 60% amplitude for 10 min could effectively increase the DH of DEA. The highest antioxidant activities as well as emulsifying properties of DAH were found after 90 min of hydrolysis. Thus, heat pre-treatment, followed by ultrasonication of DEA under appropriate condition could increase DH, antioxidant activities and emulsifying properties of DAH. As DAH conjugated with epigallocatechin gallate (EGCG) at various levels (2-5%, w/w) were prepared, the conjugation between DAH and EGCG induced the change of secondary structure and modified functional groups of DAH monitored by FTIR, total sulfhydryl group and carbonyl contents. DAH conjugated with 4% EGCG showed the highest emulsifying properties and enhanced antioxidant activities than DAH. Physical and oxidative stability of fish oil emulsion was improved when DAH- EGCG conjugate was incorporated. Thus, the conjugate between DAH and 4% EGCG could serve as an antioxidant emulsifier for enhancement of physical and oxidative stability of fish oil emulsion. The impacts of DAH-EGCG conjugate at various levels (0.5-2.0%) on gel properties, sensory characteristics, and storage stability of fish tofu during 21 days of storage at 4 °C were also evaluated. The incorporation of DAH-EGCG conjugate increased gelling properties and sensory likeness scores of fish tofu when the levels of DAH-EGCG conjugate were increased (P<0.05). Moreover, 1.5-2.0% DAH-EGCG conjugate could effectively retard lipid oxidation and microbial growth of fish tofu throughout the storage, compared to the control (P<0.05). DAH-EGCG conjugate was proven as protein cross-linker, antioxidant, and antimicrobial agent in order to prolong shelf-life and maintain quality of fish meat based emulsion products. Therefore, DEA could be used as a potential protein additive, particularly for surimi, in which gel strength could be improved, regardless of salting. It could be to prepare hydrolysate with improved functional properties and bioactivity, especially after modification with the selected polyphenol, EGCG.