Mechanistic Study of Novel Dipeptidyl Peptidase IV Inhibitory Peptides from Goat's Milk Based on Peptidomics and In Silico Analysis.

Two novel dipeptidyl peptidase IV (DPP-IV) inhibitory peptides (YPF and LLLP) were discovered from goat milk protein by peptidomics, in silico analysis, and in vitro assessment. A total of 698 peptides (<23 AA) were successfully identified by LC-MS/MS from goat milk hydrolysates (hydrolyzed by papaian plus proteinase K). Then, 105 potential DPP-IV inhibitory peptides were screened using PeptideRanker, the ToxinPred tool, Libdock, iDPPIV-SCM, and sequence characteristics. After ADME, physicochemical property evaluation, and a literature search, 12 candidates were efficiently selected and synthesized in vitro for functional validation. Two peptides (YPF and LLLP) were found to exert relatively high in vitro chemical system (IC50 = 368.54 ± 12.97 µM and 213.99 ± 0.64 µM) and in situ (IC50 = 159.46 ± 17.40 µM and 154.96 ± 8.41 µM) DPP-IV inhibitory capacities, and their inhibitory mechanisms were further explored by molecular docking. Our study showed that the formation of strong non-bonding interactions with the core residues from the pocket of DPP-IV (such as ARG358, PHE357, GLU205, TYR662, TYR547, and TYR666) might primarily account for the DPP-IV inhibitory activity of two identified peptides. Overall, the two novel DPP-IV inhibitory peptides rapidly identified in this study can be used as functional food ingredients for the control of diabetes.

Identification, characterization, and insights into the mechanism of novel dipeptidyl peptidase-IV inhibitory peptides from yak hemoglobin by in silico exploration, molecular docking, and in vitro assessment.

Dipeptidyl peptidase IV (DPP-IV) inhibitory peptides were screened and identified from yak hemoglobin for the first time by in silico analysis, molecular docking, and in vitro evaluation. Results showed that yak hemoglobin had a high potential to produce DPP-IV inhibitory peptides based on the sequence alignment and bioactive potential evaluation. Furthermore, "pancreatic elastase + stem bromelain" was the optimal combined-enzymatic strategy by simulated proteolysis. Additionally, 25 novel peptides were found from its simulated hydrolysate, among which 10 peptides had high binding affinities with DPP-IV by molecular docking. Most of these peptides were also in silico characterized with favorable physicochemical properties and biological potentials, including relatively low molecular weight, high hydrophobicity, several net charges, good water solubility, nontoxicity, acceptable sensory quality, and good human intestinal absorption. Finally, six novel DPP-IV inhibitory peptides were identified via in vitro assessment, among which EEKA (IC50 = 235.26 µM), DEV (IC50 = 339.45 µM), and HCDKL (IC50 = 632.93 µM) showed the strongest capacities. The hydrogen bonds and electrostatic attractions formed with core residues within the S2 pocket of DPP-IV could be mainly responsible for their inhibition performances. This work provided a time-saving method and broadened application for yak by-products development as sources of functional foods.

Selection of goat ß-casein derived ACE-inhibitory peptide SQPK and insights into its effect and regulatory mechanism on the function of endothelial cells.

The angiotensin I-converting enzyme (ACE)-inhibitory peptide SQPK was selected by in silico digestion and virtual screening from goat ß-casein, and its effect and regulatory mechanism on function of endothelial cells was further evaluated. The results showed that SQPK exhibited relatively good ACE inhibition capacity (IC50 = 452.7 µg/mL). Treatment with 25 µg/mL SQPK for 12 h significantly elevated nitric oxide (NO) production, stimulated eNOS expression (p < 0.05) and affected the transcriptomic profiling of EA. Hy926 cells. In particular, SQPK stimulated the expression of genes encoding inflammatory cytokines (CXCL1/2 and IL6) but depressed encoding mesenchymal markers (FN1 and CNN3). Furthermore, SQPK modified the expression of genes involved in endothelial-to-mesenchymal transition (EndMT). Therefore, the selected peptide SQPK may exert potential protective effects on the function of endothelial cells by inhibiting the EndMT.

The Effects and Regulatory Mechanism of Casein-Derived Peptide VLPVPQK in Alleviating Insulin Resistance of HepG2 Cells.

The liver plays a key role in keeping the homeostasis of glucose and lipid metabolism. Insulin resistance of the liver induced by extra glucose and lipid ingestion contributes greatly to chronic metabolic disease, which is greatly threatening to human health. The small peptide, VLPVPQK, originating from casein hydrolysates of milk, shows various health-promoting functions. However, the effects of VLPVPQK on metabolic disorders of the liver are still not fully understood. Therefore, in the present study, the effects and regulatory mechanism of VLPVPQK on insulin-resistant HepG2 cells was further investigated. The results showed that VLPVPQK exerted strong scavenging capacities against various free radicals, including oxygen radicals, hydroxyl radicals, and cellular reactive oxygen species. In addition, supplementation of VLPVPQK (62.5, 125, and 250 µM) significantly reversed the high glucose and fat (30 mM glucose and 0.2 mM palmitic acid) induced decrement of glucose uptake in HepG2 cells without affecting cell viability. Furthermore, VLPVPQK intervention affected the transcriptomic profiling of the cells. The differentially expressed (DE) genes (FDR < 0.05, and absolute fold change (FC) > 1.5) between VLPVPQK and the model group were mostly enriched in the carbohydrate metabolism-related KEGG pathways. Interestingly, the expression of two core genes (HKDC1 and G6PC1) involved in the above pathways was dramatically elevated after VLPVPQK intervention, which played a key role in regulating glucose metabolism. Furthermore, supplementation of VLPVPQK reversed the high glucose and fat-induced depression of AKR1B10. Overall, VLPVPQK could alleviate the metabolic disorder of hepatocytes by elevating the glucose uptake and eliminating the ROS, while the HKDC1 and AKR1B10 genes might be the potential target genes and play important roles in the process.

Effects of chitosan coating combined with thermal treatment on physicochemical properties, bacterial diversity and volatile flavor of braised duck meat during refrigerated storage.

The current study aimed to assess the impact of chitosan coating (0.005 g/mL) combined with thermal treatment (85 °C for 30 min) on tenderness, lipid and protein oxidation, bacterial diversity, and volatile flavor compounds in braised duck leg meat under vacuum packaging during refrigerated storage (4 °C for 15 days). The findings revealed that the three preserved treatments significantly increased tenderness from days 1 to 3. There was a substantial decrease from days 6 to 12 compared to the control, but no significant differences were observed on day 15. Compared with the control, the three preserved treatments reduced TBARS values by 25.8%-78.6% (from days 6 to 12) and total sulfhydryl concentrations by 24.1%-75.7% (from days 3 to 9). The combination treatment had the lowest values (carbonyl concentration, TVC, and TEC) compared to the chitosan coating and thermal treatment, indicating a significant synergistic effect. The proportions of the four primary spoilage organisms, Brochothrix, Weissella, Acinetobacter, and Pseudomonas, were 74.8%, 76.3%, 70.7%, and 49.7% in control, chitosan coating, thermal treatment, and combination treatment, respectively. The combination treatment produced the most volatile flavor compounds (38 compounds) at the end of storage (15 days). Hexanal, 1-nonanal, 1-octen-3-ol, and 2, 3-octanedione were the main volatile flavor compounds, and the average relative peak area was above 80. Therefore, chitosan coating and thermal treatment could be developed as synergistic methods to preserve braised duck meat.

Co-encapsulation of resveratrol in fish oil microcapsules optimally stabilized by enzyme-crosslinked whey protein with gum Arabic.

O/W emulsion and its spray-dried microcapsule contain the oil phase and the protein matrix, providing the potential to co-encapsulate different antioxidants. However, antioxidants were generally encapsulated in the oil phase of microcapsule, its protein matrix is rarely used. It is first to prove the possibility to encapsulate resveratrol in the emulsified oil droplets at high wall/core ratios. The optimal microcapsule with 1.75% surface oil was fabricated with 15% transglutaminase-crosslinked WPI (TGase-WPI) and 5% gum Arabic (GA). Resveratrol mainly located in the protein matrix of initial emulsion and reconstituted microcapsule. The effects of TGase-WPI/GA microcapsule and resveratrol co-encapsulation on DHA/EPA and lipid hydroperoxides/TBARS were different. The interfacial protein, the partition of resveratrol in the emulsified oil droplets and its storage stability and inhibitory effect on size change of reconstituted microcapsules increased as the polyphenol increased. These results expand the potential use of spray-dried microcapsules as co-encapsulation carriers.

Physicochemical property, volatile flavor quality, and microbial community composition of Jinhua fatty ham and lean ham: A comparative study.

The physicochemical property, volatile flavor compounds, and microbial community structure of Jinhua fatty ham (FH) and lean ham (LH) were investigated and compared by high-throughput sequencing and HS-GC-IMS. Results showed that FH had higher pH and slightly lighter and yellower color than LH. Meanwhile, 33 volatile flavor compounds were identified from FH and LH, among which LH showed higher abundance of total alcohols and acids, but FH had generally richer aldehydes, ketones, esters, heterocyclic, and sulfur-containing compounds. Moreover, FH and LH did not have significant difference in α-diversity of bacterial community, but LH presented a much lower α-diversity of fungal community than FH. Besides, the dominant microorganisms (relative abundance >2%) in FH were Ruminococcaceae UCG-005, Staphylococcus, Ruminococcaceae UCG-014, Meyerozyma, and Aspergillus at the genus level, while in LH were Staphylococcus, Psychrobacter, Halomonas, Propionicicella, Ruminococcaceae UCG-005, Meyerozyma, Yamadazyma, and Aspergillus. Furthermore, the analysis of Pearson's correlation and metabolic network confirmed that the discriminative flavor compounds of FH were mainly ß-oxidation and degradation products of fatty acids, while those of LH were mostly derived from the Strecker reaction or microbial metabolism of amino acids. The present study could help understand the potential pathway of characteristic microorganisms affecting flavor formation of fat-deficient dry-cured hams and provide theoretical supports for developing healthier fermented meat products.

In vitro digestion and fermentation combined with microbiomics and metabolomics reveal the mechanism of superfine yak bone powder regulating lipid metabolism by altering human gut microbiota.

The effects of superfine yak bone powder (YBP) on human gut microbiota (HGM) were investigated by in vitro digestion and fermentation combined with microbiomics and metabolomics. Results showed that size reduction and protein structural degradation during digestion allowed superfine YBP to release more Ca2+ than CaCO3 powders with similar particle size. Moreover, the indigestible YBP further influenced HGM and was associated with increased occurrence of beneficial bacteria such as Megasphaera spp., Megamonas spp., Acidaminococcus spp., and Prevotella spp. The altered HGM was associated with greater production of short-chain fatty acids with 4-6 carbon atoms. Furthermore, the indigestible YBP was associated with up-regulation of many lipid-related metabolites, including taurine, secondary bile acids, saturated long-chain fatty acids, and ω-3/ω-6 polyunsaturated fatty acids, which modulated favorably lipid metabolic pathways. These findings implied the potential activity of superfine YBP as a food fortifier in favorably altering HGM community structure and regulating lipid metabolism.

Insights into the Protein Differentiation Mechanism between Jinhua Fatty Ham and Lean Ham through Label-Free Proteomics.

Jinhua lean ham (LH), a dry-cured ham made from the defatted hind legs of pigs, has become increasingly popular among consumers with health concerns. However, the influence of fat removal on the quality of Jinhua ham is still not fully understood. Therefore, a label-free proteomics strategy was used to explore the protein differential profile between Jinhua fatty ham (FH) and lean ham (LH). Results showed that 179 differential proteins (DPs) were detected, including 82 up-regulated and 97 down-regulated DPs in LH vs. FH, among which actin, myosin, tropomyosin, aspartate aminotransferase, pyruvate carboxylase, and glucose-6-phosphate isomerase were considered the key DPs. GO analysis suggested that DPs were mainly involved in binding, catalytic activity, cellular process, and metabolic process, among which catalytic activity was significantly up-regulated in LH. Moreover, the main KEGG-enriched pathways of FH focused on glycogen metabolism, mainly including the TCA cycle, pyruvate metabolism, and glycolysis/gluconeogenesis. However, amino acid metabolism and oxidative phosphorylation were the main metabolic pathways in LH. From the protein differentiation perspective, fat removal significantly promoted protein degradation, amino acid metabolism, and the oxidative phosphorylation process. These findings could help us to understand the effects of fat removal on the nutritional metabolism of Jinhua hams and provide theoretical supports for developing healthier low-fat meat products.

Effects of micro-/nano-scaled chicken bones on heat-induced gel properties of low-salt pork batter: Physicochemical characteristics, water distribution, texture, and microstructure.

The effect and mechanism of micro-/nano-scaled (MCB/NCB) chicken bones on improved gel properties of low-salt (0.5%) pork batters (LSPB) were investigated. Results showed that the Ca2+ released from MCB/NCB induced protein conformational transition of LSPB from α-helix and ß-turn to random coil, which facilitated the orderly protein aggregation, resulting in the formation of denser and more uniform gel network. The better-organized gel network not only improved the textural properties and gel strength, but also contributed to the cooking loss reduction, water release decline, water state transition from free water into more immobilized water, and proton density increase, resulting in better sensory attributes. NCB showed more preferable effects on the property improvement of LSPB gels than MCB, because of its smaller particle size, less redness and yellowness, higher dispersibility, and higher Ca2+ release. This study could help promote the high-value utilization of poultry by-product bones and development of low-salt meat products.

Effect of κ-carrageenan addition on protein structure and gel properties of salted duck egg white.

BACKGROUND: Salted duck egg white (SDEW) is a major by-product during salted egg yolk manufacturing. Due to the high salt concentration, SDEW has not been efficiently utilized. Moreover, functional properties of SDEW are altered by salt during pickling. To improve the functional properties, the effect of κ-carrageenan (κ-CAR) addition on the protein structure and gel properties of SDEW was investigated in this study. RESULTS: The surface hydrophobicity and free sulfhydryl content of SDEW protein increased, while total sulfhydryl content decreased significantly with the addition of κ-CAR (0.02-0.10%). Fourier-transform infrared spectroscopy analysis revealed that the relative content of α-helix and ß-turn decreased, ß-sheet and random coil increased, indicating the variation tendency of protein structure from order to disorder. As κ-CAR addition increased, the texture profiles including hardness, gumminess, chewiness, springiness, cohesiveness and resilience of SDEW gel were all improved. Water holding capacity increased significantly by 32.33% in the presence of 0.10% κ-CAR addition. The scanning electron microscopy indicated that the microstructure of SDEW/κ-CAR mixed gel was more smooth and compact. CONCLUSION: The results suggested that adding κ-CAR can be an effective method to improve gel quality of SDEW. This study is expected to provide theoretical basis for modification of SDEW protein, as well as preparation of food ingredients with better gel properties from SDEW. © 2020 Society of Chemical Industry.

Microencapsulation of immunoglobulin Y: optimization with response surface morphology and controlled release during simulated gastrointestinal digestion.

Immunoglobulin Y (IgY) is an effective orally administered antibody used to protect against various intestinal pathogens, but which cannot tolerate the acidic gastric environment. In this study, IgY was microencapsulated by alginate (ALG) and coated with chitooligosaccharide (COS). A response surface methodology was used to optimize the formulation, and a simulated gastrointestinal (GI) digestion (SGID) system to evaluate the controlled release of microencapsulated IgY. The microcapsule formulation was optimized as an ALG concentration of 1.56% (15.6 g/L), COS level of 0.61% (6.1 g/L), and IgY/ALG ratio of 62.44% (mass ratio). The microcapsules prepared following this formulation had an encapsulation efficiency of 65.19%, a loading capacity of 33.75%, and an average particle size of 588.75 µm. Under this optimum formulation, the coating of COS provided a less porous and more continuous microstructure by filling the cracks on the surface, and thus the GI release rate of encapsulated IgY was significantly reduced. The release of encapsulated IgY during simulated gastric and intestinal digestion well fitted the zero-order and first-order kinetics functions, respectively. The microcapsule also allowed the IgY to retain 84.37% immune-activity after 4 h simulated GI digestion, significantly higher than that for unprotected IgY (5.33%). This approach could provide an efficient way to preserve IgY and improve its performance in the GI tract.

Prebiotic oligosaccharides change the concentrations of short-chain fatty acids and the microbial population of mouse bowel.

The purpose of this study was to clarify effects of selected oligosaccharides on concentrations of cecal short-chain fatty acids (SCFAs), total large bowel wet weight and wall weight, and cecal microbiota levels in mice. Mice were respectively given gavage of selected fructooligosaccharides (FOS), galactooligosaccharides (GOS), mannanoligosaccharides (MOS), and chitooligosaccharides (COS) [1000 mg/(kg body weight.d)]. Control group was given physiological saline solution. After 14 d treatment, SCFAs and lactate in mice cecum were significantly increased (P<0.05) by intake of oligosaccharides, especially FOS and GOS. Thus, providing these oligosaccharides as ingredients in nutritional formulas may benefit the gastrointestinal tract.

Fatty acid profiles of muscle from large yellow croaker (Pseudosciaena crocea R.) of different age.

We investigated the fatty acid profiles of muscle from large yellow croaker (Pseudosciaena crocea R.) of different age. One- and two-year-old fish were cultured in floating net cages and sampled randomly for analysis. Moisture, protein, lipid and ash contents were determined by methods of Association of Analytical Chemist (AOAC) International. Fatty acid profile was determined by gas chromatography. Crude protein, fat, moisture and ash contents showed no significant differences between the two age groups. The contents of total polyunsaturated fatty acids and docosahexaenoic acid (DHA) were significantly higher and eicosapentaenoic acid (EPA) content was significantly lower in the two-year-old large yellow croaker than in the one-year-old (P<0.05). No significant differences were observed in the contents of total saturated fatty acids and monounsaturated fatty acids, or the ratio of n-3/n-6 fatty acids among the large yellow croakers of the two age groups. We conclude that large yellow croakers are good food sources of EPA and DHA.

Effects of fish protein hydrolysate on growth performance and humoral immune response in large yellow croaker (Pseudosciaena crocea R.).

We investigated the effects of fish protein hydrolysate (FPH) on growth performance and humoral immune response of the large yellow croaker (Pseudosciaena crocea R.). One thousand and two hundred large yellow croakers [initial average weight: (162.75+/-23.85) g] were divided into four groups and reared in floating sea cages (3 m x 3 m x 3 m). The animals were fed with 4 diets: basal diet only (control) or diets supplemented with 5%, 10% and 15% (w/w) FPH. The results show that dietary FPH levels significantly influenced the growth and immunity of the large yellow croaker. Compared with the control group, total weight gain (TWG) in all treatment groups, relative weight gain (RWG) and specific growth rate (SGR) in fish fed with diets supplemented with 10% and 15% FPH were significantly increased (P<0.05). Similar results were observed in immune parameters [lysozyme activity, serum complements, immunoglobulin M (IgM)]. Lysozyme activity, complement C4 and IgM were also significantly increased (P<0.05) in fish fed with diets supplemented with 10% and 15% FPH, while complement C3 level was significantly increased (P<0.05) in all treatment groups. In general, with the supplementation of FPH, particularly at dose of 10%, the growth performance and immunity of the large yellow croaker can be improved effectively.

Influence of dietary conjugated linoleic acid on growth, fatty acid composition and hepatic lipogenesis in large yellow croaker (Pseudosciaena crocea R.).

We examined the effects of conjugated linoleic acid (CLA) on growth, fatty acid composition and enzyme activity of fatty acid oxidation in the liver of large yellow croaker. We divided 1600 fish (average initial weight 150 g) into 4 groups and reared them in 8 cages. Four dietary treatments were formulated to contain 0%, 1%, 2% and 4% (w/w) CLA, respectively. The fish were fed for 10 weeks ad libitum twice daily. We found that the dietary CLA had no effect on growth, biometric parameters and whole body proximate (P>0.05), but showed some significant effects on the fatty acid composition in both muscle and the liver. The activities of lipogenic enzymes were slightly depressed in fish fed with increasing levels of CLA when compared with control (P>0.05). Dietary CLA supplementation had no effects on liver lipid content, but significantly increased the contents of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) (P<0.05) and decreased monounsaturated fatty acid (MUFA) content in both muscle and the liver. Dietary CLA inclusion resulted in significant increases of the biologically active cis-9, trans-11 and trans-10, cis-12 isomers in both tissues (P<0.05). The total accumulation of CLA was higher in the liver (3.83%, w/w) than in muscle (3.77%, w/w) when fed with 4% (w/w) CLA. This study demonstrates that large yellow croakers are capable of absorbing and depositing CLA and long-chain n-3 PUFA in the liver and muscle, showing that this species fed with CLA could be an important human food source for these healthful fatty acids.