Identification of Novel Peptides in Distillers’ Grains as Antioxidants, α-Glucosidase Inhibitors, and Insulin Sensitizers: In Silico and In Vitro Evaluation

Distillers’ grains are rich in protein and constitute a high-quality source of various bioactive peptides. The purpose of this study is to identify novel bioactive peptides with α-glucosidase inhibitory, antioxidant, and insulin resistance-ameliorating effects from distiller’s grains protein hydrolysate. Three novel peptides (YPLPR, AFEPLR, and NDPF) showed good potential bioactivities, and the YPLPR peptide had the strongest bioactivities, whose IC50 values towards α-glucosidase inhibition, radical scavenging rates of 2,2′-azino-bis (3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were about 5.31 mmol/L, 6.05 mmol/L, and 7.94 mmol/L, respectively. The glucose consumption of HepG2 cells treated with YPLPR increased significantly under insulin resistance condition. Moreover, the YPLPR peptide also had a good scavenging effect on intracellular reactive oxygen species (ROS) induced by H2O2 (the relative contents: 102.35% vs. 100%). Molecular docking results showed that these peptides could stably combine with α-glucosidase, ABTS, and DPPH free radicals, as well as related targets of the insulin signaling pathway through hydrogen bonding and van der Waals forces. This research presents a potentially valuable natural resource for reducing oxidative stress damage and regulating blood glucose in diabetes, thereby increasing the usage of distillers’ grains peptides and boosting their economic worth.


Introduction
The global incidence of type 2 diabetes mellitus (T2DM), characterized by high blood sugar levels, is steadily rising, posing a significant challenge to the worldwide healthcare system [1].Prolonged hyperglycemia can lead to severe oxidative stress damage to blood vessels, ultimately resulting in complications such as inflammatory responses and atherosclerosis [2].In the treatment of T2DM, the key strategies for controlling blood glucose levels involve inhibiting crucial enzymes such as α-glucosidase and improving insulin resistance status; additionally, maintaining normal levels of free radicals in the body can indirectly ameliorate diabetes complications [3][4][5].Nowadays, common clinical hypoglycemic drugs include acarbose, voglibose, metformin, and miglitol [6,7].While these drugs are effective in managing T2DM, they often come with certain toxic side effects and adverse reactions for long-term use of these drugs, with severe cases even leading to patient comas [8].Hence, the pursuit of safe and effective hypoglycemic compounds and the development of high-efficiency, low-side-effect hypoglycemic functional foods have emerged as a new research focus.
Food-derived bioactive peptides are generally low-molecular-weight compounds that are safe, low in toxicity, and easily absorbed and that exhibit good antibacterial, antihypertensive, hypoglycemic, immunomodulatory, and antioxidant properties [9][10][11].Since these peptides have small molecular weights, they are more likely to be absorbed intact by the gastrointestinal tract after oral ingestion, thereby exerting systemic or local effects [12].Consequently, the study of bioactive peptides has progressively emerged as a prominent research area in recent years.Numerous studies have demonstrated that certain bioactive peptides can regulate blood glucose levels and enhance the management of type 2 diabetes through diverse pathways, such as the inhibition of α-glucosidase activity or the improvement of insulin resistance [13,14].For instance, Mora et al. (2020) identified the peptides AEEEYPDL and LGVGG from Spanish dry-cured ham, which effectively inhibited α-glucosidase activity [13].In the study conducted by Hu et al. (2019), water-extracted peptides and proteins were found to alleviate insulin resistance, thereby improving diabetes by enhancing glucose utilization [14].
Distillers' grains, by-products of rice wine production, possess abundant protein and alimentary value.Nevertheless, currently, most distillers' grains are primarily utilized as animal feed, resulting in potential resource wastage.Hence, the purpose of this study is to identify novel bioactive peptides with good α-glucosidase inhibition, antioxidant capacity, and insulin resistance-ameliorating effects from distiller's grains protein hydrolysate and to further reveal the underlying mechanisms.This research may provide new insights into the use of distillers' grains peptides as a valuable natural resource to combat oxidative stress damage and regulate blood glucose levels in diabetes, and the outcomes may hold the potential for enhancing the economic benefits derived from distillers' grains peptides.

Sample Preparation
Distillers' grains underwent lyophilization using the Christ Alpha 1-2 LD plus lyophilizer and then were finely made into a powder using the Lingdan LD-T300 grinder.The isolation of protein from the distillers' grains followed the way outlined by Wang et al. (2019) (minor modifications) [15,16].The concrete method of separating proteins is displayed in the Supplementary Materials.

Composition Analysis of Peptide by UHPLC-ESI-HRMS/MS
The molecular mass and amino acid identity of peptides in rice protein hydrolysate were determined by the Thermo Fisher Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Dreieich, Germany) combined with the Agilent Zorbax SB-C18 column (1.7 µm, 2.1 mm × 100 mm, Dr. Asp 80Maisch, Germany).According to the Tian et al. ( 2022) method (slightly modified), the mass spectrometry parameters were set [17].Details of the method are shown in the Supplementary Materials.Peak Studio 8.0 (Bioinformatics Solutions, Waterloo, ON, Canada) was used to analyze molecular mass and amino acid sequence.Peptides with an average local confidence (ALC) score exceeding 85% were deemed to be included.

Determination of α-Glucosidase Inhibitory Effect
The inhibitory effect on α-glucosidase activity of the protein hydrolysates from distillers' grains or three selected peptides was assessed following the methodology outlined by Zhang et al. (2018) with slight modifications, and the concrete method is shown in the Supplementary Materials [18].

Assessment of Scavenging Capacity against DPPH and ABTS Radicals
The antioxidant properties of vinasse hydrolysates or three screening peptides were evaluated by DPPH and ABTS free radical scavenging experiments according to the method of Sun et al. (2015) [19].Detailed information about the test method has been recorded in the Supplementary Materials.

In Silico Screening of Main Bioactive Peptides in Distillers' Grains Protein Hydrolysates
Peptide bioactivity prediction was conducted using Peptide Ranker (http://distilldeep. ucd.ie/PeptideRanker/, (accessed on 12 October 2023)) and selected peptides with a biological activity score of ≥0.6 for further analysis.It is important to highlight that a higher score indicates a higher likelihood of bioactivity [17].The detailed method of studying the interaction between distiller's grains protein hydrolysate polypeptide and αglucosidase by molecular docking technology is noted in the Supplementary Materials.For the pretreatment of peptides, refer to O'Boyle et al. (2011) [20].Given the unavailability of the 3D structure of α-glucosidase, Saccharomyces cerevisiae isomaltose (PDB Code: 3A4A) from the database RCSB PDB (http://www.rcsb.org/pdb/home/home.do,(accessed on 12 October 2023)) was employed as a substitute.The post-treatment of α-glucosidase and the subsequent molecular docking followed the approach [10].Based on the results of the molecular docking, peptides with an affinity equal to or superior to WLRL were identified as potential α-glucosidase inhibitory peptides [14].The structure and interactions of latent α-glucosidase inhibitory peptides were analyzed using PyMOL (version 2.3.1).ToxinPred (http://crdd.osdd.net/raghava/toxinpred/(accessed on 25 October 2023)) was used to appraise the toxicity of peptides [21].Additionally, the solubility of peptides was evaluated by an online website (http://www.innovagen.com/proteomicstools,(accessed on 26 October 2023)) [22].

Peptides Synthesis and Activity Verification
Peptides YPLPR, AFEPLR, and NDPF, derived from distillers' grains protein hydrolysates, were synthesized.The synthesized peptides were stored, and their corresponding activities were verified through in vitro experiments.The α-glucosidase inhibitory effects and free radical scavenging capacities were performed as described above in Sections 2.4 and 2.5.

Determination of Insulin-Resistance HepG2 Cells on Bioactive Peptides
The MTT method was adopted to assess the impact of samples and insulin on cell viability, helping determine the optimal concentrations of insulin and the samples.The experimental protocol for improving insulin resistance followed previous methods [23,24].Details are provided in the Supplementary Materials.In this experiment, various groups were established, incorporating the blank control group (K), model group (M), peptide sample groups, and the positive control group (treated with rosiglitazone).

Evaluation of Scavenging Capacity against Intracellular ROS
According to Liu et al. (2020), the H 2 O 2 -induced scavenging activity of reactive oxygen species was evaluated [25].In this experiment, various groups were established, including the blank, sample, positive control, and model groups.Refer to the Supplementary Materials for specific information.

Statistical Analysis
All experiments were conducted at least three times.The data were disposed of and analyzed by Origin 2021 (Origin Lab, Northampton, MA, USA) and SPSS 26 (Chicago, IL, USA) software.The data were represented by a median of 25% and a 75% quartile, and significance was determined by the Kruskal-Wallis test (p < 0.05).

Identification of the Peptides in Distillers' Grains Protein Hydrolysates
In the process of hydrolysis, the degree of hydrolysis of distiller's grains protein gradually augmented with the increase in time, and after hydrolysis for 2.5 h, the degree of hydrolysis gradually remained steady, indicating that the enzymatic reaction also stabilized.The final hydrolysis degree of alkaline protease was 18.49 ± 0.49%.In previous studies, the final hydrolysis degree of alkaline protease for soybean protein was 19.00% [28], and that for mung bean protein was about 25.00% [29].In the lump, 20 peptides with ALC scores > 85% were identified among the distillers' grains protein hydrolysate peptides (Table 1).These 20 peptides contained 4-8 amino acids, and their molecular weights ranged from 438-860 Da; all of them were low-molecular-weight peptides.The study of beer and spent grain peptides also showed that the main bioactive peptides were low-molecularweight peptides with relative molecular masses < 1500 Da [11,17].The better bioactivity of low molecular mass peptides is mainly due to the fact that they can bind more easily to the active sites of specific enzymes or be more readily absorbed by the gastrointestinal digestive tract and blood vessel system and eventually demonstrate physiological regulatory properties, which also shows that distillers' grains protein hydrolysate peptides may have good biological activity.And in previous studies on rice wine, 99 peptides with ALC scores > 85% were also identified from rice wine [10].The number of peptides in rice wine was higher than that in distillers' grains protein hydrolysates, mainly because many peptides were further broken down into more peptides during the fermentation of rice wine.The biological activity of distillers' grains protein hydrolysate peptides needs to be further explored and developed.

α-Glucosidase Inhibitory Effect of Distillers' Grains Protein Hydrolysates
α-glucosidase is a membrane-associated enzyme.Inhibition of α-glucosidase activity can reduce carbohydrate hydrolysis, thus reducing sugar absorption and effectively controlling diabetes.[11].As shown in Figure 1A, with the increase in concentration, the α-glucosidase inhibitive activity of distiller's grains protein hydrolysate increased, and the α-glucosidase inhibitive activity was 60.88 (58.07, 67.04) % when the concentration of distillers' grains protein hydrolysates was 5 mg/mL, with an IC 50 value of 3.76 (3.51, 3.83) mg/mL.In the study on rice wine peptides, the α-glucosidase inhibitory activity of 50% ethanol-eluted rice wine peptides had an IC 50 value of 1.71 (1.65, 1.74) mg/mL [10].And the IC 50 value of α-glucosidase depressant activity was 4.94 ± 0.07 mg/mL in the study of soy protein hydrolyzed peptide [15].The comparison indicated that the distillers' grains protein hydrolysates had α-glucosidase inhibitive activity and deserves more deep investigation and development.

Scavenging Capacity against DPPH and ABTS Radicals of Distillers' Grains Protein Hydrolysates
The oxidation stress is nearly relevant to diabetes.Reducing the production of free radicals, or directly breaking the free radicals produced by the body, can reverse the tissue damage caused by oxidative stress, thus preventing or delaying diabetes complications.While ABTS and DPPH free radicals are the most common free radicals in the body, the scavenging activities of distillers' grains protein hydrolysates on ABTS and DPPH free radicals are shown in Figure 1B,C.The scavenging activity of distillers' grains protein hydrolysates on ABTS and DPPH radicals increased gradually with increasing concentrations, with IC 50 values of 0.59 (0.52, 0.64) mg/mL and 1.85 (1.74, 1.86) mg/mL, respectively.This shows that distillers' grains protein hydrolysates have good antioxidant activity.

Screening of Main Bioactive Peptides in Distillers' Grains Protein Hydrolysates
Peptide Ranker (http://distilldeep.ucd.ie/PeptideRanker/(accessed on 12 October 2023)) was used to score the probability of biological activity for each peptide sequence, and the outcomes are shown in Table 2.The closer the probability of a peptide being biologically active predicted by Peptide Ranker to 1, the more confident Peptide Ranker is that the peptide is biologically active.Peptides with a score above or equal to 0.6 were more likely to be biologically active [16,17].Based on the scoring results, ten peptides with scores greater than 0.6 were selected, which were RFDR, PDVGHPM, YPLPR, AFEPLR, FEPLR, WLDY, FEPL, FDGVLRGP, NDPF, and WNLN.

Scavenging Capacity against DPPH and ABTS Radicals of Distillers' Grains Protein Hydrolysates
The oxidation stress is nearly relevant to diabetes.Reducing the production of free radicals, or directly breaking the free radicals produced by the body, can reverse the tissue damage caused by oxidative stress, thus preventing or delaying diabetes complications While ABTS and DPPH free radicals are the most common free radicals in the body, the scavenging activities of distillers' grains protein hydrolysates on ABTS and DPPH free radicals are shown in Figure 1B,C.The scavenging activity of distillers' grains protein hy drolysates on ABTS and DPPH radicals increased gradually with increasing concentra tions, with IC50 values of 0.59 (0.52, 0.64) mg/mL and 1.85 (1.74, 1.86) mg/mL, respectively This shows that distillers' grains protein hydrolysates have good antioxidant activity.Due to the fact that α-glucosidase is a more important and directly regulated target in the treatment of diabetes, the potential inhibitory activity of α-glucosidase was used as the main condition of inquiry for further analysis.According to a previous study, the WLRL peptide from soybean protein exhibited a good inhibitory effect toward α-glucosidase [15].Therefore, this peptide was selected as a positive control to facilitate the analysis of the current study, and its affinity grading after molecular docking with α-glucosidase was −9.1 kcal/mol.Four peptides, YPLPR, AFEPLR, NDPF, and WNLN, had affinities of −10.7, −9.6, −9.1, and −9.2 kcal/mol, respectively, which were better than WLRL, indicating that these peptides may have good α-glucosidase inhibitory potential.Therefore, these peptides can be used as potential functional candidates for α-glucosidase inhibitors for further analysis.The solubility and toxicity of bioactive peptides are closely related to the biological activity of the peptide.Before further application, it plays a large part in analyzing the toxicity of peptides.The Toxin Pred tool is used to predict whether or not peptides are toxic, and it is being utilized more widely to assess numerous bioactive peptides [30].The prediction results obtained using this tool showed that all the peptides screened in this study were safe and non-toxic.The potential efficacy of bioactive peptides is closely related to the solubility of the peptides, which was assessed by the peptide property calculator Innovagen.The results are shown in Table 2.Among the four peptides obtained by bioactivity scoring, α-glucosidase inhibition binding energy, and toxicity prediction, YPLPR, AFEPLR, and NDPF had good solubility.Therefore, YPLPR, AFEPLR, and NDPF were selected as the main potentially bioactive peptides for further study.

Improvement of Bioactive Peptides on Insulin Resistance in HepG2 Cells
Insulin is one of the main pathogenic mechanisms of type 2 diabetes mellitus, and the liver is an important site for insulin to function.HepG2 cells are derived from human embryonic hepatocytes and possess many biological characteristics of normal hepatocytes, so HepG2 cells are chosen to establish an insulin resistance model.As the cellular insulin resistance condition arises, the utilization of glucose by the cells decreases, and the consumption of glucose decreases.Therefore, insulin resistance is usually expressed by the consumption of glucose in studies [24].Using the MTT method, this study explored the effect of different concentrations of insulin on cellular glucose consumption after 24 h of action with HepG2 cells.As shown in Figure 2A, the glucose consumption of the cells gradually decreased as the concentration of insulin increased.When the insulin concentration reached 25.0 µmol/L, the cellular glucose consumption was significantly reduced compared with the group without insulin, which indicated that HepG2 cells developed insulin resistance.And MTT experiments showed that insulin was not cytotoxic at a concentration of 25.0 µmol/L.Therefore, the insulin concentration of 25.0 µmol/L was chosen to establish the insulin resistance model in this experiment.Also, MTT results showed that YPLPR, AFEPLR, and NDPF had no cytotoxicity at 1.0 mmol/mL, so the sample concentrations for subsequent experiments were all 1.0 mmol/L.

Intracellular ROS Scavenging Activity by Bioactive Peptides
Excessive ROS may attack normal cells or biological macromolecules and undergo oxidative stress, causing severe damage to cellular structures, which causes many diabetic complications and chronic diseases.Accordingly, in this study, the scavenging activities of ABTS and DPPH radicals of bioactive peptides were not only investigated by in vitro experiments, but their antioxidant activity was also further investigated at the cellular level.The cell damage rate of the H2O2-induced cell damage model was generally around In this study, the effect of YPLPR, AFEPLR, and NDPF on the glucose consumption of HepG2 cells in an insulin resistance state was investigated.As shown in Figure 2B, the glucose consumption of model group M was 6.08 (5.80, 6.81) mmol/L, which was significantly lower than that of blank control group K (p < 0.05), indicating that the insulin resistance model of this experiment was successful and the results of the relevant experiments were obtained under the condition that the cells were insulin resistant.The glucose consumption of peptides YPLPR, AFEPLR, and NDPF were 9.56 (9.37, 9.83), 10.56 (10.21, 10.71) and 9.00 (8.80, 9.33) mmol/L, respectively.The glucose consumption of these three peptides was significantly increased compared with model group M; YPLPR and AFEPLR were significantly different from the positive control rosiglitazone group (9.09 (8.29, 9.23) mmol/L) (p < 0.05).The efficacy of food-derived peptides to improve insulin resistance has been studied previously, and in a study of walnut-derived peptides, the identified peptides LVRL, LRYL, and VLLALVLLR all increased glucose consumption in HepG2 cells compared to the model group and served to improve insulin resistance [33].In a study by Gong et al. (2020), specific bioactive peptides SDIPNPIGSE, NPWDQVKR, and QEPVLGPVRGPFP extracted from goat milk casein hydrolysate have demonstrated an improvement in insulin resistance in HepG2 cells exposed to high glucose.This study establishes the potential of goat cheese protein hydrolysate as a food component for enhancing insulin sensitivity [34].Comparison with these studies indicates that the peptides YPLPR, AFEPLR, and NDPF have strong effects on improving insulin resistance, all of which can promote cellular glucose uptake, and these peptides have some development and utilization value.

Intracellular ROS Scavenging Activity by Bioactive Peptides
Excessive ROS may attack normal cells or biological macromolecules and undergo oxidative stress, causing severe damage to cellular structures, which causes many diabetic complications and chronic diseases.Accordingly, in this study, the scavenging activities of ABTS and DPPH radicals of bioactive peptides were not only investigated by in vitro experiments, but their antioxidant activity was also further investigated at the cellular level.The cell damage rate of the H 2 O 2 -induced cell damage model was generally around 50%.As shown in Figure 3A, with the increase in concentration, the cytotoxicity produced by H 2 O 2 was enhanced, and the cell viability gradually decreased.When the concentration of H 2 O 2 was 1.2 mmol/L, the cell viability rate was 56.38 (50.33, 68.00) %, so the concentration of H 2 O 2 of 1.2 mmol/L established the cell damage model in this study.And the intracellular ROS levels in the model group established by 1.2 mmol/L H 2 O 2 were significantly higher than those in the blank group (p < 0.05), indicating that the oxidative stress model of HepG2 cells induced by 1.2 mmol/L H 2 O 2 was successfully established.Consistent with 3.6, YPLPR, AFEPLR, and NDPF were used at 1.0 mmol/L for subsequent experiments.
The intracellular ROS inhibition of the different fractions was represented by flow and histograms.In the flow diagram (Figure 3B), the left-right shift of the peak indicates the magnitude of the fluorescence luminosity, and the more the peak is shifted to the right, the greater the fluorescence intensity.The peak of the model group M was significantly shifted to the right compared with the blank group K, which indicated an increase in fluorescence intensity and an increase in ROS content.As shown in Figure 3C, the relative intracellular ROS content in the M group and the blank group K were 139.46 (135.10,142.41) % and 101 (95.10, 103.50) %, respectively.The relative intracellular ROS content in the M group was significantly higher than that in the blank group K (p < 0.05), which indicated that the oxidative stress model in this batch of experiments was successful.The relative intracellular ROS content in VC, the YPLPR, AFEPLR, and NDPF groups were 92.03 (90.83, 98.11) %, 102.35 (101.38, 103.06) %, 104.11 (103.69,104.54) % and 95.47 (92.68, 99.37) %, respectively.It can be seen that the intracellular ROS content in the YPLPR, AFEPLR, and NDPF groups was relatively lower than that in the M group; among them, the YPLPR and NDPF intracellular ROS contents were not significantly different from that in the VC group and the blank group (p > 0.05).This indicated that the peptides YPLPR, AFEPLR, and NDPF played a good protective role against oxidative stress damage to cells and had a good scavenging effect on reactive oxygen species.
AFEPLR, and NDPF groups was relatively lower than that in the M group; among them, the YPLPR and NDPF intracellular ROS contents were not significantly different from that in the VC group and the blank group (p > 0.05).This indicated that the peptides YPLPR, AFEPLR, and NDPF played a good protective role against oxidative stress damage to cells and had a good scavenging effect on reactive oxygen species.

The Molecular Docking of Bioactive Peptides with α-Glucosidase
To reveal the possible inhibition mechanisms of YPLPR, AFEPLR, and NDPF towards α-glucosidase, the optimal docking postures between α-glucosidase and three peptides were shown in Figure 4, and the related results are shown in Table 3. Usually, when α-glucosidase binds to the ligand, the active cavity provides a strong hydrophobic environment and multiple hydrogen bond binding sites, which are conducive to the steadiness of the ligand.As shown in Figure 4A,B, YPLPR, AFEPLR, and NDPF can bind to amino acids in the active lumen of α-glucosidase and be completely encapsulated in the α-glucosidase active pocket, forming a relatively stable binding conformation.It has been shown that hydrogen bonding and hydrophobic interactions are important indicators of ligand-receptor binding.Among them, the number and distance of hydrogen bonds are the main drivers of docking ligand-α-glucosidase interaction, where the ligand inhibits α-glucosidase activity by binding to α-glucosidase active amino acids through hydrogen bonds and preventing substrate access to the active pocket [35].As shown in Figure 4C1,C2 and Table 3, YPLPR formed hydrogen bonds with 9 amino acid residues (Asp215, Ser240, Ser241, Asp242, Glu277, Gln279, Phe314, Arg315, and Asp352) in the α-glucosidase active pocket, and the longest of these bonds was 3.6 Å, the shortest was 1.9 Å.As shown in Figure 4D1,D2 and Table 3, AFEPLR established 11 hydrogen bonds with 7 amino acid residues in the active site of α-glucosidase, with a mean bond distance of 2.4 Å.And it also had hydrophobic interactions with 15 amino acids on α-glucosidase.In contrast, NDPF formed only 3 hydrogen bonds with Lys156, Tyr158, and Asp242 on α-glucosidase, with bond lengths of 2.2 Å, 3.1 Å, and 2.7 Å, respectively.And it had hydrophobic interactions with 8 amino acid residues on α-glucosidase (Figure 4E1,E2).The results indicated that YPLPR, AFEPLR, and NDPF were able to efficiently attach to the active site of α-glucosidase.In previous studies, Asp69, Tyr158, Asp215, Val216, Ser240, Asp242, Arg442, Gln279, Alp411, and Asn415 were identified as the primary active residues responsible for α-glucosidase inhibition [36].The comprehensive analysis shows that YPLPR interacts with the key residues that inhibit α-glucosidase activity the most, namely Asp215, Ser240, Asp242, and Gln279, which is consistent with the highest YPLPR and α-glucosidase binding scores and the best actual α-glucosidase inhibition experimental activity (3.5 Experimental Results).This result indicates that computer simulation is desirable to be used to assist experimental studies, and it can greatly reduce the cost and time of traditional biological screening, making it an effective method for screening enzyme-inhibiting peptides.The radical scavenging mechanisms of the three peptides YPLPR, AFEPLR, and NDPF on ABTS and DPPH radicals were also investigated by molecular docking.As shown in Figure 5A1,A2, the binding energy of YPLPR to the ABTS radical is −4.2 kcal/mol.ABTS, as a ligand, can form 2 hydrogen bonds with Met7 and Pi bonds with His5 and Pro6 on YPLPR.As shown in Figure 5B1-D2, the binding energies of YPLPR, AFEPLR, and NDPF to the DPPH radical are −4.2,−3.9, and −4.2 kcal/mol, respectively.DPPH as a ligand can form 2 hydrogen bonds with His5 and Pi bonds with Met7 on YPLPR (Figure 5B1,B2).The amino acids of DPPH and AFEPLR are mainly bound by Pi bonds (Leu5) and hydrophobic interactions (Ala1, Phe2, and Glu3) (Figure 5C1,C2).DPPH forms a hydrogen bond with Asp2 on NDPF with a bond distance of 2.5 Å and a Pi bond with Phe4 (Figure 5D1,D2).In summary, the results indicated that amino acid residues belonging to YPLPR, AFEPLR, and NDPF could bind to ABTS and DPPH free radicals through hydrogen bonds, π bonds, or hydrophobic interactions, which made these peptides have good ABTS or DPPH free radical scavenging activity.hydrophobic interactions (Ala1, Phe2, and Glu3) (Figure 5C1,C2).DPPH forms a hydrogen bond with Asp2 on NDPF with a bond distance of 2.5 Å and a Pi bond with Phe4 (Figure 5D1,D2).In summary, the results indicated that amino acid residues belonging to YPLPR, AFEPLR, and NDPF could bind to ABTS and DPPH free radicals through hydrogen bonds, π bonds, or hydrophobic interactions, which made these peptides have good ABTS or DPPH free radical scavenging activity.

Conclusions
The results showed that a total of 20 peptides with ALC scores >85% were identified in the distillers' grains peptides obtained by alkaline protease enzymatic digestion, and the molecular weights were concentrated between 438 and 860 Da.The distillers' grains peptides had good α-glucosidase inhibitory activity and antioxidant activity.Bioinformatics analysis showed that peptides YPLPR, AFEPLR, and NDPF were the main potentially bioactive peptides in distillers' grains peptides and the three peptides had good α-glucosidase inhibitory activity, insulin resistance-improving and antioxidant activities.And peptides YPLPR, AFEPLR, and NDPF may exhibit their bioactives by stably binding to αglucosidase, ABTS, and DPPH radicals, as well as relevant targets of the insulin signaling pathway, through strong hydrogen bonding and van der Waals forces.Generally speaking, these results obtained in the present work may offer a potential natural resource for alleviating oxidative stress damage and controlling blood glucose in diabetes, thereby The 3D structural surface images of three peptide with PPARγ (A); active site binding of three peptide with receptor protein (B), where YPLPR is in blue, AFEPLR in purple, and NDPF in red.The 3D images of the hydrogen bonding of YPLPR (C1), AFEPLR (D1), and NDPF (E1) with the amino acid residues of PPARγ (The yellow dotted line represents the hydrogen bond distance.),and the 2D images of the hydrophobic interaction of YPLPR (C2), AFEPLR (D2), and NDPF (E2) with the amino acid residues of PPARγ.

Conclusions
The results showed that a total of 20 peptides with ALC scores > 85% were identified in the distillers' grains peptides obtained by alkaline protease enzymatic digestion, and the molecular weights were concentrated between 438 and 860 Da.The distillers' grains peptides had good α-glucosidase inhibitory activity and antioxidant activity.Bioinformatics analysis showed that peptides YPLPR, AFEPLR, and NDPF were the main potentially bioactive peptides in distillers' grains peptides and the three peptides had good α-glucosidase inhibitory activity, insulin resistance-improving and antioxidant activities.And peptides YPLPR, AFEPLR, and NDPF may exhibit their bioactives by stably binding to α-glucosidase, ABTS, and DPPH radicals, as well as relevant targets of the insulin signaling pathway, through strong hydrogen bonding and van der Waals forces.Generally speaking, these results obtained in the present work may offer a potential natural resource for alleviating oxidative stress damage and controlling blood glucose in diabetes, thereby enhancing the utilization of distillers' grains peptides and augmenting their economic value.However, it should be noted that the methods used in the current study were all in vitro, which may differ from the real environment of the human body; the bioavailability, intestinal

Figure 1 .
Figure 1.α-Glucosidase inhibitory activities (A), ABTS (B), and DPPH (C) scavenging activities o protein hydrolysate from distillers' grains.The data are displayed as the median (n = 3) with the 25% and 75% quartiles and analyzed using the Kruskal-Wallis test.Significance is represented by differ ent letters (p < 0.05).

Figure 1 .
Figure 1.α-Glucosidase inhibitory activities (A), ABTS (B), and DPPH (C) scavenging activities of protein hydrolysate from distillers' grains.The data are displayed as the median (n = 3) with the 25% and 75% quartiles and analyzed using the Kruskal-Wallis test.Significance is represented by different letters (p < 0.05).

Nutrients 2024 ,
16,  x FOR PEER REVIEW 9 of 19 which can promote cellular glucose uptake, and these peptides have some development and utilization value.

Figure 2 .
Figure 2. The effects of different concentrations of insulin on glucose consumption of HepG2 cells (A); the effects of YPLPR, AFEPLR, NDPF, and rosiglitazone on glucose consumption of HepG2 cells (B).Data are presented as the median (n = 6) with the 25% and 75% quartiles; different letters indicated significant differences (p < 0.05).(K: control group; M: model group; rosiglitazone: positive control group).

Figure 2 .
Figure 2. The effects of different concentrations of insulin on glucose consumption of HepG2 cells (A); the effects of YPLPR, AFEPLR, NDPF, and rosiglitazone on glucose consumption of HepG2 cells (B).Data are presented as the median (n = 6) with the 25% and 75% quartiles; different letters indicated significant differences (p < 0.05).(K: control group; M: model group; rosiglitazone: positive control group).

Figure 3 .
Figure 3. Scavenging of H2O2-induced intracellular ROS production by YPLPR, AFEPLR, NDPF, and VC.Effects of different concentrations (A) on cell viability; ROS flow chart, R3 represents half the peak width of the blank group K. (B); histogram of ROS contents (C).Data are displayed as the median (n = 4) with the 25% and 75% quartiles; different letters indicated significant differences (p < 0.05).(K: control group; M: model group; VC: positive control group).

Figure 3 .
Figure 3. Scavenging of H 2 O 2 -induced intracellular ROS production by YPLPR, AFEPLR, NDPF, and VC.Effects of different concentrations (A) on cell viability; ROS flow chart, R3 represents half the peak width of the blank group K. (B); histogram of ROS contents (C).Data are displayed as the median (n = 4) with the 25% and 75% quartiles; different letters indicated significant differences (p < 0.05).(K: control group; M: model group; VC: positive control group).

Figure 4 .
Figure 4. Molecular docking results of YPLPR, AFEPLR, and NDPF with α-glucosidase (PDB C 3A4A).The 3D structural surface images of three peptide with α-glucosidase (A); active site bin of three peptide with α-glucosidase, (B), where YPLPR is in blue, AFEPLR in purple and ND red.The 3D images of the hydrogen bonding of YPLPR (C1), AFEPLR (D1), and NDPF (E1) wit amino acid residues of α-glucosidase (The yellow dotted line represents the hydrogen bond dis green part is α-glucosidase, and gray part is peptide.),and the 2D images of the hydrophobic action of YPLPR (C2), AFEPLR (D2), and NDPF (E2) with the amino acid residues of α-glucosi

Figure 4 .
Figure 4. Molecular docking results of YPLPR, AFEPLR, and NDPF with α-glucosidase (PDB Code: 3A4A).The 3D structural surface images of three peptide with α-glucosidase (A); active site binding of three peptide with α-glucosidase, (B), where YPLPR is in blue, AFEPLR in purple and NDPF in red.The 3D images of the hydrogen bonding of YPLPR (C1), AFEPLR (D1), and NDPF (E1) with the amino acid residues of α-glucosidase (The yellow dotted line represents the hydrogen bond distance, green part is α-glucosidase, and gray part is peptide.),and the 2D images of the hydrophobic interaction of YPLPR (C2), AFEPLR (D2), and NDPF (E2) with the amino acid residues of α-glucosidase.

Figure 6 .
Figure 6.Molecular docking results of YPLPR, AFEPLR, and NDPF with PIRTK protein (PDB Code: 1IR3).The 3D structural surface images of three peptide with receptor protein (A); active site binding of three peptide with PIRTK protein (B), where YPLPR is in blue, AFEPLR in purple, and NDPF in red.The 3D images of the hydrogen bonding of YPLPR (C1), AFEPLR (D1), and NDPF (E1) with the amino acid residues of PIRTK protein (The yellow dotted line represents the hydrogen bond distance.),and the 2D images of the hydrophobic interaction of YPLPR (C2), AFEPLR (D2), and NDPF (E2) with the amino acid residues of PIRTK protein.

Figure 6 .
Figure 6.Molecular docking results of YPLPR, AFEPLR, and NDPF with PIRTK protein (PDB Code: 1IR3).The 3D structural surface images of three peptide with receptor protein (A); active site binding of three peptide with PIRTK protein (B), where YPLPR is in blue, AFEPLR in purple, and NDPF in red.The 3D images of the hydrogen bonding of YPLPR (C1), AFEPLR (D1), and NDPF (E1) with the amino acid residues of PIRTK protein (The yellow dotted line represents the hydrogen bond distance.),and the 2D images of the hydrophobic interaction of YPLPR (C2), AFEPLR (D2), and NDPF (E2) with the amino acid residues of PIRTK protein.

Figure 7 .
Figure 7. Molecular docking results of YPLPR, AFEPLR, and NDPF with PPARγ (PDB Code: 2PRG).The 3D structural surface images of three peptide with PPARγ (A); active site binding of three peptide with receptor protein (B), where YPLPR is in blue, AFEPLR in purple, and NDPF in red.The 3D images of the hydrogen bonding of YPLPR (C1), AFEPLR (D1), and NDPF (E1) with the amino acid residues of PPARγ (The yellow dotted line represents the hydrogen bond distance.),and the 2D images of the hydrophobic interaction of YPLPR (C2), AFEPLR (D2), and NDPF (E2) with the amino acid residues of PPARγ.

Figure 7 .
Figure 7. Molecular docking results of YPLPR, AFEPLR, and NDPF with PPARγ (PDB Code: 2PRG).The 3D structural surface images of three peptide with PPARγ (A); active site binding of three peptide with receptor protein (B), where YPLPR is in blue, AFEPLR in purple, and NDPF in red.The 3D images of the hydrogen bonding of YPLPR (C1), AFEPLR (D1), and NDPF (E1) with the amino acid residues of PPARγ (The yellow dotted line represents the hydrogen bond distance.),and the 2D images of the hydrophobic interaction of YPLPR (C2), AFEPLR (D2), and NDPF (E2) with the amino acid residues of PPARγ.

Table 2 .
Peptides with the main potentially bioactive selected for in silico analyses.