Study on Corn Anti-Alcohol Peptides and Their Gastrointestinal Stability

Alcoholic liver disease is one of the leading causes of morbidity and mortality related to liver diseases worldwide [1-2]. At present, numerous hangover-relieving and liver-protecting products have emerged in the market, mainly including peptides [3], amino acids [4], natural extracts [5] and other categories. These products can rapidly reduce the ethanol content in the human body by enhancing the metabolic activity of alcohol dehydrogenase, thereby protecting hepatocytes and human nerves from ethanol toxicity. Among these functional ingredients, polypeptides are regarded as effective components for the prevention and alleviation of alcohol-induced liver injury [6].

Corn oligopeptides are low-molecular-weight food-derived oligopeptide mixtures obtained via enzymatic hydrolysis of corn gluten meal or microbial fermentation. They possess multiple biological functions such as antihypertension [7], anti-fatigue [8], acceleration of alcohol metabolism [9] and liver protection [10]. Currently, the main preparation methods for corn peptides include enzymatic hydrolysis, microbial fermentation and chemical synthesis. Enzymatic hydrolysis is performed under mild conditions yet suffers from high production costs and bitter off-flavor formation. Microbial fermentation relies on various endogenous enzymes produced by microorganisms to cleave protein chains at specific sites under mild conditions, which can efficiently utilize raw proteins to produce bioactive peptides and reduce bitter-forming substances; nevertheless, this method has a relatively low conversion rate. Chemical synthesis features cumbersome procedures, high costs and low polypeptide yield.

Free radicals are generated during the in vivo metabolism of ethanol, which disrupt the body’s antioxidant system. Corn peptides accelerate ethanol metabolism by scavenging free radicals and mitigate alcohol-induced hepatic damage. Accordingly, the hydroxyl radical scavenging rate can be adopted as one of the indicators to evaluate the hangover-relieving efficacy of corn peptides [11]. In this study, corn gluten meal was used as the raw material to prepare corn anti-alcohol peptides through a combined process of alkaline protease hydrolysis and liquid-state fermentation with Bacillus subtilis. On the basis of single-factor experiments, response surface methodology was applied to optimize the preparation technology. Furthermore, the effect of gastrointestinal digestion on the hydroxyl radical scavenging capacity of corn peptides was investigated. This research aims to provide a theoretical basis for high-value utilization and intensive processing of corn by-products.

1 Materials and Methods

Corn anti-alcohol peptides were prepared from corn gluten meal via alkaline protease hydrolysis combined with liquid fermentation of Bacillus subtilis. Using the hydroxyl radical scavenging rate as the evaluation index, the preparation parameters were optimized by response surface experiments on the basis of single-factor trials, and the gastrointestinal stability of corn anti-alcohol peptides was explored.

2 Results and Analysis

2.1 Results of Single-Factor Experiments on Corn Gluten Meal Hydrolysis

2.1.1 Effects of Solid-Liquid Ratio on Hydroxyl Radical Scavenging Rate and Degree of Hydrolysis of Corn Gluten Meal Hydrolysate

As illustrated in Figure 1, the hydroxyl radical scavenging rate first increased and then decreased with the rising solvent dosage, reaching a maximum value of 78.22% at the solid-liquid ratio of 1:10 (g/mL). The degree of hydrolysis also presented an upward-then-downward trend with the increase of solvent volume. In the initial reaction stage, the increased solvent dosage enabled sufficient swelling of corn protein, weakened intermolecular interactions and exposed more active binding sites, which accelerated the hydrolysis reaction and generated abundant bioactive peptides with hangover-relieving functions. Nevertheless, excessive solvent diluted the substrate concentration, reduced the contact probability between enzymes and substrates, slowed down the hydrolysis rate and decreased the yield of anti-alcohol bioactive peptides, eventually leading to a decline in hydroxyl radical scavenging activity [15]. The maximum values of both hydroxyl radical scavenging rate and hydrolysis degree were obtained at the solid-liquid ratio of 1:10 (g/mL). Therefore, three solid-liquid ratios (1:8, 1:10, 1:12 g/mL) were selected for subsequent response surface experiments.

2.1.2 Effects of Enzyme Dosage on Hydroxyl Radical Scavenging Rate and Degree of Hydrolysis of Corn Gluten Meal Hydrolysate

Figure 2 reveals that the hydroxyl radical scavenging rate rose first and then declined as enzyme dosage increased, peaking at 81.14% with an enzyme addition level of 2.5%. The degree of hydrolysis increased initially and then decreased slightly with the elevation of enzyme dosage. A higher enzyme dosage facilitated the binding between enzymes and substrates, generating more anti-alcohol peptide fragments. However, excessive enzyme addition triggered competitive substrate inhibition, limited available binding sites between enzymes and substrates, slowed down the hydrolysis reaction, reduced the content of functional peptides and thus lowered the hydroxyl radical scavenging capacity [16-17]. Comprehensively considering the experimental results, enzyme dosages of 2.0%, 2.5% and 3.0% were chosen for subsequent response surface optimization.

2.1.3 Effects of Enzymatic Hydrolysis Time on Hydroxyl Radical Scavenging Rate and Degree of Hydrolysis of Corn Gluten Meal Hydrolysate

As shown in Figure 3, the hydroxyl radical scavenging rate increased first and then decreased with prolonged hydrolysis time, achieving the maximum value of 80.49% at 4 h. The degree of hydrolysis kept increasing but with a slower growth rate in the later stage. Sufficient enzymatic reaction within a certain time range produced more hangover-relieving peptide fragments and enhanced the free radical scavenging capacity [18]. In contrast, excessively long hydrolysis time resulted in further degradation of functional bioactive peptides, which weakened the hydroxyl radical scavenging activity of corn peptides [19]. Hence, hydrolysis durations of 3 h, 4 h and 5 h were adopted for follow-up response surface tests.

2.1.4 Effects of Inoculation Amount on Hydroxyl Radical Scavenging Rate and Degree of Hydrolysis of Corn Gluten Meal Hydrolysate

According to Figure 4, the hydroxyl radical scavenging rate increased first and then decreased with the rising inoculation volume, reaching the highest value of 79.78% at an inoculation amount of 8%. The degree of hydrolysis showed a similar trend of initial growth followed by decline. Moderately increased inoculation facilitated sufficient fermentation of raw materials and enriched anti-alcohol bioactive peptides, thus strengthening free radical scavenging activity. Excess inoculation led to nutrient depletion and inter-strain competition, restrained the proliferation of Bacillus subtilis and weakened the fermentation efficiency toward corn protein substrates [20]. Consequently, inoculation levels of 6%, 8% and 10% were selected for subsequent response surface experiments.

2.1.5 Effects of Fermentation Time on Hydroxyl Radical Scavenging Rate and Degree of Hydrolysis of Corn Gluten Meal Hydrolysate

Figure 5 indicates that the hydroxyl radical scavenging rate increased first and then decreased slightly with extended fermentation time, with the maximum value of 74.86% observed at 36 h. The degree of hydrolysis rose rapidly at first and then remained stable. In the early fermentation stage, strains proliferated rapidly with high enzyme secretion, which promoted the degradation of macromolecular proteins into small-molecule peptides and improved antioxidant activity [21]. With prolonged fermentation, bacterial density increased, cell growth slowed down and cell apoptosis occurred, which hindered protein hydrolysis and reduced the yield of anti-alcohol functional peptides [22]. Overall, fermentation time exerted a relatively weak influence on the two indicators, so a fixed fermentation time of 36 h was adopted in subsequent response surface experiments.

2.2 Analysis of Response Surface Experimental Results

The design and results of response surface experiments are summarized in Table 2, and the analysis of variance (ANOVA) results are listed in Table 3.

Regression analysis was conducted on the experimental data in Table 2, and the regression equation for hydroxyl radical scavenging rate (Y) against solid-liquid ratio (A), inoculation amount (B), enzyme dosage (C) and enzymatic hydrolysis time (D) was established as follows:

 Y = 83.18 + 0.20A + 2.71B − 0.83C + 0.64D − 0.46AB + 2.73AC − 3.39AD − 1.09BC + 0.60BD − 1.86CD − 3.15A² − 4.71B² − 7.04C² − 5.53D²

As presented in Table 3, the regression model was highly significant (P ＜ 0.01), while the lack-of-fit term was non-significant (P ＞ 0.05), demonstrating high reliability, favorable goodness-of-fit and statistical validity of the model. The established regression equation could replace actual experimental data for result analysis. The coefficient of determination R² was 0.98 and the adjusted R² was 0.96, which further verified the excellent fitting performance of this model for the combined enzymatic and microbial fermentation process of corn anti-alcohol peptides.

Significance analysis of regression coefficients revealed that the linear terms B and C exerted highly significant effects (P ＜ 0.01), term D showed a significant effect (P ＜ 0.05), whereas term A had no significant influence (P ＞ 0.05). All quadratic terms A², B², C² and D² significantly affected the hydroxyl radical scavenging rate (P ＜ 0.01). The interaction terms AC, AD and CD were highly significant (P ＜ 0.01), BC was significant (P ＜ 0.05), and AB and BD exhibited no statistical significance (P ＞ 0.05). The influencing intensity of the four factors was ranked as follows: B (enzymatic hydrolysis time) ＞ C (enzyme dosage) ＞ D (inoculation amount) ＞ A (solid-liquid ratio).

The optimal technological parameters predicted by the regression model were as follows: solid-liquid ratio 1:9.78 (g/mL), inoculation amount 8.26% (based on the volume of corn gluten meal suspension), enzyme dosage 2.44% (based on the mass of corn gluten meal), enzymatic hydrolysis time 4.32 h, with a predicted hydroxyl radical scavenging rate of 83.69%. For practical verification experiments, the parameters were adjusted to solid-liquid ratio 1:10 (g/mL), inoculation amount 8.30%, enzyme dosage 2.50% and hydrolysis time 4.3 h. Under these optimized conditions, the degree of hydrolysis of corn gluten meal hydrolysate reached 23.71%, and the actual hydroxyl radical scavenging rate was 82.87%, with an error less than 0.98% compared with the predicted value. The high consistency between measured and predicted values confirmed that the established model possessed excellent fitting degree and prediction accuracy for the preparation process of corn anti-alcohol peptides.

2.3 Separation and Purification of Hydrolysate

As shown in Figure 6, Fraction 3 (molecular weight ＜ 1 kDa) exhibited the highest hydroxyl radical scavenging rate of 89.89%, which was significantly higher than that of the crude hydrolysate before ultrafiltration separation (82.87%, P ＜ 0.05). This result indicated that low-molecular-weight corn anti-alcohol peptides possessed stronger antioxidant activity.

2.4 In Vitro Gastrointestinal Digestive Stability of Corn Anti-Alcohol Peptides

Not all peptides can exert biological activities in vivo unless they are absorbed in their intact active form [23]. Therefore, the gastrointestinal digestive stability of polypeptides should be evaluated prior to their application as functional food ingredients. As shown in Figure 7, no significant decrease was observed in the hydroxyl radical scavenging capacity of corn peptides after 2 h simulated gastric digestion with pepsin. Nevertheless, subsequent 2 h simulated intestinal digestion with trypsin led to a remarkable reduction in antioxidant activity compared with untreated samples (P ＜ 0.05). This phenomenon was closely related to peptide molecular weight: low-molecular-weight peptides presented low susceptibility to pepsin. Trypsin could degrade polypeptides into oligopeptides (2–8 amino acid residues) and free amino acids, which reduced overall hydrophobicity and consequently weakened the hydroxyl radical scavenging ability [24].

3 Conclusions

In this study, single-factor experiments combined with response surface methodology were applied to optimize the preparation process of corn anti-alcohol peptides via integrated enzymatic hydrolysis and microbial fermentation, and their gastrointestinal stability was characterized. The optimal technological parameters were determined as follows: solid-liquid ratio 1:10 (g/mL), enzyme dosage 2.5% (w/w of corn gluten meal), hydrolysis temperature 55 ℃, pH 8.5, enzymatic hydrolysis duration 4.3 h, inoculation amount 8.3% (v/v of corn gluten meal suspension) and fermentation time 36 h. Under these conditions, the hydroxyl radical scavenging rate and hydrolysis degree of corn anti-alcohol peptides were 82.87% and 23.71%, respectively. After ultrafiltration purification, the fraction with molecular weight below 1 kDa showed the strongest antioxidant activity with a hydroxyl radical scavenging rate of 89.89%. Stability assays demonstrated that corn anti-alcohol peptides retained bioactivity after gastric digestion, whereas their functional activity was inhibited by trypsin during intestinal digestion. This study established a feasible preparation technology for corn anti-alcohol peptides and provided experimental data support for their development and utilization. Further research can be conducted to explore the structure-activity relationship of these hangover-relieving bioactive peptides in future work.

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