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Article

Preparation of Novel ACE Inhibitory Peptides from Skimmed Goat Milk Hydrolyzed by Multi-Enzymes: Process Optimization, Purification, and Identification

by
Wenjing Hu
1,
Guowei Shu
1,*,
Huan Lei
2,
Guanli Du
3,
Zhengxin Liu
4 and
Li Chen
5,*
1
School of Food Science and Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China
2
Xi’an Baiyue Gaot Milk Corp., Ltd., Xi’an 710089, China
3
Shaanxi Qinlong Dairy Group Co., Ltd., Xi’an 710089, China
4
Baiyue Goat Dairy (He shui) Guxiang Co., Ltd., Qingyang 745100, China
5
College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710119, China
*
Authors to whom correspondence should be addressed.
Catalysts 2025, 15(2), 140; https://doi.org/10.3390/catal15020140
Submission received: 13 December 2024 / Revised: 20 January 2025 / Accepted: 29 January 2025 / Published: 3 February 2025
(This article belongs to the Special Issue Enzyme and Biocatalysis Application)
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Abstract

:
This study optimizes the process conditions for preparing angiotensin-converting enzyme (ACE) inhibitory peptides from skimmed goat milk (SGM) hydrolyzed by multi-enzymes using response surface methodology. When the enzymatic hydrolysis time was 90 min, the optimal hydrolysis conditions were a pH of 8.49, enzyme-to-substrate ratio (E/S ratio) of 8.04%, and temperature of 61.54 °C. The hydrolysis degree and ACE inhibitory activity were 65.39% ± 0.01% and 84.65% ± 0.03%, respectively. After purification by ultrafiltration, macroporous resin, and gel filtration, the ACE inhibitory activity of F2-2 in the two components of F2 was higher, with the ACE inhibitory rate of 93.97% ± 0.15% and IC50 of 0.121 ± 0.004 mg/mL. The content of hydrophobic amino acids, fatty amino acids, and aromatic amino acids in component F2-2 accounts for 73.17%, 33.86%, and 33.72%, respectively. Eleven peptides were isolated and identified from the F2-2 components of the enzymatic hydrolysate of SGM, including two peptides without an established database. The peptides mainly came from β casein, αS1 casein, and αS2 casein.

1. Introduction

The number of hypertensive patients in the world is increasing year by year. The regulation of the angiotensin-converting enzyme (ACE) is a key measure for controlling hypertension [1]. The ACE is a dipeptide carboxypeptidase found in human tissues and body fluids, which converts angiotensin I to angiotensin II to raise blood pressure [2]. Thw ACE inhibitory peptide can inhibit ACE activity by inhibiting the synthesis of angiotensin II or promoting the release of bradykinin, thereby reducing blood pressure [3].
At present, ACE inhibitors synthesized through chemical methods are expensive and have certain toxic side effects [4], so people hope to extract ACE inhibitors from natural foods. The traditional methods for preparing food-derived ACE inhibitors include fermentation, chemical synthesis, and enzymatic hydrolysis. Enzymatic hydrolysis has the characteristics of high efficiency, specificity, and minimal damage to the nutritional value of protein [5]. A large number of studies have been conducted to extract ACE inhibitors from raw materials, such as bean [6,7,8], meat [9,10,11], dairy [12,13,14,15], and some by-products of waste proteins [16,17,18,19] by enzymatic hydrolysis. For example, Xu et al. [6] prepared soybean protein isolate hydrolysate by Alcalase. After optimization, the ACE inhibitory activity of the peptide increased by 10.4 times, and the novel peptide IY had the lowest IC50 (0.53 ± 0.02 μmol/L). Tang et al. [20] hydrolyzed walnut protein isolated with alkaline protease and purified the enzymatic solution to obtain an ACE inhibitory peptide. Pan et al. [21] obtained a novel ACE inhibitory peptide (LL) by hydrolyzing whey protein with trypsin. Rahimi et al. [22] obtained ACE inhibitory peptides from camel milk casein using proteinase K. Ibrahim et al. [13] found that the caseins and whey proteins of goat milk hydrolyzed by pepsin had strong ACE inhibitory activity.
Meanwhile, the substrates of enzymatic hydrolysis from dairy were mainly casein and whey protein [23,24,25,26,27]. And there are relatively few studies on the preparation of ACE inhibitory peptides using multi-enzymes from SGM. Lin et al. [28] used a two-enzyme combination approach (thermolysin + alcalase and thermolysin + proteinase K) to hydrolyzed yak milk casein, and the hydrolysates (<3 kDa) demonstrated high ACE inhibitory activity. Shanmugam et al. [29] found that pepsin–trypsin hydrolysates from buffalo casein had the highest ACE inhibitory activity of (72.55% ± 2.23%)/50 μg. The results of Ugwu et al. [30] also indicated that the product of pepsin- and trypsin-hydrolyzed casein had higher ACE inhibition activity than a single enzyme.
In our previous work, we investigated the effect of different proteases on the production of ACE inhibitory peptides in cow and goat milk [31,32]. Due to the limited enzymatic hydrolysis sites of a single enzyme, the purpose of this study is to use the ACE inhibitory rate and hydrolysis degree as indicators to further determine the optimal process for preparing ACE inhibitory peptides from SGM hydrolyzed by multi-enzymes and to separate and purify the enzymatic hydrolysate to obtain the component with the highest ACE inhibitory activity and identify it. The key findings of this study are expected to provide a good reference for the subsequent utilization of functional active peptides and the development of goat milk products.

2. Results

2.1. Response Surface Optimization of Multi-Enzymatic Hydrolysis of SGM

The Box–Behnken experiment design was used to optimize the production process of the ACE inhibitory peptide from SGM with multi-enzymatic hydrolysis, and the results are shown in Table 1.
Regression analysis was carried out on the response surface optimization test results by Design-Expert, and the quadratic regression equations of the degree of hydrolysis (Y1) and inhibitory rate of the ACE (Y2) of the enzymatic hydrolysis of SGM were obtained as follows:
Y1 = 8.26 + 0.00057A − 0.050B + 0.087C + 0.11AB − 0.00015C + 0.036BC − 0.20A2 − 0.21B2 − 0.22C2
Y2 = 8.87 − 0.12A + 0.33B + 0.68C + 0.23AB − 0.15AC + 0.40BC − 0.89A2 − 1.11B2 − 0.52C2
In the equation, A, B, and C represent pH, the E/S ratio, and temperature (T), respectively. From the absolute value of the primary term coefficient, it can be seen that when the ACE inhibitory peptide was prepared from SGM by multi-enzymatic hydrolysis, the degree of the influence of each factor on the hydrolysis degree and the ACE inhibitory rate of SGM followed the order of C > B > A.
The model coefficients of determination R2 for Y1 and Y2 were 0.9813 and 0.9542, respectively, indicating that the equation can explain 98.13% of the variation in the hydrolysis degree and 95.42% of the variation in the ACE inhibitory rate. The differences between the corrected coefficients of determination (Adj R2 = 0.9476, 0.8717) and the coefficients of determination (R2) were smaller, suggesting that the experimental data and the equation were well fitted. The p-value of the two models was both < 0.001, while the misfit term showed p > 0.1, which indicated that the models were valid and the results can be analyzed with these models (Table 2 and Table 3). The p-values of primary terms B and C, interaction AB, and secondary terms A2, B2, and C2 were all less than 0.05, indicating that their effects on the degree of the hydrolysis of SGM were significant (Table 2). The p-values of the primary terms B, C, and secondary terms A2, B2, and C2 were less than 0.05, indicating that these factors have a large effect on the ACE inhibitory activity of SGM hydrolysate (Table 3).
From the response surface diagrams (Figure 1), it can be seen that both the hydrolysis degree and ACE inhibitory rate first increase and then decrease with the increase in temperature, enzyme addition, or pH, and the extreme value appears at the inflection point.
The results of the response surface test were repeated and verified. According to the above optimal conditions (pH of 8.49, E/S ratio of 8.04%, temperature of 61.54 °C), three groups of repeated tests were conducted. The degree of hydrolysis and the ACE inhibitory rate of enzymatic hydrolysis of SGM were 65.39% ± 0.01% and 84.65% ± 0.03%, respectively. The hydrolysis degree of 68.09% and ACE inhibitory rate of 82.67% are very close to the predicted values, and there is no significant difference, proving the effectiveness of this method.

2.2. ACE Inhibitory Activity of SGM Fractions

As can be seen from Figure 2A, the linear equation between peptide concentration and absorbance is as follows: y = 0.15636x + 0.01727, R2 = 0.99894.
After ultrafiltration, four components were obtained, and the ACE inhibitory rate and IC50 of each component are shown in Figure 2B. With the decrease in molecular weight, the ACE inhibitory rate gradually increased and IC50 gradually decreased. Among the products of multi-enzymatic hydrolysis, the content of <1 kDa component polypeptide and the inhibitory activity of the ACE were the highest, with values of 40.38% ± 0.28% and 88.17% ± 0.28%, respectively.
The ACE inhibitory rate and IC50 of <1 kDa, DA201-C, F1, F2, and F3 are shown in Figure 2C,D. Among the five components, F2 had the best ACE inhibitory rate, with a value of 93.88% ± 0.33%. And then the Sephadex G-15 was used to continue to separate F2. Two components, F2-1 and F2-2, were obtained, and the gel chromatogram, ACE inhibitory rate, and IC50 of each component are shown in Figure 2E,F. The F2-2 had the best ACE inhibitory rate, with a value of 93.97% ± 0.15%.

2.3. Molecular Weight Distribution of ACE Inhibitory Peptides

The F2-1 and F2-2 components were identified by mass spectrometry. Figure 3 shows the components. From Figure 3A, it can be seen that the m/z of F2-1 components of SGM are 174, 287, 364, 434, 546, 686, 743, 856, 1176, 1303, 1417, 1585, 1734, 1866, 1958, and 2103, respectively. It is preliminarily known that F2-1 is mainly a small peptide with a molecular weight of less than 2000 Da and is composed of less than 16 amino acid residues. Figure 3B shows SGM components F2-2 m/z of 114, 227, 262, 328, 364, 436, 570, 685, 743, 802, 841, 904, 1022, and 1176, respectively. Preliminary results show that the molecular weight of F2-2 is mainly concentrated in 100–1200 Da, consisting of 1–10 amino acid residues.

2.4. Identification of ACE Inhibitory Peptide

The amino acid composition and content of the components F2-1 and F2-2 separated from Sephadex G-15 are shown in Table 4.
As can be seen from Table 4, the contents of Glu, Ala, Val, and Pro of component F2-1 and the contents of Glu, Ala, Tyr, and Phe of component F2-2 in SGM are higher. In this study, the contents of the hydrophobic amino acids and fatty amino acids of F2-1 were relatively high, while the contents of aromatic amino acids, basic amino acids, and cyclic amino acids were relatively low. The contents of hydrophobic amino acids, fatty amino acids, and aromatic amino acids of component F2-2 were relatively high, while the contents of basic amino acids and cyclic amino acids were low. The ACE inhibitory activity reached 93.97% ± 0.15%, and the corresponding IC50 was 0.121 ± 0.004 mg/mL (Figure 2F).
The primary amino acid sequence of the enzymatic hydrolysis of SGM isolated and purified by gel Sephadex G-15 (Sunresin, Xi’an, China) was determined by Q-Exactive mass spectrometry (Thermo Finnigan, San Jose, CA, USA), and the protein source and sequence of SGM were compared with the Swiss-Prot database. The structural identification results of each component are shown in Table 5.
As shown in Table 5, αS1 casein (VVAPFPE, SDIPNPIGSE) derived from SGM G15-1 was not successfully compared with the inhibitory peptide sequence in the existing databases Swiss-Prot (https://www.uniprot.org/peptide-search, accessed on 5 March 2024) and BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/en/biopep, accessed on 5 March 2024) was initially identified as the newly discovered ACE inhibitory peptide sequence. The molecular weight of the amino acids is mainly distributed in the range of 650–1050 Da. The secondary mass spectra of some of the obtained ACE inhibitory peptides are shown in Figure 4.

3. Materials and Methods

3.1. Materials and Reagents

Skimmed goat milk (SMG) (Qin Yuan Dairy Company, Weinan, China), Alcalase (EC 3.4.21.14, 2.4 AU-A/g, the optimal condition: pH of 8.5, 55 °C, Novozymes, Bagsværd, Denmark), Bacillus licheniformis protease (EC 3.4.21.62, ≥ 2.4 U/g, the optimal condition: pH of 8.0, 65 °C, Sigma-Aldrich, St. Louis, MO, USA), alkaline protease (EC 3.4.21.14, 200,000 U/g, the optimal condition: pH of 8.5–10.5, 50–60 °C, Ruiyang, Wuxi, China), Gly-Gly-Tyr-Arg, Hip-His-Leu (HHL), and ACE (Sigma-Aldrich, St. Louis, MO, USA) were acquired; all other reagents were of analytical grade.

3.2. Methods

3.2.1. Preparation of ACE Inhibitory Peptide in SGM

The SGM was dissolved in distilled water at a ratio of 1: 9 (w/v) to prepare the restored goat milk. According to the results of a single-factor test [32], we took the hydrolysis degree and ACE inhibitory rate of SGM multi-enzymatic hydrolysis as indicators, and the hydrolysis time was set to 90 min, a pH of 8.5, E/S ratio of 8%, and a temperature of 60 °C. And the enzymatic hydrolysis was carried out at the optimal ratio of multi-enzymes (EAlcalase: EBacillus licheniformis protease: Ealkaline protease = 0.388: 0.406: 0.206) (w/w) [33]. After enzymatic hydrolysis, the samples were placed at 95 °C for 15 min. Then, the pH of the samples was adjusted to 3.4–3.6, and the precipitation was removed by centrifugation at 8000 r/min for 15 min. Then, the pH of the supernatant was adjusted to 8.3, and the supernatant was obtained by centrifugation again for future use.

3.2.2. Response Surface Experimental Design

The response surface design was carried out for three factors, namely temperature, pH, and the enzyme-to-substrate ratio (E/S ratio). The optimal enzymatic hydrolysis process for the ACE inhibitory peptide in SGM was determined using the Box–Behnken method. The experimental design factors and levels are shown in Table 6.

3.2.3. Isolation and Purification of ACE Inhibitory Peptide in SGM

According to the optimal conditions, crude enzymatic hydrolysis was prepared. Then, ultrafiltration membranes (Millipore, CA, USA) with molecular weights of 10, 5, and 1 kDa were prepared sequentially at 25 °C and 0.09 MPa. The peptide content, ACE inhibitory rate, and IC50 were measured, respectively. The components with the highest inhibitory activity were desalted by DA201-C macroporous resin to obtain DA201-C.
The eluent of DA201-C macroporous resin (Sunresin, Xi’an, China) was further separated and purified by Sephadex G-25 to obtain F1, F2, and F3. The fraction with the highest ACE inhibitory rate obtained by Sephadex G-25 (Sunresin, Xi’an, China) was further purified using Sephadex G-15 to obtain F2-1 and F2-2, as shown above [34]. Sample loading conditions were as follows: sample volume was 1.5 mL, sample loading speed was 2 mL/min, concentration was 150 mg/mL, wavelength was 220 nm, distilled water was the eluent, and elution velocity was 2 mL/min. An automatic collector was then used to collect the eluent every 5 min, and the samples of each eluting peak were collected for freeze-drying.

3.2.4. Identification of ACE Inhibitory Peptides

The amino acid composition and content of each component were determined by an amino acid automatic analyzer (Hitachi 835-50, Hitachi High-Tech, Tokyo, Japan). The molecular weight distribution and sequence identification of polypeptide samples were measured and analyzed using Q-Exactive LC-MS/MS (Thermo Finnigan, San Jose, CA, USA) [33].

3.2.5. Determination of Proteolysis Degree

The degree of protein hydrolysis (DH) was tested by pH-Stat [35] (PHS-3C, Shanghai Jingke Industrial Co., Ltd., Shanghai, China). During the hydrolysis process, the pH of the system was kept unchanged by adding 0.1 mol/L of NaOH, and the volume of NaOH was recorded and the degree of hydrolysis was calculated by Formula (1).
Degree of hydrolysis (DH, %) = (B × Mb)/(α × Mp × htot) × 100%
where B is the volume of NaOH (mL); Mb is the concentration of NaOH; α is the dissociation constant of NH4+; Mp is the weight of protein (g); htot is the number of peptide bonds per gram of skimmed milk protein; and the value in goat milk is 8.35 mmol.

3.2.6. Determination of ACE Inhibitory Rate

A total of 200 μL HHL and 100 μL sample were preheated at 37 °C for 5 min; next, 20 μL ACE was added and reacted at 37 °C for 30 min, and then 250 μL 1 mol/L HCl was added to stop the reaction. After, 1.7 mL of ethyl acetate was added to each test tube, shocked for 15 s, and stood for 5 min, then 1 mL of ethyl acetate layer was absorbed, the solution was dried in the oven at 120 °C for 30 min, removed and cooled down, and dissolved in 3 mL of water. Finally, the absorbance was measured at 228 nm (UV-5300PC, Shanghai Metash Instruments Co., Ltd., Shanghai, China) [36], and the calculation formula is as follows:
ACE inhibitory activity (%) = (Ab − Aa)/ (Ab − Ac) × 100%
where Aa is the absorbance of both the ACE and its inhibitor present in the reaction; Ab is the absorbance of the reaction without the addition of ACE inhibitors; and Ac is the absorbance of ACE and HHL blank reactions.

3.2.7. Determination of Peptides Content

The standard tetrapeptide Gly-Gly-Tyr-Arg was dissolved in 5% TCA at concentrations of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mg/mL, respectively. A total of 4.0 mL biuret reagent was mixed evenly with the above standard solution (6.0 mL), left for 10 min, centrifuged at 2000 r/min for 10 min, and the absorbance of the supernatant was measured at 540 nm (the first tube was used as a blank control). The standard curve of the sample under different peptide concentration gradients was drawn.
An amount of 2.5 mL sample solution was taken, 2.5 mL 10% (w/v) TCA was added, the solution was mixed well, left for 10min, centrifuged at 4000 r/min for 15 min, the supernatant was transferred into a 50 mL volumetric bottle, the volume was adjusted with 5% TCA to the scale, and it was shaken well. An amount of 6 mL of the above solution was taken in another test tube, 4 mL of biuret reagent (sample solution: biuret reagent = 3: 2, v/v) was added, mixed well, stood for 10 min, centrifuged for 10 min at 2000 r/min, the supernatant was taken at 540 nm to determine the OD value, and the peptide content in the sample solution was calculated according to the standard curve [37].

3.3. Statistical Analysis

All tests were measured three times in parallel, and the final data were expressed as mean ± standard deviation. The regression analysis of the response surface optimization test was performed using Design-Expert 12. SPSS 26 was performed to determine significant differences between means (p < 0.05). The data were plotted using Origin 2021.

4. Discussion

Different proteases have different enzyme activities and specific cleavage sites. Meanwhile, the enzymatic hydrolysis conditions also affect the content of active peptides. In the optimization experiment, the ACE inhibitory activity of SGM enzymatic hydrolysates increased first and then decreased with the increase in temperature, E/S ratio, and pH, which may be because excessive hydrolysis produced a large number of free amino acids, which reduced the ACE inhibitory ability of protein hydrolysates. Gao et al. [38] used RSM to optimize the hydrolysis conditions of cottonseed protein by papain. When the hydrolysis temperature was 39 °C, E/S ratio was 1.0%, and pH was 7.5, the ACE inhibition rate reached 88.2%.
Ultrafiltration significantly increased the ACE inhibitory activity, and the IC50 was also decreased, which is consistent with the conclusion of Li et al. [39] and Puspitojati et al. [40]. Among the products of multi-enzymatic hydrolysis, the content of <1 kDa component polypeptide and the inhibitory activity of ACE were the highest, which were 40.38% ± 0.28%, 88.17% ± 0.28%, respectively. Shao et al. [41] also found that the content of <3 kDa had the highest ACE inhibitory activity. These results indicated that the activity of ACE inhibitory peptides in enzymatic hydrolysis was closely related to their molecular weight distribution.
The ACE inhibitory peptides purified by G-15 were mainly short peptides. Norhameemee et al. [42] obtained ACE peptides from defatted lemon basil seeds using Alcalase, and the molecular weights of the peptides LGRNLPPI and GPAGPAGL were 879.06 Da and 639.347 Da, respectively. Liu et al. [43] analyzed whey protease hydrolysates by LC-MS-MS and found that the molecular weight of the polypeptide was between 300 and 1400 Da. Li et al. [44] obtained a fraction with the ACE inhibitory activity of 90.78% and with a molecular weight range of 145–468 Da. These are close to the molecular weight range of ACE inhibitory peptides in SGM in this study.
The inhibitory activities of ACE peptides are related to their amino acid composition, and the percentage of hydrophobic amino acids in amino acids is proportional to their ACE inhibitory activity [1,4]. The ACE inhibitory activity of different peptides depends largely on their C-terminal sequence, because the C-terminal contains aromatic amino acids (W, Y, F), aliphatic groups (A, L, M), and Pro (P), and aliphatic groups (V, I, A), alkaline (R), and aromatic (Y, F) amino acid residues occur more frequently in the penultimate position of the amino acid sequence, which could improve ACE inhibitory activity [45,46]. For example, C-terminal Arg and N-terminal Tyr can increase the activity of ACE inhibitory peptides [47]. After adding Trp to the C-terminal, Xie et al. [48] found that the ACE inhibitory activity of VKW was 1450 times higher than that of the VK. When the N-terminal is a hydrophobic amino acid (V, I, L), the ACE inhibitory rate is higher, because when the branched-chain amino acids (BCAAs) such as leucine and isoleucine were at the N-terminals, the bulky side chain of the BCAA forms a hydrophobic shield that could protect the Zn–peptide chelate complex from water attacks [4]. Li et al. [49] found that N-terminal Leu could effectively improve ACE inhibitory activity by molecular docking. But when the N-terminal of the ACE inhibitory peptide contains proline, the inhibitory activity of the ACE is decreased [50]. In our study, derived from αS2-casein f (123–129), α whey protein f (35–40), β lactoglobulin f (94–100), f (114–118), β casein f (217–222), f (58–63), and f (116–120) in SGM G15-1 all conform to the distribution of C-terminal amino acid residues of the above ACE inhibitory peptides.

5. Conclusions

The experiment used the response surface method to optimize the multi-enzymatic hydrolysis degreasing and prepared goat milk ACE inhibitory peptides. When the enzymatic hydrolysis time was 90 min, the optimal hydrolysis conditions were a pH of 8.49, E/S ratio of 8.04%, and temperature of 61.54 °C. The hydrolysis degree and ACE inhibitory activity were 65.39% ± 0.01% and 84.65% ± 0.03%, respectively. This indicated that the optimized SGM enzymatic hydrolysates had an inhibitory rate on the ACE. After identified the G-15 component of the SGM, a total of 11 peptides were obtained, including two peptides without an established database. It can be considered a potential bioactive peptide with ACE inhibitory activity. This study provides a theoretical basis for the further processing and high-value utilization of goat milk and also provides a new source of information for the ACE inhibition characteristics of milk-derived active peptides.

Author Contributions

G.S. and L.C. conceived and designed the experiments. W.H., H.L., G.D. and Z.L. performed the experiments. W.H. analyzed the data and prepared the manuscript. H.L., G.D. and Z.L. contributed reagents/materials/analysis tools. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research and Development Program of Shaanxi (program no. 2024NC-GJHX-18, 2024NC-ZDCYL-03-011, 2024NC-YBXM-152), the Technology Innovation Leading Program of Shaanxi (program no. 2024QCY-KXJ-080), Heshui County Science and Technology Plan Project (no. QY-HS-2022A-03), and the National Nature Science Foundation of China (no. 32101908).

Data Availability Statement

The minimal dataset that supports the central findings of this study is available upon request from the corresponding author.

Conflicts of Interest

Huan Lei is employed by Xi’an Baiyue Gaot Milk Corp., Ltd., Guanli Du is employed by Shaanxi Qinlong Dairy Group Co., Ltd. and Zhengxin Liu is employed by Baiyue Goat Dairy (He shui) Guxiang Co., Ltd. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

SGM: skimmed goat milk; ACE: angiotensin-converting enzyme; E/S ratio: enzyme-to-substrate ratio.

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Catalysts 15 00140 g001
Figure 1. (A,C,E) Response surface plots of pH, temperature, and E/S ratio on DH for SGM. (B,D,F) Response surface plots of pH, temperature, and E/S ratio on ACE inhibitory activity for SGM.
Figure 1. (A,C,E) Response surface plots of pH, temperature, and E/S ratio on DH for SGM. (B,D,F) Response surface plots of pH, temperature, and E/S ratio on ACE inhibitory activity for SGM.
Catalysts 15 00140 g001
Catalysts 15 00140 g002
Figure 2. (A) Standard curve of Gly-Gly-Tyr-Arg. (B) Results of ACE inhibitory activity and IC50 after ultrafiltration. (C) Separation and purification of DA201-C elution by Sephadex G-25. (D) Results of ACE inhibitory activity and IC50 of each component obtained by Sephadex G-25. (E) Separation and purification of DA201-C elution by Sephadex G-15. (F) Results of ACE inhibitory activity and IC50 of each component obtained by Sephadex G-15. Different letters among samples denote significant differences (p < 0.05).
Figure 2. (A) Standard curve of Gly-Gly-Tyr-Arg. (B) Results of ACE inhibitory activity and IC50 after ultrafiltration. (C) Separation and purification of DA201-C elution by Sephadex G-25. (D) Results of ACE inhibitory activity and IC50 of each component obtained by Sephadex G-25. (E) Separation and purification of DA201-C elution by Sephadex G-15. (F) Results of ACE inhibitory activity and IC50 of each component obtained by Sephadex G-15. Different letters among samples denote significant differences (p < 0.05).
Catalysts 15 00140 g002
Catalysts 15 00140 g003
Figure 3. Mass spectrum of F2-1 (A) and F2-2 (B) from hydrolysate of SGM.
Figure 3. Mass spectrum of F2-1 (A) and F2-2 (B) from hydrolysate of SGM.
Catalysts 15 00140 g003
Catalysts 15 00140 g004aCatalysts 15 00140 g004b
Figure 4. MS/MS mapping of ACE inhibitory peptides of SGM for fractions G15-1.
Figure 4. MS/MS mapping of ACE inhibitory peptides of SGM for fractions G15-1.
Catalysts 15 00140 g004aCatalysts 15 00140 g004b
Table 1. The design and results of multi-enzymatic hydrolysis for SGM by Box–Behnken design.
Table 1. The design and results of multi-enzymatic hydrolysis for SGM by Box–Behnken design.
RunA
pH		B
E/S Ratio (%)		C
Temperature (℃)		Y1
DH (%)		Y2
ACE Inhibitory Rate (%)
18.5 (0)8 (0)60 (0)69.1172.11
28.58.5 (+1)64 (+1)61.8875.79
38.7 (+1)86463.3360
48.3 (−1)7.5 (−1)6064.0146.32
58.586067.3284.21
68.7856 (−1)59.9343.16
78.57.56462.6549.47
88.78.56062.7354.74
98.385659.5947.37
108.386462.9974.74
118.77.56060.6142.11
128.38.56059.3346.32
138.586068.0980
148.58.55658.7444.21
158.57.55661.7143.37
Table 2. ANOVA of hydrolysis degree by multi-enzymatic hydrolysate from SGM by Box–Behnken design.
Table 2. ANOVA of hydrolysis degree by multi-enzymatic hydrolysate from SGM by Box–Behnken design.
SourceSum of SquaresdfMean SquareF-Valuep-ValueSignificance
Model0.5590.06129.120.0009**
A—pH2.6 × 10−412.6 × 10−40.120.7399
B—E/S ratio0.0210.029.620.0268*
C—Temperature0.0610.0628.630.0031**
AB0.04710.04722.20.0053**
AC9 × 10−819 × 10−84.26 × 10−50.995
BC5.08 × 10−315.08 × 10−32.410.1815
A20.1410.1466.780.0004**
B20.1610.1676.370.0003**
C20.1810.1886.380.0002**
Residual0.01152.11 × 10−3
Lack of Fit4.65 × 10−331.55 × 10−30.520.7077not significant
Pure Error5.91 × 10−322.95 × 10−3
Cor Total0.5614
* p < 0.05; ** p < 0.01.
Table 3. ANOVA of ACE inhibitory activity by multi-enzymatic hydrolysate from SGM by Box–Behnken design.
Table 3. ANOVA of ACE inhibitory activity by multi-enzymatic hydrolysate from SGM by Box–Behnken design.
SourceSum of SquaresdfMean SquareF-Valuep-ValueSignificance
Model13.0991.4511.570.0075**
A-pH0.1110.110.870.3934
B-E/S ratio0.8710.876.960.0461*
C- Temperature3.713.729.470.0029**
AB0.2110.211.650.2559
AC0.08610.0860.680.4459
BC0.6510.655.150.0725
A22.9212.9223.230.0048**
B24.5214.5235.980.0018**
C21117.970.037*
Residual0.6350.13
Lack of Fit0.3930.131.060.5189not significant
Pure Error0.2420.12
Cor Total13.7114
* p < 0.05; ** p < 0.01.
Table 4. The amino acid composition and content of each component obtained by gel Sephadex G-15.
Table 4. The amino acid composition and content of each component obtained by gel Sephadex G-15.
Amino AcidMolecular WeightF2
(mol/100 mol Amino Acid)
F-2-1F-2-2
Asp133.16.64 ± 0.272.62 ± 0.11
Thr119.14.92 ± 0.221.90 ± 0.06
Ser105.15.26 ± 0.174.59 ± 0.18
Glu147.119.96 ± 0.638.69 ± 0.09
Gly75.11.52 ± 0.041.58 ± 0.04
Ala89.115.26 ± 0.3317.49 ± 0.28
Val117.19.77 ± 0.215.93 ± 0.18
Met149.24.15 ± 0.184.31 ± 0.11
Ile131.22.93 ± 0.061.11 ± 0.01
Leu131.27.43 ± 0.215.02 ± 0.13
Tyr181.24.22 ± 0.1517.69 ± 0.45
Phe165.23.88 ± 0.1216.03 ± 0.39
Lys146.24.24 ± 0.093.78 ± 0.14
His155.21.69 ± 0.053.31 ± 0.12
Arg174.20.000.36 ± 0.01
Pro115.18.13 ± 0.245.60 ± 0.22
Fatty amino acid—39.55%33.86%
Aromatic amino acid—8.10%33.72%
Basic amino acid—5.93%7.45%
Cyclic amino acid—8.13%5.60%
Hydrophobic amino acid—55.78%73.17%
Note: Cys was not detected.
Table 5. Structural identification of the fractions obtained by gel Sephadex G-15 separation.
Table 5. Structural identification of the fractions obtained by gel Sephadex G-15 separation.
SourceMolecular WeightLocationSequenceName
SGM G15-1886.44174ɑS2 casein f (123–129)NPWDQVKP1
904.41592ɑS2 casein f (89–95)VDDKHYQP2
638.31442α whey protein f (35–40)KDYGGVP3
657.36784κ casein f (88–93)VRSPAQP4
775.47125β lactoglobulin f (94–100)TKIPAVFP5
641.27769β lactoglobulin f (114–118)DTDYKP6
758.40832ɑS1 caseinVVAPFPEP7
1028.48947ɑS1 caseinSDIPNPIGSEP8
686.39842β casein f (217–222)RGPFPIP9
747.35191β casein f (58–63)DELQDKP10
575.32214β casein f (116–120)TMVPKP11
Table 6. Factors and levels of response surface experiment.
Table 6. Factors and levels of response surface experiment.
FactorsLevels
−101
A—pH8.38.58.7
B—E/S ratio/(%)7.588.5
C—Temperature/(℃)566064
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Hu, W.; Shu, G.; Lei, H.; Du, G.; Liu, Z.; Chen, L. Preparation of Novel ACE Inhibitory Peptides from Skimmed Goat Milk Hydrolyzed by Multi-Enzymes: Process Optimization, Purification, and Identification. Catalysts 2025, 15, 140. https://doi.org/10.3390/catal15020140

AMA Style

Hu W, Shu G, Lei H, Du G, Liu Z, Chen L. Preparation of Novel ACE Inhibitory Peptides from Skimmed Goat Milk Hydrolyzed by Multi-Enzymes: Process Optimization, Purification, and Identification. Catalysts. 2025; 15(2):140. https://doi.org/10.3390/catal15020140

Chicago/Turabian Style

Hu, Wenjing, Guowei Shu, Huan Lei, Guanli Du, Zhengxin Liu, and Li Chen. 2025. "Preparation of Novel ACE Inhibitory Peptides from Skimmed Goat Milk Hydrolyzed by Multi-Enzymes: Process Optimization, Purification, and Identification" Catalysts 15, no. 2: 140. https://doi.org/10.3390/catal15020140

APA Style

Hu, W., Shu, G., Lei, H., Du, G., Liu, Z., & Chen, L. (2025). Preparation of Novel ACE Inhibitory Peptides from Skimmed Goat Milk Hydrolyzed by Multi-Enzymes: Process Optimization, Purification, and Identification. Catalysts, 15(2), 140. https://doi.org/10.3390/catal15020140

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