RESEARCH ARTICLE

Open Access

The purification and identification of antioxidants and dipeptidyl peptidase IV inhibitory peptides from whey protein hydrolysates

Zheng Yuanrong

State Key Laboratory of Dairy Biotechnology, Technical Center of Bright Dairy & Food Co., Ltd, Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China

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Pang Jiakun,

Pang Jiakun

State Key Laboratory of Dairy Biotechnology, Technical Center of Bright Dairy & Food Co., Ltd, Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China

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Liu Zhenmin,

Corresponding Author

Liu Zhenmin

[email protected]

State Key Laboratory of Dairy Biotechnology, Technical Center of Bright Dairy & Food Co., Ltd, Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China

Correspondence Liu Zhenmin, State Key Laboratory of Dairy Biotechnology, Technical Center of Bright Dairy & Food Co., Ltd., Synergetic Innovation Center of Food Safety and Nutrition, Bldg 2, No.1518, West Jiangchang Rd, Shanghai 200436, China.

Email: [email protected]

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Zheng Yuanrong,

Zheng Yuanrong

State Key Laboratory of Dairy Biotechnology, Technical Center of Bright Dairy & Food Co., Ltd, Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China

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Pang Jiakun,

Pang Jiakun

State Key Laboratory of Dairy Biotechnology, Technical Center of Bright Dairy & Food Co., Ltd, Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China

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Liu Zhenmin,

Corresponding Author

Liu Zhenmin

[email protected]

State Key Laboratory of Dairy Biotechnology, Technical Center of Bright Dairy & Food Co., Ltd, Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China

Email: [email protected]

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First published: 11 October 2022

https://doi.org/10.1002/fbe2.12027

Citations: 1

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Abstract

In the present study, whey protein was enzymatically hydrolyzed using an ultrahigh-pressure synergistic enzymolysis method. The antioxidant activities and DPP-IV inhibitory activities of the enzymatic hydrolysates were measured. Three-layer isolation and purification were conducted on the enzymatic hydrolysates with antioxidant activity and DPP-IV inhibitory activity by gel filtration chromatography and RP-HPLC. The amino acid sequences were determined by LC-MS/MS. The identified amino acid sequences were then synthesized, and their antioxidants and DPP-IV inhibitory activities were determined. The results showed that 3 of the 14 polypeptides of N3-8 exhibited high antioxidant activity. Among them, peptide DDQNPHSSN had both high antioxidant activity and DPP-IV inhibitory activity. When the concentration was 1 mg/mL, then the ABTS radical scavenging rate, DPPH radical scavenging rate and reducing power were prominent, reaching 91.42%, 88.76%, and 0.637%, respectively, and DPP-IV inhibitory activity reached 66.28%. Whey protease hydrolysates are expected to be commercially developed as functional peptides.

1 INTRODUCTION

Whey is a by-product produced during the production of cheese and casein. However, if whey is directly discharged into the environment without treatment, it will produce excessive pollution and cause serious environmental problems. On the other hand, since whey retains the nutrients found in raw milk, including protein, peptides, lipids, lactose, minerals, and vitamins, it has high nutritional value. Therefore, as a byproduct of production, whey is considered to have major development potential. At present, research studies have been conducted regarding whey reuse in many Western countries, and the utilization rate of whey in Europe has reached 80% (Argenta & Scheer, 2020; Nicolás et al., 2019; Zhao et al., 2017). However, in China, most whey has not been used due to the immature comprehensive utilization of whey. It has instead been directly discharged or returned to pasture-land for animal feed, which has resulted in resource waste and environmental pollution issues. Therefore, effective methods are urgently required to further strengthen the comprehensive utilization of whey, and large-scale production processes need to be quickly developed to solve the current waste and pollution problems.

Whey protein is one of the main components of whey. It has high nutritional value and functional characteristics. Whey protein has been widely used in various foods and is an ideal source of bioactive peptides. It has also been used to prepare bioactive peptides which have the effects of antioxidation; DPP-IV (dipeptidyl peptidasem IV) inhibition; angiotensin converting enzyme (ACE) inhibition; antibacterial properties; lowering of cholesterol, and so on. Embiriekah et al. (2018) hydrolyzed whey protein with trypsin to obtain enzymatic hydrolysate with antioxidant activities. Ballatore et al. (2020) obtained the peptides with high radical scavenging activity and cytoprotector effect by hydrolyzing WPC 35 with trypsin. Baba et al. (2021) identified peptides with potential anti-hypertensive properties from enzymatic hydrolysis of camel whey proteins. In another related study, Demers-Mathieu et al. (2013) hydrolyzed whey protein with trypsin and obtained enzyme hydrolysate with obvious antibacterial activities. In addition, Jiang et al. (2018) used a complex enzyme method to perform the enzymolysis of whey protein, and obtained the peptides with cholesterol lowering abilities through isolation and purification. Adjonu et al. (2013) treated whey protein with preheating treatments and protease hydrolysis to obtain doubly active peptides with antioxidant and ACE inhibitory abilities. Researchers (Mares-Mares et al., 2019; Nongonierma et al., 2017) obtained peptides displaying ACE inhibitory and DPP-IV inhibitory activities through the preparation of zymolytic whey protein. At present, the research regarding the preparation of peptides with double activities from whey proteins have generally focused on the combination of ACE inhibitory and DPP-IV inhibitory activities, as well as the combination of ACE inhibitory and antioxidant activities. However, only a few studies have concentrated on the preparation of peptides with antioxidant and DPP-IV inhibitory activities. Some of the previous studies (Corrochano et al., 2018; Jiang et al., 2019) have shown that, among the many proven bioactive peptides derived from whey protein, antioxidant activities are the most common. DPP-IV inhibitory activities effectively maintain the activity of incretin and prolong the action time of insulin. As a result, blood glucose levels can be more effectively regulated, which has become a new goal for the treatment of diabetes. Moreover, DPP-IV inhibitory peptides obtained from natural sources are currently hot topics in the development of oral hypoglycemic drugs. Therefore, effective methods of modifying whey protein to obtain peptides with both antioxidant and DPP-IV inhibitory activities have gradually attracted attention in this field.

The goals of the present study were to effectively isolate and purify whey protein hydrolysates with antioxidant and DPP-IV inhibitory activities to some extent, then to identify and synthesize the amino acid sequences of the obtained components. As a result, peptides with dual biological activities could then be obtained. Therefore, the findings of this study provided a relevant scientific basis for improving the deep processing of whey protein, as well as assisting in increasing the value and expanding the types of whey products.

2 MATERIALS AND METHODS

2.1 Materials and instruments

In the current study, the examined whey protein isolate (WPI, protein mass fraction 89%) was purchased from Müller Co.; trypsin and pepsin were purchased from Sigma (US); alkaline protease and papain were purchased from the Shanghai Yuanye Bio-Technology Co., Ltd; acid protease was purchased from Danisco; and all other reagents were analytical reagents.

An FPG7100 ultrahigh pressure instrument and an RT5 magnetic stirring apparatus were purchased from the IKA Co.; Molecular Devices (US) supplied a SepectraMax M5 microplate reader; a SPECORD-205 ultraviolet spectrophotometer was obtained from Analytikjena; Gene Vac (UK) provided the EZ-2 parallel evaporator; a Laborota 4000 rotary evaporator was acquired from Heidolph Co.; an AKTA + RID10A rapid purification chromatography system was supplied by General Electric Medical System Co., Ltd.; a Waters 2707 preparative liquid chromatograph was purchased from Waters Technologies Ltd.; and Agilent Technology Co., Ltd. provided an Agilent 1260 Infinity analytical liquid chromatograph.

2.2 Experiment method

2.2.1 Enzymatic hydrolysis of the whey protein isolates

This study referred to the methods used in previous studies (Pang et al., 2020) to prepare a 1% (w/v) WPI solution, which was treated for 30 min under 400 MPa ultra high-pressure conditions. Then, after adjusting the pH and temperature to the suitable conditions for pepsin, the solution was placed on a magnetic stirrer and pepsin was added to hydrolyze WPI for 60 min. Next, the pH and temperature were once again adjusted to the suitable conditions for acid protease, and acid protease was added to hydrolyze WPI for 60 min. The ratio of enzyme dosage of the pepsin and acid protease was 1:1. Following the completion of the hydrolysis to a predetermined time, the enzymes were terminated in boiling water for 10 min. Finally, after a cooling period, the supernatant was removed by centrifugation and lyophilized for future use.

2.2.2 Determination of antioxidant activities

The antioxidant activities of the WPI were evaluated by 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radial scavenger activities; 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities, and total reducing power.

Determination of the ABTS free radical scavenging capacity

This study referred to the method presented by Athira et al. (2015) to determine the ABTS free radical scavenging activities. In this study's experiments, 2.45 mmol/L of potassium persulfate were added to 20 ml of 7 mmol/L ABTS solution to prepare 40 ml of ABTS^•+ working solution. The solution was left to stand undisturbed for 12–16 h at room temperature before use. Then, the ABTS^•+ working solution was diluted with phosphate-buffered solution (PBS) to a suitable factor (20–50 times), in order that the absorbance at 734 nm was (0.70 ± 0.02). At this point, 5 μl of the WPI sample was extracted and added into 500 μl of the diluted ABTS^•+ working solution. Following a reaction period at room temperature of 6 min, the absorbance at 734 nm was determined. The scavenging rate was calculated using Formula (1) as follows:

\text{ABTS free radical scavenging rate}( \% )=\left(1-\frac{{As}-{Ab}}{{Ad}-{Ab}}\right)\times 100,

(1)

where As indicates the absorbance of the ABTS^•+ working solution after adding the sample; Ab represents the absorbance of the blank control group; and Ad denotes the absorbance of the ABTS^•+ working solution without the sample added.

Determination of DPPH free radical scavenging capacity

The DPPH free radical scavenging activities were determined by referring to the method proposed by Brand-Williams et al. (1995). In this study's experiments, 1 ml of the WPI sample was added to 4 ml of DPPH solution (60 mmol/L). The absorbance at 517 nm was determined following a reaction period of 30 min at room temperature. The scavenging rate was calculated using Formula (2) as follows:

\text{DPPH free radical scavenging rate}( \% )=\left(1-\frac{A1-A2}{A0}\right)\times 100,

(2)

where A₀ indicates the absorbance of the DPPH solution; A₁ is the absorbance of the sample plus the DPPH solution; and A₂ represents the absorbance of the sample solution.

Determination of total reducing power

The total reducing power of the sample was determined by referencing the method proposed by Yen and Duh (1993), in which 200 μl of the WPI sample was added to 0.5 ml of PBS (0.2 mol/L, pH 6.6) and 0.5 ml potassium ferricyanide (1%, w/v). Then, 5 ml of trichloroacetic acid (10%, w/v) was added following a 20 min reaction period at 50°C. Centrifugation was conducted for 10 min at 3000 rpm, and 0.5 ml of the supernatant was taken to mix with 0.5 ml of deionized water and 0.1 ml of ferric chloride (0.1%, w/v). Then, following a 10 min reaction period, the absorbance at 700 nm was determined. It was observed that the higher the absorbance, the stronger the reducing power.

2.2.3 Determination of DPP-IV activities

The DPP-IV inhibitory activities of the samples were determined by referring to the method proposed by Nongonierma and FitzGerald (2013). In this study, the sample was dissolved with 0.1 mol/L of Tris-HCl buffer (pH 8.0), and 100 µl of the sample were added to a 96-hole microplate (with concentrations ranging from 0.012 5 mg/ml to 1.25 mg/ml). Then, 50 µl of a 0.55 mmol/L H-Gly-Pro-pNA·HCl solution was added and mixed thoroughly. The solution was incubated at 37°C for 10 min, and then 50 µl of a 2 U/L DPP-IV solution was added, the solution was mixed thoroughly, and reacted for 60 min at 37°C. Finally, 200 µl of a 1.0 mol/L acetic acid/sodium acetate buffer solution (pH 4.0) was added to terminate the reaction. The absorbance value (A) was determined using a microplate reader at a wave-length of 405 nm. The average value of three parallel tests was taken and substituted into Formula (3) to calculate the inhibition rate (%) of the DPP-IV, and diprotein (concentration of 1.25 µg/ml, 3.6 µM) was utilized as the positive control.

\text{Inhibition rate}( \% )=\left[1-\frac{{A}_{{Pilot}{sample}}-{A}_{{Sample}{blank}}}{{A}_{{negative}{control}}-{A}_{{Negative}{blank}}}\right]\times 100 \% .

(3)

2.2.4 Isolation and purification of the bioactive peptides

Gel filtration chromatography

A protein purification system was used to conduct gel filtration chromatography of the samples. The separation conditions were as follows: The eluent was distilled water, with a flow rate of 1.0 ml/min and detection wavelength of 280 nm. According to the elution curve, the four components collected were separated and lyophilized using a vacuum freeze dryer. Next, the antioxidant and DPP-IV inhibitory activities of each component were determined, and the component with the highest biological activities was selected for further separation.

RP-HPLC separation

The components were further separated by RP-HPLC. The separation conditions were as follows: The chromatographic column was an XBridge C18; Mobile Phase A was ultra-pure water containing 0.1% trifluoroacetic acid (TFA); Mobile Phase B was acetonitrile solution containing 0.1% TFA; the injection volume was 500 μl; injection concentration was 50 mg/ml; flow rate was 1 ml/min; and detection wavelength was 280 nm. The elution conditions were 0–5 min at 100% A; 5–20 min at 100% A to 45% B; 20–30 min at 100% B; 31 min, stop. Each component was collected using an automatic collector, and the biological activities were determined after freeze-drying was completed.

LC-MS/MS identification

In the present study, the amino acid sequences of the components were identified by UPLC-MS/MS (Zhang et al., 2015). The chromatographic conditions were as follows: Mobile Phase A was ultrapure water containing 0.1% formic acid; Mobile Phase B was acetonitrile solution containing 0.1% formic acid; injection volume was 5 μl; injection mass concentration was 1 mg/ml; gradient elution condition was 0–63 min; and B was from 2% to 100%. An electrospray positive ion scanning mode was used, and the scanning range was between 0.2 and 2 ku. The two-stage MS collision voltage was 28 eV. The amino acid sequences of bovine species in the Swiss-Prot protein database were searched using PEAKS 10.0 software to obtain the amino acid sequences of the peptides. Peaks software was configured by setting the mass tolerances for precursor ions and fragment mass to 20 ppm and 0.2 Da, respectively (Xu et al., 2021). And the statistical approach of a False Discovery Rate was of less than 1% (Montandon et al., 2016).

2.2.5 Synthesis of the active peptides

The amino acid fragments of the peptides obtained by mass spectrometry were sent to a specialized biological company, and the peptides were synthesized using a solid-phase synthesis method. The peptides with purity levels higher than 90% were obtained by a cutting process and HPLC purification. The peptides were refrigerated at −20°C for future use.

2.3 Statistical analysis

All of this study's experimental processes were measured three times in parallel, and the data were expressed with the average value ± standard deviation. The drawings were completed using origin 8.0 software, then Excel and GraphPad prism 7.04 software were used for the statistical analysis of the data.

3 RESULTS AND DISCUSSION

3.1 Enzymatic hydrolysis conditions and biological activities of whey protein

The optimum conditions for enzymatic hydrolysis of the pepsin and acid protease are shown in Table 1. Under those conditions, the WPI was first hydrolyzed with pepsin for 60 min, then hydrolyzed with acid protease for 60 min. The antioxidant and DPP-IV inhibitory activities of the WPI were detected following the composite enzymatic hydrolysis of the WPI (Table 2). The results revealed that the enzymatic hydrolysate displayed both strong antioxidant and DPP-IV inhibitory activities. Therefore, this enzymatic hydrolysate was selected for the subsequent separation and purification processes.

Table 1. Optimum conditions for the enzymolysis of pepsin and acid protease

Protease	Temperature (°C)	Time (min)	E/S(%)	pH
Pepsin	40	60	1	2.5
Acid protease	55	60	1	2.5

Table 2. Antioxidant and DPP-IV inhibitory activities of the whey protease hydrolysates

Indicator	DPPH inhibition rate (%)	ABTS inhibition rate (%)	Reducing power	DPP-IV inhibitory activity (%)
Untreated WPI	12.29 ± 0.69^b	11.16 ± 0.68^b	0.141 ± 0.001^b	29.71 ± 0.63^b
Enzymatic hydrolysate	62.97 ± 1.21^a	65.18 ± 1.06^a	0.399 ± 0.007^a	62.28 ± 0.65^a

Note: In the table, the different lowercase letters indicate significant differences (p < 0.05).
Abbreviations: ABTS, 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; DPP-IV, dipeptidyl peptidasem IV.

3.2 Gel filtration chromatography of the whey protein hydrolysates

The elution curves for the gel filtration chromatography of the WPI hydrolysates are detailed in Figure 1a. The following four components were isolated from the WPI hydrolysates using a gel filtration chromatography method.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

(a) Elution curve of the gel filtration chromatography. (b) Gel filtration chromatography for the ABTS radical scavenging rates of each component. (c) Gel filtration chromatography for the DPP-IV inhibition rates of each component. In the figure, the different lowercase letters indicate significant differences (p < 0.05). ABTS, 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; DPP-IV, dipeptidyl peptidasem IV.

As shown in Figure 1a, the four components separated by gel filtration chromatography were collected and their antioxidant and DPP-IV inhibitory activities were detected. The sample mass concentration was adjusted to 1 mg/ml. The antioxidant activities were measured by the indicator of ABTS free radical scavenging rate. The ABTS free radical scavenging rates are detailed in Figure 1b as follows: The ABTS free radical scavenging rate of Component N3 was significantly higher than those of the other components (p < 0.05), reaching 71.82 ± 1.09%. Figure 1c shows the DPP-IV inhibition rates of each component. The DPP-IV inhibition rates of Components N2 and N3 were determined to be 51.94 ± 0.69% and 52.43 ± 0.78%, respectively, which were significantly higher than those of Components N1 and N4 (p < 0.05). However, there were no significant differences observed between Components N2 and N3. Following this study's comprehensive comparison of the ABTS free radical scavenging rates and DPP-IV inhibition rates, Component N3 was selected for further separation processes following the gel filtration chromatography. However, there might be a single peptide with high antioxidant activity or high content of DPP-IV inhibitory enzyme in N1 and N2, which were worthy to further study.

3.3 Reversed-phase high-performance liquid chromatography (RP-HPLC) separation and purification of the N3 component

The chromatogram of Component N3 obtained by RP-HPLC separation is shown in Figure 1c. It can be seen in Figure 2a that Component N3 was divided into 13 different components through a C18 column, and the composition is relatively complex. Next, 13 components (N3-1 to N3-13) were collected. The concentration of the sample was adjusted to 1 mg/ml, and the antioxidant activities and DPP-IV inhibitory activities were determined.

Figure 2b shows that the ABTS free radical scavenging rate of Component N3-8 was the highest (82.96 ± 2.11%), and was significantly higher than that of the other components. Figure 2c shows that the DPP-IV inhibition rate of Component N3-7 was the highest (72.94 ± 1.95%), and was significantly higher than that of the other components. Therefore, in accordance with the comprehensive comparison detailed in Figure 2b and c, it was confirmed that Component N3-8 simultaneously displayed significant ABTS free radical scavenging and DPP-IV inhibition rates. Therefore, Component N3-8 was selected in this study for the subsequent mass spectrometry identification process.

3.4 Identification and analysis of the bioactive peptides derived from the whey protein by mass spectrometry

The amino acid sequence of the peptides in Component N3-8 was identified using LC-MS/MS. Following the identifications using mass spectrometry, 14 peptides were selected for this study's chemical synthesis process according to the relationships between the structures and the functions. In addition, the ABTS free radical scavenging and DPP-IV inhibitory activities of the synthetic peptides were detected. During the detection process, the detected mass concentration of the samples was set as 1 mg/ml.

In the present research investigation, three peptides (DDQNPHSSN, HLVDEPQNLIK and DDEVDVDGTVEEDLGK) with high-ABTS free radical scavenging activities were selected from Table 3, and their DPPH free radical scavenging rates and reducing powers were respectively determined at a sample mass concentration of 1 mg/ml (Table 4). The results indicated that the ABTS free radical scavenging rates, DPPH free radical scavenging rates, and reducing power of DDQNPHSSN were significant, reaching 91.42 ± 0.63%, 88.76 ± 0.41%, and 0.637 ± 0.016%, respectively. Previous studies have found that ultrahigh pressure can significantly change the surface morphology of WPI, change the content of α-helix, β-sheet and random coil of whey protein, and affect the secondary structure of protein. Therefore, it is speculated that the production of new active peptides is because ultrahigh pressure can expose the internal cleavage sites of proteins.

Table 3. Amino acid sequence and biological activities of the peptides derived from Component N3-8

Peptide sequence	Protein source	Site		ABTS free radical scavenging rate (%)	DPP-IV inhibition rate (%)
KIDALNENKVLVLD	β-lactoglobulin	109	122	11.97 ± 0.73^j	21.33 ± 1.45^h
DDEVDVDGTVEEDLGK	Endoplasmin	22	37	81.19 ± 1.04^b	48.69 ± 0.42^de
ELKDLKGY	α-lactalbumin	30	37	56.24 ± 1.67^d	59.66 ± 0.33^bc
ETIKYLK	Glycosylation dependent cell adhesion molecule	135	141	41.66 ± 1.03^f	35.87 ± 0.62^fg
LDAQSAPLRVY	β-lactoglobulin	48	58	65.48 ± 0.94^c	44.91 ± 0.64^e
HLVDEPQNLIK	Serum albumin	402	412	83.63 ± 0.31^b	71.23 ± 0.92^a
APFPEVFGK	α-s1-casein	41	49	27.23 ± 0.29^h	7.03 ± 0.65^ij
LEILLQK	β-lactoglobulin	70	76	61.52 ± 0.18^cd	31.90 ± 0.28^g
LTKCEVFR	α-lactalbumin	22	29	53.48 ± 061^de	8.99 ± 0.32^ij
DDQNPHSSN	α-lactalbumin	82	90	91.42 ± 0.63^a	63.28 ± 0.37^b
ELQDKIHPF	β-casein	59	67	13.81 ± 0.42^j	38.76 ± 0.51^ef
LSFNPTQLEEQCHI	β-lactoglobulin	165	178	18.33 ± 1.29ⁱ	12.48 ± 0.59ⁱ
ALVSTLVPLA	Polymeric immunoglobulin receptor	631	640	47.52 ± 1.45^e	53.24 ± 1.26^cd
ALCSEKLDQW	α-lactalbumin	128	137	32.94 ± 0.89^g	45.70 ± 0.44^e

Note: In the table, the different lowercase letters indicate significant differences (p < 0.05).
Abbreviations: ABTS, 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; DPP-IV, dipeptidyl peptidasem IV.

Table 4. Antioxidant activities of the three polypeptides

Peptide sequence	ABTS free radical scavenging rate (%)	DPPH free radical scavenging rate (%)	Reducing power
DDEVDVDGTVEEDLGK	81.19 ± 1.04^b	75.82 ± 1.16^b	0.486 ± 0.011^b
HLVDEPQNLIK	83.63 ± 0.31^b	71.96 ± 0.84^c	0.519 ± 0.008^b
DDQNPHSSN	91.42 ± 0.63^a	88.76 ± 0.41^a	0.637 ± 0.016^a

Note: In the table, the different lowercase letters indicate significant differences (p < 0.05).
Abbreviations: ABTS, 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; DPP-IV, dipeptidyl peptidasem IV.

The three peptides HLVDEPQNLIK, DDQNPHSSN, and ELKDLKGY were found to display high DPP-IV inhibitory activities, respectively reaching 71.23 ± 0.92%, 66.28 ± 0.37%, and 59.66 ± 0.33% at a sample mass concentration of 1 mg/ml. Then, in accordance with the results of this study's comprehensive comparison of the two types of activities of each peptide, it was found that the DDQNPHSSN peptides not only had strong antioxidant activities, but also had displayed DPP-IV inhibitory activities with a certain intensity. Finally, the antioxidant and DPP-IV inhibitory activities of the DDQNPHSSN were determined once again. It was observed that the antioxidant activities were expressed by the Trolox equivalent (TE) and the DPP-IV inhibitory activities were expressed by the IC₅₀ value. The ABTS free radical scavenging activities of the DDQNPHSSN were 121 ± 2.48 μmol TE/g protein; the DPPH free radical scavenging activities were 92 ± 2.67 μmol TE/g protein; and the total reducing power was 34 ± 1.56 μmol TE/g protein. The IC50 of the DDQNPHSSN was determined to be 79 ± 1.18 μmol/L. The amino acid sequence of the DDQNPHSSN was obtained using a mass spectrometry analysis method, as detailed in Figure 3.

4 CONCLUSIONS

In the present study, whey protein hydrolysate which was obtained by the enzymolysis of pepsin and acid protease displayed the dual biological activities of antioxidant activities and DPP-IV inhibitory activities. The whey protein hydrolysate was isolated and purified, and the amino acid sequence of Component N3-8 was determined using LC-MS/MS. Then, 14 peptides were selected for the synthesis and detection of their biological activities. The results revealed that the peptide DDQNPHSSN had displayed both high antioxidant activities and high DPP-IV inhibitory activities. It was found that at a concentration of 1 mg/mL, the ABTS free radical scavenging rates, DPPH free radical scavenging rates, and reducing power of the DDQNPHSSN peptide were 91.42%, 88.76%, and 0.637%, respectively, and the DPP-IV inhibitory activities were 66.28%. The results obtained in this study provided strong theoretical support for the deep development of whey powder, particularly the utilization of whey protein, and provided a scientific basis for expanding the varieties of whey products in the future.

AUTHOR CONTRIBUTIONS

Yuanrong Zheng: Writing−original draft; writing−review and editing. Jiakun Pang: Writing−review and editing. Zhenmin Liu: Writing−review and editing.

ACKNOWLEDGMENTS

This study was supported by National Key Research and Development Program of a study of the specificity of whey protein hydrolysis and the development of specific functional dairy products (2018YFC1604205) and Shanghai Dairy Bioengineering Technology Research Center (19DZ2281400).

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

ETHICS STATEMENT

None declared.

REFERENCES

Adjonu, R., Doran, G., Torley, P., & Agboola, S. (2013). Screening of whey protein isolate hydrolysates for their dual functionality: Influence of heat pre-treatment and enzyme specificity. Food Chemistry, 136(3–4), 1435–1443. https://doi.org/10.1016/j.foodchem.2012.09.053
10.1016/j.foodchem.2012.09.053
CAS PubMed Web of Science® Google Scholar
Argenta, A. B., & Scheer, A.D.P. (2020). Membrane separation processes applied to whey: A review. Food Reviews International, 36(5), 499–528. https://doi.org/10.1080/87559129.2019.1649694
10.1080/87559129.2019.1649694
Web of Science® Google Scholar
Athira, S., Mann, B., Saini, P., Sharma, R., Kumar, R., & Singh, A. K. (2015). Production and characterisation of whey protein hydrolysate having antioxidant activity from cheese whey. Journal of the Science of Food and Agriculture, 95(14), 2908–2915. https://doi.org/10.1002/jsfa.7032
10.1002/jsfa.7032
CAS PubMed Web of Science® Google Scholar
Baba, W. N., Baby, B., Mudgil, P., Gan, C.-Y., Vijayan, R., & Maqsood, S. (2021). Pepsin generated camel whey protein hydrolysates with potential antihypertensive properties: Identification and molecular docking of antihypertensive peptides. LWT, 143, 111135. https://doi.org/10.1016/j.lwt.2021.111135
10.1016/j.lwt.2021.111135
CAS Web of Science® Google Scholar
Ballatore, M. B., Bettiol, M. R., Vanden Braber, N. L., Aminahuel, C. A., Rossi, Y. E., Petroselli, G., Erra-Balsells, R., Cavaglieri L. R., & Montenegro M. A. (2020). Antioxidant and cytoprotective effect of peptides produced by hydrolysis of whey protein concentrate with trypsin. Food Chemistry, 319, 126472. https://doi.org/10.1016/j.foodchem.2020.126472
10.1016/j.foodchem.2020.126472
CAS PubMed Web of Science® Google Scholar
Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT - Food Science and Technology, 28(1), 25–30. https://doi.org/10.1016/S0023-6438(95)80008-5
10.1016/S0023-6438(95)80008-5
CAS Google Scholar
Corrochano, A. R., Buckin, V., Kelly, P. M., & Giblin, L. (2018). Invited review: Whey proteins as antioxidants and promoters of cellular antioxidant pathways. Journal of Dairy Science, 101(6), 4747–4761. https://doi.org/10.3168/jds.2017-13618
10.3168/jds.2017-13618
CAS PubMed Web of Science® Google Scholar
Demers-Mathieu, V., Gauthier, S. F., Britten, M., Fliss, I., Robitaille, G., & Jean, J. (2013). Antibacterial activity of peptides extracted from tryptic hydrolyzate of whey protein by nanofiltration. International Dairy Journal, 28(2), 94–101. https://doi.org/10.1016/j.idairyj.2012.09.003
10.1016/j.idairyj.2012.09.003
CAS Web of Science® Google Scholar
Embiriekah, S., Bulatović, M., Borić, M., Zarić, D., & Rakin, M. (2018). Antioxidant activity, functional properties and bioaccessibility of whey protein hydrolysates. International Journal of Dairy Technology, 71(1), 243–252. https://doi.org/10.1111/1471-0307.12428
10.1111/1471-0307.12428
CAS Web of Science® Google Scholar
Jiang, B., Na, J., Wang, L., Li, D., Liu, C., & Feng, Z. (2019). Separation and enrichment of antioxidant peptides from whey protein isolate hydrolysate by aqueous two-phase extraction and aqueous two-phase flotation. Foods, 8, 34(1). https://doi.org/10.3390/foods8010034
10.3390/foods8010034
CAS PubMed Web of Science® Google Scholar
Jiang, C., Liu, L., Li, X., Ma, L., Du, L., Zhao, Y., Li, D., & Zhao W. (2018). Separation and purification of hypocholesterolaemic peptides from whey protein and their stability under simulated gastrointestinal digestion. International Journal of Dairy Technology, 71(2), 460–468. https://doi.org/10.1111/1471-0307.12453
10.1111/1471-0307.12453
CAS Web of Science® Google Scholar
Mares-Mares, E., Barboza-Corona, J. E., Sosa-Morales, M. E., Gutiérrez-Chávez, A. J., Gutiérrez-Vargas, S., & León-Galván, M. F. (2019). Inhibition of dipeptidyl peptidase IV by enzymatic hydrolysates derived from primary and secondary whey of fresh and oaxaca cheeses. International Journal of Dairy Technology, 72(4), 626–632. https://doi.org/10.1111/1471-0307.12623
10.1111/1471-0307.12623
CAS Web of Science® Google Scholar
Montandon, C. E., Barros, E., Vidigal, P. M., Mendes, M. T., Anhê, A. C. B. M., de Oliveira Ramos, H. J., de Oliveira, C. J. F., & de Siqueira C. L. M. (2016). Development of data for the identification and characterization of proteins found in Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri. Data in Brief, 7, 844-847. https://doi.org/10.1016/j.dib.2016.03.053
10.1016/j.dib.2016.03.053
PubMed Google Scholar
Nicolás, P., Ferreira, M. L., & Lassalle, V. (2019). A review of magnetic separation of whey proteins and potential application to whey proteins recovery, isolation and utilization. Journal of Food Engineering, 246, 7–15. https://doi.org/10.1016/j.jfoodeng.2018.10.021
10.1016/j.jfoodeng.2018.10.021
CAS Web of Science® Google Scholar
Nongonierma, A. B., & FitzGerald, R. J. (2013). Dipeptidyl peptidase IV inhibitory and antioxidative properties of milk protein-derived dipeptides and hydrolysates. Peptides, 39, 157–163. https://doi.org/10.1016/j.peptides.2012.11.016
10.1016/j.peptides.2012.11.016
CAS PubMed Web of Science® Google Scholar
Nongonierma, A. B., Lalmahomed, M., Paolella, S., & FitzGerald, R. J. (2017). Milk protein isolate (MPI) as a source of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. Food Chemistry, 231, 202–211. https://doi.org/10.1016/j.foodchem.2017.03.123
10.1016/j.foodchem.2017.03.123
CAS PubMed Web of Science® Google Scholar
Pang, J. K., Zheng, Y. R., Liu, Z. M., Bao, Y., Chen, S. Y., & Dang, H. J. (2020). Effects of ultra-high pressure on structure and antioxidant activity of whey protein isolates. Food and Fermentation Industries, 46(04), 72–77 https://doi.org/10.13995/j.cnki.11-1802/ts.022365
10.13995/j.cnki.11-1802/ts.022365
Google Scholar
Xu, D., Yu, C., Wang, J., Fan, Q., Wang, Z., Xiao, W., Duan, J., Zhou J., & Ma H. (2021). Ultrafiltration strategy combined with nanoLC-MS/MS based proteomics for monitoring potential residual proteins in TCMIs. Journal of Chromatography B, 1178, 122818. https://doi.org/10.1016/j.jchromb.2021.122818
10.1016/j.jchromb.2021.122818
CAS PubMed Web of Science® Google Scholar
Yen, G.-C., & Duh, P.-D. (1993). Antioxidative properties of methanolic extracts from peanut hulls. Journal of the American Oil Chemists' Society, 70(4), 383–386. https://doi.org/10.1007/BF02552711
10.1007/BF02552711
CAS Web of Science® Google Scholar
Zhang, Y., Chen, R., Ma, H., & Chen, S. (2015). Isolation and identification of dipeptidyl peptidase IV-Inhibitory peptides from trypsin/chymotrypsin-treated goat milk casein hydrolysates by 2D-TLC and LC-MS/MS. Journal of Agricultural and Food Chemistry, 63(40), 8819–8828. https://doi.org/10.1021/acs.jafc.5b03062
10.1021/acs.jafc.5b03062
CAS PubMed Web of Science® Google Scholar
Zhao, Z., Li, H., & Li, Q. (2017). Isolation of β-lactoglobulin from buffalo milk and characterization of its structure changes as a function of pH and ionic strength. Journal of Food Measurement and Characterization, 11(3), 948–955. https://doi.org/10.1007/s11694-017-9468-7
10.1007/s11694-017-9468-7
Web of Science® Google Scholar

Citing Literature

Volume1, Issue3-4

December 2022

Pages 298-306

The purification and identification of antioxidants and dipeptidyl peptidase IV inhibitory peptides from whey protein hydrolysates

Abstract

1 INTRODUCTION