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Effect of AAPH oxidation on digestion characteristics of seed watermelon (Citrullus lanatus var) kernels protein isolates

2020-05-26ShugangLiZhihaoLiXiutingLiPingWangXiongwiYuQinliFuSihaiGao

食品科学与人类健康(英文) 2020年4期

Shugang Li, Zhihao Li, Xiuting Li, Ping Wang, Xiongwi Yu, Qinli Fu,Sihai Gao

aSchool of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009, China

bKey Laboratory of Fermentation Engineering, Ministry of Education, Glyn O. Phillips Hydrophilic Colloid Research Center, Faculty of Light Industry, School of Food and Biological Engineering, Hubei University of Technology, Wuhan, 430068, China

cBeijing Technology and Business University, Beijing, 100048, China

dHuazhong Agriculture University, Wuhan, 430070, China

eWuhan Xudong Food Co., Ltd., Wuhan 430000, China

fDepartment of Cardiothoracic and Vascular Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China

ABSTRACT

Seed watermelon kernel is a typical complex food with high fat and protein contents. During storage and processing, it is often affected by various factors to undergo interactions between components, which lead to its quality change. In this experiment, seed watermelon kernels were used as research objects,and the effects of 2′-Azobis (2-amidinopropane) dihydrochloride (AAPH) on seed watermelon kernel protein isolates (WMP) were investigated. The structure and digestion characteristics of WMP after oxidation were studied. The results showed that with the increase of AAPH concentration (0.05−5 mol/L),WMP showed obvious aggregation, and its solubility decreased from 6.76 mg/mL to 9.59 mg/mL. The free sulfhydryl content of WMP was 18.24 mmol/g decreased to 11.25 mmol/g, α-helix decreased and β-sheet decreased in secondary structure, and its disulfide bond increased by 43.06 mmol/g from 39.57 mmol/g,enthalpy (ΔH) and denaturation temperature increased (Td) (P < 0.05). By mass spectrometry results of simulated gastric digestion products, it was found that oxidation adversely affected the digestion characteristics of WMP. It can be seen that the lipid oxidation product APPH of seed watermelon kernel can significantly affect the structure and function of the protein extracted from the seed kernel.

Keywords:

Seed watermelon kernel protein

Digestion characteristics

AAPH oxidation

1. Introduction

The seed watermelon (Citrullus lanatus var) is Cucurbitaceae,belonging to the watermelon family [1]. The protein content of its seed kernel is as high as 36%–40%, furthermore its ratio of essential amino acids is even closer to the Food and Agriculture Organization(FAO) recommendation than that of soybean, which makes the seed watermelon kernel protein a nutritionally balanced plant protein with high values to be developed [2]. The kernels of seed watermelon seeds are easily oxidized and produce metabolites (such as peroxidation-derived free radicals, lipid hydroxides and reactive aldehydes) due to the high content of unsaturated fatty acids, which could indirectly lead to the oxidation of proteins [3]. The protein oxidation is the covalent structure modification of proteins caused because of the direct action of reactive oxygen species (ROS) or secondary oxidation products on the protein [4], which would result in the loss of nutrition and deterioration of products in the process of food processing and storage.

Therefore, the research on protein oxidation in food has important practical significance for improving product quality and protecting human health. In normal food processing, the main factor leading to protein oxidation is that the active intermediate of ROS-induced lipid oxidation indirectly mediates protein oxidation,in which lipoxygenase (LOX) is a key factor in oxidation. As in many fat-rich foods, the seed melon kernel protein is susceptible to lipid peroxides catalyzed by LOX during process and storae, which could lead to multiple structural changes in proteins, including oxidation of side chain groups, backbone cleavage, unfolding, degradation and aggregation [5].

A large number of studies have con firmed that the oxidation of proteins can lead to a decrease in their functional properties[6]. Studies by Cui et al. [7] have shown that the emulsifying and foaming properties of whey proteins treated by hydroxyl radical oxidation are reduced. Chanarat et al. [8] treated tilapia myosin with hydrogen peroxide and found that its gelling properties and water holding capacity were significantly reduced. The same result was demonstrated in the study by Wu et al. [3]. Lipid peroxideinduced protein oxidation had a negative effect on its gelling properties. But the effects of oxidation on protein digestion have very different results. A large number of studies have reported that oxidation can inhibit protein digestion, however some researchers have discovered that moderate oxidation can improve protein digestion [9,10]. Among them, Lin et al. [11] studied the in vitro digestibility of catechin-modified glycinin under oxidative stress,which showed that oxidation had a positive or negative effect on protein digestibility depending on the degree of oxidation.

Because there are few reports on the chemical modification of protein digestibility by lipid peroxide products, this experiment took seed watermelon kernel protein as raw material to compare different AAPH concentrations (0, 0.05, 0.1, 0.5, 1 and 5 mmol/L)treatment of the structure and in vitro digestibility of seed watermelon kernel protein, combined with MALDI-TOF-MS analysis of digestion products. The study aimed to reveal the effect of nut lipid oxidation products on its protein structure and function, and provided theoretical support for the development and utilization of nut foods represented by seed watermelon kernels.

2. Materials and methods

2.1. Samples and materials

Seed watermelon kernel were purchased from 10th company horticultural field of 9th Regiment, 1st division of Xinjiang Construction Corps (Xinjiang, China). Bovine serum albumin (BSA)and 8-Anilino-1-naphthalenesulfonic acid (ANS) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) (CAS No. 2997-92-4)was purchased from Sigma-Aldrich (Saint Louis, America). All other chemicals were of analytical reagent grade and obtained in China.

2.2. Watermelon kernel protein isolates (WMP) preparation

The WMP was prepared according to the method described by Li et al. [12]. The shell of seed watermelon kernels was removed,and kernels were milled through a 50 mesh sieve, then 5 g of the sample was degreased with petroleum ether (30–60°C) using the material to liquid ratio of 1:30 (g/mL), and then filtered by suction, the petroleum ether was volatile under a fume hood. The degreasing process was repeated once to obtain the degreased seed watermelon kernel powder. The degreased powder was added into ultrapure water, and extracted at a material to liquid ratio of 1:30 (g/mL) for 3 h (pH 9.5, temperature at 35°C). After centrifugation at 8000 × g (Multifuge X1R, Thermo, America) for 20 min,the supernatant was collected, the residue was washed with the same extract solution and centrifuged for 10 min, this process was repeated twice. The pH of collected supernatant was adjusted to 7.0 and dialyzed against ultrapure water (MWCO: 8000–14000 Da)for 3 days. The seed watermelon kernel protein isolate (WMP) was obtained by freeze-drying and stored at 4°C.

2.3. Modification of WMP with AAPH

Oxidized WMP was prepared according to the method described by Liu et al. [13]. WMP was formulated as a 40 mg/mL protein solution (0.05% NaN3bacteriostatic) using 0.01 mol/L sodium phosphate buffer (pH 7.4). A quantity of AAPH (2′-Azobis(2-amidinopropane)dihydrochloride) was mixed with the WMP solution to a final concentration of 0, 0.05, 0.1, 0.5, 1.0, 5.0 mmol/L.After sealing at 25°C for 24 h, the temperature was lowered to 4°C and centrifuged at 8000 × g for 20 min. The supernatant was dialyzed against ultrapure water at 4°C for 24 h, lyophilized and stored at 4°C for use.

2.4. Sulfhydryl and disulfide bond

The content of free sulfhydryl groups in the oxidized WMP was determined using method by Beveridge et al. [14]. The oxidized WMP sample was placed in a solution of 5 mg/mL in 8 mol/L urea Tris-Gly buffer (pH 8.0) and protected from light for 30 min at room temperature. The suspension was centrifuged at 8000 × g for 20 min at 4°C, and the protein concentration in the supernatant was determined by Coomassie Brilliant Blue method. 3 mL of the supernatant was then reacted with 0.02 mL of DTNB reagent (4 mg/mL)dissolved in Tris-Gly buffer. The mixture was allowed to stand at room temperature for 1 h, and the absorbance was measured at 412 nm using a UV–vis spectrophotometer (TU-1900, Xipu, China).The content of sulfhydryl and total sulfhydryl groups was calculated by extinction coef ficient of 13600 L/mol.

2.5. Surface hydrophobicity (Ho)

The surface hydrophobicity of the protein was determined using the ANS fluorescent probe (F-7000, Hitachi, Japan) following the method of Kato et al. [15] with some modifications. The sample was diluted to 0.00125, 0.0025, 0.005, 0.01, 0.02 mg/mL with phosphate buffer (0.01 mol/L, pH 7.4), and 20 μL of ANS solution (8 mmol/L)was added to 4 mL of the diluted protein solution and shaken. The fluorescence intensity was measured at an excitation and emission wavelength slit width of 2.5 nm, an excitation wavelength of 390 nm, and an emission wavelength of 470 nm. The surface hydrophobicity is determined from the initial slope of the fluorescence intensity and protein concentration curve.

2.6. Fluorescence

According to the method of Lv et al. [16], the sample was dissolved in phosphate buffer (0.01 mol/L, pH 7.4), diluted to 2 mg/mL,centrifuged at 8000 × g for 20 min, and the supernatant was filtered with 0.22 μm. The sample was scanned by a fluorescence spectrophotometer at an excitation wavelength of 280 nm and a scanning wavelength of 300–420 nm.

2.7. Particle size distributions of WMP measurement

The particle size distribution was detected by dynamic light scattering (DSL) using a Zeta sizer Nano-ZS instrument (Malvern Instruments, Worcestershire, UK). 0.1 mg/mL WMP solution was prepared in phosphate buffer (0.01 mol/L, pH 7.4). The suspension was stirred with a magnetic stirrer and centrifuged at 8000 × g for 10 min. 1.5 mL of the supernatant was placed in a quartz cuvette for DLS measurement. The results were analyzed by using Dispersion Technology Software version 4.20.

2.8. Circular dichroism spectra (CD)

The CD spectrum was measured at 190–250 nm using a J-1500(J-1500, JASCO, Japan) spectropolarimeter, and the sample was placed in a 1 mg/mL solution with ultrapure water and centrifuged at 10000 × g for 10 min. The protein concentration in the supernatant was determined by the Coomassie Brilliant Blue method,and data was generated using a resolution of 0.1 nm, a response time of 1 s, and an average of 3 scans.

2.9. FTIR

The FTIR spectra of MP samples were recorded using a Nexus-470 FTIR spectrometer (Nexus-470, Mettler, USA) [17]. The dried sample was mixed with KBr at a ratio of 1:100 and compressed into disks. The spectra were recorded in the range of wavenumber from 4000 cm−1to 400 cm−1with KBr spectrum was used as a background. The curve in the wavenumber range of 1600–1700 cm−1was fitted by using Peak-Fit version 4.12 software to analyze the secondary structure of samples.

2.10. SDS-PAGE

SDS-PAGE analysis was carried out by SDS gel electrophoresis analysis electrophoresis (DYY-8C, Liu yi, China) [18]. Mix 2 mg lyophilized sample with 500 mL sample buffer, boil the sample buffer with β-mercaptoethanol for 5 min, and add 10 μL sample to polyacrylamide gel after centrifugation at 8000 × g for 10 min at 4°C. Glue was made from 12% separation gel and 5% concentrated gel.

2.11. Solubility

Solubility was determined by the method of Bera et al. [19,20].Protein samples were dispersed into phosphate buffer (0.01 mol/L,pH 7.4) and stirred at room temperature for 1 h to prepare 10 mg/mL solutions. Then centrifuged at 8000 × g for 20 min.The protein content in the supernatant was determined by the Coomassie Brilliant Blue method.

2.12. Differential scanning calorimeter

Differential scanning calorimetric study was performed using a Micro DSC III Evo thermal analyzer (Micro DSC III Evo, Setaram,France). The sample was con figured as a 10 mg/mL protein solution using ultrapure water, and 300 mg of ultrapure water was placed in the sample cell and the reference cell, respectively, and equilibrated at 25°C for 1 h. The running temperature was from 25°C to 110°C,with a rate of 5°C/min. Peak denaturation temperature (Td) and enthalpy change (ΔH) were calculated from the curves by Universal Analysis 2000, version 4.1D (TA Instruments Waters LLC).

2.13. Protein hydrolysis pro filing

The degree of hydrolysis of WMP digests was analyzed using the OPA method [21]. The OPA reagent was prepared according to the method of Chen et al. [22]. 400 μL of WMP hydrolysate was mixed with 3 mL of OPA reagent for 2 min. Then, the absorbance was measured at a wavelength of 340 nm, and the degree of hydrolysis(DH) was calculated as follows:

Where β is 0.342 mequv/g, α is 0.970 mequv/g, htotis the constant of WMP 7.8 mequv/g.

2.14. Peptide products of pepsin digestion

Peptide fractions were analyzed using a Dionex 3000 RSLC UHPLC system and a Q Exactive mass spectrometer. A 10 μL sample was loaded onto an Aeris PEPTIDE 2.6 mm XB-C18, 250 4.6 mm column. The peptide was eluted with a chromatographic gradient of 0%–60% solvent (80% acetonitrile, 0.1% formic acid) for 60 min at a flow rate of 250 mL/min. The mass spectrometer runs in the data-dependent mode and automatically switches between MS and MS/MS. MS scans (200–2000 m/z) were acquired in an orbitrap analyzer with a resolution of 70000, 400 m/z. Select the top strongest ion for MS/MS (target value is 3106 ions and maximum ion fill time is set to 50 ms). The peptide was subjected to a higher energy collision dissociation (HCD) MS/MS (standardized collision energy 1/428, 3500 resolution). The data was analyzed using Proteome Discover (version 1.4, Thermo Fisher Scientific) using the Sequent search algorithm using the following standard protein database(www.uniprot.org, UP000009136).

2.15. Statistical analysis

Statistical analysis was carried out using SPSS 19.0. Data were shown as mean ± S.D. (n = 3). Differences between means of each group were assessed using one-way analysis of variance followed by Duncan’s test. significant differences were accepted as a P < 0.05.

3. Results and discussion

3.1. Sulfhydryl and disulfide bond

The degree of oxidation of the protein is usually characterized by the content of free sulfhydryl groups and disulfide bonds, since the protein can be oxidized to a reversible form of disulfide, sulfenic acid or irreversible form of sulfinic acid, sulfonic acid under different circumstances [23]. As shown in Fig. 1, with the increase of AAPH concentration, the content of free sulfhydryl groups decreased from 18.24 mmol/g to 11.25 mmol/g, and the content of disulfide bonds increased from 39.57 mmol/g to 43.06 mmol/g.According to a study by Davies et al. [5], the free radical (ROO—)provided by AAPH can rapidly react with free sulfhydryl groups in proteins to form sulfinyl free radicals, which are then joined with molecular oxygen to form thiol free radicals. The thiol free radicals can further induce protein oxidation, resulting in a decrease in sulfhydryl content, and induces the conversion of a sulfurcontaining amino acid side chain in cysteine and methionine to a disulfide bond by oxidative stress. In the study of skeletal muscle fibril protein oxidation system by Morzel et al. [24], it was found that the free sulfhydryl group was reduced by 80% when the oxidant concentration changed from 0.2 mmol/L to 20 mmol/L.Wei et al. [3] also found a similar rule in the study of AAPH oxidation of soy protein. Since cysteine and disulfide bonds have an important Influence on the structure of proteins, oxidation induced the conversion of free sulfhydryl groups in WMP into disulfide bonds, which causes the changes of the protein structure.

Fig. 1. Sulfhydryl and disulfide bond content of WMP incubated with different concentration of AAPH for 24 h at 25°C.

Fig. 2. Surface hydrophobicity of WMP incubated with different concentration of AAPH for 24 h at 25°C. 0, 0.05, 0.1, 0.5, 1 and 5 mmol/L, WMP treated with 0, 0.05, 0.1,0.5, 1 and 5 mmol/L AAPH. Different superscript letters indicate significant difference(P < 0.05).

3.2. Surface hydrophobicity

Surface hydrophobicity is an important property for maintaining protein structure and a common method for assessing changes in protein structure. As the protein concentration increases, the linear relationship of fluorescence intensity accurately describes the hydrophobicity of the protein. It can be seen from Fig. 2 that the hydrophobicity of WMP decreased significantly with the increase of AAPH concentration, and its value decreased from 8807 to 3066 (P < 0.05). The results of this study are highly consistent with the findings of Grune et al. [25]. Low concentrations of AAPH expose the hydrophobic groups inside the WMP and form aggregates by interaction, reducing the solubility of the protein.Thereby, the hydrophobicity of the surface is first lowered and then stabilized. When the AAPH concentration is increased from 1 mmol/L to 5 mmol/L, the decrease in hydrophobicity of the protein surface may be due to the re-aggregation of unfolded protein molecules by hydrophobic associations, the covalent modification of the exposed hydrophobic residues (e.g. tryptophan residues),as well as the formation of partially hydrophilic groups (e.g. protein carbonyl groups) by structural change of oxidized surface hydrophobic amino acids [25–27].

Fig. 3. The fluorescence intensity of WMP incubated with different concentration of AAPH for 24 h at 25°C. 0, 0.05, 0.1, 0.5, 1 and 5 mmol/L, WMP treated with 0, 0.05,0.1, 0.5, 1 and 5 mmol/L AAPH.

Fig. 4. The particle size distribution of WMP incubated with different concentration of AAPH for 24 h at 25°C. 0, 0.05, 0.1, 0.5, 1 and 5 mmol/L, WMP treated with 0, 0.05,0.1, 0.5, 1 and 5 mmol/L AAPH.

3.3. Fluorescence chromatography

Since tryptophan is sensitive to the environment and is highly susceptible to oxidation, changes in the fluorescence intensity of the protein are usually used to reveal the spatial structure and degree of oxidation of the tryptophan residue. As shown in Fig. 3, as the AAPH concentration increases, the fluorescence intensity of WMP decreases, and the maximum fluorescence intensity red-shifted. This is consistent with the results of Campos et al.[28], because tryptophan has the lowest single-electron oxidation potential, and as the degree of oxidation increases, tryptophan residues were transformed by lipid peroxides into metastable tryptophan free radical, which combined with divided molecular oxygen atom to form tryptophan peroxy radicals, which were ultimately converted to kynurenine, resulting in a decrease in fluorescence intensity [29]. St˘anciuc et al. [30] demonstrated that AAPH oxidation unfolded protein structure, tryptophan residues gradually transferred from internal protein molecules to protein surface, the protein molecules were unfolded, larger protein aggregates were formed, and the protein fluorescence intensity λ max red shifted.

3.4. Particle size distributions of WMP measurement

Dynamic light scattering is commonly used to monitor the formation of protein aggregates [31]. As shown in Fig. 4, the particle size distribution of the oxidized WMP was analyzed by DLS. When the concentration of AAHP is between 0 and 1 mmol/L, the particle size of WMP increases from 190.1 nm to 396.1 nm, and a small amount of large particles appear. At higher AAPH concentrations(1–5 mmol/L), further increase in oxidation promotes the formation of insoluble components. At a concentration of 5 mmol/L, the particle size was reduced to 220.2 nm. It is possible that some of the larger soluble aggregates break down into smaller soluble peptides,which are converted into insoluble components by centrifugation,covalent and non-covalent direct interactions [32]. This is consistent with the results of Huang et al. [33]. The decrease in peaks in Fig. 4 con firms the formation of insoluble aggregates, resulting in a decrease in WMP solubility.

Fig. 5. Deconvoluted FTIR spectra in the amide I region of WMP modified at different concentration of AAPH for 24 h at 25°C. A-F, WMP treated with 0, 0.05, 0.1, 0.5, 1 and 5 mmol/L AAPH.

Fig. 6. SDS-PAGE of WMP incubated with different concentration of AAPH for 24 h at 25°C. Lanes: 1, marker proteins, 2-7, WMP treated with 0, 0.05, 0.1, 0.5, 1 and 5 mmol/L AAPH.

3.5. Secondary structure of protein

CD spectroscopy is widely used to study protein secondary structure. The secondary structure content of WMP is shown in Table 1 by spectral analysis software calculations. When the AAPH concentration was 0–1 mmol/L, the α-helix content decreased from41.62% to 33.87%, the β-sheet content increased from 4.65% to 32.1%, and the β-turn content decreased from 14.12% to 1.52%. The Random coil content decreased from 39.61% to 32.41%. When the AAPH concentration was 5 mmol/L, the solubility decreased due to an increase in the degree of aggregation of the protein, resulting in data close to the blank group (0 mmol/L).

Table 1Secondary structure table calculated by circular dichroism spectroscopy.

Table 2The denaturation temperature and enthalpy of AAPH oxidized WMP.

The Amide I band was known to be sensitive to the secondary structure of proteins [17]. The amide I band (1700–1600 cm−1)in FTIR spectra of different samples was fitted by the Gaussian model and the results shown in Fig. 5. As the AAPH concentration increased (0–5 mmol/L), the α-helix content decreased from 45.94% to 12.55%, while the β-sheet content increased from 12.39% to 39.09%, and the random coil decreased from 26.69% to 19.24%. Some studies had shown that the nutritional value and digestion characteristics of protein was related to protein secondary structure, the content of α-helix and random coil was positively associated with in vitro digestibility of protein, while β-sheet content was negatively correlated with in vitro digestibility [34]. So it indicated that the digestibility of WMP was decreased in the presence of APPH.

3.6. SDS-PAGE

SDS-PAGE is commonly used to characterize the molecular weight distribution of proteins. It can be seen from Fig. 6 that the distribution of WMP is mainly concentrated between 50 and 70 KDa. As the concentration of AAPH increases, the protein mobility decreases gradually (lanes 2–7). This indicates an increase in the molecular weight of WMP, which may be caused by peroxy radicals leading to cross-linking of covalent bonds in WMP [35]. In addition, as the AAPH concentration increases, the electrophoresis strips fade in the low molecular weight region (14.4–31 kDa) (lanes 3–6), which meant that high concentrations of free oxygen radicals could cause WMP oxidative decomposition into small molecular weight peptides fragment [36].

3.7. Solubility

Solubility is one of the indicators for evaluating the functional properties of proteins. It can largely characterize the hydration ability of proteins, and can also be used to simply characterize the aggregation and denaturation of seed watermelon kernel protein.

As shown in Fig. 7, as the concentration of AAPH increased, the solubility decreased significantly (9.59–6.76 mg/mL) (P < 0.05). At low oxidation (0–1 mmol/L), WMP produces soluble aggregates. As the degree of protein oxidation increases, covalent bond crosslinking may lead to further aggregation of these soluble aggregates,resulting in insoluble aggregates [35], resulting in WMP solubility is reduced.

Fig. 7. Solubility of WMP incubated with different concentration of AAPH for 24 h at 25°C. 0, 0.05, 0.1, 0.5, 1 and 5 mM, WMP treated with 0, 0.05, 0.1, 0.5, 1 and 5 mM AAPH. The different superscript letters indicate significant difference (P < 0.05).

3.8. Differential scanning calorimeter

DSC was widely used to characterize the stability of proteins.As illustrated in Table 2, the two peak transition temperatures (Td)of WMP were about 60 and 80°C, respectively, corresponded to the thermal denaturation temperatures of WMP components. The enthalpy value, mainly came from the undenatured protein, and the decrease of enthalpy was always accompanied by the denaturation of protein [37,38]. The Td1and Td2increased while ΔH2value and the solubility of WMP gradually decreased with the increasing AAPH concentration, indicating the protein was denatured with the addition of APPH. The value of Tdreflected the thermal stability or tertiary conformational stability of the proteins. AAPH-modified WMP exhibited higher Tdwhen compared with the control, which suggested that the denatured protein possessed a more stable structure. This could be attributed to the cross-linking between molecules caused by disulfide bonds which could enhanced the thermal stability of protein.

3.9. Characteristics of the in vitro digestion

The degree of hydrolysis is the percentage of peptide bond cleavage in protein hydrolysates, and the in vitro digestibility of proteins is often evaluated using the degree of hydrolysis of the protein [39].The hydrolysis of WMP using pepsin was shown in Fig. 8. The degree of protein hydrolysis increased as the hydrolysis time prolonged(P < 0.05). Meanwhile, the hydrolysis rate reduced, mainly due to the reduction of available hydrolysis sites and the inhibition coming from the digestion products of the enzyme [40]. According to the previous studies, the same digestion pattern was observed in the hydrolysis of soy protein by pepsin [24]. Within the first 60 min,the degree of hydrolysis of WMP increased rapidly with digestion time and stabilized around 120 min.

Fig. 8. Hydrolysis curve of WMP incubated with different concentration of AAPH for 24 h at 25°C. 0, 0.05, 0.1, 0.5, 1 and 5 mmol/L, WMP treated with 0, 0.05, 0.1, 0.5,1 and 5 mmol/L AAPH.

As the degree of oxidation increased, the DH of WMP decreased significantly (P < 0.05). Because many crosslinks between protease chains and peptide chains were formed during the protein oxidation, they masked the action sties of proteases and reduced the sensitivity of protein hydrolysis [35], and at 5 mmol/L AAPH, WMP formed oxidized aggregates, which could not be efficiently digested by pepsin. Morzel et al. [24] reported a reduction in proteolysis caused by oxidative aggregates. Santé-Lhoutellier et al. [10] also demonstrated that the digestion rate of myofibrillar protein by pepsin was reduced due to AAPH oxidation.

3.10. Peptide products of pepsin digestion

From the mass spectrometry (MALDI-TOF-MS) analysis of Fig. 9, it can be seen that the peptides after digestion by WMP without AAPH are concentrated between 1000 and 1500 Da,and the peptides after WMP digestion are treated with low concentration of AAPH (0.05–0.5 mmol/L). The segments were concentrated between 1500 and 2000 Da. The high-concentration AAPH (1–5 mmol/L) treated WMP digested peptides were concentrated between 1500 and 2500 Da. As the degree of oxidation increases, the molecular weight of the peptide segment after WMP digestion gradually increases. This is because as the degree of oxidation increases, WMP cross-links and aggregates, masking the pepsin cleavage site, thereby affecting the digestibility of WMP.

The Venn diagram (https://bioinfogp.cnb.csic.es/tools/venny/index.html) (Oliveros and Venny) intuitively displayed the common and unique peptides of each sample treated with only pepsin(Fig. 10). Among the enzymatic hydrolysates, 3135 unique peptides were identified after WMP digestion without AAPH treatment.After treatment with 0.05 mmol/L AAPH, the digested products of WMP identified 2958 unique peptides. After treatment with 0.5 mmol/L AAPH, the digested products of WMP identified 3594 unique peptides. After digestion with 5 mmol/L AAPH, the digested products of WMP identified 6174 unique peptides, of which the four treatment groups shared 91 peptides.

Fig. 9. Mass fingerprints of digested products of WMP incubated with different concentration of AAPH for 24 h at 25°C. A-F, WMP treated with 0, 0.05, 0.1, 0.5, 1 and 5 mmol/L AAPH.

Fig. 10. Peptide Venn diagram of digested products of WMP incubated with different concentration of AAPH for 24 h at 25°C. 0, 0.05, 0.5 and 5 mmol/L, WMP treated with 0, 0.05, 0.5 and 5 mmol/L AAPH.

4. Conclusions

In this study, the protein of seed watermelon kernel was treated by different concentrations of AAPH, and the structure and digestion characteristics of the isolated proteins were analyzed and characterized. The digestion products were analyzed by MALDI-TOF-MS. The results showed that under AAPH treatment conditions, the free sulfhydryl group in the seed watermelon kernel protein is converted into disulfide bond, intermolecular crosslinking, the average particle size distribution increases, the thermal stability is enhanced, the α-helix content of the protein decreases and the β-sheet content increases, and the pepsin cleavage position The point is buried inside the protein, and the digestibility of the seed watermelon kernel protein is reduced. The results of mass spectrometry of the digested product showed that the molecular weight of the peptide segment identified in the WMP digestion product gradually increased with the increase of the degree of oxidation, and was not easily absorbed by the human body. The research results provide a good theoretical basis and technical support for the development and utilization of seed watermelon kernels and storage and preservation. However, the in fluencing factors and related mechanisms are still Insufficient, and further research is needed.

Declaration of Competing Interest

The authors declare that they have no competing financial interests.

Acknowledgements

This study was supported by National Natural Science Foundation of China (No: 31760477), Beijing Advanced Innovation Center for Food Nutrition and Human Health (No: 20181007) and Youth Science and Technology Innovation, Leader in Corps (No:2016BC001).