Highlights
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Ultrasound improves protein extraction rate by 16.56% than alkaline extraction.
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Ultrasound can affect iron-chelating peptide generation by protein extraction.
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Two alcalase hydrolysates own similar iron-chelating rate and binding sites.
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Main peptide sequences have different structural characteristic from each other.
Keywords: Haliotis discus hannai Ino, Ultrasound assisted extraction, Iron-chelating peptides, Structural characteristic, Peptide sequence
Abstract
This study aims to investigate effects of ultrasound assisted extraction on the abalone viscera protein extraction rate and iron-chelating activity of peptides. The optimal conditions for ultrasound assisted extraction by response surface methodology was at sodium hydroxide concentration 14 g/kg, ultrasonic power 428 W and extraction time 52 min. Under the optimal conditions, protein extraction rate was 64.89%, compared with alkaline extraction of 55.67%. The iron-chelating activity of peptides affected by ultrasound technology was further evaluated by iron-chelating rate, FTIR spectroscopy and LC-HRMS/MS. Alcalase was the suitable enzyme for the preparation of iron-chelating peptides from two abalone viscera proteins, showing no significant difference between their iron-chelating rate of 16.24% (ultrasound assisted extraction) and 16.60% (alkaline extraction). Iron binding sites from the two hydrolysates include amino and carboxylate terminal groups and peptide bond of the peptide backbone as well as amino, imine and carboxylate from side chain groups. Moreover, 24 iron-chelating peptides were identified from hydrolysate (alcalase, ultrasound assisted extraction), which were different from the 27 iron-chelating peptides from hydrolysate (alcalase, alkaline extraction). This study suggests the application of ultrasound technology in the generation of abalone viscera-derived iron-chelating peptides which have the ability to combat iron deficiency.
1. Introduction
Pacific abalone (Haliotis discus hannai Ino) is a highly valued shellfish widely cultured in East Asia [1]. Abalone viscera, normally discarded as aquatic product processing wastes, account for 15%~25% of the total body weight of abalone [2]. Abalone viscera are reported to contain rich nutrients, whose protein content makes up a large percent. In previous studies, abalone viscera protein exhibited good properties for the production of bioactive peptides, including antioxidant peptides [3] and angiotensin I-converting enzyme inhibitory peptides [4]. It is of great importance to develop the new resource of abalone viscera protein.
Nowadays, many methods have been conducted to extract the protein. As a conventional method, alkaline extraction (AE) is applied widely because of its simple process [5]. However, compared with other protein extraction method, protein extraction yield and efficiency of alkaline extraction is lower, which limits its application [6], [7]. In recent years, great efforts have been made on improving protein extraction rate from various sources for food industry. Ultrasound technology shows the advantage on the protein extraction, as a result of its capacity to enhance the extraction efficacy by promoting mass transport and probable rupture of cell wall due to the influence of acoustic cavitation [8], [9]. Ultrasound is generally considered to be safe, inexpensive, reproducible and reliable, non-invasive and environmentally friendly, which give the use of ultrasound a major advantage over the conventional alkaline extraction. Moreover, ultrasound was also reported to be able to improve enzymatic efficiency and activities of bioactive hydrolysates greatly [10]. By now, ultrasound assisted extraction (UE) has been applied to develop many new protein resources from pork liver [11], duck liver [12], sesame bran [13] and sunflower meal [14].
Iron-chelating peptides have shown good potentiality as novel carriers to deliver iron [15]. During the process of ultrasound assisted protein extraction, some chemical and physical changes, caused by cyclic generation and collapse of cavities, might affect the protein structure [16]. Wen et al. [17] found that ultrasound treatment could significantly improve the antioxidative activities of watermelon protein hydrolysates. Zou et al. [18] studied the physicochemical property of acid-soluble collagen by ultrasound assisted extraction from calipash of soft-shelled turtle. Results showed that the ultrasound treatment did not disrupt the triple-stranded helical structures in acid-soluble collagen by ultrasound assisted extraction, which had higher thermal stability than collagen from the conventional extraction. The iron-chelating activity of peptides is greatly affected by their structures. But until now, effect of ultrasound assisted extraction on the preparation of iron-chelating peptides is not clear.
The objective of this study was to optimize the conditions for ultrasound assisted extraction of abalone viscera protein and to evaluate the effect of ultrasound technology on abalone viscera protein to generate iron-chelating peptides. Firstly, response surface methodology (RSM) with Box-Behnken design was adopted to obtain the optimized parameters for abalone viscera protein extraction. Furthermore, the effect of ultrasound technology on the iron-chelating activity of abalone viscera protein-derived peptides was studied by iron-chelating rate, FTIR spectroscopy and LC-HRMS/MS. By now, little research has been reported on this subject. The result would be of significance in the utilization of ultrasound technology for abalone viscera protein extraction as well as the application of abalone viscera-derived iron-chelating peptides as ingredients in functional foods to improve iron absorption and to prevent iron deficiency-related diseases.
2. Material and methods
2.1. Materials
Abalone viscera were obtained from Dalian Bangchuidao Food Co., Ltd. (Liaoning, China) and stored at −20 °C before use. Alcalase 2.4 L and flavourzyme 500 MG were obtained from Novozymes A/S (Bagsvaerd, Denmark). Trypsin was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Other chemical reagents used in this study were of analytical grade and commercially available.
2.2. Preparation of defatted abalone viscera
Abalone viscera were lyophilized and pulverized, whose size were <0.25 mm. Abalone viscera powder was degreased by n-hexane/ethanol (3:1, mL/mL) at 50 °C for 6 h with constant agitation. Degreasing solvent was changed every two hours. The abalone viscera powder, treated by filtration, was air-dried at room temperature as a thin layer.
2.3. Protein extraction process from abalone viscera
2.3.1. Ultrasound assisted extraction of abalone viscera protein
2.3.1.1. Ultrasound treatment
Defatted abalone viscera powder (10 g) was mixed with sodium hydroxide solution at a ratio of 1:20 g/mL in a conical flask (250 mL). The ultrasound assisted extraction was carried out at 20 kHz using an ultrasound generator (Scientz-950E, Scientz Biotechnology Co., Ltd, Ningbo, China) with a 6 mm flat tip probe (pulse duration of on-time 3 s and off-time 3 s). The probe was immersed 2 cm into the liquid.
2.3.1.2. Single factor experiment
The protein extraction from abalone viscera was conducted by ultrasound treatment as described in Section 2.3.1.1. Sodium hydroxide concentration, ultrasonic power, extraction time and extraction temperature were evaluated separately in order to get the maximum value for protein extraction rate (Table 1). Sodium hydroxide concentration was assessed under the fixed condition of ultrasonic power 400 W, extraction time 1 h and extraction temperature 40 °C. Ultrasonic power was investigated under the fixed condition of sodium hydroxide concentration 15 g/kg, extraction time 1 h and extraction temperature 40 °C. Extraction temperature was studied under the fixed condition of sodium hydroxide concentration 15 g/kg, ultrasonic power 400 W and extraction time 1 h. Extraction time was evaluated under the fixed condition of sodium hydroxide concentration 15 g/kg, ultrasonic power 400 W and extraction temperature 40 °C.
Table 1.
Factors affecting protein extraction rate.
| Factor | Level | ||||
|---|---|---|---|---|---|
| NaOH concentration (g/kg) | 5 | 10 | 15 | 20 | 25 |
| Ultrasonic power (W) | 100 | 200 | 300 | 400 | 500 |
| Extraction temperature (℃) | 30 | 40 | 50 | 60 | 70 |
| Extraction time (min) | 15 | 30 | 45 | 60 | 75 |
2.3.1.3. Experimental design of response surface methodology
A Box-Behnken experimental design with 3 variables, including sodium hydroxide concentration (X1), ultrasonic power (X2) and extraction time (X3), was used to determine the optimum combination of variables. The factors at 3 variation levels were shown in Table 2. The extraction process was conducted by ultrasound treatment according to the description of Section 2.3.1.1. Other condition was fixed at 40 °C extraction temperature and a defatted abalone viscera powder/sodium hydroxide solution ratio of 1:20 g/mL.
Table 2.
Factors and their levels employed in Box-Behnken design.
| Factor | Level |
||
|---|---|---|---|
| −1 | 0 | +1 | |
| X1/NaOH concentration (g/kg) | 10 | 15 | 20 |
| X2/Ultrasonic power (W) | 300 | 400 | 500 |
| X3/Extraction time (min) | 30 | 45 | 60 |
2.3.2. Alkaline extraction of abalone viscera protein
The alkaline extraction of abalone viscera protein was investigated as a classical extraction process, commonly used in order to have a kind of reference. For alkaline extraction of abalone viscera protein, defatted abalone viscera powders were mixed with sodium hydroxide solution (14 g/kg) at a ratio of 1:20 g/mL. The extraction was conducted at a controlled temperature of 40 °C for 52 min with constant agitation. Subsequently, the mixture was centrifuged at 10,000g for 30 min. The supernatant was carefully collected for further treatment.
2.3.3. Total protein content and protein extraction rate
Total protein content of defatted abalone viscera powders was measured using Kjeldahl method. Soluble protein content in the extracts from UE or AE process was measured by the Bradford method using an automatic microplate reader (Tecan Infinite M200, Mannedorf, Switzerland). Protein extraction rate was calculated as (PS/P0) × 100%, where P0 was total protein content in sample and PS was protein content in supernatant.
2.3.4. Isoelectric point measurement of abalone viscera protein
In order to measure isoelectric point of abalone viscera protein, the pH of extracts was adjusted to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0, respectively. Afterwards, protein extracts were left at 4 °C for 1 h. Subsequently, the extract was centrifuged at 10,000g for 15 min, and the supernatant was collected for further use. Isoelectric point of abalone viscera protein was determined by precipitation rate of abalone viscera protein at different pH value. Precipitation rate was calculated as [(C0 − CS)/C0] × 100%, where C0 was protein content of abalone viscera extracts and CS was protein content of the supernatant.
2.4. Effect of ultrasound assisted extraction on iron-chelating activity
2.4.1. Preparation of iron-chelating peptides from abalone viscera protein
Iron-chelating peptides were prepared according to our previous study [19]. Proteins obtained by ultrasound assisted extraction or alkaline extraction were hydrolyzed with three commercial enzymes (trypsin, 37 °C, pH 7.5; alcalase, 55 °C, pH 8.0; flavourzyme, 50 °C, pH 7.0) for 240 min under protein concentration of 20 mg/mL and enzyme/substrate ratio of 40 mg/g, respectively. Hydrolysates were heated in boiling water bath for 10 min to inactivate the protease. Subsequently, the mixture was cooled to room temperature and centrifuged at 10,000g for 20 min. The supernatants were collected and lyophilized.
2.4.2. Determination of iron-chelating rate
Iron-chelating rate was determined using a modified colorimetric assay [19]. Lyophilized hydrolysate (250 µL, 1 mg/mL), dissolved in sodium acetate buffer (0.05 mol/L, pH 5.0), was added into the 96-well plate and pre-incubated for 5 min at 37 °C in a thermostat chamber. With the addition of FeSO4 (20 μL, 0.25 mmol/L), the reaction was conducted for 30 min at 37 °C. Ferrozine (15 μL, 2.5 mmol/L) was added to terminate the reaction. After incubation for 10 min at room temperature, the free Fe2+ remaining in solution, able to complex with ferrozine, was recorded at 562 nm using an automatic microplate reader (Tecan Infinite M200, Mannedorf, Switzerland). Deionized water was used as the blank. The iron-chelating rate was calculated as [(A0 − AS)/A0] × 100%, where A0 was absorbance of the blank and AS was absorbance of the sample.
2.4.3. Fourier transform infrared (FTIR) spectroscopy
FTIR spectra were recorded in KBr discs using an infrared spectrophotometer (PerkinElmer, Salem, MA, USA). The iron-peptide complexes were obtained by adding FeSO4 (0.4 mL, 0.25 mmol/L) into the peptide solution (5 mL, 1 mg/mL), respectively. The reaction was conducted at 37 °C and pH 5.0 for 1 h with constant agitation. Subsequently, lyophilized sample (1 mg), mixed with dried KBr (100 mg), was ground and pressed into a transparent pellet. The spectra were recorded over a wavenumber region from 4000 cm−1 to 400 cm−1 at a resolution of 4 cm−1.
2.4.4. Identification of peptide by LC-HRMS/MS and peptide sequencing
Samples were analyzed on a LC-30A UPLC system (Shimadzu, Kyoto, Japan) coupled with a Triple TOF 5600 + system (SCIEX, Singapore). The mass spectrometer was operated in the positive ESI mode with a Duo Spray source. Peptide sequence was identified using Protein Pilot software with Uniprot database. The false discovery rate was set to 0.01.
2.5. Statistical analysis
Data were presented as means ± standard deviation (SD) of triplicate determinations. A comparison of means was performed by Duncan’s multiple range test with the confidence level set at P < 0.05 using SPSS 20.0 software (Chicago, IL, USA).
3. Results and discussion
3.1. Effect of different factors on protein extraction rate from abalone viscera
Previous studies showed that sodium hydroxide concentration, ultrasonic power, extraction temperature and extraction time were regarded as main factors affecting protein extraction rate from different sources [20], [21].
Sodium hydroxide solution can be applied to extract protein as a result that sodium hydroxide is able to improve protein solubility in the solution by destroying the hydrogen bonds, dissociating some polar groups and changing the surface charge of protein [22]. With sodium hydroxide concentration increasing from 5 g/kg to 15 g/kg, protein extraction rate of abalone viscera had an increase up to a maximum value of 65.22 ± 0.32% at sodium hydroxide concentration 15 g/kg (Fig. 1A). But when the concentration of sodium hydroxide is too high, some adverse reactions, including peptide bond cleavage and Maillard reactions, may take place, which can lower protein extraction rate [23]. In this study, as sodium hydroxide concentration increased from 15 g/kg to 25 g/kg, protein extraction rate experienced a decline and fell to 59.23 ± 0.76%.
Fig. 1.
Protein extraction rate from abalone viscera affected by sodium hydroxide concentration (A), ultrasonic power (B), extraction temperature (C) and extraction time (D), respectively.
As ultrasonic power varied from 100 W to 500 W, Fig. 1B showed that protein extraction rate reached the maximum value of 65.18 ± 0.14% at ultrasonic power of 400 W. However, with the increase of ultrasonic power, protein extraction rate increased rapidly as a result of the strengthened interaction of protein molecule with water molecule. Cavitation and mechanical effects caused by ultrasonic action are able to promote the protein dissolution. But when ultrasonic power reaches a certain level, the too high ultrasonic power may cause the aggregation of dissolved protein molecules, which reduces the increasing speed of protein extraction rate [24].
With extraction temperature increasing from 30 °C to 40 °C, protein extraction rate went rapidly up to the value of 65.23 ± 0.82% (Fig. 1C). As extraction temperature continued to increase to 50 °C, protein extraction rate reached 64.84 ± 0.32%, showing no significant difference from protein extraction rate of 40 °C extraction temperature. As extraction temperature changed from 50 °C to 70 °C, protein extraction rate begun to decline. This result can be explained as follows. The improved extraction temperature can accelerate the movement of protein molecules and expand its structure. As a result, proteins can be isolated from raw materials more quickly, which results in improved protein extraction rate. However, excessive extraction temperature can cause protein denaturation and gelation, which makes adverse effect on protein extraction rate of abalone viscera [25].
Extraction time also plays an important part in the protein extraction process. As shown in Fig. 1D, with the prolonged extraction time from 15 min to 75 min, the maximum protein extraction rate was 65.83 ± 1.10% at extraction time of 45 min. The suitable extraction time is beneficial for protein dissolution from raw materials. Excessive extraction time may lead to lower protein extraction rate, resulting from protein aggregation and denaturation [24].
3.2. Optimization of the protein extraction procedure by RSM
3.2.1. Model fitting and statistical analysis
Response surface methodology was adopted to optimize the protein extraction condition for abalone viscera. Sodium hydroxide concentration (X1), ultrasonic power (X2) and extraction time (X3) were chosen as factors for further optimization of the protein extraction procedure by Box-Behnken design, resulting from their obvious effects on the protein extraction from abalone viscera. Protein extraction rate under a three-level three-factor factorial design was shown in Table 3. The protein extraction rate ranged from 58.37% to 65.87%. The highest protein extraction rate (65.87%) was obtained via the combination of sodium hydroxide concentration 15 g/kg, ultrasonic power 400 W and extraction time 45 min.
Table 3.
Box-Behnken design matrix.
| No. | X1/NaOH concentration (g/kg) | X2/Ultrasonic power (W) | X3/Extraction time (min) | Y/Protein extraction rate (%) |
|
|---|---|---|---|---|---|
| Measured Value | Predicted Value | ||||
| 1 | 10 | 400 | 60 | 63.66 | 63.51 |
| 2 | 10 | 500 | 45 | 63.55 | 63.28 |
| 3 | 10 | 400 | 30 | 60.37 | 60.39 |
| 4 | 15 | 300 | 30 | 58.97 | 58.55 |
| 5 | 15 | 400 | 45 | 65.52 | 65.04 |
| 6 | 20 | 300 | 45 | 59.58 | 59.85 |
| 7 | 10 | 300 | 45 | 60.95 | 61.35 |
| 8 | 20 | 400 | 60 | 62.61 | 62.59 |
| 9 | 15 | 500 | 60 | 63.75 | 64.17 |
| 10 | 15 | 400 | 45 | 64.55 | 65.04 |
| 11 | 20 | 400 | 30 | 58.37 | 58.52 |
| 12 | 15 | 400 | 45 | 64.93 | 65.04 |
| 13 | 15 | 400 | 45 | 64.31 | 65.04 |
| 14 | 15 | 300 | 60 | 63.23 | 62.98 |
| 15 | 15 | 500 | 30 | 61.19 | 61.44 |
| 16 | 20 | 500 | 45 | 62.39 | 61.99 |
| 17 | 15 | 400 | 45 | 65.87 | 65.04 |
Regression analysis was conducted to determine effect of factors as well as likely interactions between them and to evaluate statistical significance of the model. The regression equation for protein extraction rate (Y) was given as follows:
The statistical analysis of model equation and the significance of coefficients were estimated by F-value and P-value as summarized in Table 4. The observed model was significant for fitting the response, representing that this model could suitably describe the relation between response and factors. Moreover, three linear terms (X1, X2 and X3) and three quadratic terms (X12, X22 and X32) were all significant (P < 0.05), while three interaction terms (X1X2, X1X3 and X2X3) were all not significant (P > 0.05). The determination coefficient was 0.9683, indicating that 96.83% of the total variation could be explained by quadratic regression model.
Table 4.
Analysis of variance for the second-order polynomial model.
| Source | Sum of squares | Df | Mean square | F value | P value |
|---|---|---|---|---|---|
| Model | 82.34 | 9 | 9.15 | 23.79 | 0.0002 |
| X1/NaOH concentration | 3.89 | 1 | 3.89 | 10.12 | 0.0155 |
| X2/Ultrasonic power | 8.30 | 1 | 8.30 | 21.59 | 0.0024 |
| X3/Extraction time | 25.74 | 1 | 25.74 | 66.93 | < 0.0001 |
| X1X2 | 0.011 | 1 | 0.011 | 0.029 | 0.8703 |
| X1X3 | 0.23 | 1 | 0.23 | 0.59 | 0.4688 |
| X2X3 | 0.72 | 1 | 0.72 | 1.88 | 0.2128 |
| X12 | 16.43 | 1 | 16.43 | 42.72 | 0.0003 |
| X22 | 8.77 | 1 | 8.77 | 22.80 | 0.0020 |
| X32 | 13.76 | 1 | 13.76 | 35.79 | 0.0006 |
| Residual | 2.69 | 7 | 0.38 | ||
| Lack of Fit | 0.99 | 3 | 0.33 | 0.77 | 0.5666 |
| Pure Error | 1.70 | 4 | 0.43 | ||
| Cor Total | 85.03 | 16 |
R2 = 0.9683 RAdj2 = 0.9276.
3.2.2. Analysis of response surface model and contour plots
The full model resulting from regression equation was used to generate 3D response surface plots and contour plots for predicting the relationships between the independent and dependent variables. Fig. 2 showed a function of any two factors, and the third factor is kept fixed. With the increase of sodium hydroxide concentration (10–20 g/kg) and ultrasonic power (300–500 W), protein extraction rate increased at the beginning and decreased afterwards (Fig. 2A). Fig. 2B showed effect of sodium hydroxide concentration (10–20 g/kg) and extraction time (30–60 min) on protein extraction rate, and the maximum value of protein extraction rate was obtained around sodium hydroxide concentration 15 g/kg and extraction time 45 min. In Fig. 2C, sodium hydroxide concentration was kept at 15 g/kg, and protein extraction rate obtained the maximum value around ultrasonic power 400 W and extraction time 45 min. The predicted optimum condition obtained by Design-Expert 10.0 software (Minneapolis, USA) was the combined level of sodium hydroxide concentration 14 g/kg, ultrasonic power 428 W and extraction time 52 min, which was predicted to provide the maximum protein extraction rate of 65.64%.
Fig. 2.
Response surface plots (A-1, B-1 and C-1) and contour plots (A-2, B-2 and C-2) showing the interactive effects of sodium hydroxide concentration (X1), ultrasonic power (X2) and extraction time (X3) on protein extraction rate of abalone viscera by ultrasound assisted extraction.
3.2.3. Model validation and comparison with alkaline extraction method
To verify the mathematical model determined from RSM, protein extraction rate was measured under the predicted optimum parameters. The major aim of this approach is to estimate the levels of factors which could give the highest protein extraction rate. The combined levels of sodium hydroxide concentration 14 g/kg, ultrasonic power 428 W and extraction time 52 min were predicted to provide the highest protein extraction rate of 65.64%. The measured value of protein extraction rate at optimal points was 64.89 ± 0.29%. The measured protein extraction rate was found to be comparable to the predicted value created by the model. Therefore, the mathematical model was suitable for optimizing protein extraction rate of abalone viscera.
In comparison, conventional alkaline extraction method was used to extract abalone viscera protein under the condition of sodium hydroxide concentration 14 g/kg and extraction time 52 min. Protein extraction rate by AE was 55.67 ± 0.62%, which was lower than protein extraction rate obtained with UE process (64.89 ± 0.29%).
3.3. Measurement of isoelectric point of abalone viscera protein
As shown in Fig. 3, the precipitation rate of abalone viscera protein increased with the increase of pH value. With the pH value increasing from 2.0 to 4.0, protein precipitation rate increased and reached the maximum value of 77.62 ± 0.10% at pH 4.0. Subsequently, protein precipitation rate decreased rapidly. At the pH value of 6.0, protein precipitation rate fell to 13.52 ± 0.15%. The isoelectric point of abalone viscera protein was 4.0.
Fig. 3.
Isoelectric point of abalone viscera protein.
3.4. Effect of ultrasound assisted extraction on the iron-chelating activity
3.4.1. Effect of different proteases on the iron-chelating rate of hydrolysates
Compared with alkaline extraction, ultrasound assisted extraction can affect protein characteristic from abalone viscera, which may further have an influence on the iron-chelating rate of peptides. In order to evaluate effect of ultrasound assisted extraction on the generation of iron-chelating peptides, two abalone viscera proteins were hydrolyzed for 240 min using three commercial enzymes (trypsin, alcalase and flavourzyme) at their optimal conditions outlined in Section 2.4.1, respectively. During the preparation of iron-chelating peptides, abalone viscera protein obtained by UE showed similar characteristics with abalone viscera protein obtained by AE (Table 5). Alcalase and trypsin showed the similar ability to produce iron-chelating peptides from the two abalone viscera proteins. The iron-chelating rate of hydrolysate (alcalase, alkaline extraction, PAE) was 16.60 ± 0.22%, which had no significant difference from the iron-chelating rate of hydrolysate (alcalase, ultrasound assisted extraction, PUE) (16.24 ± 0.27%) and hydrolysate (trypsin, alkaline extraction) (16.25 ± 0.31%). The iron-chelating rate of hydrolysate (trypsin, ultrasound assisted extraction) was 16.19 ± 0.13% with no significant difference from the iron-chelating rate of PUE and hydrolysate (trypsin, alkaline extraction). However, among the three commercial enzymes, hydrolysates prepared by flavourzyme owned the lowest iron-chelating rate. The iron-chelating rate of hydrolysate by UE and hydrolysate by AE was 15.72 ± 0.09% and 15.78 ± 0.16%, respectively. In this study, alcalase was selected for enzymatic hydrolysis of abalone viscera protein to prepare iron-chelating peptides. In previous studies, alcalase was also used to generate mineral-chelating peptides. Zhu et al. [26] prepared defatted wheat germ protein hydrolysates by alcalase, flavourzyme and papain. Results showed that hydrolysates prepared by alcalase had the highest iron-chelating ability. Xie et al. [27] used alcalase to obtain rapeseed protein hydrolysates, from which four zinc-chelating peptides were identified.
Table 5.
Iron-chelating rate of different hydrolysates.
| Project | Trypsin | Alcalase | Flavourzyme |
|---|---|---|---|
| Ultrasound assisted extraction | 16.19 ± 0.13%b | 16.24 ± 0.27%ab | 15.72 ± 0.09%c |
| Alkaline extraction | 16.25 ± 0.31%ab | 16.60 ± 0.22%a | 15.78 ± 0.16%c |
Groups with different superscripts are significantly different at P < 0.05, examined by Duncan’s multiple range tests.
3.4.2. FTIR spectroscopy of iron-chelating peptides and iron-peptide complexes
FTIR spectroscopy can supply information about the interaction of ligand groups in peptide with mineral ion, which was of great use in studying formation and composition of mineral-peptide complexes [28]. As shown in Fig. 4, PUE showed different FTIR spectrum from PAE, which indicated that the composition of PUE was different from that of PAE. The amide A bands of PUE and PAE were observed at 3287 cm−1 and 3294 cm−1, respectively, which were assigned to stretching vibrations of N—H bond. In FTIR spectra of iron-peptide complexes, the amide A bands shifted to the higher frequency region (iron-PUE, 3310 cm−1; iron-PAE, 3307 cm−1). The absorption bands of PUE at 1620 cm−1 and PAE at 1611 cm−1 were characterized as amide I bands, which were associated with stretching vibrations of carbonyl group (C O) of amide (peptide bond). The amide II bands of PUE (1530 cm−1) and PAE (1532 cm−1) resulted from C-N stretching vibrations coupled with N–H bending vibrations. When iron-peptide complexes formed, amide I bands moved to the higher frequency region (iron-PUE, 1658 cm−1; iron-PAE, 1660 cm−1), and amide II bands of PUE (1530 cm−1) and PAE (1532 cm−1) shifted to the higher frequency region of 1550 cm−1 (iron-PUE) and 1533 cm−1 (iron-PAE), respectively. Moreover, the bands for carboxylate group (COO−) shifted from 1403 cm−1 (PUE) and 1402 cm−1 (PAE) to 1399 cm−1 (iron-PUE) and 1397 cm−1 (iron-PAE), respectively.
Fig. 4.
FTIR spectra of iron-chelating peptides and iron-peptide complexes in the region from 4000 cm−1 to 400 cm−1.
Therefore, PUE and PAE both could bind with ferrous ions through the amino and carboxylate terminal groups and peptide bond of the peptide backbone. Nevertheless, the side chain groups of some amino acids, including amino, imine and carboxylate, supplied additional iron binding sites.
3.4.3. Mass spectrometry identification and sequence analysis
The iron-chelating activity of two hydrolysates was affected by structural characteristic of their iron-chelating peptides [15], [29]. In order to make clear effects of ultrasound assisted extraction and alkaline extraction on the further preparation of iron-chelating peptides, two hydrolysates were analyzed and sequenced by LC-HRMS/MS.
As shown in Table 6, 24 iron-chelating peptides were identified from PUE, whose amino acid number was from 7 to 20. Additionally, 27 iron-chelating peptides, with amino acid number range from 6 to 20, were identified from PAE (Table 7). The main peptide sequences of PUE were different from that of PAE. Molecular weight is an important factor affecting iron-chelating activity. In previous studies, the identified iron-chelating peptides, with a high affinity to iron, own the molecular weight between 300 Da and 1500 Da [15]. The molecular weight of main peptide sequences from PUE was from 716.4069 Da to 1799.0054 Da. Additionally, the molecular weight of main peptide sequences from PAE was from 716.4432 Da to 1656.8260 Da. Most of identified iron-chelating peptides have the molecular weight as previous study described, except for Pro-Gly-Asn-Arg-Gly-Ser-Thr-Gly-Pro-Ala-Gly-Ile-Arg-
Table 6.
Main peptide sequences of PUE.
| No. | Amino acid sequence | Amino acid number | Molecular weight (Da) |
|---|---|---|---|
| 1 | Glu-Ala-Gly-Thr-Val-Leu-Lys | 7 | 716.4069 |
| 2 | Gln-Val-Pro-Val-Leu-Asp-Asp | 7 | 784.3967 |
| 3 | Pro-Val-Pro-Val-Thr-Thr-Gln-Asn | 8 | 854.4498 |
| 4 | Gly-Arg-Ile-Gln-Gly-Asn-Pro-Glu | 8 | 869.4355 |
| 5 | Thr-Val-Glu-Gln-His-Ile-Gly-Asn | 8 | 896.4352 |
| 6 | Lys-Pro-Lys-Pro-Lys-Pro-Lys-Pro | 8 | 918.6015 |
| 7 | Val-Glu-Lys-Ser-His-Pro-Gly-Pro-Asn | 9 | 963.4774 |
| 8 | Thr-Gln-Ser-His-Gln-Ala-Ala-Leu-Met | 9 | 985.4651 |
| 9* | Glu-Leu-Gly-Pro-Lys-Gly-Thr-Gln-Gly-Thr | 10 | 986.5033 |
| 10 | Leu-Asn-Asn-Lys-Val-Lys-Ile-Leu-Val | 9 | 1039.6754 |
| 11 | Lys-Asp-Ser-Gln-Ser-Asn-Phe-Lys-Asn | 9 | 1066.5044 |
| 12 | Gln-Asn-Pro-Pro-Gly-Ala-Pro-Pro-Asn-Val-Val | 11 | 1088.5614 |
| 13* | Tyr-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly | 14 | 1242.6033 |
| 14 | Thr-Ser-Ala-Ala-Thr-Thr-Gly-Gly-Pro-His-Arg-Thr-Val | 13 | 1254.6317 |
| 15 | Glu-Leu-Lys-Glu-Asp-Ser-Leu-Trp-Ser-Ala-Lys | 11 | 1304.6613 |
| 16 | Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Ala-Val-Val-Asn | 15 | 1308.6826 |
| 17 | Lys-Ser-Val-Arg-Ile-Ser-Glu-His-Val-Arg-Asn | 11 | 1323.7372 |
| 18 | Leu-Ala-Ser-Pro-Arg-Thr-Ala-Val-Ala-Pro-Ser-Ala-Val-Asn | 14 | 1352.7412 |
| 19* | Gly-Pro-Pro-Gly-Lys-Pro-Gly-Pro-Gln-Gly-Pro-Thr-Gly-Asp-Asn | 15 | 1374.6528 |
| 20 | Thr-Pro-Pro-Pro-Gly-Thr-Thr-Ala-Pro-Pro-Pro-Pro-Gly-Pro-Ala-Ser | 16 | 1440.7249 |
| 21* | Pro-Gly-Asn-Arg-Gly-Ser-Thr-Gly-Pro-Ala-Gly-Ile-Arg-Gly-Pro-Asn | 16 | 1506.7651 |
| 22 | Ala-Pro-Ser-His-His-His-Thr-Val-His-Leu-Gly-Ser-Ser-Ala-Pro | 15 | 1533.7437 |
| 23 | Arg-Ala-Val-Arg-Ser-Ser-Asp-Glu-Val-Gln-Asn-Ser-Arg-Ser | 14 | 1589.7870 |
| 24* | Leu-Ala-Gly-Glu-Pro-Gly-Lys-Pro-Gly-Ile-Pro-Gly-Leu-Pro-Gly-Arg-Ala-Gly-Gly-Val | 20 | 1799.0054 |
* stands for collagen peptides.
Table 7.
Main peptide sequences of PAE.
| No. | Amino acid sequence | Amino acid number | Molecular weight (Da) |
|---|---|---|---|
| 1 | Glu-Ile-Val-Lys-Thr-Lys | 6 | 716.4432 |
| 2 | Leu-Asn-Met-Thr-Lys-Leu | 6 | 718.4047 |
| 3 | Thr-Tyr-Pro-Glu-Leu-Thr | 6 | 722.3487 |
| 4 | Ser-His-Gly-Pro-Lys-Thr-Asn | 7 | 739.3613 |
| 5 | Pro-Thr-Tyr-Pro-Gly-Pro-Asn | 7 | 744.3442 |
| 6 | Thr-Asn-Ala-Thr-His-Ala-Gly-Asn | 8 | 784.3464 |
| 7 | Val-His-Arg-Cys-Val-Asn | 6 | 784.3650 |
| 8 | Lys-Asn-His-Gly-Tyr-Pro-Asn | 7 | 828.3879 |
| 9 | Gly-Lys-Phe-Ser-Lys-Ser-Ala-Asn | 8 | 837.4344 |
| 10 | Thr-Leu-Ala-Phe-Met-Ala-Val-Asn | 8 | 865.4368 |
| 11 | Gln-Thr-Arg-His-Met-Ser-Pro-Gly | 8 | 912.4236 |
| 12 | Tyr-Gly-Gln-Val-Leu-His-Ser-Asn | 8 | 916.4403 |
| 13 | Leu-Asn-Lys-Val-Asn-Lys-Val-Ile | 8 | 926.5913 |
| 14 | Gly-Pro-Lys-Thr-Ala-Ser-Asp-Lys-Ser-Asp | 10 | 1004.4775 |
| 15 | Thr-Pro-Gly-Ala-Gly-Gly-Thr-Ala-His-Arg-Arg | 11 | 1079.5585 |
| 16 | Ile-Glu-Thr-His-Glu-Gln-Lys-Ile-Ser | 9 | 1083.5560 |
| 17 | Phe-Ile-Asn-Gly-His-Lys-Met-Asp-Gln | 9 | 1088.5073 |
| 18 | Gly-Pro-Lys-Pro-Gly-Pro-Gly-Pro-Gly-Gly-Pro-Lys-Gly-Gly | 14 | 1158.6145 |
| 19 | Ala-Thr-Asn-Gly-Lys-Asp-Lys-Ser-Gly-Asp-Asn | 12 | 1206.5476 |
| 20 | Lys-Ala-Leu-Gln-Thr-Ala-Lys-Ala-Ser-Pro-Pro-Asn | 12 | 1224.6826 |
| 21 | Thr-Tyr-Lys-Thr-Pro-Gln-Tyr-Thr-Lys-Asn | 10 | 1242.6245 |
| 22* | Asn-Lys-Gly-Ala-Pro-Gly-Pro-Thr-Gly-Pro-Pro-Gly-Gly-Pro-Gly | 15 | 1259.6259 |
| 23* | Pro-Gly-Pro-Pro-Gly-Tyr-Pro-Gly-Lys-Gln-Gly-Pro-Asn | 13 | 1264.6200 |
| 24 | Lys-Pro-Gly-Arg-Arg-Ala-Thr-Thr-Gln-Thr-Val-Arg | 12 | 1369.7903 |
| 25* | Gly-Pro-Pro-Gly-Pro-Pro-Gly-Tyr-Pro-Gly-Lys-Gln-Gly-Pro-Asn | 15 | 1418.6942 |
| 26 | Glu-Met-Lys-Ala-Leu-Lys-Glu-Leu-Gly-Arg-Phe-Ser-Ile-Leu | 14 | 1633.9226 |
| 27* | Ala-Gly-Val-Ala-Gly-Pro-Phe-Gly-Pro-Pro-Gly-Ala-Pro-Gly-Thr-Pro-Gly-Pro-Pro-Gly | 20 | 1656.8260 |
*Stands for collagen peptides.
Gly-Pro-Asn (1506.7651 Da), Ala-Pro-Ser-His-His-His-Thr-Val-His-Leu-Gly-Ser-
Ser-Ala-Pro (1533.7437 Da), Arg-Ala-Val-Arg-Ser-Ser-Asp-Glu-Val-Gln-Asn-Ser-
Arg-Ser (1589.7870 Da) and Leu-Ala-Gly-Glu-Pro-Gly-Lys-Pro-Gly-Ile-Pro-Gly-
Leu-Pro-Gly-Arg-Ala-Gly-Gly-Val (1799.0054 Da) from PUE as well as Glu-Met-Lys-Ala-Leu-Lys-Glu-Leu-Gly-Arg-Phe-Ser-Ile-Leu (1633.9226 Da) and Ala-Gly-Val-Ala-Gly-Pro-Phe-Gly-Pro-Pro-Gly-Ala-Pro-Gly-Thr-Pro-Gly-Pro-Pro-
Gly (1656.8260 Da) from PAE. Viewing peptide sequences from Tables 6 and 7, the C-terminus of peptide sequences from PUE mainly was Asn and Val, and the C-terminus of peptide sequences from PAE mainly was Asn and Gly. Storcksdieck genannt Bonsmann et al. [30] identified Trp-Ala-Ala-Phe-Pro-Pro-Asp-Val-Ala-
Gly-Asn, Gln-TyrP-Gln-Thr-Pro-Leu-Phe-Val-Trp-SerP-Val and Gly-Val-Phe-Phe-
Pro-Gly-Gly-Leu-Gly-Val, whose C-terminus was Asn or Val. Lee et al. [31] identified a peptide sequence of Asp-Leu-Gly-Glu-Gln-Tyr-Phe-Lys-Gly, with the C-terminus of Gly. Ser-Met-Arg-Lys-Pro-Pro-Gly identified by Zhang et al. [32] also has the C-terminus of Gly. Ultrasound technology has affected protein proteolysis process for the production of iron-chelating peptides, which was beneficial to supply more abundant iron-chelating peptides with high iron-chelating activity. This can be attributed to the reason that ultrasound technology makes an effect on protein extraction process, leading to the result that proteins extracted by UE and AE have different amino acid compositions [11]. Additionally, ultrasound technology can accelerate the protein proteolysis process, which also can affect the structural characteristic of PUE [21], [33]. However, the iron-chelating peptides, identified from PUE and PAE, shared some similarities. For example, some amino acids with strong iron-chelating ability (Arg, Asn, Asp, Gln, Glu, His, Lys and Tyr) were abundant in both of the main peptide sequences of PUE and PAE. Moreover, as shown in Table 6, five collagen peptides were identified from PUE, including Glu-Leu-Gly-Pro-Lys-Gly-Thr-Gln-Gly-Thr, Tyr-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly, Gly-Pro-Pro-Gly-Lys-Pro-Gly-Pro-Gln-Gly-Pro-Thr-Gly-Asp-Asn, Pro-Gly-Asn-Arg-Gly-Ser-Thr-Gly-Pro-Ala-Gly-Ile-Arg-Gly-Pro-Asn and Leu-Ala-Gly-Glu-Pro-Gly-Lys-Pro-Gly-Ile-Pro-Gly-Leu-Pro-Gly-Arg-Ala-Gly-Gly-Val. Four collagen peptides were identified from PAE (Table 7), including Asn-Lys-Gly-Ala-Pro-Gly-Pro-Thr-Gly-Pro-Pro-Gly-Gly-Pro-Gly, Pro-Gly-Asn-Arg-Gly-Ser-Thr-Gly-Pro-Ala-Gly-Ile-Arg-Gly-Pro-Asn, Gly-Pro-Pro-Gly-Pro-Pro-Gly-Tyr-Pro-Gly-Lys-Gln-Gly-Pro-Asn and Ala-Gly-Val-Ala-Gly-Pro-Phe-Gly-Pro-Pro-Gly-Ala-Pro-Gly-Thr-Pro-Gly-Pro-Pro-Gly. This result illustrated that collagen accounted for a certain percentage in the two abalone viscera proteins. However, collagen has been proved to be a good source for the production of iron-chelating peptides [19].
4. Conclusions
This study investigated effects of ultrasound technology on protein extraction process and the iron-chelating activity. Results demonstrated that ultrasound assisted extraction was an efficient technology for the development of abalone viscera protein, which was a good source for the production of iron-chelating peptides. Compared with conventional alkaline extraction, ultrasound assisted extraction could improve protein extraction rate. Alcalase showed high efficiency in generating iron-chelating peptides. PUE and PAE shared some same iron binding sites and no significant differences in iron-chelating rate. However, main peptide sequences of PUE were different from that of PAE. Based on peptides with high iron-chelating activity from PUE and PAE, further studies are required for a better understanding of iron-peptide binding mode as well as promotive effects of iron bioavailability by iron-chelating peptides.
CRediT authorship contribution statement
Wenfei Wu: Conceptualization, Investigation, Methodology, Funding acquisition, Supervision, Writing - review & editing. Jiao Jia: Investigation, Writing – original draft. Chengrong Wen: Conceptualization, Investigation, Methodology. Cuiping Yu: Conceptualization, Investigation, Methodology. Qi Zhao: Conceptualization, Investigation, Methodology. Jiangning Hu: Conceptualization, Methodology, Supervision.
Declaration of Competing Interest
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.
Acknowledgements
This work was financially supported by the Doctoral Startup Fund by Science and Technology Bureau of Liaoning Province (No. 2019-BS-017), Dalian High-level Talent Innovation Support Program of China (No. 2019RQ003) and Central Funds Guiding the Local Science and Technology Development (No. 2020JH6/10500002).
Contributor Information
Wenfei Wu, Email: winfywu@163.com.
Jiangning Hu, Email: hjiangning@163.com.
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