Optimization of ACE inhibitory peptides from black soybean by microwave-assisted enzymatic method and study on its stability

Accepted Manuscript Optimization of ACE inhibitory peptides from black soybean by microwave-assisted enzymatic method and study on its stability Meiqing Li, Shanwei Xia, Yijun Zhang, Xueling Li PII:

S0023-6438(18)30698-4

DOI:

10.1016/j.lwt.2018.08.045

Reference:

YFSTL 7357

To appear in:

LWT - Food Science and Technology

Received Date: 23 April 2018 Revised Date:

16 July 2018

Accepted Date: 23 August 2018

Please cite this article as: Li, M., Xia, S., Zhang, Y., Li, X., Optimization of ACE inhibitory peptides from black soybean by microwave-assisted enzymatic method and study on its stability, LWT - Food Science and Technology (2018), doi: 10.1016/j.lwt.2018.08.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Optimization of ACE inhibitory peptides from black soybean by

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microwave-assisted enzymatic method and study on its stability

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Meiqing Lia, Shanwei Xiaa, Yijun Zhanga, Xueling Lia

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230036, Anhui, China

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School of Tea and Food Science & Technology, Anhui Agricultural University, Hefei

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E-mail: [email protected]; Tel: +86 13605699537 1

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Abstract To optimize the preparation of ACE inhibitory peptides from black soybean (Glycine max (L.)

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Merr.), three enzymatic methods were compared, with the technical conditions optimized by response

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surface analysis. The black soybean protein hydrolysate inhibited 70.38% of the ACE activity. The

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ACE inhibitory peptides were isolated from black soybean protein hydrolysates by macro-porous resin,

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ultrafiltration, and Sephadex G-15. Four fractions were obtained, with each fraction having some ACE

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inhibitory activity. Fraction III exhibited the highest activity of 90.78%, and a molecular weight range

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of 145-468 Da. The ACE inhibitory peptides were stable across a range of pH values (2-10), at

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temperatures <40 ℃, and in the presence of metal ions (Ca2+, K+, Mg2+), but had little resistance to

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digestive enzymes. These results indicated that the ACE inhibitory peptides of black soybean are

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substrate-type inhibitory peptides.

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Keywords

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Black soybean, ACE inhibitory peptides isolates, Microwave-assisted enzymatic, Stability, Functional

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food

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1. Introduction

The Angiotensin I-Converting Enzyme (ACE, a dipeptidyl carboxypeptidase, EC 3.4.15.1) plays

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important roles in the regulation of blood pressure and cardiovascular function. ACE converts the

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inactive decapeptide angiotensin I into the potent vasoconstricting octapeptide angiotensin II, and also

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inactivates the vasodilator bradykinin (Li, Wan, Le, & Shi, 2006). Hypertension affects around one

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billion people worldwide and contributes to approximately 9.4 million cardiovascular disease deaths

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each year (Ganne, Arora, Dotsenko, Mcfarlane, & Whaley, 2007). Peptides that inhibit ACE activity

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can help regulate blood pressure and have been obtained through hydrolysis of animal and plant

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proteins under mild conditions. Most of these peptides contain 2-15 amino acids (Li, Le, Shi, &

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Shrestha, 2004). Food-derived ACE inhibitors are gaining attention because their effects are mild,

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specific, and durable, safe, and they do not affect the blood pressure of people with normal blood

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pressure (Chen, Chi, Zhao, & Xu, 2012; Hu et al., 2012).

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Currently available ACE inhibitory peptides are derived from casein (Corrons, Liggieri, Trejo, &

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Bruno, 2017; Lin, Zhang, Han, & Cheng, 2017), plants (Li, Wan, Le, & Shi, 2006), fish (Elavarasan,

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Shamasundar, Badii, & Howell, 2016; García-Moreno et al., 2015), and meat proteins (Jang & Lee,

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2005). Using various enzymes, Yak milk casein derived from Qula, a traditional Tibetan acid curd

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cheese, was hydrolyzed and then fractioned into two molecular weight ranges using a 3 kDa

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ultrafiltration membrane. The highest ACE inhibitory activity, an IC50 of 8.75 µg/mL, was in the low

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molecular weight fraction (< 3 kDa) of the isolate prepared by hydrolysis with thermolysin (Lin et al.,

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2017). In a different report, the proteolytic enzymes from Maclura pomifera were used to hydrolyze

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bovine caseins. Peptides with ACE inhibitory activity were partially purified from the hydrolysate by

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chromatographic

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YQEPVLGPVRGPFPIIV and RFFVAPFPE (Corrons, Liggieri, Trejo, & Bruno, 2017). Fourteen novel

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ACE-inhibitory peptides were identified by MS spectroscopy, with the peptide VAMPF identified as

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one of the most promising (García-Moreno et al., 2015). The peptide sequence VLAGNTL from beef

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hydrolysates inhibited 30.1% of the ACE activity. This potent ACE inhibitor might be used to develop

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beef containing a bioactive peptide that lowers blood pressure (Jang & Lee, 2005).

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techniques.

Black soybeans (Glycine max (L.) Merr.) not only contain abundant protein, fat, and essential

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nutrients (Wang et al, 2008), but are also believed to reduce blood pressure in China and Southeast Asia.

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The current search for bioactive substances within black soybean has focused primarily on proteins,

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pigments, polysaccharides and fatty acids, including globulin (Ajibola, Malomo, Fagbemi, & Aluko,

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2016), peroxide (POD) and superoxide dismutase (SOD) (Liu et al., 1996), and antifungal proteins

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(Ngai & Ng, 2003). However, there has been little research on the ACE inhibitory peptides within black

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soybean.

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The objectives of this study were to prepare black soybean ACE inhibitory peptides via

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microwave-assisted Alcalase hydrolysis, to optimize the technological parameters by response surface

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methodology, and to then analyze the molecular weight distribution and stability of the prepared

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peptides.

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2. Materials and methods

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2.1. Materials

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Black soybeans were purchased from a local supermarket. Alcalase (200000 U/g) was purchased

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from Jiang Su Ruiyang Biotech Co., Ltd., Jiang Su, China. Porcine pepsin (806.3 U/mg), bovine 4

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Furanacrylic acid phenylalanine-glycine-glycine (FAPGG) were obtained from Sigma-Aldrich

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Company, USA. Sephadex G-15 was purchased from Pharmacia Company, USA. DA201-C

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macroporous resin was purchased from Hangzhou Puxiu Biological Technology Co. Ltd. Hangzhou,

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China. TSKgel G2000SWXL was purchased from TOSOH company, Japan. The relative molecular

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weight standard was derived from bovine serum albumin (MW 66430), cytochrome C (MW 12500),

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and Glycine- Glycine- Glycine (MW 189), which were obtained from the National Institute of Control

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of Pharmaceutical and Biological Products, China. Chromatographic grade acetonitrile was purchased

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from Tedia Company, Inc., USA. Other reagents used were of analytical grade.

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Equipment used included a 752 UV spectrophotometer, Shanghai Tian Mei Scientific Instrument

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Co., Ltd; a microwave drying oven, PyNN corporation; a vacuum freezing drying oven, Ningbo Scientz

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Biotechnology Co., Ltd; an IKA RV 10 digital vacuum rotary evaporator, Prima Technology Group Co.,

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Ltd; a computer automatic fraction collector, Shanghai Qingpu Huxi Instrument; a MAX 190

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microplate reader, Molecular Devices; and a Waters 600 high performance liquid chromatography

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system, Waters Company.

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2.2. Preparation of black soybean protein

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Protein was isolated from black soybean by extraction with alkaline water (pH 8.5, 1:10 w/v flour:

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water ratio) for 30 min at 55 ℃. The resulting suspension was centrifuged at 4000 rpm for 15 min. The

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supernatants were combined, and the pH was adjusted to 4.3-4.5 with 1 mol/L HCl. After 20 minutes,

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the acidic protein precipitate was separated by centrifugation. The precipitate was washed twice with

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distilled water and then lyophilized.

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2.3. Preparation of black soybean peptides

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2.3.1.

Selection of the enzymatic hydrolysis method

The precipitated black soybean proteins were hydrolyzed by Alcalase in three different ways to

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investigate which would be the best enzymatic hydrolysis method, with the degree of hydrolysis and

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ACE inhibitory activity as assessment indices. Black soybean protein (10 g) was mixed with 100 mL of

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distilled water. The enzymatic hydrolysis was terminated by heating for 10 min in a 95 ℃ water bath.

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After the solution cooled to room temperature, the pH was adjusted to 9.0, and a 6% Alcalase enzyme

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dosage (enzyme / substrate, w/w) was added for enzymatic hydrolysis using three methods: Method 1

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(conventional enzymatic hydrolysis method)- hydrolysis was performed for 2 h in a water bath at

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50 ℃; Method 2- hydrolysis was performed using a microwave drying oven at 30 ℃, 300 W for 15

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min; Method 3- hydrolysis was performed using a microwave drying oven at 30 ℃, 300 W for 15 min,

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followed by 105 min in a water bath at 50 ℃.

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2.3.2.

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Single factors experiments of preparation technology

The basic enzymatic parameters were as follows: microwave power, 325 W; microwave enzymatic

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hydrolysis time, 15 min; solid-to-liquid ratio, 2%; dosage of enzyme, 6%; pH, 10.0. Each of these

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parameters were varied as follows: microwave power: 130, 227.5, 325, 422.5, 520 W; microwave

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enzymatic hydrolysis time: 10, 15, 20, 25, 30 min; solid-to-liquid ratio: 1, 2, 3, 4, 5%; dosage of

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enzyme: 2, 4, 6, 8, 10%; pH: 9.0, 9.5, 10.0, 10.5, 11.0.

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2.3.3.

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Optimization of technological parameters by response surface methodology

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ACCEPTED MANUSCRIPT Based on the single factor experiments, a Box-Behnken design was used to determine the optimal

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conditions of microwave-assisted enzymolysis, with the degree of hydrolysis as the response value, and

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the microwave power as factor A, the microwave enzymatic hydrolysis time as factor B, and the dosage

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of the enzyme as factor C.

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2.4. Separation and purification

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Black soybean protein hydrolysate was prepared by microwave-assisted enzymolysis using the

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predicted optimal conditions determined by response surface methodology. The enzymatic hydrolysis

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was terminated by heating for 5 min in a water bath at 90 ℃. The protein hydrolysate was centrifuged

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at 4000 r/min for 10 min. The resulting supernatant was collected and then lyophilized.

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2.4.1.

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Desalting through macroporous resin

The DA201-C macroporous resin was loaded into a chromatographic column (1.6 × 30 cm). The

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elution conditions for the macroporous resin were optimized in preliminary experiments to maximize

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the adsorption rate and the adsorption capacity. The black soybean protein hydrolysate was loaded at a

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sample concentration of 14 mg/mL (adjust pH to 4.0 with HCl), the flow rate was 0.5 mL/min, the

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eluent was 70% ethanol, the elution flow rate was 1.5 mL/min, and the elution volume was 30 mL. The

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fractions collected from the macroporous resin were freeze-dried and subjected to an ACE inhibition

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assay (Section 2.8). The desalination rate of each active fraction was calculated with the following

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equation:

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Desalination rate (%) = (1-D2/D1) × 100

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Where D1 represented ash content of sample before desalination (g/100g); D2 represented ash 7

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content of desalted sample (g/100g). The desalination rate of the above formula was up to 90.46%.

2.4.2.

Ultrafiltration

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The desalted peptide fraction was adjusted to 200 µg/mL and added to an ultrafiltration tube (3

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kDa), which was then centrifuged at 5000 r/min for 20 min. Each fraction (filtrate and filter wash) was

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lyophilized and then assayed for ACE inhibitory activity (Section 2.8).

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2.4.3.

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The lyophilized powder after size fractionation was reconstituted in distilled water to a

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concentration of 55 mg/mL. A portion of the sample (2 mL) was loaded onto and separated by

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Sephadex G-15 (1.6 × 60 cm), with ultrapure water as the eluent and an elution flow rate of 0.3 mL/min.

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The elution was monitored at 280 nm. Fractions (5 mL) were collected from at least six Sephadex G-15

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runs and individually freeze-dried into powder. Each fraction was subjected to ACE inhibition assay

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(Section 2.8) and protein content measurement.

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2.5. Characterization of stability

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The Sephadex G-15 Fraction III was further characterized, including a study of its stability, as

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described below.

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2.5.1.

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Effect of pH

Fraction III was dissolved in acetic acid-sodium acetate buffer solution at pH 2, 4, 6, 8, or 10 (to a concentration of 10 mg/mL) for 2 h before assay of its ACE-inhibiting activity.

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2.5.2.

Effect of Temperature

Fraction III was prepared to 10 mg/mL in distilled water, held at 20, 40, 60, 80, or 100 ℃ for 2 h

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before assay of its ACE-inhibiting activity.

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2.5.3.

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Effect of Metal Ions in Solution

Fraction III was prepared to 10 mg/mL in distilled water. Dry CaCl2, KCl, or MgCl2 was added

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and dissolved to concentrations of 0, 2, 4, 6, or 8 mmo1/L. After incubation at room temp for 2 h, the

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ACE-inhibiting activity was measured.

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2.5.4.

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Effect of Digestive Enzymes in vitro

In vitro digestion was carried out according to the method described in (Escudero, Mora, & Toldrá,

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2014), with slight modification. Briefly, a pepsin solution in 1 mol/L HCl (pH 2.0) was added to black

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soybean peptide extract at a 1:100 enzyme to substrate ratio. The digestion was at 37 ℃ for 2 h under

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continuous stirring, after which the reaction was heated to 100 ℃ for 5 minutes, to inactive the

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enzyme, and then cooled to room temperature. The pH was adjusted to 8.3 with 1 mol/L NaOH before

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centrifugation at 5000 r/min for 20 min. The centrifugal supernatant was assayed for ACE inhibition.

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The supernatant was digested with 2% trypsin at 37 ℃ for 2.5 h, before inactivation by heating at

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95℃ for 10 min. The digestion mixture was centrifuged at 5000 r/min for 20 min. This final

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supernatant was assayed for ACE inhibitory activity.

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2.6. Determination of the peptide content

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The peptide content was measured by UV absorbance difference at 215 and 225 nm (Murphy &

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Kies, 1960).

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2.7. Determination of the degree of hydrolysis

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The degree of hydrolysis (DH) of the hydrolysates was measured using the pH-stat method as

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described (Adlernissen, 1986) previously with minor modifications. The equation was as follows:

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Where B was the volume of NaOH solution consumed during the reaction (mL), Nb was the

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concentration of NaOH solution used during the reaction (mol/L), α was the average dissociation

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degree of α-NH2 (1/α = 1.01 for Alcalase), mp was the protein mass , htot was the total number of

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peptide bonds in protein substrate (htot =7.8).

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2.8. Assay for ACE-inhibiting activity

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ACE inhibition activity was determined according to Shalaby (Shalaby, Zakora, & Otte, 2006)

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with slight change. FAPGG was used as substrate and measured on an enzyme scale. The ACE solution

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was ready for use. The 1 mL distilled water was slowly injected into a glass bottle of 0.25 U ACE,

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mixed evenly and ready for use. The specific methods were as follows: 10 µL ACE aqueous solution

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(0.25 U/mL) and 10 µL ACE inhibitory peptide solution (10 mg/mL) were not mixed in the enzyme

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labelled plate, and then added the 150 µL substrate (1 mmol/L FAPGG was dissolved in 50 mmol/L

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Tris-HCl of pH=7.5, including 0.3mol/L NaCl) of preheating (37 ℃, 5min), made them start to react.

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The microplate was rapidly placed into the enzyme labelling instrument, recording the absorbance A1 at

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340 nm, and then measuring the absorbance A2 at 340 nm after 30 min reaction. The blank control used

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10 µL buffer solution (50 mmol/L Tris-HCl of pH=7.5, including 0.3 mol/L NaCl) instead of ACE

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inhibitory peptide solution. The blank initial absorbency was recorded as A01, and the reaction was

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recorded as A02. The inhibition rate of ACE was calculated with the change of absorbance (Ainhibitor = A1

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- A2, A control = A01 – A02). The extent of inhibition was calculated as follows:

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ACE inhibition activity (%) = (1- Ainhibitor / Acontrol)×100%

2.9. Determination of relative molecular weight distribution in the hydrolysate

The molecular weight range of the peptides was determined by HPLC using a Waters 600

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high-performance liquid chromatography equipped with a 2487 UV detector, a column containing

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TSKgel G2000SWXL (7.8 × 300 mm) running a mobile phase of acetonitrile: water: trifluoroacetic

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acid (45:55:0.1, v/v). Fractions were tracked at a wavelength of UV 220 nm. The flow rate was 0.5

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mL/min; the column temperature was 30 ℃. Data were automatically analyzed by Waters GPC.

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Standards for the calibration curve of relative molecular weight were: Bovine Serum Albumin

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(MW 66430), cytochrome C (MW 12500) and Aminoacetic acid-Aminoacetic acid-Aminoacetic acid

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(MW 189). The regression equation between molecular weight (MW) and the retention time (T) was as

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follows: LgMW = 7.0753-0.2161T, R2 = 0.9909.

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2.10.

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Data Processing Method

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All experimental data was the average of three parallel tests. The error was calculated by analysis

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of variance in the SPSS 17.0 software.

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3.

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3.1. Molecular weight distribution of black soybean protein isolate

Results and discussion

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ACCEPTED MANUSCRIPT The protein from the black soybean flour, released using alkaline solution and precipitated with

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acid, showed a molecular weight distribution centered on 31697 Da, which accounted for about 91.69%

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of the protein (Fig.A.1).

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3.2. Comparison of enzymatic hydrolysis methodologies

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The ACE inhibitory activity of the acid-precipitated protein and of the isolates further hydrolyzed

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by three different methods were determined (Table 1). The ACE inhibitory activity increased (from

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18.6% of the crude protein) after any of the three hydrolysis methods (to 58-70%). While there was no

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significant difference in the degree of hydrolysis (P > 0.05) among the three hydrolysis methods, there

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were significant differences (P < 0.05) among the rate of ACE inhibition, with method 2 resulting in a

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much higher activity. Method 2, microwave-assisted Alcalase hydrolysis, was chosen for further

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optimization. Microwave radiation can directly and quickly transfer energy to both biomass and

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catalyst, increasing reaction efficiency and shortening reaction time (Zhang, Yang, & Mester, 2012).

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3.3. Optimization through single factor experiments

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Five factors within Method 2, namely microwave power, microwave enzymatic hydrolysis time,

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solid-to-liquid ratio, enzyme dosage, and pH, were varied in the hydrolysis reaction. The degree of

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hydrolysis (DH) was measured following each of these wet experiments (Fig.A.2).

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3.3.1.

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Influence of microwave power on DH

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Five different levels of microwave power were tested during hydrolysis of black soybean protein

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(Fig.A.2a). The degree of hydrolysis increased, then dropped a little, with increasing microwave power,

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with the highest the degree of hydrolysis at 227.5 W. This could be because increased irradiative power 12

ACCEPTED MANUSCRIPT stimulates the catalytic reaction, resulting in improved yield (Wang, Yang, & Huang, 2014), until the

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temperature exceeds the optimum temperature of the enzyme, after which the enzyme activity

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(Onderková, Bryjak, Vaňková, & Polakovič, 2010) and thus, the degree of hydrolysis decreased.

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Therefore, 227.5 W was selected as the best microwave power.

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3.3.2.

Influence of microwave enzymatic hydrolysis time on DH

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Five different incubation times for microwave-assisted enzymatic hydrolysis were tested during

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hydrolysis of black soybean protein (Fig.A.2b). The degree of hydrolysis increased, peaked at 20 min,

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then dropped with increased time, which was due to a gradual reduction in enzymatic activity over time

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(Su, Wang, Shi, & Yang, 2013).

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Influence of the solid-to-liquid ratio on DH

Five different solid-to-liquid ratios were tested during hydrolysis of black soybean protein

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(Fig.A.2c). The degree of hydrolysis increased with increasing solid-to-liquid ratio. This may indicate

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that a low substrate concentration leads to enzymes not combined with the substrate, which would

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result in a low degree of hydrolysis. At increased substrate concentrations, more enzymes could

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combine with the substrate, increasing the overall rate, and, thus, the degree of hydrolysis (Ruan et al.,

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2013). Once all of the enzymes and substrates were bound, the reaction rate and degree of hydrolysis

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would reach a maximum value, beyond which increased substrate concentration did not improve the

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degree of hydrolysis, indicating enzyme saturation. The optimal solid-to-liquid ratio was set at 4%.

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Because the ratio of solid-to-liquid did not significantly influence the DH, it was not included as a

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response surface test factor.

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3.3.4.

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Influence of enzyme dosage on DH 13

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Increasing the amount of the enzyme from 2% to 4% increased the degree of hydrolysis, but

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further increases in the enzyme dose did not (Kwiatkowska, Bennett, Akunna, Walker, & Bremner,

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2011) (Fig.A.2d), which was attributed to a reaching enzyme saturation at 4%.

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3.3.5.

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Influence of pH on DH

The optimum pH of Alcalase was reported to be between pH 9 and pH 11 (Liu & Wang, 2011).

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There was little change in the degree of hydrolysis of black soybean proteins at these high pH levels

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(Fig.A.2e), indicating that there was no effect within this pH range. The optimum pH was set at pH 9.

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Because the pH had no significant influence on DH, it was not furthered as a response surface test

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factor.

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3.4. Response surface analysis modeling

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Response surface design

Based on the optimization of single factor experiment results, three factors in the hydrolysis were

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varied at three levels in a Box-Behnken design (BBD) and for analysis by response surface analysis.

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The three varied factors were microwave power (A), hydrolysis time (B), and enzyme dosage (C)

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(Table 2). The experimental data were analyzed with Design-Expert 8.0.5, using the following

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quadratic polynomial regression equation for the degree of hydrolysis:

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Y=42.78 + 1.14A − 0.5B + 0.8C + 0.65AB − 1.25AC + 0.6BC − 2.66A2 −0.81B2 − 1.91C2

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In Table 3, the quadratic regression equation had a high significance (P < 0.0001) and a low

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lack-of-fit (0.2812 > P 0.05). The correlation coefficient (R2) of this model was 0.9764, the adjusted R2

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was 0.8538, indicating that the model was good and had high precision. This model was able to analyze

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and predict the microwave-assisted enzymatic preparation of black soybean peptides under various

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designs. The influence of the interaction between microwave power and enzyme dosage (AC) on the

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degree of hydrolysis was extremely significant (P < 0.01; Table 3). While the interaction between

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microwave power and hydrolysis time was significant (P < 0.05), there was no significant interaction

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between hydrolysis time and enzyme dosage (BC; P > 0.05). The influence of microwave power (A2)

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on the degree of hydrolysis was very significant (P < 0.01), and the influences of hydrolysis time (B2)

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and enzyme dosage (C2) were significant (P < 0.05). The effect of the factors on the degree of

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hydrolysis was also obtained by the F-value, which ranked microwave power > dosage of enzyme >

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hydrolysis time.

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3.4.2.

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Response surface contour analysis

When either hydrolysis time or enzyme dosage were fixed, and power varied, the degree of

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hydrolysis increased, then gradually decreased, with increasing microwave power (Fig 1A and 1B).

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Since microwave action would be based on temperature, the degree of hydrolysis would increase until

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the optimum temperature of the Alcalase enzyme was reached, while excessive microwave power

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would lead to a temperature that exceeded the optimum enzyme temperature, resulting in loss of

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enzyme activity, and thus, reduced hydrolysis.

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3.4.3.

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Verification test

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The optimum conditions for preparation of black soybean ACE inhibitory peptides by

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microwave-assisted enzyme hydrolysis were predicted by the response-surface model to be: microwave 15

ACCEPTED MANUSCRIPT power of 235.48 W, microwave enzymatic hydrolysis time of 19.51 min, and enzyme dosage of 4.12%.

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The predicted hydrolysis rate was 42.97%. Taking into account realistic conditions of operation, the

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optimal parameters were amended to: a solid-to-liquid ratio of 4%, pH 9, microwave power of 235.5 W,

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microwave enzymatic hydrolysis time of 19.50 min, and 4% enzyme dosage. Using the amended

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conditions, the achieved degree of hydrolysis was 41.92%, close to the predicted value. Therefore, the

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response surface method predicted accurate and reliable optimum parameters for microwave-assisted

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enzymatic preparation of black soybean ACE inhibitory peptides.

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The black soybean peptides from the optimized hydrolysis reaction inhibited 72.38% of the ACE

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activity. This was an improvement of 53.83 points, compared to the 18.55% ACE inhibitory activity of

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the black soybean protein isolate (Table 1). Furthermore, the optimized protocol reduced the

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preparation time, to only 15 minutes, compared to the conventional enzymatic hydrolysis time of 2 h.

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The optimized hydrolysis slightly increased the ACE inhibitory activity of the hydrolyzed black

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soybean peptides over the non-optimized Method 2 (70.37%).

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3.5. The relative molecular weight distribution of black soybean peptides

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Microwave-assisted enzymatic hydrolysis decreased the occurrence of larger proteins (Table A.1

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and Fig 2) and resulted in 92.88% of the proteins being less than 1500 Da in size. In particular, 82.26%

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of the microwave-assisted hydrolysates were less than 500 Da (Fig 2a), a higher rate of smaller proteins

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than the 45.42% in the conventional enzymatic hydrolysates (Fig 2b). The ACE inhibitory activity of

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the microwave-assisted hydrolysate was 14.18% higher than that prepared by conventional hydrolysis.

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This result is consistent with reports that ACE inhibitory peptides are mostly 2-15 amino acids in length,

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indicating that a smaller average molecular weight preparation (Li, Le, Shi, & Shrestha, 2004) would

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have higher inhibitory activity (Balti, Nedjar-Arroume, Adje, Guillochon, & Nasri, 2010). Microwave-assisted Alcalase hydrolysis could become an effective method for the preparation of

328

ACE inhibitory peptides from black soybeans. It is worth further study to determine if the increase in

329

the rate of ACE inhibition is related to the greater yield of peptides less than 500 Da in size.

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3.6. ACE inhibition activity of hydrolysate after macroporous resin desalting and ultrafiltration

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After microwave-assisted hydrolysis, desalting through macroporous resin and separation by

332

ultrafiltration were added to see the effect on ACE inhibitory activity of the hydrolysate (Figure 3).

333

Inhibition of the ACE activity increased significantly after the hydrolyzed black soybean proteins were

334

desalted through the DA201-C macroporous resin and separated by size through ultrafiltration. The

335

unprocessed black soybean hydrolysate inhibited 72.38% of ACE activity, which increased to 74.45%

336

after desalting. While this is a small improvement in activity. After separation by ultrafiltration, the

337

black soybean peptides less than 3 kDa in size inhibited 80.53% of the ACE activity, while larger

338

proteins (> 3 kDa) only inhibited 14.89% of the ACE activity. Therefore, the < 3 kDa fraction of black

339

soybean peptides was chosen for further separation and purification.

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3.7. Sephadex G-15 separation

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The desalted and size-filtered hydrolysate was fractionated into four individual fractions

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(Fractions I-IV) by Sephadex G-15 column chromatography (Figure 4A). Over several separate

343

collections, the elution peaks were similar in both retention time and size. After pooling peaks, the ACE

344

inhibitory activity was tested for each fraction (Figure 4C). The ACE inhibitory activity was observed

345

in all fractions, however, Fraction III exhibited the highest activity of 90.78%. Compared with previous

346

reports on ACE inhibitory peptides, this preparation from black soybean seemed to possess higher ACE 17

ACCEPTED MANUSCRIPT 347

inhibitory activity, and thus, is worth studying further. The molecular weight distribution within Fraction III (Figure 4B) showed that most peptides were

349

below 500 Da, with the largest amount between 145~468 Da. This indicated that Fraction III was a

350

mixture that could be further separated and purified.

351

3.8. Stability of black soybean ACE inhibitory peptides

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Polypeptide substances have the disadvantages of poor stability, low oral utilization, short half-life

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in the body and poor permeability of biofilm. The development of peptide drugs is limited, and the poor

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stability is the main problem facing the research and development of polypeptide drugs.

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3.8.1.

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pH Stability

Changes in pH did not significantly affect the ACE inhibitory activity of the black soybean

357

peptides (Figure 5a). This indicated that the isolated ACE inhibitory peptides had good pH stability,

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therefore, human gastric acid maybe not destroy its activity, which is conducive to the production

359

process.

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3.8.2.

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Temperature Stability

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Moderate temperatures below 40 ℃ did not significantly affect the ACE inhibitory activity of the

362

black soybean peptides (Figure 5b). However, higher temperatures did decrease the ACE inhibition

363

activity, with only 33% of the ACE activity inhibited at 100 ℃, a decrease of about 50 points. Higher

364

temperatures likely increased the cleavage and degradation of the black soybean peptides. These data

365

suggest that ACE inhibitory peptides isolated from black soybean are stable when the temperature

366

remains at or below body temperature. 18

ACCEPTED MANUSCRIPT 367

3.8.3.

Metal ion Stability

Addition of metal ions did little to affect the ACE-inhibiting activity of the black soybean peptides

369

(Figure 5c). The peptides maintained inhibition at 85-90%, even at the highest metal ion concentration,

370

indicating that Ca2+, K+ and Mg2+ do not affect the stability or activity of the ACE inhibitory peptides

371

from black soybeans. When black soybean ACE inhibitory peptides play a role in organisms, it is not

372

affected by the constant metal ions in the body and lost activity.

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3.8.4.

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Stability after in vitro treatment with digestive enzymes

Sequential treatment with pepsin, chymotrypsin, and trypsin diminished the ACE inhibitory

375

activity of black soybean peptides (Figure 5d). These digestive enzymes may partially hydrolyze the

376

ACE inhibitory peptides, thus, reduce their activity. This indicated, at least some, black soybean

377

ACE-inhibiting peptides were substrate-type ACE inhibitory peptides (Zhang, Tatsumi, Ding, & Li,

378

2006), and they were relatively stable in the gastrointestinal tract.

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The results of our study on the stability of ACE inhibitory peptides are in some ways similar to

380

those of some other scholars. The ACE inhibitory peptides from black soybean have high tolerance to

381

pH and digestive enzymes, which is in agree with the ACE inhibitory peptides from tilapia (Tidarat

382

Toopcham, sittiruk Roytrakul, ＆ Jirawat Yongsawatdigul，2015) and mushroom (Pin Zhang, Sittiruk

383

Roytrakul, ＆ Manote Sutheerawattananoda, 2017). These are attributed to the ACE inhibitory

384

peptides with low molecular weight (Rim Nasri, Islem Younes, Mourad Jridi, ＆ et al, 2013). Because

385

small molecular peptides are not susceptible to the effects of pH and digestive enzymes (Min Chen, ＆

386

Bo Li, 2012, ), they are able to maintain structural integrity and high bioactivity in gastrointestinal

387

digestive juice. At the same time, some scholars have reported that alkali and heat seriously inhibited

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ACCEPTED MANUSCRIPT the activity of ACE inhibitory peptides (Wei et al., 2014), which seems to be somewhat similar to our

389

research results. As we all know, the pH value of the gastrointestinal environment is not more than 9.0,

390

so the ACE inhibitory peptides are stable in the human body. But the stability of ACE inhibitory

391

peptides to metal ions is rarely reported. The existing research results, which are consistent with ours,

392

show that metal ions have no effect on the stability of ACE inhibitory peptides (Xiaorong Zhang,

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2013).

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4.

Conclusions

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The black soybean proteins were hydrolyzed using microwave-assist the Alcalase enzyme to

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prepare ACE inhibitory peptides. The optimum preparation conditions were as follows: a 4% solid -to-

398

liquid ratio, pH 9, 235.5 W of microwave power for 19.50 min, and a 4% (w/w) enzyme dosage. These

399

conditions resulted in black soybean proteins with a 42.97% degree of hydrolysis and an ACE

400

inhibitory activity of 70.38%. The majority of the black soybean ACE inhibitory peptides were less

401

than 1500 Da (92.88%), with 82.26% of them less than 500 Da. The microwave-assisted enzymatic

402

hydrolysis generated a greater amount of low molecular weight peptides, and increasing the ACE

403

inhibitory activity by 14.18% compared to the conventional enzymatic hydrolysis. Four active fractions

404

were obtained from column chromatography, with Fraction III exhibiting the highest activity (90.78%)

405

and a molecular weight concentrated in the 145~468 Da range. The ACE inhibitory peptides isolated

406

from black soybean hydrolysates showed stability across a range of pH values (2-10), at temperatures <

407

40 ℃, and in the presence of metal ions (Ca2+, K+, Mg2+). The peptides were somewhat sensitive to

408

digestion, indicating that amongst the preparation were substrate-type ACE inhibitory peptides. These

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ACCEPTED MANUSCRIPT results provided a starting point for further research on ACE inhibitory peptides from black soybeans.

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Acknowledgements

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Thanks to Hefei Agricultural Product Processing Research Institute for providing equipment support.

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Acta, 744(18), 54.

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Figure caption

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Fig. 1. Response Surface Methodology shows the interaction among A, Microwave power and time and

536

B, Microwave Power and enzyme dose, on the degree of hydrolysis of black bean proteins.

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Fig. 2. Molecular weight distribution of ACE inhibitory peptides from black soybean isolated by

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microwave-assisted (a) and conventional enzymatic hydrolysis (b) using Alcalase

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Fig. 3. ACE inhibitory activity of acid-precipitated black bean proteins after hydrolysis by Alcalase and

553

further purification by desalting on DA201-C macroporous resin and size separation through

554

ultrafiltration.

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Fig. 4. A Gel-filtration chromatography of ACE inhibitory peptides from black soybean on Sephadex

564

G-15; B Molecular weight distribution of Fraction Ⅲ; C. ACE inhibitory activity of peptide fractions

565

obtained on Sephadex G-15.

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568

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Fig. 5. Effects of pH (a), temperature (b), concentration of metal ions (c) and digestive enzymes (d) on

572

ACE inhibitory activity of the purified black soybean peptides.

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Table 1. Comparison of black soybean protein fractions after various microwave-assisted enzymatic methods (x±s,

581

n=6) Samples

Protein

Method 1

Method 2

-

43.00±0.36a

41.90±0.61a

42.63±0.61a

18.55

58.20±0.91c

70.37±0.57a

60.23±0.60b

a

hydrolysis (%) ACE inhibitory activity (%) a

Protein isolate after Section 2.2.

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582

583

587

588

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Method 3

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isolate Degree of

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589

590

30

ACCEPTED MANUSCRIPT 591

Table 2. Box-Benhnken design (BBD) and computational results of microwave-assisted preparation of ACE

592

inhibitory peptides from black soybean flour B

C

Y

microwave power

microwave time

enzyme dosage

DH

(W)

(min)

(%)

(%)

1

177.5

17.5

4

39.825

2

277.5

17.5

4

40.145

3

177.5

22.5

4

37.175

4

277.5

22.5

4

40.095

5

177.5

22.5

3

34.7

6

277.5

22.5

3

40.12

7

177.5

22.5

5

38.79

8

277.5

22.5

5

39.12

9

227.5

10

227.5

11

227.5

12

227.5

13

227.5

14

227.5

15 16

594

595

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40.185

22.5

3

38.335

17.5

5

40.575

22.5

5

41.125

22.5

4

41.98

22.5

4

42.982

227.5

22.5

4

42.97

227.5

22.5

4

42.976

227.5

22.5

4

42.973

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593

3

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17

17.5

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Model No.

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A

596

597 31

ACCEPTED MANUSCRIPT 598

Table 3.

Analysis of the response surface methodology regression and variance Quadratic

Degree of

Mean square

F-value

Pro＞F

significance

sum

freedom

model

79.06

9

8.78

32.67

< 0.0001

**

A（microwave power）

10.31

1

10.31

38.32

0.0004

**

B（microwave time）

2

1

2

7.44

0.0295

*

C（enzyme dosage）

5.06

1

5.06

AB

1.69

1

1.69

AC

6.25

1

BC

1.44

A2

0.0034

**

6.28

0.0406

*

6.25

23.24

0.0019

**

1

1.44

5.35

0.0539

29.75

1

29.75

110.62

< 0.0001

**

2.75

1

2.75

10.22

0.0151

*

C2

15.41

1

15.41

57.3

0.0001

**

Residual

1.88

7

0.27

Lack of fit

1.09

3

Net error

0.79

4

Total dispersion

80.95

16

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18.8

B

0.36

1.83

0.2812

0.2

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** represents extremely significant difference (P < 0.01), * represents significant difference (P < 0.05).

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Microwave-assisted Alcalase hydrolysis was used to prepare black soybean protein hydrolysate.

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The hydrolysis parameters were optimized using response surface methodology. . The hydrolysates exhibited an excellent stability in a wide variety of conditions.

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ACE inhibition peptides with the highest activity were obtained by purification.