Identification of angiotensin-converting enzyme inhibitory peptides from peanut meal (Arachis hypogaea Linn) fermented by Lactobacillus pentosus using MALDI-TOF–MS and LC–MS/MS

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Received: 25 October 2023 Revised: 12 December 2023 Accepted: 15 December 2023

DOI: 10.1002/fft2.352

LETTER

Identification of angiotensin-converting enzyme inhibitory

peptides from peanut meal (Arachis hypogaea Linn) fermented

by Lactobacillus pentosus using MALDI-TOF–MS and LC–MS/MS

Wenjun Li1Yexia Guan1Lin Shi2Yang Chen1Huang Huang1

Haiyin Zhen3Ping Wu3Chao Wang1Qian Wu1Wei Li1

1Hubei Key Laboratory of Industrial

Microbiology, Cooperative Innovation Center

of Industrial Fermentation (Ministry of

Education & Hubei Province), Key Laboratory

of Fermentation Engineering (Ministry of

Education), National “111” Center for Cellular

Regulation and Molecular Pharmaceutics,

Hubei University of Technology, Wuhan,

Hubei, P. R. China

2Wuhan Caidian District Public Inspection and

Testing Center, Wuhan, Hubei, P. R. China

3Hubei Yizhi Konjac Biotechnology Co., Ltd,

Yichang, Hubei, P. R. China

Correspondence

Wei Li, Qian Wu, Hubei Key Laboratory of

Industrial Microbiology, Cooperative

Innovation Center of Industrial Fermentation

(Ministry of Education & Hubei Province), Key

Laboratory of Fermentation Engineering

(Ministry of Education), National “111” Center

for Cellular Regulation and Molecular

Pharmaceutics, Hubei University of

Technology, Wuhan 430068, Hubei, P. R. China.

Email: wesley@hbut.edu.cn;

wuqian@hbut.edu.cn

Funding information

Natural Science Foundation of Hubei Province,

Grant/Award Number: 2022CFB452; the

foundation for National Innovation and

Entrepreneurship Center for College Students

(CSIE) of China, Grant/Award Number:

20230100115; The central government guides

local funds for scientific and technological

development, Grant/Award Number:

2021BGE045

Abstract

This study focused on the production of angiotensin-converting enzyme inhibitory

peptides (ACEIPs) from peanut meal (Arachis hypogaea Linn) fermented by Lactobacillus

pentosus. The fermentation process was optimized using the response surface method-

ology with ACE inhibitory activity as the experimental indicator. ACEIPs were further

purified after fermentation using ultrafiltration and Sephadex G-25 gel chromatogra-

phy. The effect of different molecular weights (ranging from 0.5 to 1.5 kDa) of ACEIP

on ACE inhibitory activity was investigated, and a maximum inhibitory rate of 48.83%

was achieved. The content of ACEIP was 78.95%. Amino acid analysis revealed that

the hydrophobic amino acids accounted for 43.09% of the total content. Among the

identified amino acids, glutamic acid had the highest content of 14.94%, followed by

leucine and aspartic acid. Matrix-assisted laser desorption/ionization time-of-flight–

mass spectrometry (MS) and liquid chromatography–tandem MS were used to identify

the molecular weights of the selected ACEIPs, yielding six ACEIPs with good stabil-

ity and high hydrophilicity. Flexible docking of the six ACEIPs with ACE was simulated

using AutoDock Vina (v1.5.7). The result showed that the ACEIPs formed 11, 8, 7, 9,

7, and 6 hydrogen bonds with ACE residues, and the lowest binding energies between

them were −9.8, −8.1, −9.0, −9.3, −8.2, and−9.1 kcal/mol, respectively. Among them,

GFGINAENNHRIF exhibited superior ACE inhibitory activity and binding stability.

KEYWORDS

angiotensin-converting enzyme inhibitory peptides, molecular docking, molecular structure,

peanut meal, response surface methodology

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided

the original work is properly cited.

University, Fujian Agriculture and Forestry University

820 wileyonlinelibrary.com/journal/fft2 Food Frontiers. 2024;5:820–832.

LI ET AL.821

1INTRODUCTION

In recent years, improved living standards and the rapidly aging popu-

lation have contributed to the increasing prevalence of hypertension,

thus significantly negatively impacting lives. The International Soci-

ety of Hypertension in 2020 announced at its annual conference that

approximately 1 billion individuals globally suffer from hypertension,

accounting for 26% of the adult population. Angiotensin-converting

enzyme inhibitory peptides (ACEIPs) are polypeptides known for

their inhibitory effects on ACE (Santiago et al., 2023). They typically

comprise 2–20 amino acid residues. ACEIPs can prevent the forma-

tion of angiotensin II and promote the production of bradykinin and

enkephalin by binding to ACE, thus exhibiting antihypertensive effects

(Grden & Jakubczyk, 2023; Ramlal et al., 2023;Ren,2021). Some

researchers suggest that ACEIPs derived from food sources can poten-

tially serve as alternative therapies for patients with hypertension or

related disorders (Chang et al., 2023; Kralova et al., 2023; Yang et al.,

2023).

Peanut meal (Arachis hypogaea Linn), a byproduct from peanut oil

extraction, is a rich source of plant protein with protein content

exceeding 50%. However, limitations in their oil extraction technolo-

gies significantly denature the protein in peanut meal, thus restricting

its application in food processing (Hu et al., 2019; Li et al., 2020).

Studies have shown that microbial fermentation can influence the pro-

tein molecular structure, and various microbes can produce different

enzyme systems and functional factors (Hu et al., 2023; Zambrano-

Cervantes et al., 2023). L. pentosus are premium lactic acid bacteria that

can ferment pentose sugars to produce high levels of lactic acid; this

creates a fermentation microenvironment conducive to their growth

(Garcia-Gonzalez et al., 2022; Zhang et al., 2023). In addition, L. pento-

sus produce various enzymes to hydrolyze proteins, endowing peanut

proteins with improved nutrition and flavor profiles (Zhang et al.,

2020, 2023). Studies have shown that L. pentosus can produce quorum-

sensing auto-inducing peptides with a molecular weight of less than

3000 Da and is highly hydrophobic (Suo et al., 2022; Zhang et al.,

2015). The ACE inhibitory peptide has certain similarities in structure,

that is, it contains more hydrophobic amino acids and aromatic amino

acids, such as anti-oxidized peptide contains Pro (P) and Tyr (Y), the

N-terminal of the ACE inhibitory peptide contains Val (V), Ile (I), and

Leu (L), and the C-terminal contains Val (V), Ala (A), and Phe (F) or Pro

(P) residues, when the N-terminus is hydrophobic valine (Val), leucine

(Leu), isoleucine (Ile), or the basic amino acid arginine (Arg). The pep-

tides of lysine (Lys) and histidine (His) have strong affinity with ACE

and have the highest ACE inhibitory activity (Qiao et al., 2022). The use

of L. pentosus to ferment peanut meal and produce functional factors,

including ACEIPs, is a promising strategy for developing food–medicine

homologous dietary supplements (Ou et al., 2011). The application of

L. pentosus is a promising strategy for extending the peanut industry

chain, adding value to peanut byproducts, and enhancing the economic

benefits of the peanut industry (Li et al., 2019).

In this research, we used L. pentosus to ferment defatted peanut

meal. Using ACE inhibitory activity as an experimental metric, we

explored the production process of peanut meal ACEIPs. Matrix-

assisted laser desorption/ionization time-of-flight (MALDI-TOF) and

liquid chromatography–tandem mass spectrometry (LC–MS/MS)

were concurrently applied for separating, purifying, and identifying

inhibitory peptides. This study aimed to provide technical support for

the deep processing of defatted peanut meal and the comprehensive

utilization of peanut protein.

2METHODS

2.1 Activation and cultivation of Lactobacillus

pentosus

The De Man Rogosa Sharpe (MRS) liquid medium was formulated with

1% peptone, 0.5% yeast extract, 0.2% diammonium citrate, 2% glucose,

0.3% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.058%

magnesium sulfate, 0.025% manganese sulfate, 1% beef extract, and

0.1% Tween 80. The medium was sterilized using an autoclave (MLS-

3781L-PC, Shanghai Medical Nuclear Inst.) at 115◦C for 20 min.

Afterward, the medium was stored at room temperature for subse-

quent use. Strains previously stored at −80◦C in the preservation

tubes of the laboratory were retrieved and inoculated into 50 mL of

the prepared MRS liquid activation medium. This culture was then

incubated at 37◦C for 24 h in a biochemical constant temperature incu-

bator (ZWYR-D2403, Shanghai Zhicheng Analytical Inst. Co., Ltd.) and

reserved for future procedures.

2.2 Production process of ACE inhibitory

peptides (ACEIP) from peanut meal

Equal masses of peanut meal and distilled water were mixed and steril-

ized in an autoclave at 121◦C for 15 min and then cooled to 25◦C. The

sterilized peanut meal was fermented under the following conditions.

After the peanut meal was fermented, the mixture was centrifuged at

10,000 ×gfor 20 min at 4◦C. Drawing from the approach adopted by

Sun et al. (2019), we designed and implemented a single-factor test for

ACE inhibitory peptides.

2.2.1 Single-factor experiment

Inoculation rate: Under laminar flow hood conditions, different inoc-

ulation rates of L. pentosus broth (1%, 3%, 5%, 7%, and 9%) were

introduced in the samples. The samples were incubated at 32◦Cina

shaker incubator at 200 ×g for 48 h.

Duration: Under laminar flow hood conditions, 5% of the volume of

the L. pentosus broth was introduced in the sample. Then, the mixture

was incubated at 32◦C in a shaker incubator at 200 ×gfor different

times: 12, 24, 36, 48, and 60 h.

Temperature: Under laminar flow hood conditions, a 5% inoculation

rate of L. pentosus broth was introduced in the samples. The mixture

was incubated in a shaker incubator at 200 ×gfor 48 h at different

temperatures: 28, 30, 32, 34, and 36◦C.

26438429, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/fft2.352 by CochraneChina, Wiley Online Library on [29/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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Identification of angiotensin-converting enzyme inhibitory peptides from peanut meal (Arachis hypogaea Linn) fermented by Lactobacillus pentosus using MALDI-TOF–MS and LC–MS/MS

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