Peptidomics- inspired discovery and activity evaluation of antioxidant peptides in multiple strains mixed fermentation of Porphyra yezoensis

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Food Chemistry 455 (2024) 139779

Available online 23 May 2024

Peptidomics- inspired discovery and activity evaluation of antioxidant

peptides in multiple strains mixed fermentation of Porphyra yezoensis

Jie Yang

, Pengpeng Zhao

, Qiqi Wang

, Feng Xu

, Yaxuan Bai

, Saikun Pan

Wenbin Wang

, Doris Ying Ying Tang

, Pau Loke Show

Jiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, 222005,

China

Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, China

Jiangsu Marine Resources Development Research Institute, Jiangsu Ocean University, Lianyungang 222005, China

Department of Chemical & Petroleum Engineering, Khalifa University of Science and Technology, PO Box 127788, Abu Dhabi, United Arab Emirates

ARTICLE INFO

Keywords:

Porphyra yezoensis

Peptidomics

Antioxidant activity

Phycobiliprotein

Enzyme

ABSTRACT

This study investigated the production of antioxidant peptides from Porphyra yezoensis through fermentation

with three strains of microorganisms: Lactiplantibacillus plantarum L13, Bacillus amyloliquefaciens MMB-02, and

Saccharomyces cerevisiae A8. The crude peptides were extracted by aqueous acid precipitation and puried by

Sephadex G-25 gel column to produce highly active antioxidant components with molecular weight of <4000 Da.

The LC-MS/MS result revealed that the fermentation group contained more hydrophobic amino acids and oli-

gopeptides, which were mainly originated from phycobiliproteins and algal blue proteins. Finally, the antioxi-

dant activity of Porphyra yezoensis was determined with DPPH⋅ and ABTS⋅ scavenging rates of 54.87% and

57.39%, respectively. The ferric ion-reducing power (FRAP) and enzyme activities of SOD and CAT were

signicantly higher than those of the control group. This study provides a scientic foundation for the deep

processing of striped seaweed and contributes to the theoretical understanding of synthetic antioxidant

substitutes.

1. Introduction

Porphyra yezoensis (P. yezoensis) is a special variety of warm

temperate coldwater seaweed in China, mainly distributed in regions

such as Jiangsu, Guangdong, Zhejiang and other places. Its annual

output accounts for >70% of the national production of Porphyra (Q. F.

Wang, Zhang, Zhang, & Hou, 2022). P. yezoensis is rich in protein, and

the protein content is much higher than that of general vegetables and

other edible seaweed. Phycobilin which is unique to Porphyra is a nat-

ural pigment active protein, which can be classied into four categories

that are phycoerythrin, phycocyanin, allophycocyanin and phycoery-

thrin. It can be used as the natural pigment dye and exhibits medicinal

properties such as antioxidant, antitumor, hypoglycemic and other

biological activities (Syama, Anjali, & Basil, 2023). Recent study has

reported six peptides from Chlorella pyrenoidosa. Among them,

SISISVAGGGR (T1) had been found to attenuate weight gain and lipid

accumulation induced by a high-fat diet (HFD), reduce fasting blood

glucose and low-density lipoprotein (LDL), and elevate high-density li-

poprotein (HDL) levels, with lipid-lowering and maintenance of intes-

tinal microbial stability properties, making it a potential prebiotic (W. X.

Liu et al., 2024; Liu, Shen, Weng, Wu, & Liu, 2024). Meanwhile, it has

been found that phycobiliprotein is an ideal protein source of antioxi-

dant active peptides. In a previous study, three novel antioxidant pep-

tides, MHLWAAK, MAQAAEYYR, and MDYYFEER, were isolated and

puried from the tryptic hydrolysate of C-PC, a bivalent protein of

cyanobacteria, and were found to have a high scavenging capacity of

DPPH⋅ and ABTS

⋅. They had the ability to protect the zebrash em-

bryos from H

-induced oxidative damage without toxic effects (F. H.

Xu et al., 2022).

The rigid cell wall composition of seaweed or striped nori presents a

* Corresponding author at: Department of Chemical & Petroleum Engineering, Khalifa University of Science and Technology, PO Box 127788, Abu Dhabi, United

Arab Emirates

** Corresponding author at: Jiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean

University, Lianyungang, 222005, China.

E-mail addresses: yangjie@jou.edu.cn (J. Yang), PauLoke.Show@ku.ac.ae (P.L. Show).

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2024.139779

Received 4 January 2024; Received in revised form 12 April 2024; Accepted 19 May 2024

Food Chemistry 455 (2024) 139779

challenge in efciently extracting its nutrient content using existing

processing approaches, resulting in signicant resource wastage. Cur-

rent cell wall breaking methods, such as repeated freezing and thawing,

ultrasonic treatment, or acid-base treatment, not only cause excessive

energy consumption, but also affect the composition of active substance.

Therefore, there is an urgent need to search better extraction methods to

signicantly increase the extraction rate of antioxidant actives from

seaweed. Microbial fermentation has emerged as an effective method to

cleave the cell wall and obtain new secondary metabolites. It has been

reported that the use of safe enzymes secreted by microbial organisms

and physiological processes within the organisms alters the physico-

chemical properties of the fermentation substrate and obtains novel

metabolites. Exploring the biological activities of these fermentation

products and their potential application in the elds of health care and

medicine represents a future direction in modern fermentation engi-

neering (Ciechanowska et al., 2024). Therefore, fermentation provides a

practical method for breaking down the cell walls of algae, such as

striped nori, which is abundant in high-quality protein. This method not

only facilitates complete cell wall disruption, but also decompose and

metabolise the macromolecules, thereby producing antioxidant pep-

tides, amino acid derivatives and other active substances. At the same

time, these active products can be further separated and puried, and

peptidomics analysis can be carried out with the help of mass spec-

trometry technology to study their potential biological activities. This

integrated approach not only achieve the purpose of resource utiliza-

tion, but also enhance the nutritional value of Porphyra, and hence,

improve the economic viability of the Porphyra industry.

Probiotics, such as lactic acid bacteria and yeasts, have demonstrated

the ability to secrete proteases and cellulases in a short period of time,

which can break down seaweed and release various active metabolites

(Ozaeta, Araújo, Estrada, Puente, & Regefalk, 2024). Yu et al. (2024)

isolated component FB-2 A from Lactiplantibacillus plantarum FB-2,

further puried and characterised it to obtain the puried component

AMP KMY 15. Co-culturing with RAW264.7 macrophages revealed that

AMP KMY 15 could uniquely act on the bacterial membrane, leading to

leakage of cell contents, and effectively inhibit the growth of Staphylo-

coccus aureus ATCC 6538 in milk (Yu et al., 2024). Gao et al. (2022)

utilized Lactobacillus plantarum MMB-05 to ferment a sandwich seaweed

product made from P. yezoensis, and found that the fermentation

signicantly increased its antioxidant activity. The fermentation process

also increased the contents of avouring nucleotides and volatiles (Gao

et al., 2022). Thinzar and Jong-Bang (2021) employed yeast and bac-

terial fermentation of a mixture of Porphyra striata and kombucha to

produce a novel functional kombucha (K-IE), which exhibited higher

total phenolic and avonoid contents as compared to black tea kom-

bucha (K-BT) and green tea kombucha (K-GT). The ferric ion reducing

ability (FRAP) of K-IE were signicantly higher than those of K-BT and

K-GT (Thinzar & Jong-Bang, 2021). It has been reported that Bacillus

subtilis can produce various dextranase enzymes specialized in the

cleavage of the cell wall, showcasing excellent ability in decomposing

organic matter and thus, holding a great potential to be used in the food

industry (M. W. Liu, Hao, et al., 2024; Liu, Shen, et al., 2024). Previous

research indicated that the fermentation of Chlorella pyrenoidosa

(C. pyrenoidosa) using Bacillus velezensis SW-37 signicantly increased

the protein extraction rate and hydrolysis degree of C. pyrenoidosa. It

provides an effective fermentation method to improve the extraction

rate of active substances from C. pyrenoidosa (R. L. Zhang, Song, Liu, &

Gao, 2023).

Relatively few studies have been conducted on mixed microbial

fermentation of seaweed and subsequent preparation of antioxidant

peptides. In a previous study, Miyu et al. (2019) investigated the

fermentation of blue-green algae Aphanizomenon os-aquae (AFA) using

L. plantarum AN7 and Lactococcus lactis subsp. The ndings revealed that

the DPPH⋅ radical scavenging capacity of the substances with molecular

weight <3 kDa and 30–100 kDa in AFA increased after fermentation. In

addition, the O

−

radical scavenging capacity and Fe

reduction

capacity of of substances with molecular weights <3 kDa were improved

(Miyu et al., 2019). Kong, Feng, and Sun (2023) isolated crude peptides

MWCO-1 and A from fermented sausages inoculated with L. plantarum

CD101 and S. simulans NJ201. These peptides were assayed for their

protective effects against oxidative damage in Caco-2 cells and were

found to exhibit slight cytotoxicity. Further purication of fraction A led

to the identication of 14 antioxidant peptides. Among them, SDEEVEH

and FAGDDAPR showed strong DPPH⋅ radical scavenging activity, while

ALELDSNLYR and QEYDESGPSIVHR showed strong ABTS

⋅ scavenging

activity (Kong et al., 2023).

Only a few studies to date have examined on the preparation of

antioxidant peptides using three different strains of fermented

P. yezoensis. Previous studies targeted or focused on the optimal strains

for fermentation of seaweed were targeted from 10 different microor-

ganisms based on parameters for example viable bacterial count, pH,

cell wall-breaking rate, hydrolysis degree, protein extraction rate of

aqueous extract, and antioxidant activity. The best strains identied for

fermentation of striped seaweed were found to be B. amyloliquefaciens

MMB-02, L. plantarum L13 and S. cerevisiae A8. It would be of special

interest to investigate the fermentation process using these 3 microor-

ganism strains and understand the mechanisms underlying the

fermentation process. Therefore, this goal of this study is to study or

examine the synergistic effect of B. amyloliquefaciens MMB-02, L. plan-

tarum L13 and S. cerevisiae A8, in the fermentation of striped nori. The

crude polypeptide is extracted by aqueous acid precipitation and puri-

ed to obtain the high antioxidant activity component. Next, undiffer-

entiated analysis of the target peptide is conducted using LC-MS/MS

technique. The potential antioxidant activity of the peptides is discov-

ered through the joint use of multiple databases and their antioxidant

activity was determined. These ndings offer technical support for the

rational utilization of striped laver, contribute to the deep processing

and establish theoretical foundations for the research on the synthesis of

the antioxidant substitutes.

2. Materials and methods

2.1. Materials and main reagents

P. yezoensis, a variety of “Yu Dongxiang”, was purchased from Lia-

nyungang Agricultural trade mall. After arriving at the laboratory, the

porphyroid was crushed into ne powder using an ultrane grinder and

stored in the dryer for subsequent use. L. plantarum L13 (CGMCC

NO.27398), B. amylolyticus MMB-02 (CGMCC NO.27399), S. cerevisiae

A8 were kept in the laboratory. Various culture media (MRS Liquid

medium and MRS Solid medium, PDA solid medium and PDA liquid

medium), yeast extract, tryptone, sodium chloride, agar was purchased

from Beijing Road and Bridge Technology Co., LTD. 95% ethanol and

hydrochloric acid were purchased from Nanjing Chemical Reagent Co.,

LTD., while chemical kits were acquired from Jiengcheng Biotechnology

Co., LTD. (Nanjing, China).

2.2. Sample preparation

L. plantarum L13, B. amyloliquefaciens MMB-02 and S. cerevisiae A8

were inoculated into MRS solid culture medium, LB solid culture me-

dium and PDA solid culture medium respectively from the glycerol

tubes. A single colony from each culture was selected and inoculated

into the corresponding liquid culture medium, passaged and activated

twice. To prepare the fermentation substrate, 100.0 g dried nori powder

was accurately weighed and mixed with 120 mL distilled water to form

the paste. The mixture was stirred evenly, autoclaved at 121 ◦C for 30

min, and then cooled to room temperature. Design Expert software

(version 8.0.6.1, Stat-Ease, UK) was utilized to generate mixed

fermentation experiments of L. plantarum L13 (X1), S. cerevisiae A8 (X2)

and B. amyloliquefaciens MMB-02 (X3) using a simplex lattice mix

design. The function model of these three strains with the strongest

J. Yang et al.

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