The targeted development of collagen-active peptides based on composite enzyme hydrolysis: a study on the structure–activity relationship

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Food &

Function

PAPER

Cite this: Food Funct., 2024, 15, 401

Received 18th October 2023,

Accepted 1st December 2023

DOI: 10.1039/d3fo04455f

rsc.li/food-function

The targeted development of collagen-active

peptides based on composite enzyme hydrolysis:

a study on the structure–activity relationship†

Xinnuo Hu,

a,b

Yanjun Yang,*

a,b

Cuihua Chang,*

a,b

Junhua Li,

a,b

Yujie Su

a,b

and

Luping Gu

a,b

Fish collagen, derived from sustainable sources, oﬀers a valuable substrate for generating peptides with

diverse biofunctionalities. In this study, alkaline, papain, and ginger protease were used to enzymatically

hydrolyze ﬁsh skin collagen. The peptide molecular weight distribution and sequence were measured

using HPLC and ICP-MS-MS, with papain/alkaline protease (AP) and papain/alkaline/ginger protease

(APG) hydrolyzed samples compared. As the results showed, the incorporation of ginger protease was

useful for increasing the degree of hydrolysis, with the content of <400 Da peptides increasing from

49.82% to 58.56%. The identiﬁed peptide sequence in the APG sample had more proline at the

C-terminal. The peptides were separated into two components (diﬀerent in molecular weight) using gel

column chromatography. The molecular weight distribution, amino acid composition, ACE inhibitory

activity, and ﬁbroblast proliferation activity of the collected components were measured. In comparison,

the contents of proline and hydroxyproline in the larger peptides decreased obviously after combined

hydrolysis by ginger protease, reﬂecting the formation of a peptide sequence of smaller molecular weight

containing glycine and hydroxyproline. The combined hydrolysis of ginger protease was beneﬁcial for the

improvement of the ACE inhibitory activity of the sample. However, the ﬁbroblast proliferation activity of

AP was higher than that of APG, indicating that further hydrolysis by ginger protease may destroy the

hydroxyproline at the end of the peptide sequence. This study proposed a creative directional hydrolysis

method and provided practical guidance for the production of collagen peptides with enhanced func-

tional activity.

1. Introduction

According to various estimates, waste products from fish pro-

cessing can be up to 85% of the total processing capacity. A

significant percentage of the waste products (about 30%) is

skin, bones, and scales with a high collagen content.

These

resources put a lot of pressure on the environment if they are

discarded and buried. However, they can be processed into

valuable products.

Edible aquatic animal-derived collagen has

weaker structural and thermal stability than mammalian col-

lagen, due to the lower content of amino acids, such as proline

and hydroxyproline.

However, edible aquatic animal-derived

collagen is more easily hydrolyzed by enzymes than mamma-

lian collagen and is more suitable for the preparation of bio-

active peptides.

Studies on fish collagen peptides have mainly

been focused on their functional eﬀect on skin health, such as

diminishing inflammation, promoting fibroblast proliferation,

and enhancing hyaluronic acid production in human skin.

Recent research has demonstrated that biomaterials derived

from fish skin have the potential to be used as scaﬀold

materials owing to their good biodegradability and biocompat-

ibility in human tissues.

Furthermore, the biocompatibility

and cell proliferation bioactivity of fish skin collagen can fit

the standards of medical materials.

Another biological func-

tion of fish collagen peptide is ACE inhibitory activity. As

reported, tripeptides and tetrapeptides have been rec-

ommended as ideal candidates for seeking potential peptide

additives in functional foods with satisfactory ACE binding.

In addition, 380–920 Da fish collagen peptides are responsible

for exhibiting ACE-inhibitory activity.

Some research studies

also describe the bone health-maintaining function of fish col-

lagen peptides, such as accelerating bone fracture healing

†Electronic supplementary information (ESI) available. See DOI: https://doi.org/

10.1039/d3fo04455f

School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu,

214122, PR China. E-mail: yangyj@jiangnan.edu.cn, chang.cuihua@jiangnan.edu.cn

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,

Jiangsu, 214122, China

This journal is © The Royal Society of Chemistry 2024 Food Funct.,2024,15,401–410 | 401

Published on 01 December 2023. Downloaded on 5/20/2024 9:51:37 AM.

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through the stimulation of osteoblast calcification or providing

novel candidate implants for bone repair, and boosting sus-

tainable utilization of bio-resources.

10,11

The degree of hydrolysis is closely related to the character-

istics of collagen peptides, as the functionality and bioactivity

of peptides rely on the size, amino acid type and sequence of

the hydrolysates. The special triple helix structure of collagen

leads to its indigestibility, while over-hydrolysis may release

peptides with no functional or bioactive properties,

indicat-

ing the importance of regulating the hydrolysis degree to maxi-

mize the functionality of peptides. A large number of recent

studies have proposed that small molecular peptides obtained

after collagen hydrolysis are more easily absorbed by the body

and detected in the blood.

13,14

Shoko et al. reported that inges-

tion of small molecular collagen peptides can improve the

eﬃciency of peptide absorption into the blood.

This suggests

that active transporters, such as oligopeptide transporters or

PepT1, might be involved in the permeation of small peptides

across the intestinal epithelial layer.

16,17

However, this does

not mean the higher degree of hydrolysis would result in

higher biological activity of collagen peptide. A medium

degree of enzymatic hydrolysis would be appropriate for good

antioxidant activity while small peptides are essential to

obtain high ACE-inhibitory activity,

18,19

A5–10 kDa fraction of

the collagen peptides obtained would be good tyrosinase and

collagenase inhibitors with the best potential antiaging

activity.

Therefore, the relationship between the degree of

hydrolysis and the function of collagen is complex, which

needs further research work to explore potential collagen pep-

tides with high physiological activity.

Each strand of collagen consists of the repeating sequence

Gly-Xaa-Yaa, where Xaa and Yaa are often proline (Pro) resi-

dues. The post-translational hydroxylation of the proline resi-

dues would generate 4(R)-hydroxy-L-proline (4-Hyp) residues or

3(S)-hydroxy-L-proline (3-Hyp) residues.

Enzyme selection is

critical for collagen hydrolysis degree and activity. In this

study, alkaline protease and papain were selected as the non-

specific enzymes to hydrolyze collagen. Alkaline protease has

been documented as the most cost-eﬀective for hydrolyzing

fish proteins, which can eﬀectively degrade the triple-helix

structure of collagen within a short time, breaking peptide

bonds from nonterminal amino acids randomly and facilitat-

ing further protein hydrolysis.

In addition, some researchers

conducted peptide cutter analysis with BIOPEP, suggesting

that papain can release the highest number of potential angio-

tensin converting enzyme (ACE)-inhibitory peptides from

alpha collagen. However, it is diﬃcult for non-specific

enzymes to hydrolyze the proline C-terminal, limiting the

increase in the hydrolysis degree and release of terminal

proline.

23,24

Therefore, in order to increase the hydrolysis

degree of collagen, specific enzymes are required for peptide

chains that are diﬃcult to hydrolyze using non-specific

enzymes. The ginger protease used in our research is a

product containing a proline-specific endopeptidase, which

can theoretically further increase the hydrolysis degree of col-

lagen and obtain peptides ending in proline.

This study aims to explore the eﬀect of compound enzymatic

hydrolysis of papain, alkaline protease and ginger protease on

the hydrolysis degree, peptide sequence distribution and func-

tionality of fish skin collagen. The obtained hydrolysates were

separated into two collections using TSK-gel PW column chrom-

atography according to molecular weight, to evaluate the func-

tional properties of collagen peptides with various lengths and

sequences. The research is meaningful for the production of col-

lagen peptides with higher yield and bio-activity.

2. Materials and methods

2.1 Materials and chemicals

Fish gelatin was purchased from Wuhan Lanabai

Pharmaceutical Chemical Co., Ltd (Wuhan, China). Alcalase (1

×10

−1

) was purchased from Angel Yeast Co., Ltd

(Yichang, China). Papain (2 × 10

−1

) was purchased from

Nanning Pangbo Biological Engineering Co., Ltd (Nanning,

China). Ginger protease (2 × 10

UmL

−1

) was purchased from

Royal DSM Co., Ltd (The Netherlands). Gly-Pro-Hyp standard

was purchased from Shanghai Qiangyao Biological Technology

Co., Ltd (Qingdao, China). Angiotensin converting enzyme and

N-hippuryl-His-Leu hydrate were purchased from Sigma-

Aldrich Trading Co., Ltd (Shanghai, China). Mouse embryonic

fibroblast cells (NIH-3T3) were kindly provided by Stem Cell

Bank, Chinese Academy of Sciences. CCK-8 kits were pur-

chased from Beyotime Biotechnology Co., Ltd (Shanghai,

China). All the other reagents used were of analytical grade.

2.2 Collagen hydrolysate preparation

The purpose of the preparation was to study the functional

activity of small molecule peptides. In order to achieve a high

degree of hydrolysis, we selected the following processes: the

fish collagen solution (5%, w/v) was treated with papain (3000

−1

protein) at pH 6.0 with an incubation time of 2 h at

55 °C, and then treated with alcalase (3000 U g

−1

protein) at

pH 10.0 with an incubation time of 2 h at 55 °C. Afterward, the

sample was treated with ginger protease (3000 U g

−1

protein)

at pH 4.5 with an incubation time of 2 h at 55 °C. The

obtained samples were recorded as AP. Another group of APGs

was obtained by enzymatic hydrolysis: the same concentration

of the substrate solution was treated under the reaction para-

meters of protease 3000 U g

−1

protein, alcalase protease (pH

10.0), and papain protease (pH 6.0), and the incubation time

was 2 h at 55 °C, while without ginger protease. The enzymatic

hydrolysates obtained above were inactivated at 90 °C for

10 min. After centrifugation, the supernatant was collected for

subsequent experiments.

2.3 Peptide sequences

HPLC-ESI-MS/MS (MALDI SYNAPT MS, Waters, USA) was used

to identify the structure of characteristic collagen peptide

sequences. Instrument parameters were set based on our pre-

vious experiments. Then, MassLynx V4.1 was used to analyze

the identified peptide sequences to further verify the

Paper Food & Function

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