YANG Fu-jia ,CHEN Xu ,HUANG Mu-chenYANG QianCAI Xi-xi ,CHEN Xuan ,DU Ming,HUANG Jian-lian ,WANG Shao-yun

1 Key Laboratory of Refrigeration and Conditioning Aquatic Products Processing of Ministry of Agriculture and Rural Affairs,Xiamen 361022,P.R.China

2 College of Biological Science and Technology,Fuzhou University,Fuzhou 350108,P.R.China

3 College of Chemical Engineering,Fuzhou University,Fuzhou 350108,P.R.China

4 School of Food Science and Technology,Dalian Polytechnic University,Dalian 116034,P.R.China

5 Fujian Anjoy Foods Co.,Ltd.,Xiamen 361022,P.R.China

Abstract Peptides are functional active fragments of proteins which can provide nutrients needed for human growth and development,and they also have unique physiological activity characteristics relative to proteins.Bioactive peptides contain a great deal of development potential.More specifically,food-derived bioactive peptides have the advantages of a wide variety of sources,unique structures,high efficiency and safety,so they have broad development prospects.This review provides an overview of the current advances regarding the preparation,functional characteristics,and structure–activity relationships of food-derived bioactive peptides.Moreover,the prospects for the future development and application of food-derived bioactive peptides are discussed.This review may provide a better understanding of foodderived bioactive peptides,and some constructive inspirations for further research and applications in the food industry.

Keywords:food-derived proteins,bioactive peptides,molecular characteristics,structure–activity relationship

1.Introduction

Proteins are important bioactive natural substances that are present in many species (Rizzelloet al.2016).Peptides are functional active fragments of proteins which can provide nutrients needed for human growth and development,and they also have unique physiological activity characteristics relative to proteins.Peptides are defined as small isolated fragments of proteins,typically consisting of 2 to 20 amino acids linked together by peptide bonds (Bhandariet al.2020).Generally,peptides can be divided into endogenous peptides and exogenous peptides according to their different sources.The number of studies on bioactive peptides that provide some physiological health benefits is increasing considerably.Bioactive peptides are known for their positive healthpromoting effects due to their small size and high rate of utilization in the body.Peptides are metabolized more easily than amino acids,which can effectively optimize

Food-derived bioactive peptides refer to the functional peptides taken by organisms from outside the body,directly or indirectly derived from food proteins,and they generally exist in dietary proteins with specific amino acid sequences (Bhatet al.2015;Karamiet al.2015).Foodderived peptides are very safe and easy to produce industrially,so they currently receive particular attention in the field of nutrition and food science.More than 3 000 different bioactive peptides have been reported in the BIOPEP-UMW database,mostly from food sources,and research on them continues to grow (Baratiet al.2020).Candidate proteins containing potential biological activities are found in milk,eggs,meat and fish,as well as various plant protein sources such as soybeans and wheat.The development of peptides with functional ingredients is an innovative strategy for functional foods,nutraceuticals and pharmaceuticals.

A large amounts of food-derived protein leftover and waste generated during processing are usually used as fertilizers or discharged directly,which not only leads to resource wastage,but also causes environmental pollution.To increase the yield and study the physiological functions of bioactive peptides,it is therefore important to properly select effective preparation methods for peptide activity research.The introduction of laborand time-saving approaches to the preparation of foodderived bioactive peptides will considerably accelerate the development of this field.

Antioxidant peptides (Yanget al.2019;Chenet al.2020a;Singhet al.2020;YangQet al.2020b),antimicrobial peptides (Zhuet al.2015;LiuLet al.2020),antifreeze peptides (Wanget al.2014;Caoet al.2016) and immunomodulatory peptides (Santiago-Lopezet al.2016b;Kanget al.2019) are some of the most common bioactive peptides.Antioxidant peptides exert cytoprotective effectsviathe detoxification of free radicals,including reactive oxygen species (ROS) (Ghassemet al.2017).Antimicrobial peptides have a broad-spectrum bactericidal effect,reach the site of infection easily,and are expected to be a substitute for antibiotics without the development of drug resistance (Qinet al.2014).Antifreeze peptides are a class of small proteins or protein hydrolysates that can protect the organism from harm when it is frozen or sub-frozen (Qinet al.2014;Chen Xet al.2019a).Immunomodulatory peptides can regulate the body’s immune function in various ways,and improve the body’s immunity,thereby preventing the occurrence of diseases (Chalamaiahet al.2014).A wide variety of peptide activities have been described.Moreover,some known peptides are versatile and can exert more than one of these functions.Therefore,they have great application prospects as potential functional ingredients in the food industry and are of great significance for improving the quality of consumers lives.

Amino acids are the basic building blocks of proteins.The complex biological structures and diverse functions of peptides mainly lie in their amino acid composition,number,arrangement order and the spatial structure of the peptide chain.The functions of bioactive peptides can also be affected by other factors such as process conditions,protein sources,pH and chemical treatments,molecular weight and charge distribution (Wonget al.2020).Thus,this paper describes the preparation process of bioactive peptides,and takes antioxidant,immunomodulatory,antimicrobial and antifreeze peptides as examples to summarize the various factors that govern the functional properties of bioactive peptides from the structural aspect.

2.Preparation of bioactive peptides

Numerous methods have been used to release bioactive peptides from food proteins,including:(1) enzymatic hydrolysis using plant,microbial and/or digestive-derived enzymes,(2) hydrolysis produced by food processing,including microbial fermentation,heat treatment or other technologies,and (3) chemical synthesis,which is conducive to continuing scientific research (Callmannet al.2020).Some studies have also combined these methods to produce bioactive peptides (Lee and Hur 2017).It is extremely important to select the most appropriate preparation methods to obtain the target bioactive peptides economically and effectively.

2.1.Enzymatic hydrolysis

Enzymatic hydrolysis is an effective mean to produce food-derived bioactive peptides because of its advantage of process stability,high catalytic efficiency and product yields (Chaiet al.2017;Esfandiet al.2019).Fig.1 shows the steps of enzymatic hydrolysis in preparing bioactive peptides.

Fig.1 The steps in the enzymatic preparation of bioactive peptides.

The primary factor in the preparation of bioactive peptides derived from food by enzymatic hydrolysis is the selection of the enzymes.Different proteases have specific enzyme cleavage sites (Table 1),which can produce a variety of functional bioactive peptides.Some commercially available proteases are often used to produce bioactive peptides,such as alcalase (Honget al.2019;Gomezet al.2020),flavorzyme (Elavarasan and Shamasundar 2017),neutrase (Renet al.2018),trypsin (Guet al.2015),pepsin (Carrasco-Castillaet al.2012),protamex (Shenget al.2019) and papain (Liu M Cet al.2016;Xuet al.2019).In some cases,more than one proteolytic enzyme is required to produce the desired bioactive peptides (Shenget al.2019).There are also studies that use pepsin-pancreatin sequential proteolysis to simulate the hydrolysates produced during gastrointestinal digestion (Torres-Fuenteset al.2015;Indiano-Romachoet al.2019).

Table 1 Preferential cleavage positions of various proteases

Various conditions,such as enzyme specificity,hydrolysis time,enzyme/substrate ratio,and substance concentration,contribute to the yield,composition and functional activity of the bioactive peptides produced.The protein material is usually hydrolyzed at the optimum temperature and pH of the selected protease (Aluko 2015;Heet al.2016;Silvaet al.2017).The substrate hydrolysis rate tends to decrease as hydrolysis time increases,and eventually reaches a stable point (Tavano 2013).The enzymatic hydrolysis reaction can be divided into four stages:rapid ascending stage,slow growth stage,stable stage,and slight declining stage.The target peptide is usually obtained in the highest amount during the stable stage.In order to obtain a great degree of hydrolysis,the ratio of enzyme to substrate is an important factor to consider.Peptide sequences and their biological activities may vary depending on the type of enzyme used (Mojica and de Mejia 2016).

Enzymatic hydrolysis to produce bioactive peptides is often preferable to other methods due to the short reaction time,ease of scalability and predictability.The preparation of bioactive peptides by enzymatic hydrolysis with mild reaction conditions and a low cost is conducive to the industrial large-scale production of peptides.In addition,it can make full use of food processing waste,thereby increasing the value of by-products and improving resource utilization.

2.2.Microbial fermentation

Microbial fermentation can be used to produce various active peptides with different amino acid sequences and molecular masses through the metabolic and fermentation activities of microorganisms (Mhinaet al.2020).The method of microbial fermentation for the production of bioactive peptides can be used for products which allow the direct intake of the peptides in the fermented food,which has high safety and can avoid the need to purify the enzyme-generated peptides and reduce production costs.The enzymes produced by the microbial fermentation process may also cause the decomposition of fat and fishy substances in the raw materials,making the products have improved sensory qualities or a unique fermentation aroma.The preparation of bioactive peptides by microbial fermentation can effectively develop and utilize the waste materials generated during industrial food processing,which brings economic benefits.Fig.2 shows the steps of microbial fermentation in the preparation of bioactive peptides.

Fig.2 Preparation of bioactive peptides by microbial fermentation.

Some bacteria have proteolytic systems which are the source of bioactive peptide production during fermentation(Korhonen 2002).Microbial fermentation can be mediated by cell wall-bound proteases or intracellular peptidases,mainly endopeptidases,aminopeptidases,tripeptidases and dipeptidases (Christensenet al.1999;Ivanov 2017).The microorganisms used for fermentation need to be cultivated to the exponential growth phase.After collecting the microbial culture solution,it needs to be washed and added to a glucose solution in sterile distilled water,and then applied to the fermentation of protein substrates (Aguilar-Toalaet al.2017;Rizzelloet al.2017).The degree of hydrolysis depends on the fermentation time,microbial strain,and the protein source.In terms of microbial strains,high protease-active bacteria,molds and yeasts are the most commonly used.Different strains have varying effects on the protein materials.Sanjuktaet al.(2015) proved that soybean proteins fermented byBacillus subtilisMTCC5480 produced a higher degree of hydrolysis compared withB.subtilisMTCC1747.The selection of different protein materials and fermentation conditions is another key to fermentation.Zhenget al.(2018) successfully prepared an ACE inhibitory peptide by solid-state fermentation of defatted walnut residue withB.subtilis.Five main factors that affect fermentation include inoculation amount,pH,substrate concentration,fermentation time and temperature.The optimal fermentation conditions are always determined by the response surface analysis method or orthogonal design on the basis of the optimized parameter range obtained by single-factor experiments.Recently,food processing with mixed culture and fermentation using two or more types of microorganisms has also been reported(Santoset al.2014;Shiet al.2017;Tangyuet al.2019).The interactions between mixed culture microorganisms are very complex (Smid and Lacroix 2013).The characteristics of fermentation metabolism are particularly important in the synergy of the microorganisms,such as the production levels of enzymes and active ingredients,which can effectively improve the safety and functionality of the fermentation bioactive peptides (Cuiet al.2020).

2.3.Chemical synthesis

Bioactive peptides can also be synthesized chemically.Since the amounts of bioactive peptides found in nature are very low,the interest in producing synthetic bioactive peptides continues to grow (Espitiaet al.2012).Chemical synthesis is a method commonly used in laboratories to prepare peptides with specific amino acid sequences.Chemical synthesis can be divided into liquid phase synthesis methods and solid-phase synthesis methods.Liquid phase synthesis is used to synthesize peptides in a homogeneous solution.However,this method requires the product to be separated,purified or crystallized after each peptide synthesis step to remove unreacted raw materials and by-products.This clean-up step takes a long time and has high technical requirements.Peptide synthesis is a process of the repeated addition of individual amino acids.Solid-phase synthesis sequences are usually synthesized from the C-terminus to the N-terminus.The advantage of solid-phase synthesis is that all reactions can be performed in a single vessel.After the coupling step,unreacted reagents and byproducts can be easily removed by washing,thereby eliminating the intermediate purification step,which greatly reduces the effort required for product purification in each step (Madeet al.2014).At the same time,in order to prevent the occurrence of adverse reactions,the amino acid side chains involved in the reaction need to be protected,while the C-terminus is free and activated before the reaction.Solid phase synthesis is an effective method for small-scale synthesis of 10 to 100 residue peptides.

Selecting appropriate preparation methods to obtain the target bioactive peptides has great value regarding the economy and social benefits.The advantages and disadvantages of several commonly used methods for the preparation of food derived bioactive peptides introduced above are summarized in Table 2.

Table 2 The advantages and disadvantages of several commonly used methods for the preparation of food derived bioactive peptides

3.Molecular characteristics and structure–activity relationship

3.1.Antioxidant peptides

Oxygen is an extremely reactive non-metallic and oxidizing agent which can readily react with most elements and other chemical compounds to form oxides.In the ground state,molecular oxygen possesses two unpaired electrons,hence oxygen can accept a pair of electrons contributed by other molecules (Parham and lhami 2018;Gulcin 2020).Such oxidation-reduction reactions of electron transfer represent an indispensable metabolic reaction process in the living body.But problems may emerge when unpaired single electron transfers occur,generating free radicals.Free radicals can be derived from three elements:oxygen,nitrogen and sulfur.Reactive oxygen species (ROS) are well-known as oxygen-centered free radicals,such as superoxide (),hydroxyl (HO·) and hydrogen peroxide (H2O2) (Unget al.2017).

In general,metabolic pathways in the human body produce ROS or free radicals,which lead to the damage of starch,fat,proteins and nucleic acids.In addition to endogenous free radicals,when the human body is stimulated by various factors in the external environment(e.g.,tobacco,radiation,toxins,excessive minerals and air pollutants),the levels of ROS or free radicals in the body will increase (Kumar 2014).During the evolution of life,aerobic organisms have produced endogenous antioxidant systems to prevent oxidative damage.It is particularly important for the body to maintain a balance between free radicals and endogenous antioxidant systems (Girgihet al.2014;Qiuet al.2020).However,the maintenance of this balance is subject to many factors,including aging,fatigue and high-fat diets.Under these circumstances,the immunity of the body will be weakened and oxidative stress is prone to occur,which causes cellular damage and various diseases,such as cancer and Parkinson’s disease (Kimet al.2015).Therefore,antioxidant peptides are a good choice for enhancing the body’s antioxidant capacity,protecting the body and promoting health by means of exogenous antioxidant ingestion.

Antioxidants are attracting a growing interest due to their protective actions in food and pharmaceutical products against oxidation during storage and in the human body against the oxidative stress which causes pathological processes.A large number of natural and synthetic antioxidants have been discovered thus far.Natural exogenous antioxidants include carotenoids,lycopene,vitamins A,C and E,natural flavonoids,lutein and various other compounds.Synthetic antioxidants mainly include butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA),octyl gallate (OG) and tert-butyl hydroquinone (TBHQ) (Gulcin 2012).During the practical application process,synthetic antioxidants are unhealthy and may have potential risks compared with natural antioxidants (Liu Ret al.2016).Hence,researchers are devoting increasing efforts to developing natural antioxidants derived from food which can be used to prevent oxidation in place of the synthetic antioxidants with side effects.

Antioxidant peptides extracted from food or food hydrolysates are a good source of natural antioxidants.Antioxidant peptides,typically composed of 2–20 aminoacids,are inactivated and encrypted in the parent proteins,so they need to be released by enzymatic hydrolysis or gastrointestinal digestion to exert their activities (Choiet al.2012).The action mechanism of antioxidant peptides is relatively complicated and many factors affect the antioxidant activity of peptides.For example,the activities of antioxidant peptides are related to molecular mass,hydrophobicity,amino acid composition and amino acid sequence (Sarmadi and Ismail 2010).

In general,low molecular weight peptides have stronger antioxidant activities than their parent proteins because they are more accessible to lipid-free radicals,allowing them to inhibit free radical-mediated lipid peroxidation.Kimet al.(2007) found that the antioxidant capacity is closely related to the molecular weight of the peptide.The low molecular weight component (1–3 kDa) of theHokiframe protein hydrolysate has a higher antioxidant capacity than other higher molecular weight components.Similarly,Wanget al.(2019) reported that peptides derived from Pacific herring protein hydrolysate with molecular weights below 3.5 kDa showed stronger antioxidant activities than the higher molecular weights fractions.Moreover,the short peptide (<3 kDa) ofSchizochytriumsp.protein hydrolysate prepared by alcalase and flavourzyme exhibited stronger antioxidant activityin vivothan other peptides (Caiet al.2017).

Several studies have demonstrated that the amino acid composition and its position in the peptide sequence play a crucial role in the antioxidant activity of peptides.Furthermore,aromatic,acidic,basic and hydrophobic amino acids are the active sites of antioxidant peptides(Rivalet al.2001;Ghassemet al.2017).For example,one study identified a specific antioxidant peptide purified from black sharkskin protein hydrolysates as Ala-Thr-Val-Tyr (ATVY).Amino acid mutation experiments revealed that the crucial antioxidant interaction site of ATVY that scavenges ABTS radicals was Tyr in the N-terminal(YangQet al.2020b).Moreover,Sunet al.(2019)demonstrated that Tyr residues at the C-terminal region were critical in the antioxidant activity of Arg-Asp-Tyr because Tyr contains phenolic hydroxyl groups,which provide protons for the free radicals (Saitoet al.2003;Guet al.2012).The sources and activities of several other food-derived peptide sequences are summarized in Table 3.It was found that isolated antioxidant peptide sequences derived from different food sources generally have aromatic,basic,acidic or hydrophobic amino acids at the C- or N- terminal regions.Therefore,the amino acid residues of the peptide chain at the C- and Nterminal regions play an important role in exerting the antioxidant effects.

Table 3 Sources and structure–activity relationships of food-derived antioxidant peptides

3.2.Antimicrobial peptides

Antimicrobial resistance (AMR) refers to the ability of microorganisms (such as bacteria) to become increasingly resistant to their previously susceptible antimicrobials(Nikaido 2009).AMR has become an increasingly serious public health crisis worldwide,involving clinical,veterinary,agricultural and other fields,eroding the effective application of antimicrobials (Davies and Wales 2019).Although AMR is the inevitable result of microbial evolution and adaptation,the unbridled use and misuse of antibiotics in humans has led to the rapid and widespread emergence of drug resistance in a wide variety of pathogens and symbiotes (Penget al.2015).The gradual expansion of human demand for animal protein has become the most significant dietary trend of this era.As a result,the annual consumption of antimicrobials in animals is currently threefold the level of humans,which ensures that large-scale animal protein production is feasible (Mc Nultyet al.2016).However,it is concerning that food acts as a vehicle for transferring antimicrobial resistant bacteria and antibiotic-resistant genes to humans.The latest findings support the view that there is a link between the use of antibiotics in agricultural production and AMR of human pathogens,in which food may be one of the key transfer pathways (Silbergeldet al.2008;Thanneret al.2016).Collectively,the present situation of AMR is serious,and there is an urgent need to develop new antibiotics or alternative therapies.

Antimicrobial peptides (AMPs) are a promising substitute for traditional antibiotics (Xu and Lai 2015).AMPs are natural proteins that protect the host from pathogenic organisms,which are derived from bacteria,insects,plants and animals (Abedinzadehet al.2015).The primary structures of AMPs,from different sources are so different that it is difficult to classify them according to sequence similarity or dissimilarity (Anderssonet al.2016).In addition,many AMPs share similar characteristics such as hydrophobicity,amphipathicity,and a positive net charge,thus it is not appropriate to classify them based solely on physical characteristics.As an alternative,therefore,the secondary structures of AMPs,the conformations formed when they interact with bacterial membranes,have become a useful tool for classification (Sperstadet al.2011).

AMPs can be classified into four groups based on their secondary structures:α-helical,β-sheet,extended peptides and loop.The α-helical and β-sheet peptides are the focus of much research,and AMPs endowed with α-helical structures are the most studied so far.The α-helical AMPs are linear in aqueous solution and exhibit amphiphilic helix structures when they interact with bacterial membranes or are dissolved in organic solvents(Fig.3-A).This transformation induces the segregation in space of the hydrophilic/charged amino acids from the hydrophobic residues,which results in an amphipathic structure that is recognized as a prerequisite for the action of AMPs on the membrane (de la Fuente-Nunezet al.2017;Koehbach and Craik 2019).Piscidin-1,cecropin-1 and magainin-2 are typical examples of peptides that belong to this group (Ahmed and Hammami 2019).

Besides α-helical,β-sheet is another major secondary structure of AMPs,which can be induced in a simulated membrane environment.Moreover,in aqueous solution under the simulated membrane environment,the conformation of most β-sheet peptides changes from unstructured to a β-sheet structure (Fig.3-B).In general,β-sheet peptides are composed of two to ten cysteine residues and from one to five disulfide bridges as conformational constraints to stabilize their bioactive conformations (Ahmed and Hammami 2019).Additionally,peptides that contain disulfide bonds universally form a cyclic β-hairpin conformation,such as cathelicidins and hepcidins (Koehbach and Craik 2019).

Fig.3 Representative structural classes of antimicrobial peptides (AMPs).A,α-helix (Lactoferricin H,PDB:1Z6V).B,β-sheet (Hepcidin,PDB:1S6W).C,loop (Lactoferricin B,PDB:1LFC).D,extended (Indolicidin,PDB:1G89) AMPs.Disulfide bond are yellow-colored.Source:Ahmed and Hammami (2019).

Loop AMPs have a stable loop structure formed by disulfide,amide or isopeptide bonds (Fig.3-C).Thanatin,isolated from the spined soldier bug,is a representative member of this group,and its structure is stabilized by a single disulfide bond between residues 11 and 18 (Di Sommaet al.2020).Extended AMPs are rich in certain amino acids,including tryptophan,arginine,proline,glycine and histidine residues,and lack the conventional secondary structures that are stabilized only by the hydrogen and van der Walls interactions with membrane lipids (Chaturvediet al.2018).At the same time,they may contain extended coils (Fig.3-D).Indolicidin and apidaecin are typical representatives of the extended AMPs (Wanget al.2018).

The bacterial membrane is the target of most AMPs.More importantly,compared with the cell membranes of plants,invertebrates and vertebrates,bacterial membranes are selective targets of AMPs (Valeroet al.2020).The existence of lipopolysaccharides (LPS),anionic lipids or teichoic acids leads to the negative charges of bacterial membranes,including Grampositive/negative bacteria,which attract antimicrobial peptides through electrostatic interactions (Matsuzaki 2009).In addition,the key structural feature of AMPs is amphipathicity,which plays an important role in the perturbation between antimicrobial peptides and bacterial membranes (Wanget al.2018).In the process of bacterial membrane destabilization caused by AMPs,there are three main models:Barrel-stave model,Toroidal-pore model,and Carpet-like model.It should be mentioned that the bacterial membrane is not the only target of AMPs (Thapaet al.2020).Under some special conditions,AMPs can also kill bacteria without changing the integrity of the bacterial membrane by inhibiting the synthesis of DNA,RNA,enzymes or proteins (Fig.4)(Mookherjeeet al.2020).For example,a study showed that thanatin disrupts the outer membrane of NDM-1-producing bacteria by competitively displacing the divalent cations on the outer membrane and inducing the release of LPS (Maet al.2019).Unlike the action mechanism of traditional antibiotics,the interaction between AMPs and the bacterial membrane is not focused on a specific intracellular target,so AMPs are less likely to facilitate the microbial evolution of drug resistance.

Fig.4 Models of antibacterial mechanisms of antimicrobial peptides (AMPs) (Source:Mookherjee et al. 2020).

The direct killing of microorganisms was initially considered to be the primary function of AMPs.With this ability,AMPs exhibit direct cytotoxic activity against bacteria,fungi,viruses,parasites and even cancer cells(Mookherjeeet al.2020).However,a growing body of evidence shows that as an important substance in protecting the host from pathogenic organisms,AMPs plays other roles in addition to directly killing the microorganisms.Under physiological conditions,AMPs play a role in regulating immunity and immune cells(Hilchieet al.2013).This is another key role of AMPs in the host.The molecular mechanisms by which AMPs regulate immunity and immune cells are very complex,and include (but are not limited to):directly or indirectly recruiting antigen-presenting cells to the infected site by inducing chemokine to enhance the antibacterial effect,changing the signal pathway mediated by endotoxin,promoting the activation of extracellular trap (Nets) of neutrophils,inducing anti-inflammatory cytokines,affecting the differentiation of dendritic cells and polarizing T cells,inhibiting pro-inflammatory cytokines,and enhancing the phagocytosis and pro-inflammatory response to nucleic acids (Hilchieet al.2013;Mookherjeeet al.2020).Therefore,the selective enhancement of host immune responses through AMPs may be a development direction of anti-infective therapy,which will reduce the probability of developing AMR.

In general,natural AMPs are sensitive to protein hydrolysis and generalized hydrolysis,high cytotoxicity and poor tissue distribution (Diet al.2020).Therefore,the development of modified synthetic peptides with unique properties has become a major focus of current research.However,understanding the structure–activity relationships of natural AMPs is a precondition for the design and development of synthetic peptides with unique properties.For example,Mihailescuet al.(2019)determined how histidine-related amphiphilic changes affected the permeabilization properties of piscidins-1 and piscidins-3 through complementary methods,including NMR-monitored titrations,oriented CD,X-ray and neutron diffraction,quartz crystal microbalance with dissipation,and molecular dynamics.Tracing the functional roles of the amino acids of the key antimicrobial region of piscidins provides a basis for designing AMPs with unique properties.In addition,the development of AMP mimics,AMP homologues,cyclopeptides,immobilized AMP,AMP conjugates and hybrid AMP are representative methods to overcome the limitations of natural AMPs (Maiet al.2017).

3.3.Immunomodulatory peptides

The specific amino acid sequences within their overall structures may have multiple immunomodulatory effects,which positively impact bodily functions and effectively inhibit inflammatory disorders.A hexapeptide (VEPIPY),found in human casein by enzymatic digestion,was the first peptide approved by Parkeret al.(1984) in 1984 for immunomodulatory application.In vitro,this hexapeptide stimulated the phagocytosis of opsonized sheep red blood cells by murine peritoneal macrophages.Many other peptides derived from food have been approved for immunomodulatory treatment since then.Plantand animal-derived proteins have been considered as important sources of immunomodulatory peptides(Santiago-Lopezet al.2016a).The immunomodulatory peptides depend on their specific amino acid sequences and their bioavailability.However,the origin and role of immunomodulatory peptides are uncertain,and their immunomodulatory activity is independent of their source.They are revealed only after the degradation of the native proteins during the digestion processes.

The immune system resists the invasion of pathogens and protects the body from damage.However,it is also susceptible to various factors which are difficult to clearly define,such as age,stress,and an unhealthy lifestyle.The treatment of adjusting the immune system with drugs is not only expensive but also has side effects,and most drugs are not suitable for the treatment and prevention of chronic diseases.Food-derived immunomodulatory peptides have effects on both innate immune responses and adaptive immune responses,including either enhancing or weakening immunity by up-regulating antior pro-inflammation cytokines,stimulating the proliferation of spleen lymphocytes,increasing the phagocytic ability of macrophages,and enhancing the activity of NK cells.Food-derived immunomodulatory peptides are ideal functional food ingredients and offer hope for the replacement of the drugs with high side effects for disease treatment.

The immunomodulatory capacity of bioactive peptides is related to their physicochemical properties,such as amino acid composition,sequence,length,charge,hydrophobicity,degree of hydrolysis and the structure of the peptide molecule.Chalamaiahet al.(2018) summarized the immunomodulatory peptides that had been isolated from several hydrolyzed food proteins and concluded that food-derived peptides with immunomodulatory effects tend to be composed of 2–10 amino acids and hydrophobic in nature.Table 4 presents the immunomodulatory peptides reported in the past five years,and each of them contains hydrophobic amino acids.Furthermore,the sequences of immunomodulatory peptides generally consist of basic amino acid residues such as Arg,a negatively charged amino acid (Glu)and neutral amino acids (Tyr,Ser),which is similar to the findings of Mercieret al.(2004).Jacquotet al.(2010) showed through analysis of regression that the proliferative effect of peptides was greatly influenced by positive charge,hydrophobicity and the length of the peptide chain.Vogelet al.(2002) also concluded that the immunomodulatory and anti-inflammation activities of lactoferricin peptides were more related to a positively charged region of the peptide by binding and activating the chemokine receptors on immune cells.Wuet al.(2016)hydrolyzed the defatted wheat germ globulin (DWGG)with alcalase,neutrase,papain,pepsin and trypsin,and determined the degree of hydrolysis (DH),molecular weight distribution,hydrophobicity,secondary structures and immunomodulatory activities of the hydrolysate.The results showed that the DH and hydrophobicity were correlated with immunomodulatory activity.Mercieret al.(2004) also considered the hydrophobic value as a key factor in the immunomodulatory activity of whey protein hydrolysates,which may improve the immunomodulatory activity by enhancing the interaction of peptides with cell membranes.Zhang Set al.(2019) found that pulsed electric field (PEF) treatment can improve the immunomodulatory activation of hexapeptide RGAVLH.Further research demonstrated that the primary structure of the RGAVLH was unchanged after the PEF treatment,but the secondary structure changed with the variation of the electric field intensity.After analyzing the changes of the peptide solution with respect to the spatial state and the spatial force treated by the PEF,the authors speculated that Ala,Val and Leu played a vital role in the improvement of the immunomodulatory activity of RGAVLH.Although the research on the structure–activity relationships of immunomodulatory peptides is currently incomplete and mostly based on speculation and inference,it is undeniable that the immunomodulatory activity of peptides is indeed affected by their physicochemical properties.

3.4.Antifreeze peptides

Antifreeze peptides (AFPs) are low-molecular-weight proteins or protein hydrolysates that can control the growth of ice crystals and inhibit recrystallization between ice crystals,and they play a role in low-temperature protection for organisms under freezing or sub-freezing conditions (Chen Xet al.2019a;Chenet al.2020b).Peptides with natural antifreeze activity have been found in various organisms.For example,in adapting to the cold environment,many fish in nature produce a class of AFPs that protect them from damage due to freezing,which gives them a specific cold resistance.According to the amino acid composition and structure,AFPs can be divided into four types:(a) alanine-rich repeat sequences,with an α-helix structure of the peptides;(b) cysteinerich,with a β-sheet structure of the peptides;(c) different from the above two peptide types,but without a specific structure;and (d) antifreeze glycoprotein peptides (Kun and Mastai 2007;Baet al.2013;Mochizuki and Molinero 2018;Chen and Wang 2019).Additionally,according to different preparation methods,AFPs can be divided into naturally extracted AFPs,synthetic AFPs and enzymeconverted AFPs (Chenet al.2020d).Naturally extracted AFPs mainly come from Type I AFPs and antifreeze glycopeptides (AFGPs) in the serum of polar fish.Enzyme-converted AFPs are mainly obtained from foodderived collagen by screening for peptide fragments with antifreeze activity through enzymatic hydrolysis,also known as collagen-based AFPs (Chen Xet al.2019b;Chenet al.2020b).Antifreeze peptides play an important role in the cryoprotection of frozen foods,biomedicine,and low-temperature tissue engineering (Chenet al.2017,2020c;Damodaran and Wang 2017).

Type I antifreeze peptidesType I AFPs have been found in winter flounder and sculpins.There are two different organ/tissue distributions of Type I AFPs in fish(Sicheri and Yang 1995).One type enters into the blood circulation after being secreted by the liver,which is called liver Type I AFPs.The other non-secretory type,known as cutaneous Type I AFPs,is widely distributed in the skin,gills,fins,stomach and other tissues in direct contact with the environment,and is even expressed in the brain.Both the sculpins and the winter flatfish contain both of these different branch types of AFP-I.Currently,a number of individual Type I AFPs have been isolated from winter flounder and sculpins.

Type I AFPs have a regular α-helix structure,and the function and mechanism of Type I AFPs have been thoroughly studied.Type I AFPs have a molecular weight in the range of 3.3–4.5 kDa,usually contain about 40 amino acid residues,and have a repeat sequence of 11 residues beginning with Thr.The sequences of (-Thr-X(2)-Asx-X(7)-) are connected in series,where the X is mostly alanine (Fig.5) (Loewenet al.1999).The spatial structure of Type I AFPs has a high helix content.The length of the helix conformation is an important factor that determines the strength of its antifreeze activity.The longer the length,the higher thermal hysteresis activity (THA) and the stronger the antifreeze activity.As the temperature rises,the α-helix content decreases significantly,and some helices have amphiphilic characteristics.

Fig.5 Type I AFP variant sequences and 3D structure.A,the wild-type sequence is shown with putative ice-binding motifs (IBMs)overlined and Thr identified by residue numbers.B,X-ray crystal structure of HPLC-6.C,side view of the helix with the salt bridge and four IBM regions identified (Source:Sicheri and Yang 1995;Loewen et al. 1999).

Antifreeze glycopeptidesThe primary structure of the antifreeze glycopeptide (AFGP) is composed of the repeat sequence of alanine-alanine-threonine (Ala-Ala-Thr),where the hydroxyl group of Thr is glycosylated with β-Dgalactosyl-(1,3)-α-N-acetyl-D-galactosamine (Fig.6).

Fig.6 Structure of AFGP8.A,sequence of AFGP8,Ala-Ala-T＊-Ala-Ala-T＊-Pro-Ala-T＊-Ala-Ala-T＊-Pro-Ala,where T＊ denotes the glycosylated Thr.B,three-dimensional structure of AFGP8,with the peptide backbone in the PPII helix conformation (Source:Mochizuki and Molinero 2018).

Extensive structure–activity relationship research has been conducted on AFGP.Most structure–activity relationship studies of AFGP are based on the naturally purified form from the polar fish.Through a series of systematic chemical modifications,as well as enzymatic and chemical degradation,certain basic structural requirements have been shown to be critical for the ability of AFGP to inhibit the growth of ice crystals.The circular dichroism (CD) spectrum shows that AFGP has an extended random coil structure in solution.Subsequent experimental studies combining NMR and CD proposed a conformation similar to the polyproline II (PPII) helix.The PPII helix is a left-handed helix conformation and contains three residues per revolution.As the temperature decreases,the PPII content increases.This shows that the antifreeze activity of AFGP strongly depends on the presence of a specific sequence of spirals similar to the PPII spirals (Mochizuki and Molinero 2018).

To understand the relationship between the structure of AFGP and its antifreeze activity that inhibits the growth of ice crystals,many studies and assumptions have been made.The sugar groups in AFGP are not only high in content,but also the main group involved in the antifreeze activity.Therefore,in recent years,international research has begun to develop antifreeze glycopeptide analogs based on glycosylation modifications,which is one of the research hotspots (Chen and Wang 2019).For example,an antifreeze peptide analog formed by four repeating Ser-Gly-Gly tripeptide peptide chains as the backbone and a galactosyl group connected through the carbon terminal of the Ser residue was found to have strong recrystallization inhibitory activity (Liu and Ben 2005).In addition,the types of amino acid residues have a significant effect on antifreeze activity.Since proline has a limited rotation angle,proteins or peptides containing it can have an obvious secondary structure in solution,and some analogs containing proline also have strong lowtemperature protection activity (Treviñoet al.2018).

Enzyme-converted antifreeze peptidesEnzymeconverted antifreeze peptides are mostly prepared from the collagen in animal skin,fish scales,and fish bones or edible gelatin as raw materials (Du and Betti 2016;Chenet al.2020b;Yuet al.2020).In recent years,a great deal of research has been conducted on the structure of antifreeze peptides.Studies have shown that enzymeconverted AFPs usually have a specific peptide chain length,and the molecular weight is generally less than 2 000 Da (Chen Xet al.2019b).Moreover,enzymeconverted AFPs have been found to have a specific composition of amino acids including Ala,Gly,and Pro,which may be largely responsible for the antifreeze activity(Damodaran 2007).The molecular structures of these enzyme-converted AFPs are generally characterized by Gly-Pro-X or -Gly-Z-X- tripeptide repeat sequences,or with GTPG-GPP (OH) G,and Pro-Ala-Gly-Tyr structural features (Wanget al.2014).

In order to explore the action mechanism of enzymemodified AFPs,molecular modeling and energy minimization applications were performed by molecular dynamics.Several gelatin-based AFPs were used as models to determine the molecular mechanism that explains why the molecular mass of high-activity AFPs is generally less than 2 000 Da.The results of molecular dynamics calculations indicate that AFPs of less than 2 000 Da are most likely to form rigid collagen-type spiral rods,which may allow them to superimpose these ice nucleus rods with the oxygen triplet facing the prismatic surface of the ice nucleus (Fig.7).However,the gelatin AFPs with a molecular mass larger than 2 000 Da may have reduced hardness,and steric hindrance may prevent the peptides from correctly accumulating on the prismatic surface of the ice nucleus,thereby reducing their ability to inhibit ice crystal growth (Damodaran 2007).

Fig.7 Energy-minimized structure of model gelatin peptide Gly-Pro-Thr-Gly-Pro-Leu-Gly.A,0° projection.B,90° projection.The oxygen atoms in the oxygen triad plane and the O-O distances are shown (Source:Damodaran 2007).

4.Conclusion and future perspectives

The role of various food compounds in promoting health has received much attention,and peptides are one of the food compounds with biological activity in the body.This review provides the research progress on the preparation and functional characteristics of food-derived bioactive peptides,and concludes that the conformation of peptides plays a decisive role in the biological activity of these

molecules.Peptides are proteinogenic and therefore have unique net charges,hydrophilic/hydrophobic properties,different molecular weights,and the ability to form secondary and tertiary structures.These characteristics also give peptides the inherent ability to show structure–property relationships.

With the discovery and in-depth study of various food-derived bioactive peptides,the following four key problems restricting the research and application of foodderived bioactive peptides have become increasingly prominent.

(1) The food-derived bioactive peptides with nontoxic effects and high stability penetrate into tissues easily due to their low molecular weight.Food-derived bioactive peptides have numerous modulatory and regulatory physiological activities.New food-derived bioactive peptides are still awaiting discovery and exploration.

(2) The development and exploration of food-derived bioactive peptides still have significant limitations.At present,most of the research on food-derived bioactive peptides is still in the laboratory stage.The development of bioactive peptide separation equipment suitable for industrial production can enable large-scale production of food bioactive peptides beyond the laboratory level.

(3) Food-derived bioactive peptides have broad application prospects as functional foods and medicines.Although they are considered relatively safe,some of them may trigger allergic responses in a small proportion of the population as the protein hydrolysates might contain immunogenic peptides (Francket al.2002;Akiyamaet al.2006;Liu Let al.2020).The activities and safety of peptidesinvivoexperiments must be carried out before the final products can be put on the market.Large-scale food development should be undertaken only after it is confirmed that adding such peptides to food would not cause negative effects,and that these peptides still retain activity when used as food ingredients.

(4) More in-depth work needs to be undertaken on the isolation,characterization,and definition of the mechanisms of action.It is possible to consider chemical modification of the obtained food bioactive peptide structures,thereby the activity can be improved and the performance can be made more stable,both of which are conducive to market development and utilization.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (U1905202,31972017,and 31771922),the National Key R&D Program of China(2018YFD0901006),the Fujian Major Project of Provincial Science &Technology Hall,China (2020NZ010008),the Open Project of the Key Laboratory of Refrigeration and Conditioning Aquatic Products Processing,Ministry of Agriculture and Rural Affairs,China (KLRCAPP2021-03),and the Quanzhou Science &Technology Project,China(2019C085R).

Declaration of competing interest

The authors declare that they have no conflict of interest.