Sources, chemical synthesis, functional improvement and applications of food-derived protein/peptide-saccharide covalent conjugates: a review

Critical Reviews in Food Science and Nutrition ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/bfsn20

Sources, chemical synthesis, functional improvement and applications of food-derived protein/peptide-saccharide covalent conjugates: a review Mengge Zhao, Hui He, Aimin Ma & Tao Hou To cite this article: Mengge Zhao, Hui He, Aimin Ma & Tao Hou (2022): Sources, chemical synthesis, functional improvement and applications of food-derived protein/peptide-saccharide covalent conjugates: a review, Critical Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2022.2026872 To link to this article: https://doi.org/10.1080/10408398.2022.2026872

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Review

Sources, chemical synthesis, functional improvement and applications of food-derived protein/peptide-saccharide covalent conjugates: a review Mengge Zhaoa,b, Hui Hea,b, Aimin Maa,b and Tao Houa,b a College of Food Science and Technology, Huazhong Agricultural University, Wuhan, China; bMinistry of Education, Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Wuhan, China

ABSTRACT

Proteins/peptides and saccharides are two kinds of bioactive substances in nature. Recently, increasing attention has been paid in understanding and utilizing covalent interactions between proteins/peptides and saccharides. The products obtained through covalent conjugation of proteins/ peptides to saccharides are shown to have enhanced functional attributes, such as better gelling property, thermostability, and water-holding capacity. Additionally, food-derived protein/ peptide-saccharide covalent conjugates (PSCCs) also have biological activities, such as antibacterial, antidiabetic, anti-osteoporosis, anti-inflammatory, anti-cancer, immune regulatory, and other activities that are widely used in the functional food industry. Moreover, PSCCs can be used as packaging or delivery materials to improve the bioavailability of bioactive substances, which expands the development of food-derived protein and saccharide resources. Thus, this review was aimed to first summarize the current status of sources, classification structures of natural PSCCs. Second, the methods of chemical synthesis, reaction conditions, characterization and reagent formulations that improve the desired functional characteristics of food-derived PSCCs were introduced. Third, functional properties such as emulsion, edible films/coatings, and delivery of active substance, bio-activities such as antioxidant, anti-osteoporosis, antidiabetic, antimicrobial of food-derived PSCCs were extensively discussed.

1. Introduction Proteins/peptides and saccharides are two important biomolecules in food materials and play important roles in affecting food textures. Protein is not only an essential ingredient in the diet, but also exhibits functional properties because of its specific native structure, including enzymatic activities, hydration properties, interfacial properties, and intermolecular interactions with other biomolecules (Foegeding and Davis 2011). Bioactive peptides usually contain 2–20 amino acid residues per molecule, but in some cases may consist of more than 20 amino acids, and molecular masses of less than 6000 Da (Sarmadi and Ismail 2010). Compared to proteins, peptides have superior physiological activity, high permeability, high solubility at any pH range, increased emulsion stability, high digestion and absorption rate (Amrita and Mann 2011; Zohreh and Behrouz 2019). Saccharides have the abilities to thicken and hold water and to regulate intestinal flora to promote intestinal health (Wong et al. 2006). Currently, these two biomolecules are used to prepare conjugates in order to improve their functional properties and bio-activities. The binding modes between proteins and saccharides mainly include electrostatic and covalent bindings, and covalent bonds are much more stable in nature than ionic bonds. Protein/peptide-saccharide covalent conjugates (PSCCs), also known as glycoproteins/ glycopeptides, are a type of saccharide whose oligosaccharide CONTACT Tao Hou

Bio-activities; functional properties; protein-saccharide covalent conjugates; sources; structures; synthetic methods

chain is connected to the hydroxyl or carboxyl group of some special amino acid residues in the protein chain through covalent bonds. Glycopeptides may exist in nature as such, and/or be released from glycoproteins by chemical/ enzymatic hydrolysis. Moreover, glycopeptides are formed by covalent bonding between oligosaccharides and amino acids or peptides as well. Compared with glycoproteins, glycopeptides have smaller molecular weights, and the constituents are simple. Although there are many types of natural glycoproteins/glycopeptides, such as sialylglycoprotein/ sialylglycopeptide (SGP) and caseinomacropeptide (CGMP), the two most common types are: N-glycoproteins/glycopeptides and O-glycoproteins/glycopeptides (Marcelo et al. 2012; Mezei and Csonka 2015). At present, the primary methods of glycopeptide synthesis mainly include chemical synthesis (direct synthesis, liquid and solid phase formation, native chemical ligation), enzymatic synthesis, and chemoenzymatic synthesis. Protein/peptide-saccharide covalent conjugates possess various bio-activities such as antioxidant, antibacterial, immunoregulatory, and anticancer activities (Wang et al. 2018c). PSCCs are also known to have enhanced functional attributes compared to their precursors (emulsifying, gelling, and foaming capacities; thermal stability; delivery properties, and release of bioactive substances) (Nooshkam and Varidi 2020). It has been widely documented that PSCCs via the Maillard reaction can improve many important functional

Figure 1. Classification, synthesis methods, functional properties, and biological activities of food-derived protein/peptide-saccharide covalent conjugates.

properties of food proteins, as reviewed by Oliver et al. (Oliver, Melton, and Stanley 2006). However, these studies mainly focus on the Maillard reaction, and pay less attention on other reactions, such as transglutaminase (TGase)catalyzed reaction. Additionally, the improvement of functional and physicochemical properties and structure of PSCCs was also the research focus. The comprehensive review about sources and structures of natural food-derived PSCCs, synthesis methods, the enhancement of functional attributes, biomedical functions, wide applications in functional food industry and potential hazards are lacking. Thus, this review first summarizes the sources, types, and classification of food-derived PSCCs as well as the synthesis methods of these conjugates. Second, the functional properties such as emulsion, edible films/coatings, and delivery of active substance, and biological activities such as antioxidant, anti-osteoporosis, antidiabetic, antimicrobial of food-derived PSCCs and their applications in food and pharmaceutical industries, especially in the delivery and release of active substances field, are reviewed (Figure 1). We hypothesized that this review could provide new theoretical guidance and research ideas for the production and the utilization of novel food resources in food industry.

2. Natural sources of protein/peptide-saccharide covalent conjugates (glycoproteins/glycopeptides) To date, protein/peptide-saccharide covalent conjugates have been isolated and characterized from a variety of sources, including glycoproteins/glycopeptides from plants such as potato, soybean, aquatic organisms, and their byproducts such as oysters (Rhopilema esculehtum), sea urchins, and edible fungi such as Ganoderma lucidum mushroom. Additionally, glycoproteins/glycopeptides have been also generated from egg and whey proteins. According to the types of cross-linking between proteins and saccharides, glycoproteins/glycopeptides are mainly divided into two categories: N- and O-linked glycoproteins/ glycopeptides. N-glycoproteins/glycopeptides contain an amide bond between the anomeric carbon atom of the

N-acetylamino saccharide and γ-amide nitrogen atom of asparagine. Sialylglycoprotein/sialylglycopeptide is a common N-glycoprotein/glycopeptide and is abundant in hen egg yolk (Zou et al. 2012), which has an A2G2S2 structure (A stands for GlcNAc, G stands for galactose, and S stands for N-acetylneuraminic acid) (Alagesan and Kolarich 2019). O-glycoproteins/glycopeptides are formed by the connection of the anomeric carbon atom of the saccharide with the hydroxyl oxygen atom of the hydroxyl amino acid in the peptide, commonly threonine, serine, 4-hydroxyproline, and 5-hydroxylysine (Mezei and Csonka 2015). One glycoprotein can contain two glycopeptide bonds (Garrido, Dallas, and Mills 2013), and the hydroxyl group of the saccharide in glycoprotein/glycopeptide can be modified by groups such as sulfate ester and phosphate ester. Bovine milk glycomacropeptide is derived from the action of κ-casein, with exclusively O-linked glycosylation, and can promote the growth of probiotics (O’Riordan et al. 2018). Moreover, C-glycopeptides are formed as a result of C-C linkage between the saccharide chain and tryptophan (cysteine, lysine) residue of the peptide chain. For example, in phosphatidylinositol (GPI)-anchored glycopeptides, the GPI moiety of the saccharide is linked to the carboxyl end of the peptide by amide bond. S-glycopeptides are formed as a result of S-C/N linkage between the saccharide chain and peptide chain (Thayer et al. 2005). However, fewer studies are found about these two glycopeptides (C- and S- types).

2.1. Animal sources Sialylglycoprotein/Sialylglycopeptide, like low-density lipoproteins, lipophosphoproteins, and highly phosphorylated proteins, are the main components of egg yolk (Alagesan and Kolarich 2019). SGP is a complex sialic acid oligosaccharide chain with a complete branching type of double-sialylation. The amino acid composition of its peptide chain is lysine-valine-alanine-asparagin e-lysine-threonine (Lys-Val-Ala-Asn-Lys-Thr), in which Asn is modified by a double sialic acid saccharide chain (Zou

et al. 2012). Bovine milk glycomacropeptide is derived from the action of κ-casein, with exclusively O-linked glycosylation, and can promote the growth of probiotics (O’Riordan et al. 2018). Caseinomacropeptide is produced by hydrolyzing κ-casein of bovine milk with chymosin, and approximately 30%–50% of caseinomacropeptide exists in the glycosylated form with threonine residues at positions of 131, 133, 135, 136 and 142 as the sites for glycosylation, known as caseinomacropeptide (Ming et al. 2015). CGMP is rich in several neutral amino acids such as threonine, serine, and isoleucine, but lacks phenylalanine, tryptophan, and tyrosine (aromatic amino acids) (Liebenberg et al. 2018); therefore, it can be used as a dietary choice for patients with phenylketonuria, a genetic disease affecting the metabolism of aromatic amino acids) (Abdel-Salam and Effat 2010). Shikov et al. (2019) recently reported a novel bioactive glycopeptide from the internal organs of green sea urchins. A shotgun proteomic approach and high-performance liquid chromatography with refractive index detection were used to identify the glycopeptide, and the major monosaccharides identified were fucose and glucose. 2.2. Plant sources Soybean protein is the primary source of plant-based protein, and its main component is β-conglycinin, which is a glycoprotein containing mainly high-mannose moieties (Li et al. 2016). Soybean glycopeptide is prepared through size exclusion chromatography following the alcalase digestion of β-conglycinin. Its mannose substructure can prevent bacteria from contacting human colon adenocarcinoma cells and has the potential to protect against bacterial infection (Yang et al. 2008). Patatin, a storage protein found in potato tubers with a molecular weight of 39–45 kDa, is shown to have antioxidant activity, and the saccharides in patatin are mainly rhamnose, mannose, glucose and galactose (Acharjee et al. 2018). 2.3. Edible fungus sources Polysaccharopeptide (TPSP) from Trametes versicolor is one of the main active ingredients of Ganoderma lucidum mushroom. The saccharides of TPSP are connected by β-1, 3, and α-1,4 glucosidic linkages, mainly glucose, galactose, xylose, arabinose, and others. The peptide part of TPSP consists of 18 amino acids, most of which are acidic amino acids, among which aspartic acid and glutamic acid are the most abundant (Pallav et al. 2014). Moreover, TPSP is a bioactive macromolecule with anti-tumor and immune-enhancing activities. (Wang et al. 2019b).

3. Synthetic reaction of protein/peptide-saccharide covalent conjugates in food system Strategies for the synthesis of glycopeptides commonly include those of N-glycopeptides, O-glycopeptides, 3 Figure 2. Structures of N-, O-, C-, and S-linked glycopeptides.

S-glycopeptides, and C-glycopeptides (Figure 2). Synthesis of N-glycopeptides usually involves the formation of amide bonds between the pre-protected glycosamine and free carboxyl group of pre-protected aspartic acid under the action of a condensation agent, which is similar to the formation of peptide bonds. Synthesis of O-glycopeptides is relatively easier than that of N-glycopeptides, wherein oligosaccharide donors (anomeric carbon atoms) react with protected serine and threonine, and most of the glycosyl donors can be used. Sulfur replacement of the anomeric oxygen or nitrogen atom produces the corresponding S-glycopeptide, which is chemically stable and more resistant to glycosidase. In addition, S-linked oligosaccharides, which are closely related to S-glycopeptides, can be used as enzyme inhibitors and are suggested to be more immunogenic than natural O-linked analogs. Moreover, various alternative methods have been developed for the synthesis of C-glycopeptides (Dondoni and Marra 2001). 3.1. Liquid and solid phase synthesis The synthesis of glycopeptides by liquid and solid phase methods is also known as linear synthesis. It starts with the construction of glycosylated amino acids that gradually condense with other fragments in the solution, followed by the prolongation of peptide chains using liquid or solid-phase synthesis technique to complete the synthesis of target glycopeptides. Types of solid-phase synthesis are as follows: (i) glycosylated amino acids are used as monomers to synthesize more complex glycopeptides on the solid phase. (ii) oligopeptides are initially synthesized on the resin and the active residues are masked with different protecting groups, which are later removed gradually to perform glycosylation reactions on the oligopeptides. Since the glycopeptide bond has been formed before the peptide chain is extended, this method has the advantages of the connection site, and stereoselectivity is easier to control (Baumann, Kowalczyk, and

Kunz 2008). This method has become a general method for the synthesis of large-size glycopeptides. However, protection and deprotection reactions must have excellent specificity in this method; therefore, this method is more suitable for the synthesis of glycopeptides with simple oligosaccharide chains. Maemura et al. (Maemura et al. 2005) used this method for the synthesis of glycopeptide 2, that is, the benzyl-protected core 8 O-glycan for glycopeptides was synthesized stereo-selectively by the glycosyl fluoride method. Then, a glycopeptide containing two O-glycan was obtained by solid-phase synthesis. Finally, the synthesized glycopeptide was separated from the resin with a reagent and glycopeptide 2 was obtained by subsequent debenzylation. 3.2. Direct synthesis In the direct synthesis method, glycopeptide bonds are formed through a condensation reaction between the oligosaccharide and polypeptide chains that are constructed separately (Dudkin, Miller, and Danishefsky 2004). Compared with the solid and liquid phase synthesis, the direct synthesis has the following advantages: first, there is no need of protecting O-glycosidic bonds under acidic conditions as in the process of peptide chain extension during solid-phase synthesis; secondly, protecting groups are not required; finally, the loss of oligosaccharide is quite small and expensive glycosyl glycopeptides can be synthesized. However, the formation of glycosidic bonds requires non-polar and anhydrous conditions for the synthesis of O-glycopeptides. Moreover, the solubility of the long peptide and condensation yield of glycopeptides is not high under such conditions. Dudkin et al. (Dudkin, Miller, and Danishefsky 2004) prepared a complex N-glycopeptide from simple monosaccharide precursors. 3.3. Native chemical ligation The native chemical ligation (NCL) method was first proposed in the 1990s (Yan and Dawson 2001). The mechanism of NCL is that peptide A with a cysteine residue at the N-terminus and peptide B with an α-thioester at the C-terminus are coupled by a transthioester reaction in a buffer solution with pH 7 to form an unstable intermediate. Then, irreversible intramolecular rearrangement occurs spontaneously to form natural peptide bonds. Both the N-terminus and the cysteine residues in the peptide chain can form thioesters, but only cysteine residues can be rearranged at the N-terminus to form peptide bonds. The NCL method can be performed in both liquid and solid phases, which makes up for the defect that conventional solid and phase synthesis can only synthesize peptides with less than 50 amino acids. However, cysteine is rarely found in natural peptides and proteins. Peptide thioesters are essential tools for the total synthesis of proteins using native chemical ligation. Premdjee et al. (Premdjee, Adams, and Macmillan 2011) synthesized N-glycopeptides by NCL, demonstrating excellent compatibility of thioester formation via N-S acyl transfer of native N-glycopeptides.

3.4. Enzymatic and chemoenzymatic synthesis Natural glycoproteins or purified glycoproteins are made to react with exoglycoside hydrolases to obtain oligosaccharide chains, which are then utilized for the synthesis of target glycopeptides. Chemoenzymatic synthesis requires that the structure of oligosaccharide chains is uniform and that the high-mannose oligosaccharide and complex oligosaccharide chains can be transferred. Moreover, glycosidyltransferase can control the linkage reaction of oligosaccharides with asparagine residues. A sialyl T-antigen-linked glycopeptide has been synthesized through a combined method of chemical synthesis and enzymatic synthesis (Ajisaka and Miyasato 2000). The above-mentioned techniques for the synthesis of glycoproteins/glycopeptides are suitable when the protein/peptide and saccharide components are relatively simple and are applicated in the pharmaceutical field for the synthesis of vaccines and drugs. However, the protein composition of food sources is complex. For example, egg white proteins mainly include ovalbumin, ovotransferrin, lysozyme, flavin protein, and protease inhibitors (Liu et al. 2018). Therefore, some synthesis methods of glycoproteins from complex proteins and saccharides components are studied like Maillard reaction and transglutaminase-catalyzed reaction(Yang et al. 2019). The formation and applications of food-derived PSCCs obtained via Maillard and TGase reactions are shown in Table 1. 3.5. Maillard reaction The Maillard reaction is widely used in the food industry due to its ease of use and cost-effectiveness. In general, the Maillard reaction is mainly divided into three reaction stages (Figure 3a). The reaction product in the primary stage is colorless and has no ultraviolet absorption. It includes ammonia condensation and Amadori rearrangement products (Akhtar and Dickinson 2007; Regan and Mulvihill 2009; Wu et al. 2014). The intermediate stage includes different routes, for example, sugar dehydration, decomposition, and amino acid degradation. At the same time, some fluorescent products and brown pigments are produced, but the concentration is generally low (Liu, Ru, and Ding 2012). In the final stage, it generates several end products such as complex nitrogenous polymeric compositions and melanoidins polymers, which are water-insoluble (Ren et al. 2015). Based on the irreversible Amadori rearrangement step, the corresponding Amadori product can be produced. For example, casein phosphopeptides (CPP) and soluble dietary fibers (SDF) can form CPP-SDF covalent conjugates as a calcium delivery system through an Amadori-type linkage between the lysine residues of CPP and the reducing end carbonyl group of SDF. CPP-SDF covalent conjugates could significantly promote the calcium-binding capacity and restrain Ca2+ release in the stomach to improve calcium absorption in the intestine (Gao et al. 2018a; 2018b). The most commonly used methods of protein and saccharide conjugates via the Maillard reaction include

Table 1. Formation and application of food-derived protein/peptide-saccharide covalent conjugates obtained via the Maillard and TGase reaction. Protein/peptide Whey protein Whey protein isolate Saccharide Inulin Maltodextrin

Formation methods Dry-heating Maillard reaction. Dry-heating Maillard reaction. Application Antioxidant activity. Improve thermal stability. Soy protein isolate Maltodextrin Dry-heating Maillard reaction.

Soy protein isolate Ribose Dry-heating Maillard reaction. Gelatin Gum arabic-maltodextrin Dry-heating Maillard reaction. Corn protein hydrolysate Carboxymethyl chitosan Dry-heating Maillard reaction. Desalted duck egg white peptides Casein

Chitosan oligosaccharides Dextran Wet-heating Maillard reaction. Ovalbumin Dextran Wet-heating Maillard reaction. Crab shell bioactive peptides Fructose Wet-heating Maillard reaction. Canola protein isolate Rice protein

Gum Arabic Dextran Wet-heating Maillard reaction. Wet-heating Maillard reaction. Whey protein isolate Maltodextrin Wet-heating Maillard reaction. Soy protein isolate Maltose Whey protein Glucose/trehalose

Mung bean protein isolates Glucose Rice dreg protein Sodium alginate Bovine serum albumin Dextran Quinoa protein Chitosan

Wet-heating Maillard reaction in the medium of ionic liquid. High pressure-high temperature processing on wet-heating Maillard reaction. Ultrasound treatment on wet-heating Maillard reaction. Microwave treatment on wet-heating Maillard reaction. Pulsed electric field treatment on wet-heating Maillard reaction. High-intensity ultrasound combined with TGase reaction.

Improve solubility, emulsibility. Reduce surface hydrophobicity. Enhance elastic properties and increase viscosity. As wall material to encapsulate stearidonic acid soybean oil and improve its antioxidant capacity As nanoparticles delivery systems to improve the absorption of rutin As delivery system to promote calcium absorption. As carrier system to improve the stability and radical scavenging activity of curcumin As nanogels to improve oral curcumin bioavailability Antioxidant and antibacterial activities. Improve solubility. Improve solubility, foaming capacity and emulsifying capacity Increase gel firmness and water-holding capacity. Reduce gel swelling capacity. Reduce surface hydrophobicity.

Silk peptide Antimicrobial peptides (melittin and warnerin) Casein phosphopeptides TGase reaction. TGase reaction. Apo-red bean seed ferritin Carboxymethyl chitosan Quaternized chitosan derivative Chitosan oligosaccharides Oligochitosan

Soy protein isolate Chitosan TGase reaction. Gelatin Chitosan TGase reaction. Bovine serum albumin Ribose Two-step process: TGase reaction, followed by wet-heating Maillard reaction. Wet-heating Maillard reaction.

TGase reaction. TGase reaction.

Reference (Wang et al. 2019c) (Wang and Zhong 2014) (Xue et al. 2013) (Zhen-Zhen, Guo-Qing, and Jun-Xia 2015) (Ifeduba and Akoh 2016) (Han et al. 2019) (Zhao et al. 2020) (Wu and Wang 2017) (Feng, Qi, and Liu 2016a) (Wei et al. 2018) (Pirestani et al. 2017) (Yun-Hui et al. 2018) (Bahareh et al. 2019) (Xu and Zhao 2019)

Reduce the Browning. (Ruiz et al. 2016) Improve solubility, emulsibility and surface hydrophobicity. (Wang et al. 2016) Improve solubility, emulsibility. Immunomodulatory property. (Meng et al. 2019a)

Antioxidant activity. (Guan et al. 2010)

Increase thermal stability, water vapor permeability, thickness, tensile strength and decrease in elongation percentage of composite edible films. Improve solubility, emulsifying and foaming properties, apparent viscosity and intense viscoelastic character. Antioxidant activity. Antibacterial activity.

(Vera, Tapia, and Abugoch 2020)

As delivery system to promote calcium absorption. As nanoparticles delivery systems to improve the thermal stability of rutin. As wall material to encapsulate algal oil and improve its antioxidant capacity. As injectable hydrogel to deliver doxycycline. Increase gel strength and reduce the Browning.

(Zhu et al. 2020) (Shu-Juan and Xin-Huai 2011) (Liu et al. 2017) (Chudinova et al. 2016) (Yang et al. 2019) (Yuan et al. 2017) (Tormos, Abraham, and Madihally 2015) (Gan, Alkarkhi, and Easa 2009) 6 M. ZHAO ET AL.

Figure 3. Reaction scheme of Maillard reactions (a), and transglutaminase-catalyzed reactions (b). dry-heating conditions and wet-heating conditions. Dry-heating is the most common method for preparing protein-saccharide covalent conjugates by the Maillard reaction. The first step is to dissolve proteins and saccharides in water or buffer solutions separately, mix the two at a certain ratio, and freeze lyophilization. Then, the freeze-dried powder is placed in a closed container at a certain temperature (below the denaturation temperature of the protein, generally ranging from 40 to 80 °C, commonly at 60 °C) and certain relative humidity (ranging from 63% to 79%, usually at 79%) to form a covalent complex under a certain reaction time (Wang et al. 2019c; Wang and Zhong 2014; Xue et al. 2013; Zhen-Zhen, Guo-Qing, and Jun-Xia et al. 2015). The reaction time for conjugate formation depends on the type and conformation of the protein as well as the type of reducing sugar. Generally, lowering the temperature can suppress the progress of this reaction. Although the dry heat method is easy to perform, the reaction time is typically long, perhaps up to several weeks (Miralles et al. 2007; Chen et al. 2019a). Therefore, some scholars have studied the synthesis of soy protein isolate-maltodextrin conjugates by a dry-heat Maillard reaction under high-temperature (90, 115, 140 °C) and short-term (2 h) conditions (Lan, Yang, and Zhang 2014). However, the dry-heating reaction is not suitable for large-scale production because samples need to be predried, and humidity and temperature need to be controlled during the reaction, which has limitations in practical applications. In the wet-heating method, proteins and saccharides are mixed in a certain ratio in an aqueous solution via a closed device, through a water bath or oil bath for heating. After the reaction is completed, it is terminated by rapid cooling with an ice bath. Compared with the dry-heating method, the reaction time of wet-heating is

shorter, and the reaction temperature is lower (Pirestani et al. 2017). Maillard reaction in an aqueous solution has a significant effect on the protein structure in the wet-heating system, while no significant change in protein structure is observed when performed in a dry-heating system (de Oliveira et al. 2016). Furthermore, wet-heating in the Maillard reaction involves physical means to assist the process, such as ionic liquid used as the reaction medium (1-butyl-3-methylimidazolium chloride) (Xu and Zhao 2019), high-pressure and high-temperature (Ruiz et al. 2016), ultrasound (Wang et al. 2016), microwave (Meng et al. 2019), supercritical carbon dioxide treatment (Casal et al. 2006), pulsed electric field (Guan et al. 2010), and high hydrostatic pressure (Ma et al. 2017). These protein-polysaccharide conjugates via the Maillard reaction present several advantages: non-cytotoxicity, good biocompatibility (Lingli et al. 2019), biodegradability, good amphiphilic (Feng, Qi, and Liu 2016a), thermal stability (Xinguang et al. 2018) and functional properties such as water solubility (Zhong-He et al. 2017), gelling ability (Bahareh et al. 2019), foaming capacity (Yun-Hui et al. 2018), emulsifying capacity (Ge et al. 2016), inoxidizability (Lian-Zhou et al. 2017), antibacterial (Wei et al. 2018). Biologically active compounds are easily prone to decomposition during the production, storage, and severe gastrointestinal tracts. Protein-polysaccharide conjugates via Maillard-type can improve stabilization and control release of bioactive compounds (Xinguang et al. 2018; Zhu et al. 2020). Moreover, there are potential applications of protein-polysaccharide conjugates by the Maillard reaction for designing delivery systems, such as, ovalbumin-dextran nanogels were fabricated via the Maillard reaction followed by a heat gelation process, then curcumin was loaded into

nanogels through a pH-driven method and to improve oral curcumin bioavailability through simulated mouth and gastrointestinal digestion (Feng et al. 2016b).

3.6. Transglutaminase-catalyzed reaction Transglutaminases (EC 2.3.2.13) are responsible for acyl group transfer, deamidation or cross-linking between intraor inter-molecular glutamine (acyl donor) and ε-amino group of lysine peptide residues (acyl acceptor) (Romeih and Walker 2017). The reaction scheme can be elucidated in three ways (Figure 3b) (three kinds of acyl acceptor) (Fatima and Khare 2018):When the substrate is the ε-amino group of lysine residues, intramolecular or intermolecular interactions can be enabled between proteins or peptides through the formation of ε- (γ-glutamine) lysine heteropeptide bonds, then a stable protein network structure can be fabricated. However, only proteins with the same polarities are more prone to cross-linking, as those with different polarities cannot reach the active center of the enzyme at the same time, thus affecting the process of catalytic reaction. This reaction is carried out preferentially among 3 reactions and continues until there are no more glutamine and lysine in the substrate (De Góes-Favoni and Bueno 2014).When the lysine residues in the reaction are replaced by the primary amine group, saccharides containing a primary amino group can be cross-linked with proteins through covalent bonds to form protein-saccharide conjugates. At this time, the primary amine group of saccharides is the acyl receptor, and the reaction process is similar to that of I.When free lysine residues or primary amines are absent, TGase hydrolyzes the γ-formamide group of glutamine residues and water becomes an acyl acceptor to undergo a deamidation reaction and forms glutamic acid. This reaction changes the isoelectric point and solubility of the protein. The reactions catalyzed via TGase commonly include chitosan (Fang-Li et al. 2019), chitosan oligosaccharide (Song and Zhao 2014), soluble dietary fiber (Xia et al. 2018), glucosamine (Yuan et al. 2018), dextran (Zhang et al. 2014), cyclodextrin and galactosamine (Xiao-Jie et al., 2019). The formation of protein-saccharide covalent conjugates by the Maillard reaction has the disadvantages of long reaction time, high temperature, difficult to control conditions, easy browning of products, loss of nutrients, and formation of toxic terminal glycosylation compounds (Hrynets, Ndagijimana, and Betti 2014). Fabrication of protein-saccharide covalent conjugates through a transglutaminase-catalyzed reaction can refrain from the above problems. The degree of grafting of proteins and saccharides is also affected by the reaction time, the amount of TGase added, and the ratio of acyl donor and acceptor. Moreover, numerous factors can be used to facilitate transglutaminase-catalyzed reactions. For example, high-intensity ultrasound combined with transglutaminase treatment improved the mechanical, barrier, and physicochemical properties of quinoa protein/chitosan composite edible films (Vera, Tapia, and Abugoch 2020). Bovine serum albumin-ribose gels were prepared using a two-step process: the first step was a transglutaminase-catalyzed

cross-linking reaction, followed by heat treatment via the Maillard reaction, that results in high gel strength, neutral pH, and reduced browning (Gan, Alkarkhi, and Easa 2009). In addition, other oxidoreductases have been chosen to catalyze the residues with saccharide such as tyrosine through intra- and intermolecular covalent crosslinking of proteins. Gelatin-chitosan covalent conjugates were fabricated by microbial transglutaminase and tyrosinase catalyzed reactions to improve the thermal stability, tensile strength, stability in aqueous solutions, and in vitro antibacterial properties (Wang et al. 2015). Tyrosinase can use molecular oxygen as an oxidant to convert tyrosine residues of protein into quinones, which are active and can diffuse from the active site of tyrosinase to perform non-enzymatic reactions with chitosan. In a previous study, gelatin-conjugation gels modified by a tyrosinase-catalyzed reaction demonstrated slightly lower strength than those modified by transglutaminase (Chen et al. 2003).

4. Structural characterization of protein/peptidesaccharide covalent conjugates It’s necessary to adopt a series of qualified assays or analytical techniques to investigate what type of natural glycoprotein/glycopeptide is involved, or to determine whether covalent binding of protein/peptide and saccharide occurs. Therefore, in this section, we focus on characterizing the structural properties of PSCCs. Each structural characterization method has its own specific testing principle, key features and scope of application, which are described in Table 2. For the identification of natural glycoprotein/glycopeptide types, the most convenient way is to use the β-elimination reaction. N-glycopeptide bonds are stable to alkali, while O-glycopeptide bonds can undergo β-elimination reaction in the presence of NaOH, producing significant ultraviolet (Rozenberg et al. 2019) spectrum absorption at 240 nm (serine and tryptophan on the glycopeptide chain are converted to α-amino-acrylic acid and α-amino-butenoic acid, respectively) (Zhang et al. 2021b). Fourier transform infrared (FTIR) spectroscopy is also broadly used to analyze the covalent bond of PSCCs. The glycopeptide bond linked by the Millard reaction is essentially a covalent bond formed between protein amino residues (NH 2) and reducing saccharide carbonyl groups (C = O), and therefore the primary structure (mainly C-N, N-H) of protein. In general, the absorption spectrum at 1700–1600 cm−1 and 1600–1500 cm−1 were amide I region (C = O stretching vibration of peptide linkage) and amide II region (N-H bending vibration and C-N stretching of amino groups), respectively, which are the most sensitive region related to protein conformation (Bourbon, Cerqueira, and Vicente 2016). After the formation of PSCCs, the shift of amide I band from 1652 to 1648 cm−1 and the disappearance of amide II (1540 cm−1) suggesting that the partial protein denaturation (Feng, Qi, and Liu 2016a). The appearance of the absorption peak at 2364 cm−1 was caused by the stretching vibration of C≡N, which was the characteristic absorption peak of the Maillard reaction, and

Table 2. Structural characterization of s natural food-derived protein/peptide-saccharide covalent conjugates (glycoproteins/glycopeptides). Characterization UV FTIR CD ESI-MS MALDI-MS

Testing principle N-glycopeptide bonds are stable to alkali, while O-glycopeptide bonds can undergo β-elimination reaction in the presence of NaOH, producing significant ultraviolet spectrum absorption at 240 nm. Characteristic stretching or deformation vibration absorption bands representing the characteristic functional groups can be reflected in the infrared spectrum. According to Lambert-Beer law, the change in the nature of polarized light at a specific absorption wavelength of the optically active compounds, resulting in circular dichroism. Ionization of compounds using different modalities, then accurately determine the molecular mass, analyze the structural level and identify glycosylation sites with soft ionization mass spectrometry.

Key features Fast, convenient, and simple Less sample preparation steps Poor accuracy and limitations Scope of application •Identify as N-glycopeptide bonds or O-glycopeptide bonds

Fast, convenient, and extensive range Require strong background in spectrogram knowledge Large error, low sensitivity, not quantitative analysis Fast, simple, and extensive range Accurately measured and calculated the changes in the secondary structure during covalent bond formation

•Direct to analyze the changes in the characteristic functional groups of protein/peptide and saccharide, and to identify the covalent bond of PSCCs. •The secondary structure mainly manifested as the increase and decrease of α-helix, β-sheet, β-turns and random coils of protein/peptide, saccharide and PSCCs.

Direct, reliable, and powerful analysis ability Allow simultaneous analysis of complex protein/peptide and saccharide mixtures Usually used in conjunction with the HPLC Mass spectrum analysis needs to be performed by consulting the corresponding mass spectrometry library (convenient but potentially limited) Complex sample preparation steps, expensive and not widely application in food field

The technologies provide the average extent of covalent bonds formation, a distribution profile of the protein glycoforms, the average degree of substitution per protein molecule, the molecular mass and the change in primary structure of PSCCs.

reflected that the Maillard reaction existed in the PSCCs (Gao et al. 2018a). On the other hand, some typical chemical bonds can be formed during the Amadori reaction, such as the C = O in Amadori compounds and the C = N in Schiff base, which are rightly reflected by the FTIR. For instance, the absorption bands used to characterize the desalted duck egg white peptides-chitosan oligosaccharide copolymer also had a stronger absorption peak at 1656 cm−1 caused by the stretching vibration of C = N (Zhao et al. 2020). The glycopeptide bond linked by the TGase reaction is essentially a covalent bond formed between glutamine or ε-amino group of lysine residues of proteins and amino of saccharides. The intensity of the spectrum of TGase induced casein phosphopeptides-chitosan oligosaccharides copolymers decreased both at 1200 cm−1 (N-H stretching vibration of NH2 in chitosan oligosaccharides) and 980 cm−1 (N-H bending vibration of Gln residues or ε-amino groups in casein phosphopeptides). Moreover, compared to casein phosphopeptides, copolymers exited stronger absorption peaks of C = O and N-H (Zhu et al. 2020). The absorption of glycoprotein extracted from the epidermal mucus of African catfish at the range of 1078.6– 1055.0 cm−1 denoting the primary amine and C-N stretch and the absorption at 989.2–731.1 cm−1 arising from O-C-N bending confirming the presence of carbohydrate portion in the glycoprotein (Abdel-Shafi et al. 2019). This region is well correlated with the presence of sugars linked to protein moiety particularly arabinose and fructose (Rozenberg et al. 2019). Moreover, the secondary structure of the glycoproteins was reflected by these bands in FTIR spectroscopy as follows: 1610–1640 cm−1 for the β-sheet; 1640–1650 cm−1 for the random coil; 1650–1658 cm−1 for the α-helix; 1660–1700 cm −1 for the β-turn (Zhang et al. 2003).

With the formation of PSCCs, the secondary structure mainly manifested as the increase and decrease of α-helix, β-sheet, β-turns and random coils. Circular dichroism (Daly et al. 2019) was used in the study. As reported by Yang et al. (2019), TGase-oligochitosan-apo-red bean seed ferritin conjugates exhibited significantly increased contents of α-helix, β-sheet and reduced contents of random coils compared to the ferritin, respectively (Yang et al. 2019). In contrast, significantly reduced content was found in the a-helix; however, the content of unfolded structures increased significantly and β-sheet increased slightly of ultrasound-assisted Maillard-ovalbumin-xylose conjugates compared with ovalbumin (Liu et al. 2021). It might be due to the binding of saccharides to protein involves condensation between the carbonyl group and the ε-amino group, which is within the α-helix region or its neighbor protein (Chen et al. 2019b). Moreover, structural analysis of PSCCs by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), nuclear magnetic resonance (Marcelo et al. 2012) are the common methods. Higher-energy collisional dissociation of glycopeptide molecules dissociates fragments to produce oxonium ions, which carry information about the saccharides structure in the fragments. Eshghi et al. (2016) investigated the association between the structures in the glycopeptide spectra and the intensity of oxonium ions to establish a spectral database of N-glycopeptides and O-glycopeptides by HPLC-MS/MS. Finally, glycopeptides derived and enriched from human serum were evaluated and efficiently screened for N-glycopeptides. Electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) can accurately determine the molecular mass and analyze the degree of cross-linking of PSCCs products. Oliver (2011)

reviewed the role that ESI- and MALDI-MS had played in advancing our understanding of the glycation of milk proteins. Analysis of intact proteins provided an overview of the average degree of glycosylation and the distribution of proteoglycan forms. Moreover, they identified site-specific glycosylation at the structural level and identified glycosylation sites. However, structural characterization of PSCCs by ESI-MS or MALDI-MS has been less studies reported yet. Bai et al. (2021) successfully adopted ESI-MS to distinguish different glycosidic bond configurations of reducing disaccharides and identify the α/β-configuration of Amadori (maltose and lactose, proline and tryptophan conjugates) compounds.

5. Biological activities and applications in health food industry Food-derived protein/peptide-saccharide covalent conjugates have many physiological functions and unique nutritional properties, which can be widely used in health- food and pharmaceuticals. Table 3 presents a list of studies on the biological activities of PSCCs.

5.1. Antioxidant capacity Oxidative stress plays a significant role in arteriosclerosis, cardiovascular diseases, diabetes, cancer, and other chronic diseases (Wen et al. 2020). Glycopeptides obtained from ginseng flowers exhibited excellent radical-scavenging capacities, including 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroxyl radicals (•OH), and superoxide anion radicals (•O2−) (Rui-Zhan et al. 2018). Similarly, mung bean protein isolate-dextran covalent compounds prepared through the hot-humid Maillard reaction displayed anti-oxidation properties and could scavenge DPPH, •OH, and •O2− radicals (Lian-Zhou et al. 2017). This antioxidant capacity is attributed to the electron transfer of

the hydroxyl and pyrrole moieties of the coupling and to the hydrogen donor potential of protein denaturation, exposing more amino acids with electron-donating capacity and thus terminating the free radical chain reaction. In addition, pyridones or pyranones in the melanin product of the Maillard reaction may provide, for example, hydroxyl and ketone group chelating donors (Wei et al. 2018). HypSys peptides, the glycopeptides contain 18–20 amino acids and are rich of hydroxyproline, were first discovered in tobacco and tomato, and were found to generate H2O2 in vivo and activate several antioxidant defensive enzymes in potato seedling leaves (Bhattacharya et al. 2013). Hydroxyproline is a precursor for the synthesis of glycine in vivo, and glycine is one of the precursors for the synthesis of glutathione in the body, which plays an important role in antioxidant and anti-inflammatory (Leon-Lopez et al. 2019). Additionally, patatin, which contains 12 free radical scavenging amino acids such as Met, Try, Tyr, Phe, Cys, and His, can serve as a hydrogen donor to break off the radical peroxidation chain reaction with their aromatic residues to scavenge DPPH free radical, reduce low-density lipoprotein peroxidation, and protect against hydroxyl free radical-induced DNA damage (Petersen et al. 2009). A glycoprotein obtained from fupenzi had DPPH and ABTS free radical scavenging ability and could considerably delay cell senescence to enhance cell vitality (Huo et al. 2020). 5.2. Anti-osteoporosis activities Osteoporosis is the most common metabolic bone disease due to the increased bone loss relative to bone formation, resulting in reduced bone mass and deterioration of bone microstructure (Leder et al. 2020). Studies have found that a combination of factors can contribute to the osteoporosis, including an imbalance in the levels of hormones (estrogen, calcitonin, parathyroid hormone), poor nutrition, poor lifestyle, lack of exercise, and genetic and psychological factors. SGP was found to upregulate osteoprotegerin (OPG) levels and downregulate those of Receptor activator of

Table 3. Sources and biological activities of natural food-derived protein/peptide-saccharide covalent conjugates (glycoproteins/glycopeptides). Name Sialylglycoprotein Sialylglycopeptide Crucian carp Egg yolk

Source

Sialylglycopeptide Casein glycomacropeptide Casein glycomacropeptide Casein glycomacropeptide Casein glycomacropeptide Casein glycomacropeptide Egg yolk Bovine milk Bovine milk Bovine milk Bovine milk Bovine milk

Casein glycomacropeptide Casein glycomacropeptide HypSys peptide Glycopeptide Bovine milk Bovine milk Potato Soybean β-conglycinin

Glycopeptide Glycopeptide Glycoprotein Glycopeptide Glycopeptide Polysaccharopeptide Polysaccharopeptide

Ginseng flowers Korean ginseng Solanum nigrum Linne Strongylocentrotus droebachiensis Strongylocentrotus droebachiensis Trametes versicolor Trametes versicolor 9

Activity Improve osteoporosis. Inhibition of Salmonella typhimurium and Escherichia coli. Inhibition of influenza virus hemagglutinin Improve oxazolone-induced ulcerative colitis. Antimanic efficacy. Control the content of phenylacetone Inhibition of influenza virus hemagglutinin Promote the proliferation of Lactobacillus and Bifidobacterium Suppress gastric secretions. Immunomodulatory property. Activate antioxidant defensive enzymes. Inhibition of Salmonella typhimurium and Escherichia coli. DPPH, •OH, •O2− radical scavenging capacity. Regulate immunosenescence. •OH, •O2− radical scavenging capacity. Anti-acute bronchitis activity. Anti-chronic bronchitis activity. Anti-tumor activity. Analgesic effects.

Reference (Wang et al. 2014) (Sugita-Konishi et al. 2004) (Makimura et al. 2006) (Ming et al. 2015) (Liebenberg et al. 2018) (Abdel-Salam and Effat 2010) (Brody 2000) (O’Riordan et al. 2018) (Fayed 2012) (Brody 2000) (Bhattacharya et al. 2013) (Yang et al. 2008) (Rui-Zhan et al. 2018) (Kim et al. 2018) (Lee and Lim 2006) (Katelnikova et al. 2018a) (Shikov et al. 2019) (Liu et al. 2014) (Wang et al. 2019b)

10 M. ZHAO ET AL. nuclear factor-κB ligand (RANKL) leading to a decreased ratio of RANKL to OPG in ovariectomy (OVX)- induced osteoporotic rats. Therefore, it was suggested that SGP could prevent bone resorption in rats and inhibit the overestimation of bone turnover to improve osteoporosis (Wang et al. 2014). SGP isolated from the eggs of Carassius auratus (Ca-SGP) significantly promoted the proliferation of MC3T3-E1 pre-osteoblasts, subsequently promoting cell differentiation and mineralization. In OVX- induced osteoporotic rats, Ca-SGP promoted the proliferation of osteoblasts by increasing the ratio of OPG/RANKL and inhibiting bone absorption, thus improving osteoporosis (Xia et al. 2015). Furthermore, Ca-SGP also accelerated the fracture healing in OVX- induced osteoporotic mice by promoting the proliferation of endochondral osteoblasts. (Wang et al., 2018a). In addition, Ca-SGP has been demonstrated to promote osteogenesis by stimulating the transformation of mesenchymal stem cells into osteoblasts (Mao et al. 2019). Sialylglycoprotein isolated from Gadous morhua eggs (Gm-SGP) significantly improved bone density, enhanced biomechanical properties of bone, and reduced serum alkaline phosphatase content in OVX- induced osteoporotic rats. Gm-SGP improved osteoporosis by downregulating bone morphogenetic protein-2 (BMP-2)/Smad and Wnt/β-catenin signaling pathways, such as BMP-2, Smad1, Smad4, LRP-5, runt-related transcription factor 2, osterix, alkaline phosphatase, collagen type I, osteocalcin, and β-catenin (Wang et al. 2018b). 5.3. Antidiabetic activities Diabetes and obesity are two chronic diseases related to metabolic syndrome. However, most obese patients have insulin resistance and are often accompanied by

complications such as hyperglycemia and hypertension. High concentration of glucose will cause pancreatic β cells to produce reactive oxygen species through the mitochondrial pathway, thereby in turn inhibiting the glucose-stimulated insulin secretion and triggering the development of diabetes. Inflammatory factors such as TNF-α and IL-1β inhibition of pancreatic islet cells and insulin secretion of pancreatic β cells, and mainly inhibit insulin signaling in adipose, muscle cells, liver cells and tissues by restraining the phosphorylation of insulin receptor, insulin receptor substrate-1 (IRS-1) and protein kinase Akt-B (Kim et al. 2012; Ballak et al. 2015). Dipeptidyl peptidase-4 (DPP-4) is an enzyme that degrades incretin hormones, such as glucagon-like peptide 1 (GLP-1), which is secreted by intestinal L cells in response to nutrient load. It is known to promote the secretion of insulin by pancreatic β cells, maintain the proliferation and regeneration of β cells, and inhibit the production of glucagon (Brunton 2014). Angiotensin (Ang) II, produced from Ang I by angiotensin-converting enzyme (ACE), has been known to inhibit insulin PI3K/Akt signaling, induce oxidative stress by activating NADPH oxidase, and active NF-κB to upregulate inflammation (Wang et al. 2019a). As shown in Figure 4, the antidiabetic function of glycoproteins/glycopeptides is closely related to the inhibitory activities of DPP-4 and ACE, antioxidant and anti-inflammatory activities. Casein glycomacropeptide hydrolysate (CGPH) has been proven to have DPP-4 inhibitory activity and can be used as a potential DPP-4 inhibitor to exert an antidiabetic effect (Qian, Hanyu, and Xueying 2015). In high-fat diet and type 2 diabetic C57BL/6J mice, casein CGPH was confirmed to promote glucose transport factor muscle glucose transport protein (GLUT-4) translocation by regulating IRS-1/phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway in skeletal muscle, thus playing antidiabetic and

Figure 4. Antidiabetic activities and related mechanisms of food-derived protein/peptide-saccharide covalent conjugates. “|” mean inhibitory effect. TNF-α, tumor necrosis factor-α; IL-1, interleukin-1; GLUT-4, glucose transport factor muscle glucose transport protein; PI3K, phosphatidylinositol 3-kinase; IRS-1, insulin receptor substrate-1; DPP-4, dipeptidyl peptidase-4; GLP-1. glucagon-like peptide 1; Ang, Angiotensin; ACE, angiotensin converting enzyme; AMPK, AMP-activated protein kinase; AMPKK, AMPK kinase.

hypolipidemic effects (Yuan et al. 2020). In addition, CGPH has been shown to be beneficial to insulin resistance. In high-fat diet C57BL/6J mice, CGPH increased the phosphorylation level of glycogen synthase kinase 3β (GSK3β) in liver tissue of mice by reducing serine phosphorylation of IRS-1 and elevating the phosphorylation level of Akt, thus promoting liver insulin sensitivity and antidiabetic activity (Song et al. 2018). Similarly, IPPKKNQDKTE derived from CGMP played a potential role in the prevention and treatment of hepatic insulin resistance and type 2 diabetes by activating AMP-activated protein kinase (AMPK), regulating IRS-1/PI3K/Akt signaling pathways, and inhibiting high glucose-induced insulin resistance in HepG2 cells (Song et al. 2017). 5.4. Immunomodulatory and anti-inflammatory capacities As a common type of allergen, native protein-based foods produce antibody responses because of protein immunogenicity, while the presence of saccharide component in natural glycoproteins could efficaciously reduce protein immunogenicity. For example, immunogenicity of CGMP was investigated using two animal models based on different routes of immunization. CGMP inhibited the proliferation of splenic cells in immune-stressed mice. Since splenic cells (splenic lymphocytes) are involved in inflammatory responses, the inhibition of their proliferation by CGMP can suppress allergic reactions and regulate the immune system (Brody 2000). Similarly, many researches focused on covalently conjugating reaction with a saccharide, which changes the original molecular structure of protein to influence the immunogenicity of protein. In immunosuppressed mice model, rice dreg protein modified with sodium alginate through the Maillard reaction by wet heating assisted with microwave treatment could improve the immunomodulatory effect, and the immunomodulation was concentration-dependent, being generally enhanced by increased concentrations (Meng et al. 2019). Bovine β-lactoglobulin was conjugated with different oligosaccharides via the Maillard reaction and exhibited reduced allergenicity (Wu et al. 2013). Nevertheless, this strategy offers an important idea to produce high-protein foods without protein antigen. In contrast, synthetic protein-saccharide covalent conjugates vaccines were designed to enhance the immunogenicity of that active substance. Researchers have found that combining capsular polysaccharides that are produced by bacteria with carrier proteins elicits the desired antibody levels in infants, while pure polysaccharide vaccines cannot (Zhou, Petrova, and Edgar 2021). A glycopeptide fraction from internal organs of green sea urchins inhibited lipopolysaccharide-induced p38 mitogen-activated protein kinase phosphorylation and cyclooxygenase2 by blocking TLR4 in vitro (Katelnikova et al. 2018a). In vivo, through formalin-induced acute and chronic bronchitis inflammatory models, the glycopeptide was confirmed to have an excellent protective ability against

bronchitis (Katelnikova et al. 2018b; Shikov et al. 2019). Moreover, GMPH exerted an anti-inflammatory effect of lipopolysaccharide through the inhibition of NF-κB activation by MAPK and Akt in RAW264.7 macrophages (Li et al. 2018). 5.5. Antimicrobial and antiviral capacity Bacterial adhesion to intestinal epithelial cells is a vital inducer of bacterial infection in the human body (Sankarganesh et al. 2018). Soybean glycopeptide and SGP could blocked the adhesion of Salmonella typhimurium and Escherichia coli bacteria to intestinal cells and exhibited antibacterial properties (Yang et al. 2008). Similarly, quaternized chitosan derivatives and antimicrobial peptides (melittin and warnerin) cross-linked by TGase showed antibacterial activity against Escherichia coli (Chudinova et al. 2016). Moreover, CGMP can be used as an antibacterial feed additive to reduce the number of Escherichia coli K88 in the intestinal contents of piglets, and relieve the inflammatory reaction caused by pathogenic bacteria (Rong et al. 2015). Human influenza virus has two kinds of membrane proteins: hemagglutinin and neuraminidase. The former is responsible for the contact of the toxic particles with the extracellular surface, while the latter is responsible for releasing the newly formed virions in invaded cells (Suzuki 2005). When the host cell is infected, the virus first binds the α-(2-6)-Sialyl-N-acetyllactosamine residue of the saccharide chain on the surface of the host cell by hemagglutinin (Makimura et al. 2006). The sialic oligosaccharide chains of SGP and CGMP competitively strong interaction to the hemagglutinin of the human influenza virus (Tsuji et al. 2020), thus, demonstrating antiviral ability. 5.6. Other biological activities Polysaccharopeptide (PSP) is a glycopeptide with an antitumor biological function (Liu et al. 2014). The underlying mechanism is to induce apoptosis of tumor cells through mitochondrial and death receptor pathways, blocking tumor cell cycle, and inhibiting cell growth. Yang et al. (Yang et al. 2005) showed that PSP acted on HL-60 cells of human promyelocytic leukemia. HL-60 cells reduced the expression of anti-apoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bax, thereby decreasing the ratio of Bcl-2/Bax to induce apoptosis of tumor cells. Lee et al. investigated the cytotoxic effect of Lobelia glycoprotein on human breast cancer MCP-7 cells and showed that 1 μg/mL of Lobelia glycoprotein I and 100 μg/mL of Lobelia glycoprotein II could produce cytotoxicity in MCP-T cells and induce NO production (Lee and Lim 2006). Moreover, patatin from potato was identified as an effective antiproliferative agent against mice melanoma B16 cells, leading to cell cycle arrest in the G1 phase (Sun, Jiang, and Wei 2013). Huang et al. showed that the morphology of murine bone marrow-derived dendritic cells was more typical and mature

Figure 5. Applications of food-derived protein/peptide-saccharide covalent conjugates in food industry.

after 24 h of the action of tea glycoprotein, and showed a dose-dependent effect in the concentration range of 0.1– 25 μg/mL (Huang et al. 2009). Panax ginseng glycoproteins efficiently suppressed SH-SY5Y cell apoptosis induced by Aβ25–35, possibly through the inhibition of Aβ-induced NO overproduction. Additionally, Panax ginseng glycoproteins significantly improved the learning and memory ability of Alzheimer’s disease rats. These findings suggest that glycoproteins derived from ginseng might be a promising neuroprotective agent to against Alzheimer’s disease (Luo et al. 2018). Some glycopeptides are capable of promoting the proliferation of probiotics (O’Riordan et al. 2018). After oral administration of glycomacropeptide in rats, the microbial contents in of feces were analyzed. It was found that the populations of Lactobacillus and Bifidobacterium in the intestine increased significantly after 3 days of treatment. Ten days after cessation of administration, the populations of Lactobacillus and Bifidobacterium remained elevating (Jimenez et al. 2016). Moreover, with the addition of some essential and semi-essential amino acids (AA), CGMP increased the concentration of Phe in the blood of children with PKU, and CGMP-AA served as a partial protein substitute for PKU patients. (Daly et al. 2019). In addition to the above biological functions, natural glycopeptides are also capable of suppressing gastric secretions (Fayed 2012).

6. Functional properties and applications in food industry The modified functionalities exhibited in PSCCs benefits from the changes in molecular structures during covalent bond formation. These PSCCs present several advantages: non-cytotoxicity, good biocompatibility (Lingli et al. 2019), low allergenicity (Bu et al. 2010), biodegradability, good amphiphilic properties (Feng, Qi, and Liu 2016a), and thermal stability (Kasuya et al. 2014; Xinguang et al. 2018).

Consequently, as exhibited in Table 1, the modified or enhanced processing-functional properties are water solubility (Shu-Juan and Xin-Huai 2011; Zhong-He et al. 2017), gelling ability (Bahareh et al. 2019; Niu et al. 2019), foaming capacity (Yun-Hui et al. 2018), and emulsifying capacity (Song and Zhao 2013; Ge et al. 2016). Figure 5 has shown the application of PSCCs in food industry. 6.1. Stabilization of emulsion system The emulsifying properties of proteins make them as food ingredients for baking, beverages and desserts to enhance their nutritional value and extend their shelf life. However, most food proteins, especially plant-based proteins, tend to limit the functional properties of proteins such as emulsifying properties due to their poor solubility and complex fractions. For example, whole soybean curd is a new type of whole soybean product with the advantages of high soybean protein utilization, comprehensive nutrient composition (Zhang et al. 2018a). However, the presence of water-soluble okara components hinders the formation of protein networks, which ultimately making whole soybean curd a coarser texture than okara-filtered tofu (Zhang et al. 2021a). Moreover, some food proteins are unstable during the actual food processing under the presence of extreme conditions, such as a high acidity or a high salt ion concentration, and organic solvents (Akhtar and Ding 2017). Therefore, grafting of saccharides on natural proteins to enhance their emulsification ability and stability has more preponderant application potentials in food systems. Kasran, Cui, and Goff (2013) have prepared covalent conjugates of coumarin and whey isolate using the Maillard reaction and found that the emulsification properties of the conjugates were significantly improved and the conjugates showed excellent emulsion stability in emulsion systems with acidity (pH 4.0), high temperature (75 °C and 85 °C) and

high ionic strength (0.5 mol/L NaCl). Hou et al. (2017) have prepared a novel emulsifier by modifying acacia polysaccharide with casein hydrophobic peptide through the Maillard reaction, which showed that the emulsification capacity of the conjugates was 46 times higher than that of acacia polysaccharide and the emulsion stability was 21 times higher than that of acacia polysaccharide. The stabilizing activity of protein-saccharide covalent conjugates is due to a combination of molecular structure, steric and electrostatic stabilization. The hydrophobic groups of proteins are anchored to the oil droplets, while the hydrophilic groups of saccharides position themselves in the aqueous phase (Karbasi and Madadlou 2018), thus enhancing the stability of the hydrophilic outside and hydrophobic inside in the aqueous solution. As the reaction proceeds, the secondary and tertiary structures of the protein change and hydrophobicity decreases, which means that the protein is more likely to form hydrogen bonds with water molecules in the aqueous environment, leading to conformational stretching and transformation of the protein α-helical structure (Li et al. 2019b). In addition, the increased flexibility of the quaternary structure makes it easier for the protein to diffuse at the water-oil interface and improves emulsification capacity (Li et al. 2019a). The emulsification performance of duck egg protein was improved by using three types of monosaccharides, including glucose, D-galactose and D-xylose in wet Maillard environment, and the addition of D-xylose had the best emulsification ability. Correlation analysis showed that the improved emulsification ability of duck egg protein-monosaccharides covalent conjugates was mainly influenced by molecular flexibility, surface hydrophobicity, changes in tertiary structure and free sulfhydryl groups (Ai, Xiao, and Jiang 2021). The charge of the conjugated molecule also plays an important role in the emulsion stability of the conjugated molecule. Inlet of negatively charged saccharides enhances the hydrophilic and hydrated layers of proteins, which means that the balance of gravitational and repulsive forces between proteins is disrupted, resulting in the aggregation of more stable glycosylated proteins. The spatial structure of the aggregated proteins changes thus affecting emulsifying properties (Setiowati et al. 2017). In some studies, black soybean protein isolate (BSPI) and chitosan oligosaccharide were used to prepare covalent conjugates via wet heating Maillard and TGase catalytic reaction, which display partly destroyed α-helix and β-sheet structures that form more open secondary BSPI structures, and the emulsification was improved compared with BSPI (Maillard and TGase were increased by 24.5% and 12.2%, respectively) (Zhang et al. 2018b). 6.2. Functional edible films/coatings for food packaging Edible films are used in many products to control moisture transfer, gas exchange or oxidation processes. Protein gels are widely used in the food industry based on their film-forming properties, including ductility, barrier property, and breaking strength, make it excellent packaging properties, and due to their ability to enhance the safety, nutritional value, and sensory qualities of food products (Assad et al. 2020). However, the film formed by natural proteins

is unstable (low water barrier characteristics) and prone to fracture, rupture and dissolution (Chen et al. 2019c). Therefore, glycosylation modification of proteins using saccharides is performed to improve the film-forming properties and ultimately the mechanical properties of the films. The gamma-aminobutyric acid-rich fermented soy protein glycosylated with chitosan via the Maillard reaction was blended to fabricate edible films (Zareie, Yazdi, and Mortazavi 2020). The films exhibited higher tensile strength and elongation at break, as well as smoother, denser, more uniform surfaces and fewer pores and cracks, and these films also showed considerable antioxidant and antibacterial activity. High-intensity ultrasound combined with TGase catalytic reaction was used to prepare quinoa protein-chitosan covalent conjugates edible films. The films exhibited a significant enhancement in thermal stability, significantly increase in thickness, decrease in elongation, increase in tensile strength and permeability to water vapor according to the comparative results of microstructure (Vera, Tapia, and Abugoch 2020). Various studies have shown that it is feasible to prepare edible films using protein-saccharide covalent conjugates to improve the mechanical strength of protein films. Oil, vegetables and meats are susceptible to oxidation and microbial spoilage during processing, transportation and storage. Fish gelatin-glucose covalent conjugates film showed a decrease in water solubility and wettability induced by the Maillard reaction. In contrast, an enhancement of some films properties was obtained including color development intensity and UV resistance. In addition, the films showed enhanced DPPH• and ABTS free radicals scavenging activity and β-carotene bleaching inhibition, and the antioxidant activity displayed unchanged under high-temperature treatment (Kchaou et al. 2019). In one study, gelatin-glucose covalent conjugates via the Maillard reaction were used as container materials to prevent oxidation of flaxseed oil to light, temperature and oxygen induced oxidation. Conjugate-based containers resulted in essentially stabilization of peroxide index during incubation 50 °C, and with a decreasing trend at day 21. The thiobarbituric acid reactive substances in the oil stored in the containers were less compared to the non-packaged oil and decreased with increasing oxidation time (Kchaou et al. 2020). In addition, the storage quality of shiitake mushrooms with chitosan-glucose covalent conjugates as a preservative was better than that of chitosan or glucose alone (Jiang, Feng, and Li 2012). Indeed, the protective effect of covalent conjugates films is due to their UV, oxygen barrier and anti-microbial properties, which suggest the potential application of covalent conjugates films instead of synthetic packaging for foods sensitive to oxidative protection, extending shelf-life of fresh fruit and vegetables, and enhancing quality of other food products.

6.3. Delivery of bioactive ingredients Biologically active compounds are prone to decomposition during production, storage, and transport in the gastrointestinal tract. PSCCs can improve the stabilization and

14 M. ZHAO ET AL. control release of bioactive compounds (Xinguang et al. 2018). Moreover, there are potential applications of protein-saccharide covalent conjugates for designing delivery systems, such as (i) oil-in-water (O/W) system is formed as a carrier of hydrophobic substances. Casein-dextran covalent conjugate micelles prepared through the Amadori rearrangement of the Maillard reaction can be used as a curcumin-carrier system to improve the stability and radical scavenging activity of curcumin (Wu and Wang 2017). Protein-saccharide covalent conjugates can be used to encapsulate active components in the microcapsule system. Biopolymer blends [gelatin-gum arabic-maltodextrin, GE-GA-MD (2:2:1, w/w/w)] were cross-linked by a dry-heating Maillard reaction, then stearidonic acid soybean oil was encapsulated into GE-GA-MD by complex coacervation to improve its antioxidant capacity (Ifeduba and Akoh 2016). (iii) Nanoparticle delivery systems to improve the absorption of biologically active compounds in oral delivery vehicles. Casein phosphopeptides/desalted duck egg white peptide-chitosan oligosaccharides by TGase-catalyzed and Maillard reactions can be used as a calcium delivery system, which can improve calcium-binding ability and promote the absorption of Ca 2+ in the intestine (Zhao et al. 2020; Zhu et al. 2020). Rutin-loaded corn protein hydrolysate-carboxymethyl chitosan conjugate nanoparticles were obtained by a dry-heating Maillard reaction for 48 h (60 °C, 79% relative humidity). The nanoparticles had a spherical morphology with a small particle size of 183.0 nm, high encapsulation efficiency (98.8%), and improved the stabilization of rutin (Han et al. 2019). As nanogels, there are several advantages such as entrapping a large amount of water without being dissolved in an aqueous solution, being sufficient stability under pH variation, long-term storage, dilution, and freeze-drying conditions. The three-dimensional network of nanogels with hydrophobic compartments can be used to deliver hydrophobic components. (Feng, Qi, and Liu 2016a). Ovalbumin-dextran nanogels were fabricated via the Maillard reaction followed by a heat gelation process, and then curcumin was loaded into nanogels through a pH-driven method and to improve oral curcumin bioavailability through the simulated mouth and gastrointestinal digestion (Feng, Qi, and Liu 2016a). Chitosan-gelatin injectable hydrogels based on transglutaminase-catalyzed reactions can be used for local cell delivery of doxycycline. The addition of TGase enhanced the stability of the hydrogel for controlled release of doxycycline from the cross-linked hydrogel (Yuan et al. 2017).

7. Prospection Natural protein/peptide-saccharide covalent conjugates are derived from a wide range of sources, and exhibit several physiological activities and beneficial properties, making them useful in the field of medicine. However, owing to the difficulties in their extraction, various methods of synthesizing food-derived PSCCs are speculate to improve

the synergistic biological activity of proteins/peptides and saccharides. PSCCs formed through chemical synthesis exhibit improved functional properties of proteins. It is also known that PSCCs have great potential for controlling the release of biologically active substances. However, toxicity and safety of synthetic PSCCs as nutritional supplements, potential therapeutic agents and oral delivery systems still need more attention. Harmful heterocyclic amines (HCA), acrylamide (AA), and advanced glycation end products (AGEs) may be produced in three stages of the Maillard reaction (shown in Figure 3a). HCA and AA compounds are carcinogenic and mutagenic in animal studies, and some exhibit potent mutagenicity in bacterial assays (Bear and Teel 2000; Cheng et al. 2009). To reduce the production of these harmful compounds, there are four strategies to consider based on the effect factors: (i) type and content of reducing saccharide and amino acids; (ii) physical parameters of reaction processes such as temperature, duration, pH; (iii) processing method. Zhang et al. (2021a, 2021b, 2021c) summarized in detail the factors contributing to the formation of harmful substances by various factors, and it has a good function in reducing the toxicity of synthetic PSCCs (Zhang et al. 2021c). Moreover, the toxicity of PSCCs could be reduced by introducing other active substances into the synthetic system via the Maillard reaction. Specifically, phenolic compounds, such as catechin, (-)-epicatechin, (-)-epigallocatechin-3-gallate, have been shown to possess inhibiting effects on the formation of AGEs and HCA. These effects have been explained by different reaction mechanisms, namely, radical scavenging, amine group blocking, scavenging dicarbonyl intermediates, or reactive carbonyls produced from amino acid degradation and lipid oxidation (Račkauskienė et al. 2019). Therefore, phenolic antioxidant-rich natural plant extracts may be useful additives in proteinaceous foods for inhibiting toxic Maillard reaction products. Nevertheless, TGase-induced synthetic PSCCs is a good method to prepare oral system instead of Maillard reaction synthetic PSCCs because TGase has the characteristics of non-toxicity. In general, further research is needed to develop improved glycoproteins and facilitate their industrial production. Overall, PSCCs have great potential in the food and pharmaceutical industry.

Author’s contributions Mengge Zhao, Hui He, Aimin Ma and Tao Hou conducted the literature research, conceptualized and synthetized information and wrote the manuscript. Tao Hou and Aimin Ma revised the manuscript and were responsible for the supervision of the whole research. All authors have proofread the manuscript and approved the final version of the paper.