Antioxidant proteins and peptides to enhance the oxidative stability of meat and meat products: A comprehensive review

ABSTRACT

Lipid oxidation is among the major flaw-grounding processes in meat and meat-based products that can affect interactions among lipids and proteins, leading to critically undesirable changes. Therefore, it is imperative to control lipid oxidation in meat allied products to enhance consumer acceptability. Moreover, lipid oxidation is somber dilemma visage by the meat processing industry, affects food constituents, leading to detrimental alterations that can impart the deleterious effects on human health upon consumption. Various synthetic (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary butylhydroquinone (TBHQ)) and natural antioxidants (vitamin C, vitamin A, tocopherols, especially vitamin E, flavonoids particularly quercetin, proteins, and peptides) as well as preservatives are employed to extend the storability of meat and resultants products; however, great consideration is paid to the utilization of natural antioxidants due to the harmful side effects imparted by synthetic counterparts. Recently, bioactive peptides are claimed to thwart lipid oxidation in meat and other products; in addition, these antioxidant peptides have also been reported to possess substantial health-promoting potential besides controlling oxidation. Therefore, the present review is intended to emphasize the sources, production methods, and applications of antioxidant proteins and peptides to control oxidative degradation in meat products and the potential health benefits of bioactive peptides. Furthermore, the techniques available for the extraction, characterization, and assessment of the antioxidant capability of bioactive peptides are discussed critically in this review.

KEYWORDS:

• Antioxidant peptides
• Fatty acids
• Free radicals
• Oxidative damage
• Quality degradation

Introduction

Meat and meat-based products can serve as excellent carriers for health-boosting food ingredients due to their versatility from minimally processed to fully cooked products as well as their intrinsic high-quality nutrients such as protein, vitamins, and minerals.^[1] Moreover, they are sources of micronutrients, with special reference to vitamins, minerals, and omega-3 fatty acids that are considered vibrant for human health.^[2] Nevertheless, meat products fulfil a major portion of the daily dietary protein requirement of individuals in industrialized economies. The consumption of these products is affected by different factors including product characteristics, consumer’s choice, and the storage conditions for keeping the meat products^[3] The polyunsaturated fatty acids (PUFA) considered beneficial for human health are prone to oxidation, which can deteriorate the flavour and nutritional quality of meat-based products.^[4] Moreover, in stored foods, oxidation of lipids is the major process responsible for quality degradation.^[[5]

Due to the enhanced knowledge of consumers regarding safe as well as nutritious food, there is increasing demand for quality meat products.^[6] Technologically, quality refers to water-holding capacity, colour intensity, firmness, and the processing yield of meat-based products.^[7] Mostly meat and meat products contain lower amounts of PUFA; however, poultry meat and meat products contain relatively greater amounts of PUFA, particularly omega-3 fatty acids, and these fatty acids in meat can trigger oxidation, which can adversely affect the colour, flavour, and storage stability of the resultant products.^[8]

Lipid oxidation and meat quality

Lipid oxidation represents one of the most important causes of deterioration in meat and meat-based products as it affects unsaturated fatty acids, particularly PUFA, in membrane phospholipids as well as cholesterol, mainly low-density lipoproteins (LDL) cholesterol. The end-products of this process impair the colour, aroma, flavour, and texture of meat and allied products and hence reduce the nutritive value.^[9] Besides nutritional deterioration, oxidation generates cytotoxic and genotoxic compounds, which are deleterious for humans health.^[10]

Oxygen is one of the essential elements mandatory for oxidative metabolic reaction involved in energy production in living organisms. Besides, it also participates in various reactions generating toxic compounds such as reactive oxygen species (ROS), considered harmful for the physiological system. The natural defence system in living beings is prone to harm produced by ROS. Numerous evidences have suggested that singlet oxygen induces damage to body tissues by producing free radicals that accumulate in tissues.^[11] The damage is associated with oxidative stress, which is mainly involved in many chronic diseases such as cancer, cardiovascular disease, immune dysfunction, and ageing. The harmful effects of oxygen are due to its ability to generate ROS, which act as free radical or pro-oxidant. These free radicals are unstable due to their reactive nature,^[12] and include hydroxyl (OH⁻), superoxide (O⁻²), nitric oxide (NO⁻), and lipid peroxyl (LOO⁻), which degrade cellular constituents including protein, lipids, and nucleic acid.^[13] It has been observed that technological processes like restructuring and grinding of meat also enhance the exposure of lipid bilayers to the air, which can trigger oxidation.^[14]

Specifically, lipid hydroperoxides, which are the primary products of lipid oxidation, have higher polarity than normal fatty acids, thereby disrupting the integral structure and function of the membrane, resulting in detrimental effects to the tissues. The aldehyde 4-hydroxynonenal, generated during lipid peroxidation, possesses cytotoxic potential in animals as it binds to protein, thereby inhibiting their function.^[15] Lipids are subjected to oxidation when catalytic systems like light, heat, enzymes, metals, metalloproteins, and microorganisms are present. The presence of intermediate reactive species and free radicals as well as these conditions lead to autoxidation, photooxidation, and thermal or enzymatic oxidation. However, oxidation of lipid caused by autoxidation is a spontaneous reaction of the substrate with oxygen through a chain reaction with further cascade.^[16] It has been well observed that lipid deterioration is slow at initiation; however, it proceeds rapidly after induction and the process involves three phases: initiation, propagation, and termination.

Mechanistic approach of lipid oxidation

The mechanistic approach regarding lipid oxidation indicates that in the presence of pro-oxidants, a hydrogen atom is abstracted from the methylene group from the hydrocarbon chain of the lipid molecule (RH), especially from unsaturated fatty acids, thereby producing free radicals (R·). These radicals tend to stabilize by the rearrangement of methylene-interrupted double bond in PUFA to generate conjugated dienes. Under aerobic conditions, conjugated dienes react with oxygen to form lipid peroxyl radicals (ROO·). Once these peroxyl radicals are generated, they are unstable due to their reactive nature and attack new lipid molecule sites, thus engaging in the rapid progression of reaction. During the propagation stage, peroxyl radicals remove hydrogen atom (H·) from the lipid molecule to form lipid hydroperoxides (ROOH), which are primary products of the oxidation process.^[17] Secondary oxidation products originating from lipid hydroperoxides include aldehydes (hexanal, 4-hydroxynonenal, malondialdheyde), ketones, alcohols, hydrocarbons, volatile organic acids, and epoxy compounds depending on the fatty acid substrates and reaction conditions. It has been documented that some secondary oxidation products have undesirable odour, which can be detected at low threshold levels such as alcohols, including pentanal and heaxnals.^[18] The oxidative damage to meat-based products results in problems like tissue damage, putrification, and loss of nutrients, enhanced free-radical generation and malonaldehydes production, which reduce the antioxidant capacity of meat-based products like nuggets, patties, sausages, etc. However, the oxidative stability of meat mainly depends on the balance of antioxidants, oxidation substrate, cholesterol, and haeme pigment.^[19]

Strategies to control lipid peroxidation

There are various approaches to control lipid oxidation in meat and meat-based products. Among these, the application of antioxidants is considered a pragmatic choice as they can retard the rate of oxidation in meat and meat products, ultimately enhancing the oxidative stability of products. Antioxidants are substances that, at low concentrations, retard the oxidative problems of oxidizable biomolecules such as lipids and proteins in meat products, thus improving their shelf stability and quality.^[20]

Antioxidants play a vital role in both food systems and the human body to reduce oxidative processes. In meat-based models, antioxidants are extremely useful to retard lipid peroxidation as well as secondary lipid peroxidation product formation, and thus help maintain the flavour, texture, and, in some cases, colour of the meat products during storage.^[21] They further reduce protein oxidation as well as the interaction of lipid-derived carbonyls with proteins, which leads to an alteration of protein functionality.^[22] The addition of antioxidants-rich formulations in various fresh and cooked meat products has the potential to reduce oxidation problems by hindering the free-radical formation.

Dietary supplementation of antioxidants is a process in which antioxidants are added via feed to be deposited later in the inner layers of phospholipids membranes or flesh of animal, which can protect live animal as well as feed from oxidation stress and enhance the storability and quality of meat.^[23] Dietary fortification of functional ingredients to various animals, especially broiler, is an ideal vehicle for delivering the target nutrient to increase the concentration of the respective compounds in muscles through deposition, thereby enhancing the functional value of the resultant meat products. In animal nutrition, use of plant and their bioactive derivatives including extracts, essential oils, as well as secondary metabolites for the development of healthier/functional meat is a growing trend. The administration of herbs and botanicals to animals regulates their feed intake, maintains a balanced microflora in the gastrointestinal tract (GIT), and exhibits antimicrobial properties, thereby enhancing the immunomodulatory and anti-inflammatory properties. The bioextracts from plants like ascorbic acid, α-tocopherol, β-carotene, flavonoids, and other phenolic compounds have the tendency to improve the antioxidant potential of the living system as well as processed meat products .^[24]

Dietary supplementation as well as the direct addition of antioxidants to meat products is used; however, dietary supplementation is a convenient strategy to distribute them in the inner and outer layers of phospholipid membranes uniformly. Moreover, the incorporation of antioxidants in blends performed better compared to a single one in living tissues and meat-based products. Various antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ), vitamin C, tocopherols, alpha lipoic acid, quercetin, other flavonoids as well as natural extracts from spices such as plant extracts like oregano, sage, rosemary, grape seed, etc. have the potential to mitigate the menaces of the oxidation process in meat-based products.^[25] Recently, there has been a trend of the application of antioxidant peptides and proteins to control oxidation problems in meat and meat products.

Antioxidant proteins and peptides

Among the various antioxidants of different origins employed in meat products, peptide antioxidants have drawn up much attention.^[26] Recent studies have documented peptide antioxidants, which can be fashioned through the enzymatic hydrolysis of different types of proteins. In hydrolysates of various proteins like soybean protein, β-lactoglobulin, ovalbumin or α-lactalbumin, some of the peptide antioxidants are identified, which can scavenge free radicals in meat products. Through enzymes, different peptides can be acquired from the solitary source of protein.^[27] Antioxidant enzymes exert their effects through several mechanistic approaches including scavenging free-radical species that pledge peroxidation, chelation of metal ions, quenching singlet oxygen, breakdown of free-radical chains, and controlling oxygen concentration, which promote the rate of oxidation^[28]]. However, the efficiency of antioxidant compounds is varied depending on different mechanisms. For instance, phenolic acids have shown their potential to scavenge free radicals, but they are not good chelating agents. On the contrary, flavonoids can effectively trap free radicals and can bind metal transition ions^[29]. As antioxidant enzymes employ varied mechanistic strategies to perform their oxidation-preventive role, a single assay cannot account for all the different modes of action of various enzymes in different food systems.

Bioactive peptides, representing the products of varied food proteins hydrolysis, are the spotlight of the current research. There was exertion of diverse biological roles, of which one of the most critical is the antioxidant activity. The overturn relationship between diseases and antioxidant intake has been standardized through various studies. The antioxidant potential of peptides can be accredited to their metal ion chelation, lipid peroxidation inhibition, and radical-scavenging properties. It also has been documented that the structure of peptides with its amino acid sequence can influence their antioxidative potential ^[30]

Proteins can restrain the lipid oxidation in meat and its products via biologically designed mechanisms (such as iron-binding proteins and antioxidant enzymes) or through some nonspecific mechanisms. The antioxidant proteins of both mentioned types add value with the endogenous antioxidant activity of some foodstuffs. Moreover, proteins have exceptional prospective as antioxidant additives in meat products as they can restrain lipid oxidation by following multiple pathways including the reduction of hydroperoxides, scavenging free radicals, ROS inactivation, prooxidative transition metals chelation, and modifying the physical properties of the meat products. The overall antioxidant capacity of proteins can be augmented by disrupting their tertiary structure, which ultimately increases the solvent convenience for amino acid residues, which further scavenge chelate prooxidative metals and free radicals. Peptide production via hydrolytic reactions poses to be the chief applicable technique for the formation of proteinaceous antioxidants as peptides have significantly advanced antioxidant potential compared with the intact proteins. Peptides and proteins have tremendous potential as antioxidants for food, especially in meat and meat products.^[31]

Production of antioxidant proteins and peptides (Table 1)

A wide range of commercial enzymes has been used for the extraction of bioactive peptides from different animal sources. The most commonly used enzymes include pepsin, trypsin, collagenase, and papain. These enzymes are usually derived from various microbial sources like Bacillus subtillus, B. polimixa, and Streptomyces.^[32] Antioxidant peptides are extracted through ultrafiltration using special membranes called molecular-weight cut-off (MWCO) membranes. Later, these peptides are tested for their antioxidant potential through various assays such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, thiobarbituric acid reactive substance assay (TBARS), metal-chelating activity, hydroxyl radical-scavenging activity, superoxide radical-scavenging activity, peroxyl radical-scavenging activity, peroxide value or carbonyl values. After evaluating the antioxidant activity of bioactive peptides, these are subjected to further purification steps.^[33]

Table 1. Sources, production and activity of antioxidant peptides

Sources of antioxidant peptides	Enzymes used for production	Activity
Rice endosperm proteins and frog skin	Neutrase	Inhibits auto-oxidation and has hydroxyl radical-scavenging activity
Peanut proteins	Esperase	Prevents LDL oxidation
Peanut proteins, corn gluten, alfalfa leaves	Alcalase	Controls peroxidation and possesses chelating properties
Yam tubers, zein hydrolysate	Pepsin	Contains radical chelating properties and has DPPH-scavenging activity
Sunflower protein	Pancreatin	Shows copper-chelating activity
Peptide from frog skin	Trypsin	DPPH and superoxide scavenger
Animal proteins	Papain	Inhibition of lipid peroxidation
Animal skin	Chymotrypsin	Has DPPH, superoxide and hydroxyl-scavenging activity
Marine sources	Papain, pepsin	Control oxidation and possess strong chelating properties

For the purification of antioxidant bioactive peptides, several techniques have been employed to date. The most frequently used purification methods include gel filtration chromatography (GFC), ion-exchange chromatography (IEC), and high-performance liquid chromatography (HPLC). Among these chromatographic techniques, HPLC has gained more importance because of its high potential to separate bioactive peptides compared with the other methods. After separation, these peptide fractions are further analysed for their antioxidant efficiency followed by sequencing through mass spectrometry (MS).^[34]

The most commonly deployed methods for the production of antioxidant bioactive peptides are categorized as enzymatic hydrolysis and microbial fermentation processes. In addition, these peptides can also be obtained during the processing of various foods as well as in the process of gastrointestinal (GI) digestion. These methods are briefly highlighted in the following section.

Enzymatic production of antioxidant bioactive peptides

Biologically active antioxidative peptides are chiefly prepared from precursor proteins through enzymatic hydrolysis. The most commonly used enzymes are digestive enzymes, plant-derived enzymes as well as enzymes derived from various microorganisms.^[35] Mostly, the enzymatic hydrolysis method for the production of bioactive peptides is applied to elevate the nutritional attributes and functional properties of proteins.^[36] It has been reported that the enzymatic digestion of glycinin and conglycinin increased the antioxidant potential of the derived bioactive peptides.^[37] The mechanistic approach behind this increment in antioxidant activity is more R-groups of amino acids are exposed upon hydrolysis, ultimately contributing to the elevated antioxidative activity of peptides. In addition, studies also reported hydrophobicity of the proteins increased due to enzymatic digestion due to the protein unfolding of quaternary and tertiary protein structures to both secondary and primary, making them easier to digest and utilize. Enzymatic hydrolysis is also involved in reducing the production of different allergenic agents present in these proteins. Additionally, solubility of the proteins is increased as a result of enzymatic digestion due to the breakdown of peptide linkages that enhances the levels of free amino and carboxyl groups. Furthermore, the solubility of peptides is reported to be enhanced under acidic conditions that prevailed during the digestion process ^[38]. Therefore, it is generally claimed that hydrolysis results in an increase or decrease in the hydrophobicity of the extracted peptides depending upon the nature of the precursor proteins as well as the molecular weight of the generated peptides.^[39] For example, a study was conducted on soy proteins that were enzymatically modified by incorporating hydrolysate (20 g/100 mL) with chymotrypsin and glycerol at 37°C, followed by ultrafiltration of the hydrolysates. The findings revealed that the modified hydrolysate showed lower emulsifying capacity as well as hydrophobicity ^[38]. Nevertheless, it has also been stated that extensive enzymatic hydrolysis can impose adverse effects on the functional attributes of biologically active peptides; therefore, this point is kept in mind while selecting the conditions and time duration for enzymatic hydrolysis.^[40]

The alfalfa leaf protein that is mainly used in various food and pharmaceutical formulations originally has lower solubility as well as sensory acceptability; however, these attributes of alfalfa leaf protein can be improved by deploying enzymatic hydrolysis to enhance the functional and technological properties of peptides.^[41] Protease enzyme is claimed to have positive effects on the amount and composition of peptides, which influences the antioxidant activity of the peptides.^[42] Similarly, ^[43] evaluated the effect of different enzymes on the production rate of hydrolysates from soy protein isolates and postulated that the use of a mixture of various enzymes produced a peptides mixture with varied degrees of hydrolysis and diverse levels of antioxidant potential. It has also been revealed that the use of alcalase for the enzymatic production of peptides results in the formation of peptides having diverse biological and antioxidant activities. Besides, alcalase provides a higher yield of antioxidative peptides compared with other protease enzymes and these derived peptides possess higher resistance against digestive enzymes.^[44]

Fermentation and peptidic production (Table 2)

Fermentation is known as the oldest method of food preservation, particularly in China, Korea, and Japan. It is thought that fermentation enhances the storability of food products and boosts the nutraceutical and functional values of foods mainly due to protein fragmentation to bioactive peptides by the action of microbial proteases.^[45] Studies have shown that some strains of Lactobacilli have the ability to prevent or delay oxidative deterioration, which leads to the reduction in the production and accumulation of ROS during food ingestion.^[46] To date, a few antioxidant peptides have been identified in fermented milk products. For example, k-casein-derived peptides are generated by Lactobacillus delbrueckii from fermented milk, which are shown to have antioxidant potential.^[47]

Table 2. Composition and mode of action of amino acids in relation to the antioxidant activities of bioactive peptides.

Type of amino acids	Mode of action
Aromatic amino acids	Convert free radicals to stable molecules by donating electrons Improve radical-scavenging activity
Hydrophobic amino acids	Enhance the solubility of peptides in lipids, which aids in easy access to hydrophobic free radicals
Acidic and basic amino acids	Carboxyl and amino groups provide chelating properties Act as hydrogen donor to stabilize free-radical species
Sulphur-containing amino acids (cystein)	SH group acts as free-radical scavenger Provides protection to the tissues from oxidative damage Improves glutathione activity

Bioactive peptides having high antioxidant activity are obtained from fermented food products through the action of different microorganisms and endogenous proteolytic enzymes.^[48,49] In an investigation, Rajapakse and co-workers ^[40] extracted HFGBPFH peptide having free-radical-scavenging activity from fermented mussel sauce. Similarly, several antioxidant peptides are also obtained from fermented milk products with the help of different strains of lactic acid bacteria.^[50] Likewise, several antioxidant bioactive peptides can also be derived from fermented soybean products due to the action of fungal proteases.^[51]

Antioxidative peptide production during food processing

Bioactive peptides can also be generated during the processing of various food products. For instance, cyclo (His-Pro) is a peptide with antioxidative potential that can be obtained during the processing of various foods such as dried shrimp, tuna, fish sauce, ham, noodles, potted meat, white bread, and non-dairy creamer.^[52] This peptide is cyclic in nature and has shown the potential to be absorbed readily in the GIT, leading to the reduction of oxidative stress.^[47] Likewise, the production of several antioxidative bioactive peptides has also been reported in pressure-cooked salmon cartilage.^[53]

Production of antioxidant peptides during digestion

Another possible way of producing bioactive peptides is the GI digestion of food proteins through the action of digestive or microbial enzymes.^[54] These peptides have the ability to exert their antioxidant potential directly in the intestine or other targeted locations in the body with the aid of receptors present in the cells or cell signalling in the gut. These peptides generally have the ability to pass through various cell membranes. For instance, oligo-peptides have the potential to permeate through the upper intestinal tract depending on their structural properties. Studies pointed out that the LVGDEQAVPAVCVP peptide generated during the GI digestion of mussel proteins depicted a strong potential to inhibit the oxidation of PUFA.^[55] Sannaveerappa et al.^[56] investigated that herring proteins prevent LDL oxidation, which is usually derived from enzymatic hydrolysis in the intestine. Zhu and peers^[57] testified that the increase or decrease in the antioxidant potential of bioactive peptides depends on the types of enzymes present in the digestive tract and the period of hydrolysis. Hence, the probability of alteration or breakdown of bioactive peptides produced after digestion is one of the most imperative factors to be considered while assessing the potential of these peptides for human health promotion.

Methods to evaluate the antioxidant activity of peptides

The significance of oxidative stress and the preventative roles of antioxidant motivate the interest to investigate the antioxidant efficacy of food products, which led to the development of different methods to evaluate the antioxidant potential of different functional ingredients present in food. These methods are also helpful for researchers in determining the quantity of antioxidant compounds in various food products and to estimate their ability to fight against the oxidative deterioration of food products.^[58]

Methods for the determination of the antioxidant ability of different bioactive peptides are categorized as: a) methods based on hydrogen atom transfer (HAT) and b) methods that depend on electron transfer (ET). The HAT-based methods usually involve competitive reactions in which antioxidant is compared with the substrate for the production of peroxyl radicals. The most common examples of HAT-dependent assay include oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and carotene bleaching assay. On the contrary, in ET-based methods, the capability of a particular antioxidant is evaluated against a certain oxidant.^[59] The evaluation methods based on ET are trolox equivalent antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), and DPPH radical-scavenging capacity .^[60]

The type of assay used for evaluating the antioxidant capacity of bioactive peptides is of significant importance. Studies confirm that in vitro measurements to analyse the antioxidant potential of bioactive peptides is less reliable compared to in vivo analysis. The main reason behind that is in vitro analysis does not provide information about the bioavailability of peptides, their in vivo reactivity, stability, and storage in body tissues. Moreover, in vitro systems have no or very little similarity to biological systems. Consequently, a comprehensive in vivo monitoring is mandatory to support the antioxidant efficiency of a particular bioactive peptide. However, despite the availability of several methods for the detection of the antioxidant efficiency of bioactive peptides, none of them can be used as an official standardized method due to the several limitations associated with each method. Hence, it is usually proposed that each evaluation be done by various methods of measurement in different oxidation conditions.^[61]

A study was carried out to purify and identify antioxidant peptides from the extract of Chinese dry cured Jinhua ham. Accordingly, the extracts were divided into five fractions (A–E) using size-exclusion chromatography, followed by every fraction subjected to simulated GI digestion system. The results showed 33 peptides were identified, and among these, GKFNV indicated the highest DPPH radical-scavenging activity as 92.7% antioxidant activity at a concentration of 1 mg/mL. Similarly, LPGGGHGDL reported the highest hydroxyl radical-scavenging activity and LPGGGT and HA showed a strong inhibition activity against erythrocyte haemolysis (about 45%) before digestion. Additionally, KEER may contribute to Fe (2+)-chelating ability in fraction C after GI digestion ^[62].

Fishery waste and its by-products are valuable sources for extracting useful antioxidant and bioactive peptides. Accordingly, the backbone of rastrelliger kanagurta (Indian mackerel) was evaluated for its antioxidant potential. The result of the antioxidant assay indicated that the DPPH radical-scavenging efficacy of fish protein hydrolysate (FPH) was similar to synthetic antioxidant butylated hydroxyl toluene (BHT) and FPH exhibited significant reducing power ability and great potential to inhibit lipid peroxidation, similar to BHT and α-tocopherol, respectively. Furthermore, the results also indicated the degree of hydrolysis for rapid proteolytic cleavage during the first 60 min of incubation^[63]. Similarly, the cod muscle protein after mechanical deboning was sequentially hydrolysed using pepsin and a trypsin+chymotrypsin combination was subjected to digestion followed by passing through 1 kDa equipped tangential flow filtration system. Later, the permeate (<1 kDa peptides) was collected as the cod protein hydrolysate (CPH). The results indicated that these peptide fractions showed significantly higher (p<0.05) ORAC values (698–942 μM Trolox equivalents, TE/g), and DPPH-scavenging activities (17–32%) than those of CPH (613 μM TE/g and 19%, respectively). The CPH and peptide fractions also reported a dose-dependent inhibition of linoleic acid oxidation^[64].

A study was conducted in which hydrolysed chicken breast proteins were extracted using papain under optimal conditions followed by the in vitro and in vivo evaluation of these proteins for antioxidant trait. The results elucidated that the reducing power and DPPH radical-scavenging activity of hydrolysate was 0.5 at 2.37 mg/mL and 1.28 mg/mL (EC50), respectively. The results of in vivo trials showed that the antioxidant enzyme activity increased in a significant manner whereas the malondialdehyde level decreased in serum and liver samples in d-galactose-induced ageing mice administrated with peptides of chicken breast protein hydrolysate^[65]. Similarly, another study concluded that chicken breast protein hydrolysate plays a significant role in reducing the oxidative stress in hepatocytes in vivo. They concluded that chicken breast protein hydrolysate exhibits significant antioxidant activity. Moreover, ^[66] postulated that antioxidant peptide purified from duck bones and meat showed potential hydroxyl radical-scavenging activity in in vitro trials.

Application of antioxidant peptides in meat products (Table 3)

Lipid oxidation is proved to be as one of the chief issues for the resultant deterioration of meat and meat products as their detection concludes the commencement of numerous detrimental changes in nutritional value, texture, and flavour. However, the lipid oxidation rate can be efficiently restrained through the utilization of antioxidant peptides and proteins.^[67] Although synthetic antioxidants were broadly employed in the meat industry, food safety concerns regarding consumers pushed the food industry to discover natural sources for their utilization. Antioxidant proteins and peptides can be used as substitutes for synthetic antioxidants as they have equivalent or even greater potential for the inhibition of lipid oxidation.^[68] Antioxidant peptides in meat products help in the protection of cells from damage resulted by electrophilic and oxidative stress through metal ion binding, superoxide scavenging or via the regeneration of various oxidized antioxidants. One of the most efficient methods for impending lipid oxidation in meat or meat products is to incorporate antioxidant proteins. They work by diversified mechanisms, including control of pro-oxidants, control of oxidation substrates, and scavenging free radicals. Much interest has been urbanized freshly in naturally occurring antioxidants, alleged to be safe and sound as they are obtained from plant foods. The petition in the application of antioxidant proteins and peptides as antioxidants is hoisted because of their impending role.^[69]

Table 3. Application of antioxidant peptides in meat preservation and additional health impacts.

Antioxidant peptides	Application in meat preservation	Additional health benefits
Casein protein-derived peptides	Prevent lipid oxidation in processed minced beef	Reduce oxidative stress in body
Whey protein peptides	Control oxidative stability during the refrigeration storage of meat, reduce cooking loss	Control onset of diabetes, release oxidative stress
Soy protein peptides	Prevent early-stage lipid oxidation in red meat	Reduce the risk of paraquat-induced oxidative stress, prevent mutation, improve liver and kidney functioning

Antioxidants peptides have ability to inhibit lipid peroxidation problems in meat besides offering defensive action against oxidative smash up to the membrane functions in biological systems. Although the peptides possess antioxidant activity but they can influence the activity of antioxidant enzymes resulting improved performance. Depending on the degree of enzyme used, the hydrolysis potential, activation, and inhibitory effects of the peptides are diverse. Peptides derived from wheat albumin with neutral protease are widely used in various food products. In one study, the enzymatic hydrolysis of cottonseed and wheat flour proteins with neutral and acid proteases has been described, which ultimately led to the construction of peptides along with antioxidant possessions. Moreover, the study also revealed that peptides that are derivative of wheat albumin along with acid proteases acquire 10–12 times superior plummeting power than the peptides consequent from neutral proteases. Therefore, the peptides derived from acid and wheat albumin proved to be more efficient in meat and meat products. Similarly in another study, peptides were derived from cottonseed albumin, resulting in good antioxidant property in meat and allied meat products.^[70] Accordingly, Bougatef et al.^[71] in 2010 extracted and purified antioxidant peptides deploying the enzymatic hydrolysis of Sardinellaaurita proteins. These peptides reported good antioxidant potential in meat and meat products. Moreover, in another study, the antioxidant potential of peptides extracted from mushroom Ganoderma lucidum (G. lucidum) was found to suppress lipid oxidation without affecting the consumer acceptability traits of the products. Polysaccharide–peptide complex, polysaccharides, and phenolics of Ganoderma lucidum were accountable for the antioxidant action. However, the study showed that the component of G. lucidum peptide (GLP) is the chief antioxidant constituent of G. lucidum, which could efficiently inhibit lipid oxidation in meat and meat products through its free-radical scavenging, metal-chelating, and antioxidant activities.^[72] However, in 2015, Nimalaratne et al.^[73] worked on the isolation, characterization, and purification of antioxidant peptides through the process of enzymatic hydrolysis from chicken egg white, which is the most promising constituent that can reduce the potential of lipid oxidation in meat or meat products. Moreover Gu et al. ^[74] documented the antioxidant potential of peptides extracted from the meal proteins of defatted walnut.

Proteins from dairy sources are also enriched with antioxidant peptides that have potential applications in meat preservation. The peptides derived from casein have demonstrated strong efficiency in preventing TBARS formation in cooked ground beef. The authors of this study proposed the mechanism involved in the antioxidative potential of casein peptides: during cooking, the chelating activity of casein bioactive peptides increases, which increases their antioxidant activity in cooked meat products. Additionally, casein calcium peptides also proved effective as an antioxidant in preventing lipid oxidation in ground beef homogenates, and the incorporation of 2% casein calcium peptides inhibited lipid oxidation in beef samples by up to 70%.^[75]

A significant amount of literature has discussed the antioxidant efficacy of bioactive peptides in preventing lipid oxidation of meat and meat-based products during processing and storage, thereby increasing the shelf life.^[76] Likewise, whey protein peptides have also shown their capability to be used as functional ingredients in meat products. Pena-Ramos and Xiong^[77] showed that the addition of 2% whey protein bioactive peptides not only suppressed the oxidative deterioration of pork meat during refrigeration storage but also reduced cooking loss during processing and storage. In addition, it is also claimed that hydrolysis improves the capability of different antioxidant peptides, prevents early-stage lipid oxidation in meat products, and delays the onset of oxidation in meat during storage. Thus, peptides obtained from partially hydrolysed proteins seem to be a predominantly attractive choice for processors to improve the quality of meat-based products by effectively preventing oxidative degradation in muscle foods.

Health benefits of meat products containing antioxidant peptides

It has also been proved through a number of studies that bioactive peptides have considerable health benefits. The generation of free radicals during oxidative stress is responsible for the progression of several other lifestyle-related ailments. For example, recent evidence proposes that elevated oxidative stress has a direct relation with the onset of type II diabetes due to elevated insulin resistance and impaired secretion insulin. Additionally, the unrestrained formation of free radicals worsens the development and expansion of hyperglycaemia and related snags.^[78] Moreover, an elevated level of oxidative stress plays a dominant role in the initiation of several cardiovascular diseases in patients suffering from metabolic syndrome.^[79] Thus, due to the strong association between oxidative stress and various metabolic disorders, prevention of oxidative stress is crucial and steps should be taken to overcome this problem in order to prevent these complications. In this regard, several components having antioxidant potential have been identified, isolated, and utilized from natural and synthetic sources to curtail oxidative stress. Besides these well-known natural and synthetic antioxidants, peptides with antioxidative characteristics are also the focus of recent research. The beneficial health effects of using bioactive peptides have also been investigated and proven through efficacy trials. For instance, the ingestion of soybean protein-based peptides has shown positive effects on liver and kidney functions by retarding oxidative stress.^[51]

Studies have also postulated that egg protein-derived bioactive peptides contribute to prevent oxidative stress by controlling lipid-based oxidative degradation and enhancing plasma radical-scavenging capacity.^[80] In another investigation, it was also reported that soy peptide ingestion reduced the risk of paraquat (PQ)-induced oxidative stress in rats. Additionally, serum TBARS concentration can also be reduced by incorporating soy proteins and peptides in the diet.^[81] Likewise, soy peptides have demonstrated a preventative role against mutation and oxidative damage.^[82]

Conclusion

Bioactive peptides have been known to be a part of human diet for several years. With the development of advanced extraction, isolation, and characterization techniques, the number of investigations studying the health impacts of bioactive peptides against several health-related disorders has increased. Accordingly this article is an attempt to describe the various sources of functional bioactive peptides, their extraction, and production methods, applications in meat preservation and potential health benefits. The discussion concludes that bioactive peptides can be effectively utilized as food ingredients in meat and other food products for enhancing their shelf life and oxidative stability. Some of the bioactive peptides have already been commercialized; however, there is need to explore the new sources through which novel peptides can be extracted and further utilized as functional ingredients in meat-based products. Additionally, further research work is needed to evaluate the antioxidant efficiency, bioavailability, and health effects of existing and unexplored bioactive peptides.

Acknowledgements

The authors extend their appreciation to the International Scientific Partnership Program ISPP at King Saud University for funding this research work through ISPP# 0023. The authors declare no conflict of interest.