Antidiabetic Food-Derived Peptides for Functional Feeding: Production, Functionality and In Vivo Evidences

foods Review Antidiabetic Food-Derived Peptides for Functional Feeding: Production, Functionality and In Vivo Evidences Fernando Rivero-Pino * , F. Javier Espejo-Carpio and Emilia M. Guadix Department of Chemical Engineering, University of Granada, 18071 Granada, Spain; [email protected] (F.J.E.-C.); [email protected] (E.M.G.) *Correspondence: [email protected]; Tel.: +34-958241329 Received: 11 June 2020; Accepted: 21 July 2020; Published: 23 July 2020   Abstract: Bioactive peptides released from the enzymatic hydrolysis of food proteins are currently a trending topic in the scientific community. Their potential as antidiabetic agents, by regulating the glycemic index, and thus to be employed in food formulation, is one of the most important functions of these peptides. In this review, we aimed to summarize the whole process that must be considered when talking about including these molecules as a bioactive ingredient. In this regard, at first, the production, purification and identification of bioactive peptides is summed up. The detailed metabolic pathways described included carbohydrate hydrolases (glucosidase and amylase) and dipeptidyl-peptidase IV inhibition, due to their importance in the food-derived peptides research field. Then, their characterization, concerning bioavailability in vitro and in situ, stability and functionality in food matrices, and ultimately, the in vivo evidence (from invertebrate animals to humans), was described. The future applicability that these molecules have due to their biological potential as functional ingredients makes them an important field of research, which could help the world population avoid suffering from several diseases, such as diabetes. Keywords: bioactivity; enzymes; glycemic index; nutraceutical; peptides; proteases; protein hydrolysates 1. Introduction 1.1. Proteins, Protein Hydrolysates and Peptides Proteins are one of the main components of human diets. These biomacromolecules are formed by the association of amino acids, through the peptidic bond between the amino group of one amino acid and the carboxyl group from the following amino acid in the chain [ 1 ]. Food proteins are an important topic in the research, due to their health benefits in human and their enormous variety. At the end of the 20th century, scientists started to focus on studying the hydrolysis of food proteins in order to determine their forming-peptides, because some of them have been proven to be bioactive, and to have beneficial consequences in the organism [ 2 ]. The gastrointestinal digestion of proteins leads to the formation of peptides in the human tract due to the action of digestive proteases, and these have beneficial effects. However, the key in this topic is that different food-grade proteases might have different specificities [ 3 ], and from the same substrate, the pool of peptides produced would be different, and would show different properties. A protein hydrolysate is the mixture of peptides that originally formed the protein, after its hydrolysis. The complex structure of proteins in the native state hides the functionality of the peptides, preventing them from exerting their bioactivity by association with some other molecules. Foods 2020,9, 983; doi:10.3390/foods9080983 www.mdpi.com/journal/foods Foods 2020,9, 983 2 of 33 The hydrolysis reaction consists of breaking the peptidic bonds and releasing different-sized peptide chains, whose properties may well vary depending on the properties of the amino acids included in each peptide [ 4 ]. The released peptides usually present improved technological and biological properties that allow the utilization of these products to improve food formula properties. Protein hydrolysis leads to an improvement in different technological aspects: solubility, emulsifying and foaming capacity, water holding capacity, oil binding capacity and lipid oxidation prevention. The technological property modification of protein hydrolysates, compared to the intact protein, is an advantage related to their use in functional feeding, because it mainly involves the facilitation of the formulation of the food. This improvement is due to the exposition of the residues of different amino acids. In this context, both the molecular weights of peptides and amino acid sequences are important. The solubility of proteins depends on the interactions between the macromolecules forming the protein [ 5 ]. Protein hydrolysis leads to polar group exposition when small peptides are released, and consequently, a high degree of hydrolysis is correlated with the higher solubility of protein hydrolysates [ 6 , 7 ]. This increase in solubility is important in the production of food and beverages intended for parenteral or gastric administration. Proteins are amphipathic molecules due to the different polarities of the amino acids that compose them [ 8 ], which are absorbed into the interface formed during the emulsification process [ 9 ], reducing the interfacial tension and stabilizing the emulsion. Notably, limited hydrolysis leads to this interfacial activity, increasing the emulsifying activity [ 10 ]. Foaming property improvement also depends on the surface activity of the proteins [ 6 ], and low degrees of hydrolysis are adequate for an increase in foaming capacity [ 11 ]. Protein hydrolysis also enhances the water holding capacity and the oil binding capacity, depending on the amino acids composing the peptides and the lengths of them. Hydrophilic groups would retain water more effectively [ 12 ], whereas for oil binding capacity, the hydrophobic residues of proteins are important due to the lipophilic character of oils, thus allowing the interaction with their hydrocarbon chains [ 13 ]. These are important parameters to consider when formulating a fortified food product, because they would affect its structure. Proteins and peptides show antioxidant activity [ 14 , 15 ]. Numerous protein hydrolysates have been reported as antioxidants, coming from different protein sources. Concerning technological improvement, it is important to remark that these peptides with antioxidant activities and emulsifying properties can be used in complex food matrices as an emulsion, those peptides being a double-action agent[ 16 , 17 ]. Assuch, thesepeptideswouldprevent thelipidoxidation processin foodformulation [ 18 ]. Another improvement provoked by hydrolysis is the increase in digestibility and the loss of antigenicity of the proteins. This is an important statement in terms of nutrition; for example, for specific groups of people, such as infants or elder. The digestibility of proteins is increased because the available N-terminal sites are increased after enzymatic hydrolysis [ 19 , 20 ], and consequently, the peptidases action is enhanced. The antigenicity of proteins is caused by epitopes, specific sequences in the allergen proteins that are potentially recognizable by the immune system and would potentially activate an allergenic response. Enzymatic hydrolysis leads to allergenic epitopes degradation, and therefore helps to reduce the immunoreactivity of the native protein [ 21 ]. Enzymatic hydrolysis, due to its reaction conditions, does not destroy amino acids, which is desirable for food formulation because the nutritional profile of the proteins is conserved. Nonetheless, one disadvantage that enzymatic hydrolysis can produce in terms of nutrition is the bitter taste of peptides, related to the release of hydrophobic residues. To overcome this limitation, the encapsulation of peptides inside different matrices (i.e., chitosan, glucose syrup) or the addition of flavor-developing proteases (i.e., flavourzyme) are adequate solutions. These techniques would avoid the disadvantages of traditional techniques, such as the adsorption of bitter peptides onto activated carbon, chromatographic removal, or selective extraction with alcohols [22]. On the other hand, the similarity of food-derived peptides to the structure of human regulatory peptides also makes them suitable for interacting with some enzymes and receptors involved in human metabolism. In this way, the most important improvement of proteins after hydrolysis, concerning Foods 2020,9, 983 3 of 33 functional food, is the bioactivity development. Lately, the proportion of the world population suffering an illness has increased, and prevention and pre-treatment are considered good options for most of them. At the economical level, the cost savings, compared to those associated with the treatment of the disease, are high [ 23 ]. Bioactive peptides are considered to be regulator molecules operating at different levels in the organism. As was previously mentioned, protein hydrolysis during digestion releases peptides that exert bioactivity in humans, but the intake of peptides with improved bioactivity, compared to those obtained naturally, is seen as a good option for humans [ 19 ]. This is due to the specificity of food-grade proteases employed in the industry, which are able to release peptides that digestive proteases cannot. The bioactivity of peptides is considered to be related to the hydrophobicity characteristics of the residues, and more precisely, to the amino acid functional groups of their sequences [ 20 ]. Focusing on the bioactivities those peptides can exert, their antioxidant, antihypertensive and antidiabetic (glycemic index regulation) activities are the most remarkable because of the diseases they would prevent, which are some of the most prevalent worldwide nowadays (cardiovascular disease, diabetes, hypertension). Recent studies, both in vitro and in vivo , show the functionality of these protein hydrolysates. In vitro analyses allow comparisons of the biological potentials of different products, by evaluating, for example, the inhibitory capacity of different enzymes involved in metabolic processes. Bioactive peptides can exert physiological effects at a cardiovascular, digestive, endocrine, immune and/or nervous level [ 24 ]. The most studied historically are antihypertensive and antioxidant, whereas there is less information regarding the antidiabetic properties of peptides coming from food proteins. Peptides are considered to be bioactive in different metabolic pathways, depending on how they interact with the human body. When it comes to inhibiting an enzyme, peptides can interact at the active site and/or outside the catalytic site of it, preventing the enzyme from interacting with the substrate. Peptides are defined as antihypertensive when they are able to inhibit the angiotensin-converting enzyme (ACE). This enzyme plays a key role in blood pressure regulation [ 25 ], and its inhibition has a positive effect on hypertensive patients [ 26 ]. In the antioxidant case, a wide variety of mechanisms and implications are involved. Beyond their ability to slow down lipid oxidation in food systems, these peptides would also prevent oxidative stress related to several diseases such as hypertension and ageing [ 27 ]. As such, peptides with both antihypertensive and antioxidant activities can be considered anti-ageing peptides. Beyond these two bioactivities, anticholesterolemic, antithrombotic and anti-inflammatory peptides have also been described [ 28 , 29 ]. Furthermore, peptides might have diverse bioactivities, and might consequently exert synergistic effects on the human body. For example, a correlation has been proposed between diabetic and hypertensive patients, so a treatment for both problems would be ideal. Ketnawa et al. [ 30 ] obtained both ACEand dipeptidyl peptidase IV (DPP-IV)-inhibitory peptides from rainbow trout. In this review, we will focus on glycemic index-regulating peptides, that is, antidiabetic peptides. 1.2. Carbohydrates Digestion Process and Diabetes The metabolism of carbohydrates is the process of transforming the carbohydrates ingested from food into glucose molecules, the most efficient source of energy. The carbohydrates in foods generally appear as polysaccharides, such as starch or cellulose, or as disaccharides, such as lactose or sucrose. Carbohydrate digestion involves different enzymes and a complex series of metabolic processes. A graphical simplification is depicted in Figure 1. Initially, when the bolus is ingested, digestive enzymes would hydrolyze these complex polysaccharides. Foods 2020,9, 983 4 of 33 Foods 2020, 9, x 4 of 34 Figure 1. Mechanisms involved in peptides and carbohydrate digestion. The broken lines recreate the digestion process of the different molecules. Color reference: Yellow—Molecular state of carbohydrate during digestion; Orange—Main digestive enzymes involved in the carbohydrate digestion; Light blue—Molecular state of proteins during digestion; Dark blue—Digestive proteases. Permission: The original picture was released into the public domain by its author (LadyofHats) and modified by authors to depict the information detailed in the document. α-Amylase (EC 3.2.1.1) hydrolyzes complex carbohydrates such as starch into oligosaccharides, which would be further hydrolyzed by α-glucosidase. This enzyme is secreted from the salivary and pancreatic glands. α-Glucosidase (EC 3.2.1.20) is a membrane-bound enzyme found in the epithelial mucosa of the small intestine (brush border of the enterocytes). It releases free glucose molecules from terminal, non-reducing (1-4)-linked α-glucose residues. Furthermore, food intake causes the release of intestinal hormones called incretins (gastric inhibitory polypeptide, GIP, and glucagon-like peptide-1, GLP-1). These two would affect numerous target tissues in the body, acting as endocrine signals to the pancreas, leading to insulin production in the β-cells and the suppression of the release of glucagon in the α-cells. These two incretins are responsible for ~70% of the insulin secretion after meal intake [31]. This results in the uptake of glucose by the muscles, as well as a lower production of glucose in the liver. The final consequence is therefore the decrease in blood glucose after ingestion, which allows the adequate regulation of postprandial blood glucose levels. At this level, the enzyme dipeptidyl peptidase IV (DPP-IV) regulates the degradation of incretins according to physiological needs. GLP-1 and GIP have halflives of approximately 2 min and 5–7, min respectively, before they are degraded by DPP-IV [32,33]. DPP-IV is a cell surface (EC 3.4.14.5) that cleaves dipeptides from the N-terminus of polypeptides, in which proline is at the penultimate position [34]. DPP-IV can largely be found on the luminal surface of enterocytes; therefore, it can interact with any of the molecules from food intake before their absorption, that can be further metabolized before the molecules’ interaction with soluble and vascular endothelial DPP-IV (the one affecting GIP and GLP-1 levels). Gut hormones released from the enteroendocrine cells play an important role in food intake regulation [35]. Figure 1. Mechanisms involved in peptides and carbohydrate digestion. The broken lines recreate the digestion process of the different molecules. Color reference: Yellow—Molecular state of carbohydrate during digestion; Orange—Main digestive enzymes involved in the carbohydrate digestion; Light blue—Molecular state of proteins during digestion; Dark blue—Digestive proteases. Permission: The original picture was released into the public domain by its author (LadyofHats) and modified by authors to depict the information detailed in the document. α -Amylase (EC 3.2.1.1) hydrolyzes complex carbohydrates such as starch into oligosaccharides, which would be further hydrolyzed by α -glucosidase. This enzyme is secreted from the salivary and pancreatic glands. α-Glucosidase (EC 3.2.1.20) is a membrane-bound enzyme found in the epithelial mucosa of the small intestine (brush border of the enterocytes). It releases free glucose molecules from terminal, non-reducing (1-4)-linked α-glucose residues. Furthermore, food intake causes the release of intestinal hormones called incretins (gastric inhibitory polypeptide, GIP, and glucagon-like peptide-1, GLP-1). These two would affect numerous target tissues in the body, acting as endocrine signals to the pancreas, leading to insulin production in the β -cells and the suppression of the release of glucagon in the α -cells. These two incretins are responsible for ~70% of the insulin secretion after meal intake [ 31 ]. This results in the uptake of glucose by the muscles, as well as a lower production of glucose in the liver. The final consequence is therefore the decrease in blood glucose after ingestion, which allows the adequate regulation of postprandial blood glucose levels. At this level, the enzyme dipeptidyl peptidase IV (DPP-IV) regulates the degradation of incretins according to physiological needs. GLP-1 and GIP have half-lives of approximately 2 min and 5–7 min respectively, before they are degraded by DPP-IV [ 32 , 33 ]. DPP-IV is a cell surface (EC 3.4.14.5) that cleaves dipeptides from the N-terminus of polypeptides, in which proline is at the penultimate position [ 34 ]. DPP-IV can largely be found on the luminal surface of enterocytes; therefore, it can interact with any of the molecules from food intake before their absorption, that can be further metabolized before the molecules’ interaction with soluble and vascular endothelial DPP-IV Foods 2020,9, 983 5 of 33 (the one affecting GIP and GLP-1 levels). Gut hormones released from the enteroendocrine cells play an important role in food intake regulation [35]. Diabetes mellitus type 2 is one of the most prevalent diseases, affecting more than 400 million people and with estimations of 700 million people becoming affected by 2045 [ 36 ]. This metabolic disorder is characterized by insulin resistance, that is, the inability of the organism to react to the insulin action, or an insufficient production of this hormone. It is especially important to control the postprandial glucose level, because the long-term consequences of high glucose levels in the bloodstream are diverse, from renal failure to neurological damage and cardiovascular disorders [ 24 , 37 ]. Regarding the causes, both genetic and environmental factors take part in diabetes development. It is believed that the main cause is obesity, which operates through several pathways including an imbalance in the concentration of hormones, cytokines and other inflammatory signals [33]. 1.3. Diabetes Prevention Strategies Numerous strategies to manage postprandial hyperglycemia, and consequently prevent the development of type 2 diabetes, have been described [ 24 ]. Insulin injection [ 38 ] is the direct treatment for this disease, positively regulating the functioning of the organism. The main disadvantage is that insulin cannot be orally ingested. In addition to this, medications involved in the metabolic pathway of digestion are also options as regards preventing and treating the disease. A graphical simplification of the most important mechanisms involved in diabetes prevention is depicted in Figure 1. Food-derived peptides from food proteins play a crucial role in the regulation of glucose homeostasis, due to their implication at different levels (e.g., glucagon-like peptide 1 regulation) and due to their capacity to inhibit digestion-related enzymes. Furthermore, some authors have described peptides as being able to enhance cholecystokinin levels, a gut hormone regulating food intake [ 39 , 40 ]. Peptides and amino acids would have an effect on body fat loss, insulin secretion and glycaemia reduction, but further research is needed in order to unravel these mechanisms. Further information regarding the peripheral regulation of food intake can be found in the following references [ 35 , 41 ], including how protein digestion products act as signaling molecules in enteroendocrine cells. Regarding the digestion process and the enzymes involved in carbohydrate metabolism, the first approach to preventing an increase in glucose blood level is to avoid the degradation of polysaccharides into glucose. Therefore, the inhibition of digestive hydrolases (amylases, glucosidases) would avoid complex polysaccharides from becoming hydrolyzed, and thus absorbed in the bloodstream. Amylases inhibition can be exerted in the saliva and in the gastrointestinal tract, lowering the blood glucose level [ 32 ]. For its part, glucosidase inhibition would essentially preclude the uptake of glucose into the blood circulation, effectively decreasing postprandial hyperglycemia [ 24 , 42 , 43 ]. Delayed carbohydrate absorption is considered an adequate contributing factor in stimulating GLP-1 secretion, which would ultimately lead to the incretin effect. Among the α -glucosidase and α -amylase main inhibitors, we find acarbone, miglitol and voglibose. However, numerous side-effects, such as gastrointestinal disturbances, stomach pain and flatulence, have been described for these drugs, and consequently this have limited their use as inhibitors [ 24 ]. The obtaining of inhibitors for these enzymes with no side effects is consequently an interesting research topic. On the other hand, if the body suffers from insulin resistance, considering that DPP-IV acts by degrading incretins [ 44 ], one of the oral antidiabetic drugs used today is the group of DPP-IV enzyme inhibitors called gliptins [ 45 ]. The discovery that the enzyme DPP-IV inactivates more than 95% of GLP-1 has put it in the spotlight as a type 2 diabetes mellitus (T2DM) management therapy [ 46 ]. When DPP-IV is inhibited, the inhibitory action it has on incretins is suppressed, and the half-life of these incretins is increased. Protein intake can also elevate plasmatic GLP-1 levels [ 47 ]. This causes insulin secretion to be stimulated, in addition to inhibiting glucagon release [ 34 ], and the blood glucose level is adequately regulated. The first gliptin approved by the Food and Drug Administration (FDA) was sitagliptin, in 2006, and since then, more synthetic DPP-IV inhibitors have been approved, in spite of Foods 2020,9, 983 6 of 33 the adverse effects they may have. Among these, we can find nasopharyngitis, nausea, hypersensitivity, headache, skin irritations and the risk of acute pancreatitis [ 48 , 49 ]. Furthermore, their long-term safety remains unclear. Although the previous strategies described are the most important in terms of health and the research related to bioactive peptides, some other ways to prevent diabetes have been described. There are different kinds of molecules acting in different organs, which also have antidiabetic effects via different mechanisms, such as insulin sensitizers, insulin secretagogues, GLP-1 mimetics or glizofins [ 32 , 33 , 50 ]. There is a need for further research since some peptides are able to stimulate incretin secretion, this effect being related (or not) to the DPP-IV inhibition. Peptides are able to interact at many physiological levels in the human body [51]. Recent studies show the importance of diabetes pretreatment in minimizing the economic impact of the disease treatment [ 52 , 53 ], beyond the health consequences it has on the patient. Bioactive peptides appear to be a good alternative for employment in functional foods as health-promoting ingredients. In the literature, the discovery of peptides coming from food proteins able to inhibit amylases, glucosidases and DPP-IV has been reported. These kinds of enzyme-inhibitory peptides are still in the basic research stage, and none have been approved by the FDA [ 32 ]. Bioactive peptides can also regulate glucose homeostasis due to their ability to regulate gut hormones [ 35 ]. As such, bioactive peptides for preventing the development of diseases are an important field of research, the interest in which is increasing, and which could have positive effects on the human health and economic levels. 2. Production of Glycemic Index-Regulating Protein Hydrolysates 2.1. Enzymatic Hydrolysis Reaction Obtaining bioactive peptides from food proteins is preferably carried out by enzymatic hydrolysis rather than chemically, or via microbial fermentation. Chemical hydrolysis requires high temperatures and an extremely acidic or basic environment in order to destabilize the bond, and consequently, some amino acids are modified or even destroyed, meaning a loss in the nutritional value of the peptides. Microbial fermentation, to produce peptides, is not a reproducible technique, since there are some uncontrollable factors (i.e., enzyme levels, metabolism of microbes, etc.). However, genetic recombinant strains could help palliate these limitations [ 54 ]. In this review, we will focus on obtaining bioactive peptides via enzymatic hydrolysis. Enzymatic hydrolysis requires mild reaction conditions, and is specific and controllable. Thereactionitself issimple, needingthe substrate(protein)andthe enzyme(s)(protease(s)). The reaction conditions (pH and temperature) are determined by the protease, and many factors, such as enzyme/substrate ratio or substrate concentration, must be taken into consideration too. The optimal conditions for obtaining highly bioactive hydrolysates are usually achieved via different kinds of experimental designs [55–57]. The enzymatic hydrolysis is generally carried out in a jacketed reactor, under stirring, in order to ensure the homogeneity and constant temperature of the reaction. In terms of large-scale production, some authors have produced protein hydrolysates at a pilot or semi-pilot plant scale. In this context, different hydrolysates have been produced from such sources as fish discards [ 58 ] for their valorization, and trials have confirmed the results of production obtained at lab scale, or from boarfish [59], which show strong DPP-IV inhibitory activities. Lately, scientists have been testing enzymatic hydrolysis carried out after or during the application of non-thermal techniques, such as high pressure, ultrasound or microwave. The global conclusions are that high hydrostatic pressure and ultrasound pre-treatment improve the efficacy of enzymatic hydrolysis and the consequent release of bioactive peptides [ 60 – 63 ]. Regarding the protein structure, the tertiary and quaternary structures are generally affected by high-pressure treatment, while the secondary structures tend to be maintained. Nonetheless, we must consider the possibility that pressure treatment may lead to the denaturation of proteins, but also to aggregation or precipitation [ 64 ]. Foods 2020,9, 983 7 of 33 With respect to the primary structure, the application of pressure does not affect the covalent bonds, and so the sequences of amino acids are not lost [65,66]. The main parameter for characterizing protein hydrolysates is the degree of hydrolysis. This is defined as the proportion of cleaved peptide bonds compared to the original protein. The higher the degree of hydrolysis, the smaller the peptides size would be in the product obtained after the hydrolysis reaction. It is generally reported that bioactive peptides have a length of 2 to 20 amino acids. 2.2. Proteases The enzymes responsible for protein hydrolysis are called proteases (EC 3.4.X.X), and they can be classified via where they catalyze the hydrolysis of bonds. They can be considered (a) endopeptidases, if the cleavage site is inside the protein, or (b) exopeptidases, if the cleavage sites are located at or near the ends of chains. The active site of the protease determines its substrate specificity, that is, the position where the hydrolysis will take place [ 67 ]. Then, the choice of the protease employed is essential, since it will define the degree of hydrolysis and the profile of released peptides [68]. Endopeptidases can be classified depending on their catalytic mechanism and their tertiary structure, considering the amino acid or metal present in the active site, such as aspartate, cysteine, metallo or serine-proteases. Exopeptidases can be classified as aminopeptidases, carboxypeptidases or dipeptidases [ 3 , 69 ]. Some examples of proteases widely employed in the industry are Subtilisin, a non-specific endo-peptidase, exerting its proteolytic activity over hydrophobic amino acids [ 70 ], Trypsin, a specific endo-peptidase, exerting its proteolytic activity over arginine and lysine residues [ 71 ], and Flavourzyme, a complex mixture of endoand exo-peptidases, exerting its proteolytic activity over lineal chains, releasing small peptides and free amino acids [ 72 ]. Depending on the type of bioactive peptide desired, certain enzymes have been tested and considered as adequate proteases for obtaining these molecules. For example, a combination of Alcalase and Flavourzyme has been reported as a good enzymatic treatment for obtaining DPP-IV inhibitory peptides [ 73 , 74 ]. For α -glucosidase inhibitory peptides, trypsin has been reported as an adequate protease [ 75 ], and so has Alcalase [ 76 ]. Further research should be carried out, since different proteins might lead to different bioactive protein hydrolysates. 2.3. Protein Source The substrates usually employed for protein hydrolysis are of natural origin, usually with a high protein percentage. The most-studied protein substrates for obtaining antidiabetic peptides to date are milk [ 42 , 77 , 78 ] and soy proteins [ 34 , 79 ], due to their high biological value compared to other proteins. One such example is seen in Lacroix and Li-Chan [ 80 ], who described the formation of DPP-IV inhibitors from dairy proteins, using 11 enzymes and different substrates. Another valuable protein source is marine species. The literature reporting fish peptides with antidiabetic activity has been recently stated [ 59 , 81 – 83 ]. In this context, the use of fishing discards as protein sources for value-added products is important [ 55 , 59 , 83 , 84 ]. The scientific community considers enzymatic hydrolysis as a helpful option for revaluing these low-quality products and increasing their potential, as they have no side effects on the patient’s health. In addition, by-products of the food industry, such as whey or gluten, with adequate protein content are also possible options for generating value-added products. Recently, vegetable protein sources such as peas or lupine have also been used, given the lower ecological impact they have [ 85 ]. Similarly, insect or algae proteins are being used today in the food industry for the production of food products [ 86 , 87 ]. These sources of proteins are reported to be sustainable sources with great potential for use in the food industry. There is already literature concerning the production of DPP-IV inhibitory peptides from these kinds of sources, such as Palmaria Palmata and brewers’ spent grain [75,88]. The main differences among substrates are their protein structure complexities and their amino acid sequences. Table 1shows the amino acid profiles of different substrates considered as novel protein sources, such as insects. It can be observed that in some cases, the difference in the amount Foods 2020,9, 983 8 of 33 of a certain amino acid (g/100 g of substrate) is remarkable, and consequently the bioactivity related to the peptides released after hydrolysis is expected to be considerable. This statement is based on the fact that the bioactivity of peptides is mainly related to their amino acid sequence characteristics, that is, their hydrophobicity and/or length. The enormous diversity of substrates that are currently being used to obtain peptides with antidiabetic capacity is summarized in Table 2. It can be seen that marine, vegetable, insect or dairy sources are used, among others. The choice of the protein source used for the production of peptides must consider, in addition to the resulting bioactivity of the peptides, the environmental and economic factors during its production, via a life cycle assessment. Table 1. Amino acid content of some vegetable, insect and fish proteins (g/100 g of substrate). Amino Acid Quinoa Lentil Protein Isolate Brewer Spent Grain Mealworm Larvae Meal Silkworm Pupae Meal Mussel Meal Herring Essential H 2.2 2.0 3.6 2.9 2.6 1.9 2.1 I 0.8 3.1 4.2 4.7 5.1 4.5 3.3 L 2.5 6.9 7.2 8.0 7.5 7.2 7.9 K 2.3 5.6 3.1 6.3 7 8.3 10.1 M 0.3 0.6 1.4 1.4 3.5 2.6 3.3 F+Y 2.8 7.1 9.7 9.5 11.1 8.7 5.9 T 5.7 3.0 3.2 4.3 5.1 5.3 4.0 W 1.0 - 1.2 0.9 1.0 - V 1.0 3.5 6.0 8.5 5.5 4.6 4.6 C 0.1 0.5 1.4 0.8 1 1.0 1.1 Non-essential R 3.0 7.4 5.9 5.4 5.6 7.6 7.5 G 3.0 3.1 3.8 5.5 4.8 6.6 7.6 E 8.7 15.5 24.8 10.6 13.9 14.0 17.1 D 3.7 10.5 6.6 7.8 10.4 11.3 9.3 P 1.8 2.9 9.7 6.0 5.2 4.2 4.7 S 1.7 5.2 4.1 4.6 5.0 5.4 4.3 A 2.2 3.4 4.3 8.4 5.8 5.1 7.1 Ref. [89] [90] [91] [92] [93] [94] [95] A=alanine, R =arginine, D =aspartic acid, C =cysteine, E =glutamic acid, G =glycine, H =histidine, I =isoleucine, L=leucine, K =lysine, M =methionine, F =phenylalanine, P =proline, S =serine, T =threonine, W =tryptophan, Y=tyrosine, V =valine. Foods 2020,9, 983 9 of 33 Table 2. Summary of recent publications concerning antidiabetic bioactive peptides (from Scopus, 2018–2020). In Vitro Cellular Assay In Vivo Substrate Enzymatic Treatment ID Verification B-A DPP-IV GIA AMY Cell line Model Ref Rainbow trout (Oncorhynchus mykiss) Alcalase No No No Yes No No No No [30] Camel whey protein PTN 6.0S Yes Yes Yes Yes No No No No [57] Boarfish (Capros aper)Alcalase 2.4 L, Flavourzyme 500 L; Simulated digestion Yes Yes No Yes No No Caco-2; BRIN-BD11 No [59] Blue whiting (Micromesistius poutassou)Alcalase 2.4 L and Flavourzyme 500 L Simulated digestion Yes No No Yes No No BRIN-BD11, GLUTag, 3T3-L1 NIH Swiss mice [81] Cricket (G. sigillatus)Alcalase Simulated digestion No No No Yes No No No No [96] Soybean (Glycine max) Simulated digestion Yes No No Yes Yes Yes No No [97] Luffa cylindrical seed Alcalase, trypsin No No No No Yes Yes No No [76] Salmon (Salmo salar)Alcalase 2.4 L, Alcalase 2.4 L and Flavourzyme 500 L, and Promod 144 MG Yes No No Yes No No BRIN-BD11; GLUTag No [73] Boarfish (Capros aper)Alcalase 2.4 L, Flavourzyme 500 L Simulated digestion No No No Yes No No Caco-2; BRIN-BD11; GLUTag; 3T3-L1 Mice [98] Mealworm (Tenebrio molitor) Alcalase, trypsin, ficin, flavourzyme No No No Yes No No No No [74] Tropical banded crickets (Gryllodes sigillatus) Protamex Simulated digestion No No No Yes No No No No [99] Hempseed (Cannabis sativa) Pepsin, trypsin No No No Yes No No Yes Ex-vivo [100] Bovine whey Trypsin Yes Yes No Yes No No No No [101] Sea cucumber (Stichopus japonicus) Simulated gastrointestinal digestion Yes No Yes Yes No No 3T3-L1, HepG2 No [102] Casein PROTIN SD-NY10 No No No No Yes Yes No No [103] Walnut (Juglans mandshurica) Alcalase 2.4 L Yes Yes Yes No Yes Yes HepG2 cells No [104] Mealworm (T. molitor), locust (Schistocerca gregaria), cricket (G. sigilatus)Simulated digestion Yes Yes No No Yes No No No [105] Corn germ Alcalase, flavourzyme, trypsin No No No Yes Yes Yes No No [106] Millet grains (Panicum miliaceum) Simulated digestion Yes No No No Yes Yes No No [107] Cowpea (Vigna unguiculata) Alcalase +Flavourzyme No No No Yes Yes Yes No No [108] Beans (Phaseolus vulgaris) Simulated digestion No No No No Yes Yes No Male wistar rats [109] Basil seeds (Ocimum tenuriflorum) Pepsin No No No No No Yes No No [110] Brewers’ spent grain Alcalase +Flavourzme Simulated digestion Yes Yes No Yes No No No No [111] Porphyra dioica extracted protein Alcalase +Flavourzyme Yes Yes No Yes No No No No [112] Red Seaweed (Porphyra spp) Alcalase, neutrase, pepsin, and trypsin Yes Yes No No No Yes No No [113] Soybean (Glycine max) Trypsin Yes Yes Yes No Yes No No Mice [114] Foods 2020,9, 983 16 of 33 The stability of peptides can be improved by different techniques. Gianfranceschi et al. [ 185 ] reviewed the biochemical peculiarities that can enhance the nutraceutical functionality of peptides, that is, their ability to be actually bioactive at their active site. Some techniques for the chemical modification of amino acids would prevent them from being digested by proteases, and consequently, peptide structure would be maintained, and the peptides would be expected to exert their physiological activity. Beyond that, trapping peptides inside different matrices increases their bioavailability too. For example, chitosan is a polymer able to increase the paracellular permeability of peptide drugs across mucosal epithelia [ 186 ]. Research on peptide absorption lacks studies regarding the influence of food matrices. It is important to investigate the influence of coexisting food components on the absorption of food-derived peptides. Harnedy-Rothwell et al. [ 187 ] subjected DPP-IV inhibitory peptides to simulated digestion in different matrices (tomato soup and juice), and verified that bioactivity was conserved. Different food matrices influenced protein and peptide digestibility during gastrointestinal digestion and absorption, so this must be considered a major factor in characterizing the bioaccessibility and bioavailability of peptides [174,188]. Unravelling the mechanisms that explain how nutrients might have physiological effects on the human body would allow to design or optimize the production of molecules with adequate molecular features for enhancing the bioactive properties of the ingredients [ 41 ]. In this sense, protein hydrolysates, sometimes poorly characterized, might have different bioactivities with synergistic effects responsible for the antidiabetic effect that they exert on humans. Related research to be remarked upon includes the observation of a reduction in the gastrointestinal hydrolysis of a peanut protein isolate in the presence of polysaccharides, which is suggested to be due to the non-specific interactions between the polysaccharides and the peptides [ 189 ], or the reporting that almond flour inside a chocolate mousse and a sponge cake reduces protein degradation by pepsin [ 190 ]. In this regard, the effects of sugar-containing matrices could lead to Maillard product formation, and this would have an effect on the digestibility of proteins, since some amino acids are destroyed [191,192]. 5. Stability and Functionality in Food Matrices The food processing operations currently employed in the industry include thermal treatments (sterilization, pasteurization), non-thermal treatments (high-pressure homogenization or processing, ultrasound), storage (freezing and frozen), drying (dehydration, spray drying, freeze-drying) and separation (membrane processes). Some of these processes may well affect food protein functionality, due to physical and chemical changes. Proteins and peptides are prone to interact between one another, and with other molecules. The processing of food products containing proteins and peptides could, in consequence, reduce, maintain or enhance their bioactivity [ 54 ]. The amino acid residues would interact with molecules in different ways, also depending on the location of the peptides in the food matrix, ultimately affecting their native and denatured polymeric state [ 193 , 194 ]. It has been reported that high-fiber food matrices are adequate to carry these bioactive peptides, because chemical interactions are not likely to occur. A fiber network would avoid the aforementioned bitter taste of hydrophobic peptides, improving the sensorial acceptability of functional foods, including peptides [188,195]. There are not too many studies on how food processing and/or storage modify peptide structure, and consequently their functionality and bioactive properties. Graves et al. [ 196 ] analyzed the bioactivity of a rice bran peptide described as anti-cancer, during its 6-month storage inside an orange juice. Contreras et al. [ 197 ] reported some antihypertensive peptides’ resistance to atomization, homogenization and pasteurization, plus their retained bioactivity after incorporation into liquid yoghurt. Similar results concerning antioxidant and antihypertensive peptides’ resistance to food processing techniques were reported by Rivero-Pino et al. [198]. As aforementioned, some authors have reported that non-thermal treatments, such as ultrasound or high pressures, enhance protein enzymatic hydrolysis. However, there is a lack of information Foods 2020,9, 983 17 of 33 regarding how these processing techniques would affect peptides employed as ingredients in food formulation. These techniques are nowadays seen as less aggressive in terms of nutritional loss of ingredients [ 199 ], and are potentially employed for commercial sterilization or emulsifying processes. Non-thermal processing technologies have been described to produce hypoallergenic foods due to structural epitopes changes [ 200 ], same as enzymatic hydrolysis [ 201 ]. The food industry can take advantage of this knowledge to fabricate hypoallergenic products without heat treatments. However, food processing may also affect amino acid composition, by forming derivatives such as lysinoalanine, d-amino acids and biogenic amines, which are usually related to undesired physiological consequences in the human body if consumed [202]. Hydrophobic amino acids tend to be more stable [203,204]. The consequences of known high-pressure treatments mainly affect the protein structure, leading to denaturation, aggregation or precipitation [ 64 ]. The effect must be studied for each case, considering the fact that the residue characteristics of peptides would be crucial in determining the result. Ultrasound reduces the size and hydrodynamic volume of the proteins, leading to better physical-chemical and emulsifying properties [ 205 ]. Drying processes improve the stability of products, extending the shelf-life of products by reducing water activity [206]. Another important example of chemical reaction is the formation of Maillard compounds, products of the non-enzymatic glycosylation of proteins. Sugar is a widely employed ingredient in the food industry due to its sweet flavor. The combination of reduced sugar with proteins or peptides at a high temperature leads to the formation of these compounds [ 207 ], affecting oxidative stability [ 208 ] and improving the antihypertensive or antioxidant bioactivities of protein hydrolysates [ 198 , 209 – 212 ]. Nonetheless, to the best of our knowledge, there is no literature reporting the increased DPP-IV inhibitory activity of Maillard reaction products coming from protein hydrolysates. There are some techniques to avoid or slow down the effect of the digestion process, and to increase the peptides’ stability when introduced into food matrices. The most widely employed technique is encapsulation [213] with polymers or hydrogels [100]. Many factors are also involved in the potential loss or gain of bioactivity via the modification of peptides’ structure, or the aggregation of them. The state of the protein determines its properties [ 214 ], but the primary structure is not affected by the denaturation caused by most physical processes, [ 201 ], whereas in a more complex aggregation can occur. It is expected that protein hydrolysates, as a mixture of defined peptides, would not suffer further modifications, since the linear sequences are affected by sequence decomposition processes, such as hydrolysis itself or fermentation. The heterogeneous chemical composition of a food, as well as its molecular structure, is related to different chemical reaction behaviors [215] and, in consequence, its functionality. Once the product containing peptides is formulated and its bioactivity maintained, it should also be ensured that bioactivity is not lost during its life as a commercial product. Chemical reactions might occur during the storage, depending on the formulation of the product and the temperature of it. The Maillard reaction has been described to occur at high temperatures, but long periods might lead to the appearance of Maillard reaction products too. Guyomarc’h et al. [ 216 ] reported the occurrence of the Maillard reaction within refrigerator-stored milk powder at 4 ◦ C, whereas Albal á -Hutado et al. [ 217 ] reported it in liquid infant’s milk at room temperature. Recently, Harnedy-Rothwell et al. [ 187 ] fortified different food products (tomato-based soup and juice products) that were subjected to thermal treatments (sterilization and pasteurization) and stored at refrigerated temperature for 30 days. No modification of bioactivity was reported, indicating this treatment’s potential use on foods that could contain the bioactive protein hydrolysates. Furthermore, peptides and proteins might tend to aggregate or precipitate over time, due to some other interactions, such as the van de Waals interaction, hydrogen bonding or a hydrophobic interaction. Hence, when considering the use of a protein hydrolysate as a bioactive ingredient, its stability during the food formulation, and its stability during storage, should be established. Foods 2020,9, 983 18 of 33 6. Bioactivity Analysis 6.1. Bioactivity Initial Approaches Nowadays, considering the novelty of the research subject, literature concerning in vivo analysis with animals and humans is extremely highly needed, but unfortunately, also scarce. Evidently, this research point is the most important, and is the one that offers authentic evidence concerning the implementation of these bioactive peptides as nutraceutical ingredients. The formulation of foods with legal claims to being a glycemic index-regulator due to the presence of these bioactive peptides would be the final step. For this purpose, plenty of evidence and verification in humans is required. The literature currently available on protein hydrolysates and bioactive peptides focusses mainly on in vitro analysis. In this regard, for the antidiabetic analysis, different analyses can be carried out, concerning the different metabolic routes involved in the disease. The most reported bioactive peptides with antidiabetic properties are those with amylases, glucosidases and DPP-IV inhibitory properties. Concerning cell assays, among the cell lines generally used (Table 2) for the evaluation of the functionality of antidiabetic peptides, we found: - BRIN-BD11: insulin-secreting cells (pancreatic B cells) in response to glucose, to analyze the effect of the compounds on insulin secretion [218]; - GLUTag: enteroendocrine cells that allow the secretion of GLP-1 (intestinal hormone regulated by the DPP-IV enzyme) to be measured using the ELISA technique [219]; - 3T3-L1: adipocyte cells that allow the measurement of glucose absorption by fluorimetry [220]; - STC-1: intestinal secretin tumor cell line that expresses and secretes gut hormones in response to physiological stimuli [221]. Different studies have employed these cellular lines in exploring DPP-IV inhibitory peptides, as can be observed in Table 2. For example, Harney et al. [ 81 ] showed that a blue whiting hydrolysate mediated insulin and glucagon-like peptide-1 (GLP-1) release from BRIN-BD11 and GLUTag cells, respectively, and Li et al. [ 122 ] observed the inhibition by a spirulina hydrolysate of the DPP-IV activity expressed by Caco-2 cells. Nonetheless, in vitro , in situ and ex vivo approaches are not enough for the scientific community to establish claims about the functionality of food peptides. 6.2. In Vivo Analysis In vivo analysis should be carried out to verify effectiveness, and to establish the required dose that should be consumed for the protein hydrolysate to effectively exert its biological activity. In these analyses, different markers are evaluated that indicate the physiological influence that these hydrolysates have on the subject [ 145 , 222 ]. In these investigations, model organisms, such as cell cultures or experimental animals, are used, while clinical studies in humans are less frequent. The results published so far are promising, since they show that, indeed, these protein hydrolysates have beneficial properties for the organism. 6.2.1. Invertebrates Models The use of Caenorhabdtis elegans as the model organism [ 223 ] in examining the functionality of bioactive peptides is not extensively reported in literature. Wang et al. [ 224 ] and Zhou et al. [ 225 ] reported a delay in senescence and stress resistance, and lifespan extension, respectively, through the antioxidant activities of bioactive peptides from Angelica sinensis protein and mussels (Mytilus edulis). Focusing on the antidiabetic activity of peptides, Zhu et al. [ 226 ] proposed an integrated microfluidic device, that resembles the hyperglycemic condition in diabetics, using this nematode as a model, and thereby investigated the responses after exposition to continuously high glucose concentrations in a physiologically relevant manner. These first approaches suggest that this easy-to-work nematode could also be employed [227]. Foods 2020,9, 983 19 of 33 Another model organism employed in the research is Drosophila [ 228 ]. In the same way, scarce information is available in the bioactive peptides field. Chen et al. [ 229 ] reported the up-regulation of antioxidant-related genes, a prolonged lifespan and the reduction of the accumulation of peroxide products when feeding the animal with crimson snapper scale peptides. To the author’s knowledge, no studies have been published concerning the antidiabetic properties of food-derived peptides in Drosophila. 6.2.2. Vertebrates Models The easiest animal models to work with in vivo are rats and mice. In these assays, different biological parameters are measured. In the case of antihypertensive peptides, blood pressure and plasma ACE and renin concentrations are measured [ 230 ]. Animal models of type 2 diabetes usually reflect insulin resistance and/or beta cell failure. Furthermore, many of them are obese, reflecting the human condition, wherein obesity is closely linked to type 2 diabetes development [ 231 ]. This latter author summarizes numerous examples of these animal models as related to diabetes. In regard to the literature dealing with this topic, Harnedy et al. [ 81 ] and Parthsarathy et al. [ 98 ] reported a protein hydrolysate from blue whiting and boarfish with in vitro and in vivo antidiabetic properties, using cell cultures and mice. Similar research was carried out by Jung et al. [ 232 ] with silk fibroin hydrolysate, and by Hsieh et al. [ 162 ] with milk proteins. Mochida et al. [ 233 ] reported that zein-derived peptides induced glycemic regulation via GLP-1 secretion, and DPP-IV inhibition in rats, whereas Ishikawa et al. [ 234 ] obtained similar results by employing rice-derived peptides. Valencia-Mej í a et al. [ 109 ] studied the antihyperglycemic and hypoglycemic activity of naturally occurring peptides and protein hydrolysates from beans in male Wistar rats. D’Souza et al. [ 235 ] introduced an α -amylase inhibitor peptide into Lactococcus lactis, a bacteria usually employed to produce a yogurt, and diabetic mice fed with it showed a reduction in blood glucose levels after 20 days. Along the same line, Wang et al. [ 236 ] improved glycemic control in diabetic rats vis administration of fish skin gelatin hydrolysates, with a DPP-IV inhibitory capacity and a GLP-1 stimulation capacity. Drotningsvik et al. [ 237 ] showed that fish protein hydrolysates could affect different metabolic parameters, such as postprandial glucose regulation and lipid metabolism in obese Zucker rats. Swinehavebeenusedasamodelduetotheirsimilaritiestohumanspecies. Wesharecardiovascular anatomies and functions, metabolisms, lipoprotein profiles, tendencies to obesity, etc., making swine adequate for testing the functionality of molecules altering metabolism [ 238 ]. The use of swine as an animal model for diabetes is stated [ 239 , 240 ]. However, studies wherein peptides are included in their diets do not focus on the bioactive effects on these features, but on the palatability or feed efficiency, in order to improve their nutritional status and gut function [177]. 6.2.3. Humans Finally, the authentic evidence that bioactive peptides are adequate for employment in the food industry as nutraceuticals must overcome the clinical analysis carried out in humans. Peptides have extensive applications in medicine nowadays. The Food and Drug Administration (FDA) has approved more than 60 peptide drugs for marketing, and thousands of preclinical studies are being carried out for numerous peptides [ 241 ]. Concerning the regulatory requirements of protein hydrolysates from food proteins, different countries have developed different protocols to approve them as health-promoting ingredients [ 242 ]. For instance, at the European level, the European Food Safety Authority approved some angiotensin-converting enzyme inhibitory peptide products as a functional food ingredient, but no glycemic index-regulator peptides have been approved so far. Concerning the bioactive peptides from food protein hydrolysates, studies are mainly carried out by employing dairy or fish proteins hydrolysates, since these are the most studied ones. Focusing on dairy proteins, the large amount of proline residues in casein makes this protein exceptional for the production of DPP-IV inhibitory peptides [ 24 ]. There are numerous studies reporting the efficacy of Foods 2020,9, 983 20 of 33 casein protein hydrolysates in humans, as a pretreatment for diabetes [ 145 , 243 – 247 ], which involve the observing of different parameters related to an adequate regulation of glucose blood level in type 2 diabetes patients. Recently, Saleh et al. [ 248 ] studied the effect of casein protein hydrolysate (a twice-daily dose of 8.5 g) in patients with gestational diabetes, concluding a moderate reduction of plasma glucose levels, suggesting the potential functionality of protein hydrolysate in the prevention of diabetes. Along the same line, whey [ 249 – 252 ] and egg [ 253 ] protein hydrolysates have been proven to have a positive effect on postprandial blood glucose, both in T2DM subjects and in healthy subjects. Calbet and Holst [ 254 ] reported that milk protein hydrolysates elicited about 50% more gastric secretion than the native protein, plus higher GIP plasma levels during the first 20 min of the gastric emptying process. On the other hand, fish proteins are also seen as an adequate protein source [ 255 ]. Along this line, Hovland et al. [ 256 ] showed the effectiveness of milk and different fish protein hydrolysates (2.5 g/day of proteins) in affecting glucose regulation and acting as markers of insulin sensitivity in overweight adults, in a randomized, double blind study. Fish species, such as cod [ 257 ] or boarfish, proteins [ 258 ] have also been employed in human studies concerning diabetes prevention. However, Curran et al. [ 259 ] showed the need for further precise nutrition analysis, after showing the ability of a casein hydrolysate to improve glycemic function only in some of the individuals analyzed. All the aforementioned evidence shows that the enzymatic hydrolysis of food proteins is an adequate methodology for obtaining a mixture of peptides that are potentially bioactive. Historically, casein protein is the most widely studied protein, and there are currently food products including it as an ingredient. However, this ingredient in food products is not stated as a bioactive compound, but as a nutritionally improved protein. For example, Arla Foods Ingredients offers a range of whey protein hydrolysates, described as being more quickly absorbed into the blood, and Abbott declare that collagen protein hydrolysate, in their Promod ® Liquid Protein, helps to improve pressure ulcer healing [ 260 ]. Concerning antidiabetic hydrolysates, Nutripeptin TM by Copalis Sea Solutions ® is described as a glycemic index-reducing peptide extracted by enzymatic hydrolysis from fresh or fresh-frozen fillets of codfish. Protein hydrolysates’ functionalities as ingredients are currently an important topic. The biggest drawback concerning the bioactive properties of protein hydrolysates would be that the in vivo results show differences among individuals. Metabotyping individuals is an important step when considering which subgroups of people could benefit from protein hydrolysates as a functional food [261]. 7. Conclusions The available literature on bioactive peptides highlights their relevance to nutrition. The potential of bioactive peptides as antidiabetic agents to be employed in food formulation is a relevant field of research. A protein hydrolysate is a source of peptides capable of modulating different physiological processes. The choice of the protein source is essential, considering not only the bioactivity of peptides but also the environmental, economic and social factors. Then, its potential use as an ingredient must include the evaluation of its stability during storage, and its sensory properties via technical studies. Furthermore, there is a need to validate the antidiabetic properties of food-derived peptides through well-designed clinical trials with cellular assays, in animals and humans, to ensure their effectiveness and safety. These studies would describe the actual activity of protein hydrolysates with the purpose of being commercially developed in the food industry for functional feeding. Funding: This research was funded by SPANISH MINISTRY OF SCIENCE, INNOVATION AND UNIVERSITIES and the RESEARCH GROUP BIO-110 from University of Granada, project CTQ2017-87076-R. Conflicts of Interest: The authors declare no conflict of interest. Foods 2020,9, 983 21 of 33 References 1. Nelson, D.L.; Cox, M.M. Lehninger: Principles of Biochemistry; W H Freeman & Co: New York, NY, USA, 2008; ISBN 9788578110796. 2. Möller, N.P.; Scholz-Ahrens, K.E.; Roos, N.; Schrezenmeir, J. Bioactive peptides and proteins from foods: Indication for health effects. Eur. J. Nutr. 2008,47, 171–182. [CrossRef] 3. McDonald,J.K.Anoverviewofproteasespecificityandcatalyticmechanisms: Aspectsrelatedtonomenclature and classification. Histochem. J. 1985,17, 773–785. [CrossRef] [PubMed] 4. Adler-Nissen, J. Enzymic Hydrolysis of Food Proteins; Elsevier Applied Science Publishers: London, UK, 1986; ISBN 0-85334-386-1. 5. Wouters, A.G.B.; Rombouts, I.; Fierens, E.; Brijs, K.; Delcour, J.A. Relevance of the Functional Properties of Enzymatic Plant Protein Hydrolysates in Food Systems. Compr. Rev. Food Sci. Food Saf. 2016 ,15, 786–800. [CrossRef] 6. Klompong, V.; Benjakul, S.; Kantachote, D.; Shahidi, F. Antioxidative activity and functional properties of protein hydrolysate of yellow stripe trevally (Selaroides leptolepis) as influenced by the degree of hydrolysis and enzyme type. Food Chem. 2007,102, 1317–1327. [CrossRef] 7. Kristinsson, H.G.; Rasco, B.A. Fish protein hydrolysates: Production, biochemical, and functional properties. Crit. Rev. Food Sci. Nutr. 2000,40, 43–81. [CrossRef] [PubMed] 8. Damodaran, S.; Parkin, K. Fennema’s Food Chemistry; CRC Press: Boca Raton, FL, USA, 2017. 9. Genot, C.; Kabri, T.H.; Meynier, A. Stabilization of omega-3 oils and enriched foods using emulsifiers. In Food Enrichment with Omega-3 Fatty Acids; Woodhead Publishing: Cambridge, UK, 2013; pp. 151–193. 10. Lam, R.S.H.; Nickerson, M.T. Food proteins: A review on their emulsifying properties using a structure–function approach. Food Chem. 2013,141, 975–984. [CrossRef] 11. Hall, F.G.; Jones, O.G.; O’Haire, M.E.; Liceaga, A.M. Functional properties of tropical banded cricket (Gryllodes sigillatus) protein hydrolysates. Food Chem. 2017,224, 414–422. [CrossRef] 12. Zayas, J.F. Water Holding Capacity of Proteins. In Functionality of Proteins in Food; Springer: Berlin/Heidelberg, Germany, 1997; pp. 76–133. 13. Zieli´nska, E.; Kara´s, M.; Baraniak, B. Comparison of functional properties of edible insects and protein preparations thereof. LWT 2018,91, 168–174. [CrossRef] 14. Elias, R.J.; Kellerby, S.S.; Decker, E.A. Antioxidant activity of proteins and peptides. Crit. Rev. Food Sci. Nutr. 2008,48, 430–441. [CrossRef] 15. Aluko, R.E. Amino Acids, Peptides, and Proteins as Antioxidants for Food Preservation; Elsevier Ltd.: Amsterdam, The Netherlands, 2015; ISBN 9781782420972. 16. Padial-Dom í nguez, M.; Espejo-Carpio, F.J.; Garc í a-Moreno, P.J.; Jacobsen, C.; Guadix, E.M. Protein derived emulsifiers with antioxidant activity for stabilization of omega-3 emulsions. Food Chem. 2020 ,329, 127148. [CrossRef] 17. Morales-Medina, R.; Tamm, F.; Guadix, A.M.; Guadix, E.M.; Drusch, S. Functional and antioxidant properties of hydrolysates of sardine (S. pilchardus) and horse mackerel (T. mediterraneus) for the microencapsulation of fish oil by spray-drying. Food Chem. 2016,194, 1208–1216. [CrossRef] [PubMed] 18. Lin, C.C.; Liang, J.H. Effect of antioxidants on the oxidative stability of chicken breast meat in a dispersion system. J. Food Sci. 2002,67, 530–533. [CrossRef] 19. Hannu, K.; Pihlanto, A. Bioactive peptides: Production and functionality. Int. Dairy J. 2006 ,16, 945–960. [CrossRef] 20. Acquah, C.; Di Stefano, E.; Udenigwe, C.C. Role of hydrophobicity in food peptide functionality and bioactivity. J. Food Bioact. 2018,4, 88–98. [CrossRef] 21. Garc í a-Moreno, P.J.; P é rez-G á lvez, R.; Espejo-Carpio, F.J.; Ruiz-Quesada, C.; P é rez-Morilla, A.I.; Mart í nez-Agust í n, O.; Guadix, A.; Guadix, E.M. Functional, bioactive and antigenicity properties of blue whiting protein hydrolysates: Effect of enzymatic treatment and degree of hydrolysis. J. Sci. Food Agric. 2017,97, 299–308. [CrossRef] 22. FitzGerald, R.J.; O’Cuinn, G. Enzymatic debittering of food protein hydrolysates. Biotechnol. Adv. 2006 ,24, 234–237. [CrossRef] 23. Li, R.; Zhang, P.; Barker, L.E.; Chowdhury, F.M.; Zhang, X. Cost-effectiveness of interventions to prevent and control diabetes mellitus: A systematic review. Diabetes Care 2010,33, 1872–1894. [CrossRef] Foods 2020,9, 983 22 of 33 24. Patil, P.; Mandal, S.; Tomar, S.K.; Anand, S. Food protein-derived bioactive peptides in management of type 2 diabetes. Eur. J. Nutr. 2015,54, 863–880. [CrossRef] 25. Skeggs, L.T.; Kahn, H.R.; Shumway, N.P. The preparation and function of the hypertension-converting enzyme. J. Exp. Med. 1956,103, 295–299. [CrossRef] 26. Mizuno, S.; Matsuura, K.; Gotou, T.; Nishimura, S.; Kajimoto, O.; Yabune, M.; Kajimoto, Y.; Yamamoto, N. Antihypertensive effect of casein hydrolysate in a placebo-controlled study in subjects with high-normal blood pressure and mild hypertension. Br. J. Nutr. 2005,94, 84–91. [CrossRef] 27. Hajieva, P.; Behl, C. Antioxidants as a potential therapy against age-related neurodegenerative diseases: Amyloid Beta toxicity and Alzheimer’s disease. Curr. Pharm. Des. 2006,12, 699–704. [CrossRef] 28. Suleria, H.A.R.; Masci, P.P.; Addepalli, R.; Chen, W.; Gobe, G.C.; Osborne, S.A. In vitro anti-thrombotic and anti-coagulant properties of blacklip abalone (Haliotis rubra) viscera hydrolysate. Anal. Bioanal. Chem. 2017 , 409, 4195–4205. [CrossRef] [PubMed] 29. Meram, C.; Wu, J. Anti-inflammatory effects of egg yolk livetins ( α , β , and γ -livetin) fraction and its enzymatic hydrolysates in lipopolysaccharide-induced RAW 264.7 macrophages. Food Res. Int. 2017 ,100, 449–459. [CrossRef] [PubMed] 30. Ketnawa, S.; Suwal, S.; Huang, J.-Y.; Liceaga, A.M. Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates. Int. J. Food Sci. Technol. 2018, 1–12. [CrossRef] 31. Nauck, M.A.; Baller, B.; Meier, J.J. Gastric inhibitory polypeptide and glucagon-like peptide-1 in the pathogenesis of type 2 diabetes. Diabetes 2004,53. [CrossRef] 32. Yan, J.; Zhao, J.; Yang, R.; Zhao, W. Bioactive peptides with antidiabetic properties: A review. Int. J. Food Sci. Technol. 2019,54, 1909–1919. [CrossRef] 33. Tahrani, A.A.; Bailey, C.J.; Del Prato, S.; Barnett, A.H. Management of type 2 diabetes: New and future developments in treatment. Lancet 2011,378, 182–197. [CrossRef] 34. Lammi, C.; Bollati, C.; Ferruzza, S.; Ranaldi, G.; Sambuy, Y.; Arnoldi, A. Soybean-and lupin-derived peptides inhibit DPP-IV activity on in situ human intestinal Caco-2 cells and ex vivo human serum. Nutrients 2018 ,10, 1082. [CrossRef] 35. Caron, J.; Domenger, D.; Dhulster, P.; Ravallec, R.; Cudennec, B. Protein digestion-derived peptides and the peripheral regulation of food intake. Front. Endocrinol. (Lausanne) 2017,8. [CrossRef] 36. International Diabetes Federation. Diabetes Atlas de la FID; International Diabetes Federation: Brussels, Belgium, 2017; Volume 8, ISBN 2-930229-80-2. 37. Li, L.; Hölscher, C. Common pathological processes in Alzheimer disease and type 2 diabetes: A review. Brain Res. Rev. 2007,56, 384–402. [CrossRef] 38. Howard-Thompson, A.; Khan, M.; Jones, M.; George, C.M. Type 2 Diabetes Mellitus: Outpatient Insulin Management. Am. Fam. Physician 2018,97, 29–37. [PubMed] 39. Sufian,M.K.N.B.; Hira,T.; Miyashita,K.; Nishi,T.; Asano,K.; Hara,H.Porkpeptonestimulatescholecystokinin secretion from enteroendocrine cells and suppresses appetite in rats. Biosci. Biotechnol. Biochem. 2006 ,70, 1869–1874. [CrossRef] [PubMed] 40. Nishi, T.; Hara, H.; Asano, K.; Tomita, F. The soybean β -conglycinin β 51-63 fragment suppresses appetite by stimulating cholecystokinin release in rats. Biochem. Mol. Actions Nutr. 2003 ,133, 2537–2542. [CrossRef] [PubMed] 41. Santos-Hern á ndez, M.; Miralles, B.; Amigo, L.; Recio, I. Intestinal Signaling of Proteins and Digestion-Derived Products Relevant to Satiety. J. Agric. Food Chem. 2018,66, 10123–10131. [CrossRef] [PubMed] 42. Konrad, B.; Anna, D.; Marek, S.; Marta, P.; Aleksandra, Z.; J ó zefa, C. The evaluation of dipeptidyl peptidase (DPP)-IV, α -glucosidase and angiotensin converting enzyme (ACE) inhibitory activities of whey proteins hydrolyzed with serine protease isolated from asian pumpkin (Cucurbita ficifolia). Int. J. Pept. Res. Ther. 2014 , 20, 483–491. [CrossRef] [PubMed] 43. Ibrahim, M.A.; Bester, M.J.; Neitz, A.W.H.; Gaspar, A.R.M. Structural properties of bioactive peptides with α-glucosidase inhibitory activity. Chem. Biol. Drug Des. 2017,91, 370–379. [CrossRef] [PubMed] 44. Kshirsagar, A.D.; Aggarwal, A.S.; Harle, U.N.; Deshpande, A.D. DPP IV inhibitors: Successes, failures and future prospects. Diabetes Metab. Syndr. Clin. Res. Rev. 2011,5, 105–112. [CrossRef] [PubMed] 45. Marya; Khan, H.; Nabavi, S.M.; Habtemariam, S. Anti-diabetic potential of peptides: Future prospects as therapeutic agents. Life Sci. 2018,193, 153–158. [CrossRef] Foods 2020,9, 983 23 of 33 46. Thoma, R.; Löffler, B.; Stihle, M.; Huber, W.; Ruf, A.; Hennig, M. Structural Basis of Proline-Specific Exopeptidase Activity as Observed in Human Dipeptidyl Peptidase-IV. Structure 2003 ,11, 947–959. [CrossRef] 47. Hutchison, A.T.; Feinle-Bisset, C.; Fitzgerald, P.C.E.; Standfield, S.; Horowitz, M.; Clifton, P.M.; Luscombe-Marsh, N.D. Comparative effects of intraduodenal whey protein hydrolysate on antropyloroduodenal motility, gut hormones, glycemia, appetite, and energy intake in lean and obese men. Am. J. Clin. Nutr. 2015,102, 1323–1331. [CrossRef] 48. Liu, R.; Cheng, J.; Wu, H. Discovery of Food-Derived Dipeptidyl Peptidase IV Inhibitory Peptides: A Review. Int. J. Mol. Sci. 2019,20, 463. [CrossRef] [PubMed] 49. Juillerat-Jeanneret, L. Dipeptidyl peptidase IV and its inhibitors: Therapeutics for type 2 diabetes and what else? J. Med. Chem. 2014,57, 2197–2212. [CrossRef] [PubMed] 50. Hsia, D.S.; Grove, O.; Cefalu, W.T. An Update on SGLT2 Inhibitors for the Treatment of Diabetes Mellitus. Curr. Opin. Endocrinol. Diabetes Obes. 2017,24, 73–79. [CrossRef] [PubMed] 51. Caron, J.; Cudennec, B.; Domenger, D.; Belguesmia, Y.; Flahaut, C.; Kouach, M.; Lesage, J.; Goossens, J.F.; Dhulster, P.; Ravallec, R. Simulated GI digestion of dietary protein: Release of new bioactive peptides involved in gut hormone secretion. Food Res. Int. 2016,89, 382–390. [CrossRef] [PubMed] 52. Mata-Cases, M.; Rodr í guez-S á nchez, B.; Mauricio, D.; Real, J.; Vlacho, B.; Franch-Nadal, J.; Oliva, J. The association between poor glycemic control and health care costs in people with diabetes: A population-based study. Diabetes Care 2020,43, 751–758. [CrossRef] [PubMed] 53. Hewage, S.S.; Wu, S.; Neelakantan, N.; Yoong, J. Systematic review of effectiveness and cost-effectiveness of lifestyle interventions to improve clinical diabetes outcome measures in women with a history of GDM. Clin. Nutr. ESPEN 2020,35, 20–29. [CrossRef] 54. Daliri, E.; Oh, D.; Lee, B. Bioactive Peptides. Foods 2017,6, 32. [CrossRef] 55. P é rez-G á lvez, R.; Morales-Medina, R.; Espejo-Carpio, F.J.; Guadix, A.; Guadix, E.M. Modelling of the production of ACE inhibitory hydrolysates of horse mackerel using proteases mixtures. Food Funct. 2016 ,7, 3890–3901. [CrossRef] 56. Espejo-Carpio, F.J.; P é rez-G á lvez, R.; Guadix, A.; Guadix, E.M. Artificial neuronal networks (ANN) to model the hydrolysis of goat milk protein by subtilisin and trypsin. J. Dairy Res. 2018,85, 339–346. [CrossRef] 57. Nongonierma, A.B.; Cadamuro, C.; Le Gouic, A.; Mudgil, P.; Maqsood, S.; FitzGerald, R.J. Dipeptidyl peptidase IV (DPP-IV) inhibitory properties of a camel whey protein enriched hydrolysate preparation. Food Chem. 2019,279, 70–79. [CrossRef] 58. V á zquez, J.A.; Fraguas, J.; Mir ó n, J.; Valc á rcel, J.; P é rez-Mart í n, R.I.; Antelo, L.T. Valorisation of fish discards assisted by enzymatic hydrolysis and microbial bioconversion: Lab and pilot plant studies and preliminary sustainability evaluation. J. Clean. Prod. 2020,246, 119027. [CrossRef] 59. Harnedy-Rothwell, P.A.; McLaughlin, C.M.; O’Keeffe, M.B.; Le Gouic, A.V.; Allsopp, P.J.; Mcsorley, E.M.; Sharkey, S.; Whooley, J.; McGovern, B.; O’Harte, F.P.M.; et al. Identification and characterisation of peptides from a boarfish (Capros aper) protein hydrolysate displaying in vitro dipeptidyl peptidase-IV (DPP-IV) inhibitory and insulinotropic activity. Food Res. Int. 2020,131, 108989. [CrossRef] [PubMed] 60. Wali, A.; Ma,H.; Shahnawaz,M.; Hayat,K.; Xiaong,J.; Jing, L.Impactofpowerultrasound onantihypertensive activity, functional properties, and thermal stability of rapeseed protein hydrolysates. J. Chem. 2017 ,2017. [CrossRef] 61. Knezevic-Jugovic, Z.D.; Stefanovi´c, A.B.; Žuža, M.G.; Milovanovi´c, S.L.; Jakoveti´c, S.M.; Manojlovi´c, V.B.; Bugarski, B.M. Effects of sonication and high-pressure carbon dioxide processing on enzymatic hydrolysis of egg white proteins. Acta Period. Technol. 2012,43, 33–41. [CrossRef] 62. Yu, H.-C.; Tan, F.-J. Effect of ultrasonic pretreatment on the properties of porcine liver protein hydrolysates. Int. J. Food Sci. Technol. 2017,52, 1392–1399. [CrossRef] 63. Perreault, V.; H é naux, L.; Bazinet, L.; Doyen, A. Pretreatment of flaxseed protein isolate by high hydrostatic pressure: Impacts on protein structure, enzymatic hydrolysis and final hydrolysate antioxidant capacities. Food Chem. 2017,221, 1805–1812. [CrossRef] 64. Galazka, V.B.; Dickinson, E.; Ledward, D.A. Influence of high pressure processing on protein solutions and emulsions. Curr. Opin. Colloid Interface Sci. 2000,5, 182–187. [CrossRef] 65. Muntean, M.-V.; Marian, O.; Barbieru, V.; C ă tunescu, G.M.; Ranta, O.; Drocas, I.; Terhes, S. High Pressure Processing in Food Industry–Characteristics and Applications. Agric. Agric. Sci. Procedia 2016 ,10, 377–383. [CrossRef] Foods 2020,9, 983 24 of 33 66. Marciniak, A.; Suwal, S.; Naderi, N.; Pouliot, Y.; Doyen, A. Enhancing enzymatic hydrolysis of food proteins and production of bioactive peptides using high hydrostatic pressure technology. Trends Food Sci. Technol. 2018,80, 187–198. [CrossRef] 67. Turk, B. Targeting proteases: Successes, failures and future prospects. Nat. Rev. Drug Discov. 2006 ,5, 785–799. [CrossRef] 68. Tavano, O.L. Protein hydrolysis using proteases: An important tool for food biotechnology. J. Mol. Catal. B Enzym. 2013,90, 1–11. [CrossRef] 69. Tavano, O.L.; Berenguer-Murcia, A.; Secundo, F.; Fernandez-Lafuente, R. Biotechnological Applications of Proteases in Food Technology. Compr. Rev. Food Sci. Food Saf. 2018,17, 412–436. [CrossRef] 70. Adamson, N.J.; Reynolds, E.C. Characterization of casein phosphopeptides prepared using alcalase: Determination of enzyme specificity. Enzym. Microb. Technol. 1996,19, 202–207. [CrossRef] 71. Olsen, J.V.; Ong, S.-E.; Mann, M. Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues. Mol. Cell. Proteom. 2004,3, 608–614. [CrossRef] [PubMed] 72. Segura Campos, M.R.; Chel Guerrero, L.A.; Betancur Ancona, D.A. Angiotensin-I converting enzyme inhibitory and antioxidant activities of peptide fractions extracted by ultrafiltration of cowpea Vigna unguiculata hydrolysates. J. Sci. Food Agric. 2010,90, 2512–2518. [CrossRef] 73. Harnedy, P.A.; Parthsarathy, V.; McLaughlin, C.M.; O’Keeffe, M.B.; Allsopp, P.J.; McSorley, E.M.; O’Harte, F.P.M.; FitzGerald, R.J. Atlantic salmon (Salmo salar) co-product-derived protein hydrolysates: A source of antidiabetic peptides. Food Res. Int. 2018,106, 598–606. [CrossRef] 74. Rivero-Pino, F.; P é rez G á lvez, A.R.; Espejo-Carpio, F.J.; Guadix, E.M. Evaluation of Tenebrio molitor protein as source of peptides modulating physiological processes. Food Funct. 2020,11, 4376–4386. [CrossRef] 75. Connolly, A.; Piggott, C.O.; FitzGerald, R.J. In vitro α -glucosidase, angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory properties of brewers’ spent grain protein hydrolysates. Food Res. Int. 2014,56, 100–107. [CrossRef] 76. Arise, R.O.; Idi, J.J.; Mic-Braimoh, I.M.; Korode, E.; Ahmed, R.N.; Osemwegie, O. In vitro Angiotesin-1-converting enzyme, α -amylase and α -glucosidase inhibitory and antioxidant activities of Luffa cylindrical (L.)M. Roem seed protein hydrolysate. Heliyon 2019,5, e01634. [CrossRef] 77. Horner,K.; Drummond,E.; Brennan,L.Bioavailabilityofmilkprotein-derivedbioactivepeptides: A glycaemic management perspective. Nutr. Res. Rev. 2016,29, 91–101. [CrossRef] 78. Miralles, B.; Hern á ndez-Ledesma, B.; Fern á ndez-Tom é , S.; Amigo, L.; Recio, I. Health-related functional value of dairy proteins and peptides. In Proteins in Food Processing; Elsevier Ltd.: Amsterdam, The Netherlands, 2017; pp. 523–568. ISBN 9780081007297. 79. Montesano, D.; Gallo, M.; Blasi, F.; Cossignani, L. Biopeptides from vegetable proteins: New scientific evidences. Curr. Opin. Food Sci. 2020,31, 31–37. [CrossRef] 80. Lacroix, I.M.E.; Li-Chan, E.C.Y. Dipeptidyl peptidase-IV inhibitory activity of dairy protein hydrolysates. Int. Dairy J. 2012,25, 97–102. [CrossRef] 81. Harnedy, P.A.; Parthsarathy, V.; McLaughlin, C.M.; O’Keeffe, M.B.; Allsopp, P.J.; McSorley, E.M.; O’Harte, F.P.M.; FitzGerald, R.J. Blue whiting (Micromesistius poutassou) muscle protein hydrolysate with in vitro and in vivo antidiabetic properties. J. Funct. Foods 2018,40, 137–145. [CrossRef] 82. Huang, S.L.; Jao, C.L.; Ho, K.P.; Hsu, K.C. Dipeptidyl-peptidase IV inhibitory activity of peptides derived from tuna cooking juice hydrolysates. Peptides 2012,35, 114–121. [CrossRef] 83. Neves, A.C.; Harnedy, P.A.; O’Keeffe, M.B.; FitzGerald, R.J. Bioactive peptides from Atlantic salmon (Salmo salar) with angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory, and antioxidant activities. Food Chem. 2017,218, 396–405. [CrossRef] 84. Rivero-Pino, F.; Espejo-Carpio, F.J.; Guadix, E.M. Production and identification of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from discarded Sardine pilchardus protein. Food Chem. 2020 ,328, 127096. [CrossRef] 85. Tilman, D.; Clark, M. Global diets link environmental sustainability and human health. Nature 2014 ,515, 518–522. [CrossRef] 86. Nongonierma, A.B.; FitzGerald, R.J. Unlocking the biological potential of proteins from edible insects through enzymatic hydrolysis: A review. Innov. Food Sci. Emerg. Technol. 2017,43, 239–252. [CrossRef] Foods 2020,9, 983 25 of 33 87. Admassu, H.; Gasmalla, M.A.A.; Yang, R.; Zhao, W. Bioactive Peptides Derived from Seaweed Protein and Their Health Benefits: Antihypertensive, Antioxidant, and Antidiabetic Properties. J. Food Sci. 2018 ,83, 6–16. [CrossRef] 88. Harnedy, P.A.; FitzGerald, R.J. In vitro assessment of the cardioprotective, anti-diabetic and antioxidant potential of Palmaria palmata protein hydrolysates. J. Appl. Phycol. 2013,25, 1793–1803. [CrossRef] 89. Escuredo, O.; Gonz á lez Mart í n, M.I.; Wells Moncada, G.; Fischer, S.; Hern á ndez Hierro, J.M. Amino acid profile of the quinoa (Chenopodium quinoa Willd.) using near infrared spectroscopy and chemometric techniques. J. Cereal Sci. 2014,60, 67–74. [CrossRef] 90. Aryee, A.N.A.; Boye, J.I. Comparative Study of the Effects of Processing on the Nutritional, Physicochemical and Functional Properties of Lentil. J. Food Process. Preserv. 2017,41, 24–30. [CrossRef] 91. Connolly, A.; Piggott, C.O.; FitzGerald, R.J. Characterisation of protein-rich isolates and antioxidative phenolic extracts from pale and black brewers’ spent grain. Int. J. Food Sci. Technol. 2013 ,48, 1670–1681. [CrossRef] 92. Azagoh, C.; Ducept, F.; Garcia, R.; Rakotozafy, L.; Cuvelier, M.-E.; Keller, S.; Lewandowski, R.; Mezdour, S. Extraction and physicochemical characterization of Tenebrio molitor proteins. Food Res. Int. 2016 ,88, 24–31. [CrossRef] [PubMed] 93. Makkar, H.P.S.; Tran, G.; Heuz é , V.; Ankers, P. State-of-the-art on use of insects as animal feed. Anim. Feed Sci. Technol. 2014,197, 1–33. [CrossRef] 94. Breternitz, N.R.; Bolini, H.M.A.; Hubinger, M.D. Sensory acceptance evaluation of a new food flavoring produced by microencapsulation of a mussel (Perna perna) protein hydrolysate. LWT-Food Sci. Technol. 2017 , 83, 141–149. [CrossRef] 95. Sathivel, S.; Bechtel, P.J.; Babbitt, J.; Smiley, S.; Crapo, C.; Reppond, K.D.; Prinyawiwatkul, W. Biochemical and Functional Properties of Herring (Clupea harengus) Byproduct Hydrolysates. J. Food Sci. 2003 ,68, 2196–2200. [CrossRef] 96. Hall, F.; Johnson, P.E.; Liceaga, A. Effect of enzymatic hydrolysis on bioactive properties and allergenicity of cricket (Gryllodes sigillatus) protein. Food Chem. 2018,262, 39–47. [CrossRef] 97. Gonzalez-Montoya, M.; Hern á ndez-Ledesma, B.; Mora-Escobedo, R.; Martinez-Villaluenga, C. Bioactive peptides from germinated soybean with anti-diabetic potential by inhibition of dipeptidyl peptidase-IV, a-amylase, and a-glucosidase enzymes. Int. J. Mol. Sci. 2018,19, 2883. [CrossRef] 98. Parthsarathy, V.; McLaughlin, C.M.; Harnedy, P.A.; Allsopp, P.J.; Crowe, W.; McSorley, E.M.; FitzGerald, R.J.; O’Harte, F.P.M.M. Boarfish (Capros aper) protein hydrolysate has potent insulinotropic and GLP-1 secretory activity in vitro and acute glucose lowering effects in mice. Int. J. Food Sci. Technol. 2018 ,54, 271–281. [CrossRef] 99. Nongonierma, A.B.; Lamoureux, C.; Fitzgerald, R.J. Generation of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides during the enzymatic hydrolysis of tropical banded cricket (Gryllodes sigillatus) proteins. Food Funct. 2018,9, 407–416. [CrossRef] [PubMed] 100. Lammi, C.; Bollati, C.; Gelain, F.; Arnoldi, A.; Pugliese, R. Enhancement of the stability and anti-DPPIV activity of hempseed hydrolysates through self-assembling peptide-based hydrogels. Front. Chem. 2019 ,7, 670. [CrossRef] [PubMed] 101. Jia, C.-L.; Hussain, N.; Joy Ujiroghene, O.; Pang, X.-Y.; Zhang, S.-W.; Lu, J.; Liu, L.; Lv, J.-P. Generation and characterization of dipeptidyl peptidase-IV inhibitory peptides from trypsin-hydrolyzed α -lactalbumin-rich whey proteins. Food Chem. 2020,318, 126333. [CrossRef] [PubMed] 102. Gong, P.X.; Wang, B.K.; Wu, Y.C.; Li, Q.Y.; Qin, B.W.; Li, H.J. Release of antidiabetic peptides from Stichopus japonicas by simulated gastrointestinal digestion. Food Chem. 2020,315, 126273. [CrossRef] [PubMed] 103. Megrous, S.; Al-Dalali, S.; Zhao, X.; Chen, C.; Cao, Y.; Bourouis, I.; Mekkaoui, A.; Yang, Z.; Yang, Z. Evaluation of Antidiabetic Activities of Casein Hydrolysates by a Bacillus Metalloendopeptidase. Int. J. Pept. Res. Ther. 2020, 1–9. [CrossRef] 104. Wang, J.; Wu, T.; Fang, L.; Liu, C.; Liu, X.; Li, H.; Shi, J.; Li, M.; Min, W. Anti-diabetic effect by walnut (Juglans mandshurica Maxim.)-derived peptide LPLLR through inhibiting α -glucosidase and α -amylase, and alleviating insulin resistance of hepatic HepG2 cells. J. Funct. Foods 2020,69, 103944. [CrossRef] 105. Zieli´nska, E.; Kara´s, M.; Baraniak, B.; Jakubczyk, A. Evaluation of ACE, α -glucosidase, and lipase inhibitory activities of peptides obtained by in vitro digestion of selected species of edible insects. Eur. Food Res. Technol. 2020,246, 1361–1369. [CrossRef] Foods 2020,9, 983 32 of 33 227. Schlotterer, A.; Kukudov, G.; Bozorgmehr, F.; Hutter, H.; Du, X.; Oikonomou, D.; Ibrahim, Y.; Pfisterer, F.; Rabbani, N.; Thornalley, P.; et al. C. elegans as model for the study of high glucose-mediated life span reduction. Diabetes 2009,58. [CrossRef] 228. Brandt, A.; Vilcinskas, A. The Fruit Fly Drosophila melanogaster as a Model for Aging Research. In Yellow Biotechnology I: Insect Biotechnologie in Drug Discovery and Preclinical Research; Vilcinskas, A., Ed.; Springer: Berlin/Heidelberg, Germany, 2013; pp. 63–77. ISBN 978-3-642-39863-6. 229. Chen, S.; Yang, Q.; Chen, X.; Tian, Y.; Liu, Z.; Wang, S. Bioactive peptides derived from crimson snapper and in vivo anti-aging effects on fat diet-induced high fat Drosophila melanogaster. Food Funct. 2020 ,11, 524–533. [CrossRef] 230. Su á rez, S.; Aphalo, P.; Rinaldi, G.; Añ ó n, M.C.; Quiroga, A. Effect of amaranth proteins on the RAS system. In vitro, in vivo and ex vivo assays. Food Chem. 2020,308, 125601. [CrossRef] 231. King, A.J.F. The use of animal models in diabetes research. Br. J. Pharmacol. 2012,166, 877–894. [CrossRef] 232. Jung, H.; Kim, Y.Y.; Kim, B.; Nam, H.; Suh, J.G. Improving glycemic control in model mice with type 2 diabetes by increasing superoxide dismutase (SOD) activity using silk fibroin hydrolysate (SFH). Biochem. Biophys. Res. Commun. 2017,493, 115–119. [CrossRef] 233. Mochida, T.; Hira, T.; Hara, H. The corn protein, zein hydrolysate, administered into the ileum attenuates hyperglycemia via its dual action on glucagon-like peptide-1 secretion and dipeptidyl peptidase-IV activity in rats. Endocrinology 2010,151, 3095–3104. [CrossRef] 234. Ishikawa, Y.; Hira, T.; Inoue, D.; Harada, Y.; Hashimoto, H.; Fujii, M.; Kadowaki, M.; Hara, H. Rice protein hydrolysates stimulate GLP-1 secretion, reduce GLP-1 degradation, and lower the glycemic response in rats. Food Funct. 2015,6, 2525–2534. [CrossRef] 235. D’Souza, R.; Pandeya, D.R.; Rahman, M.; Lee, H.S.; Jung, J.K.; Hong, S.T. Genetic engineering of Lactococcus lactis to produce an amylase inhibitor for development of an anti-diabetes biodrug. New Microbiol. 2012 ,35, 35–42. [PubMed] 236. Wang, T.Y.; Hsieh, C.H.; Hung, C.C.; Jao, C.L.; Chen, M.C.; Hsu, K.C. Fish skin gelatin hydrolysates as dipeptidyl peptidase IV inhibitors and glucagon-like peptide-1 stimulators improve glycaemic control in diabetic rats: A comparison between warm-and cold-water fish. J. Funct. Foods 2015 ,19, 330–340. [CrossRef] 237. Drotningsvik, A.; Mjos, S.A.; Pampanin, D.M.; Slizyte, R.; Carvajal, A.; Remman, T.; Hogoy, I.; Gudbrandsen, O.A. Dietary fish protein hydrolysates containing bioactive motifs affect serum and adipose tissue fatty acid compositions, serum lipids, postprandial glucose regulation and growth in obese Zucker fa/fa rats. Br. J. Nutr. 2016,116, 1336–1345. [CrossRef] [PubMed] 238. Bellinger, D.A.; Merricks, E.P.; Nichols, T.C. Swine models of type 2 diabetes mellitus: Insulin resistance, glucose tolerance, and cardiovascular complications. ILAR J. 2006,47, 243–258. [CrossRef] 239. Baek, S.Y.; Chung, H.J.; Kim, K.W.; Cho, K.H.; Choi, I.; Lee, H.T. Potential use of transgenic domestic pigs expressing recombinant human erythropoietin in diabetes translation research. Anim. Cells Syst. 2019 ,23, 42–49. [CrossRef] 240. Ribel, U.; Larsen, M.O.; Rolin, B.; Carr, R.D.; Wilken, M.; Sturis, J.; Westergaard, L.; Deacon, C.F.; Knudsen, L.B. NN2211: A long-acting glucagon-like peptide-1 derivative with anti-diabetic effects in glucose-intolerant pigs. Eur. J. Pharmacol. 2002,451, 217–225. [CrossRef] 241. Fosgerau, K.; Hoffmann, T. Peptide therapeutics: Current status and future directions. Drug Discov. Today 2015,20, 122–128. [CrossRef] 242. Chalamaiah, M.; Keskin Ulug, S.; Hong, H.; Wu, J. Regulatory requirements of bioactive peptides (protein hydrolysates) from food proteins. J. Funct. Foods 2019,58, 123–129. [CrossRef] 243. Manders, R.J.F.; Hansen, D.; Zorenc, A.H.G.; Dendale, P.; Kloek, J.; Saris, W.H.M.; Van Loon, L.J.C. Protein co-ingestion strongly increases postprandial insulin secretion in type 2 diabetes patients. J. Med. Food 2014 , 17, 758–763. [CrossRef] 244. Manders, R.J.; Koopman, R.; Sluijsmans, W.E.; van den Berg, R.; Verbeek, K.; Saris, W.H.; Wagenmakers, A.J.; van Loon, L.J. Co-Ingestion of a Protein Hydrolysate with or without Additional Leucine Effectively Reduces Postprandial Blood Glucose Excursions in Type 2 Diabetic Men. J. Nutr. 2006,136, 1294–1299. [CrossRef] 245. Jonker, J.T.; Wijngaarden, M.A.; Kloek, J.; Groeneveld, Y.; Gerhardt, C.; Brand, R.; Kies, A.K.; Romijn, J.A.; Smit, J.W.A. Effects of low doses of casein hydrolysate on post-challenge glucose and insulin levels. Eur. J. Intern. Med. 2011,22, 245–248. [CrossRef] [PubMed] Foods 2020,9, 983 33 of 33 246. Geerts, B.F.; Van Dongen, M.G.J.; Flameling, B.; Moerland, M.M.; Kam, M.L.D.; Cohen, A.F.; Romijn, J.A.; Gerhardt, C.C.; Kloek, J.; Burggraaf, J. Hydrolyzed casein decreases postprandial glucose concentrations in T2DM patients irrespective of leucine content. J. Diet. Suppl. 2011,8, 280–292. [CrossRef] [PubMed] 247. Koopman, R.; Crombach, N.; Gijsen, A.P.; Walrand, S.; Fauquant, J.; Kies, A.K.; Lemosquet, S.; Saris, W.H.; Boirie, Y.; van Loon, L.J. Ingestion of a protein hydrolysate is accompanied by an accelerated in vivo digestion and absorption rate when compared with its intact protein. Am. J. Clin. Nutr. 2009 ,90, 106–115. [CrossRef] [PubMed] 248. Saleh, L.; Schrier, N.L.; Bruins, M.J.; Steegers, E.A.P.; van den Meiracker, A.H.; Visser, W. Effect of oral protein hydrolysate on glucose control in patients with gestational diabetes. Clin. Nutr. 2018 ,37, 878–883. [CrossRef] 249. Petersen, B.L.; Ward, L.S.; Bastian, E.D.; Jenkins, A.L.; Campbell, J.; Vuksan, V. A whey protein supplement decreases post-prandial glycemia. Nutr. J. 2009,8, 47. [CrossRef] 250. Frid, A.H.; Nilsson, M.; Holst, J.J.; Björck, I.M.E. Effect of whey on blood glucose and insulin responses to composite breakfast and lunch meals in type 2 diabetic subjects. Am. J. Clin. Nutr. 2005 ,82, 69–75. [CrossRef] 251. Sartorius, T.; Weidner, A.; Dharsono, T.; Boulier, A.; Wilhelm, M.; Schön, C. Postprandial Effects of a Proprietary Milk Protein Hydrolysate Containing Bioactive Peptides in Prediabetic Subjects. Nutrients 2019 , 11, 1700. [CrossRef] 252. Goudarzi, M.; Madadlou, A. Influence of whey protein and its hydrolysate on prehypertension andpostprandial hyperglycaemia in adult men. Int. Dairy J. 2013,33, 62–66. [CrossRef] 253. Plat, J.; Severins, N.; Mensink, R.P. Improvement of pulse wave velocity and metabolic cardiovascular risk parameters through egg protein hydrolysate intake: A randomized trial in overweight or obese subjects with impaired glucose tolerance or type 2 diabetes. J. Funct. Foods 2019,52, 418–423. [CrossRef] 254. Calbet, J.A.L.; Holst, J.J. Gastric emptying, gastric secretion and enterogastrone response after administration of milk proteins or their peptide hydrolysates in humans. Eur. J. Nutr. 2004,43, 127–139. [CrossRef] 255. Zhu, C.F.; Li, G.Z.; Peng, H.-B.; Zhang, F.; Chen, Y.; Li, Y. Treatment with marine collagen peptides modulates glucose and lipid metabolism in chinese patients with type 2 diabetes mellitus. Appl. Physiol. Nutr. Metab. 2010,35, 797–804. [CrossRef] [PubMed] 256. Hovland, I.H.; Leikanger, I.S.; Stokkeland, O.; Waage, K.H.; Mjøs, S.A.; Brokstad, K.A.; McCann, A.; Ueland, P.M.; Slizyte, R.; Carvajal, A.; et al. Effects of low doses of fish and milk proteins on glucose regulation and markers of insulin sensitivity in overweight adults: A randomised, double blind study. Eur. J. Nutr. 2020,59, 1013–1029. [CrossRef] [PubMed] 257. Dale, H.F.; Jensen, C.; Hausken, T.; Lied, E.; Hatlebakk, J.G.; Brønstad, I.; Lihaug Hoff, D.A.; Lied, G.A. Effect of a cod protein hydrolysate on postprandial glucose metabolism in healthy subjects: A double-blind cross-over trial. J. Nutr. Sci. 2018,7, e33. [CrossRef] [PubMed] 258. Crowe, W.; McLaughlin, C.M.; Allsopp, P.J.; Slevin, M.M.; Harnedy, P.A.; Cassidy, Y.; Baird, J.; Devaney, M.; Fitzgerald, R.J.; O’Harte, F.P.M.; et al. The effect of boarfish protein hydrolysate on postprandial glycaemic response and satiety in healthy adults. Proc. Nutr. Soc. 2018,77. [CrossRef] 259. Curran,A.M.; Horner, K.; O’Sullivan, V.; Nongonierma, A.B.; LeMaux, S.; Murphy, E.; Kelly,P.; Fitzgerald, R.J.; Brennan, L. Variable Glycemic Responses to Intact and Hydrolyzed Milk Proteins in Overweight and Obese Adults Reveal the Need for Precision Nutrition. J. Nutr. 2019,149, 88–97. [CrossRef] 260. Lee, S.K.; Posthauer, M.E.; Dorner, B.; Redovian, V.; Maloney, M.J. Pressure ulcer healing with a concentrated, fortified, collagen protein hydrolysate supplement: A randomized controlled trial. Adv. Skin Wound Care 2006,19, 92–96. [CrossRef] 261. Hillesheim, E.; Brennan, L. Metabotyping and its role in nutrition research. Nutr. Res. Rev. 2020 ,33, 33–42. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).