Introduction

Incidences of diabetes are on a perpetual rise around the globe. In 2013, the number of diabetic persons was 382 million, and this is expected to be increased to 592 million by 2035 [1]. Diabetes mellitus (DM), a chronic metabolic disorder, is caused due to defective insulin production or action [2]. It is characterized by hyperglycemia, a condition in which surplus of sugar is present in the blood stream. Prevalence of diabetes mellitus is increasing markedly because of aging, population growth, increasing urbanization, incidences of obesity, and more sedentary lifestyles [3]. Type 1 diabetes (T1D) and Type 2 diabetes (T2D) are the main two types of diabetes. Though the later is much more common and accounts for 90–95 % of all diabetes. A number of factors, such as insulin resistance, hyperinsulinemia, impaired insulin secretion, reduced insulin mediated glucose uptake, and utilization convoluted the treatment of T2D [4]. Postprandial blood glucose levels could be a better indicator of glycemic control than fasting blood glucose levels in T2D patients [5].

It is important to have a normal blood glucose level, both in fasting and postprandial conditions. However, in preventing the T2D, it is important to control postprandial glucose level [6, 7], because in the long term, this will be leading to serious complications, including hypertension, cardiovascular disorders, blindness, and renal failure [8]. For the management of T2D, various strategies are being used such as the regular use of different antidiabetic medications, manipulation of diet, change in lifestyle, and regular physical exercise [9, 10]. One therapeutic approach for management of T2D is through inhibition of carbohydrate hydrolyzing enzymes such as alpha-amylases and alpha-glucosidases in digestive organ [11–14]. Another therapeutic approach for the management of T2D is the use of glucose-lowering agent, i.e., dipeptidyl peptidase-IV (DPP-IV) inhibitors [15, 16]. Currently, in the field of diabetes research, the main focus is on the development of the antihyperglycemic agents that are from natural source and safe without any side effects [17–19]. Nutritional intervention has been established as a main element in the prevention and management of T2D [20]. In humans, several biomarkers of diabetes are affected by dietary intake of food protein and food protein hydrolysates. It was investigated that milk proteins, mainly, milk protein-derived peptides and amino acids have also been associated with the regulation of postprandial glycemia and insulin secretion in normal and T2D patients [21].

Bioactive peptides are specific protein fragments that have a positive impact on body functions or conditions and may ultimately influence health [22]. Fermented dairy products and other foods containing bioactive peptides appear to have the potential to offer specific health benefits to consumers. Milk protein-derived active biological constituents and bioactive peptides have received much attention for their biological significances, and are currently the subject of intensive research. The intrinsic bioactivities of the peptides encrypted in major milk proteins are latent until released and activated by enzymatic hydrolysis in vitro or in vivo [23]. Biologically active peptide fragments are formed during the degradation of the milk proteins (whey proteins and casein) by digestive enzymes in the gastrointestinal tract and by proteolytic lactic acid bacteria (LAB) during fermentation of milk. In these processes, varieties of peptides containing 2–20 amino acid residues are formed. Once liberated and absorbed, these bioactive peptides may exert a physiological effect on the cardiovascular, digestive, endocrine, immune, and nervous systems of the body [24]. The activity of these peptides is based on their inherent amino acid composition and length of the sequence [25]. Milk protein-derived peptides exert multiple physiological activities, including antimicrobial, antioxidant, antithrombotic, opioid, antihypertension activity, modulation of digestive enzymes, nutrient absorption, and immune responses (Fig. 1) [26–32]. Many milk-derived peptides reveal multifunctional properties, i.e., specific peptide sequences may exert two or more different biological activities. These regions known as “strategic zones” are partially protected from further proteolytic breakdown [25]. Because of their physiological and physicochemical versatility, milk peptides are reckoned as very important constituents for incorporation in functional and novel foods, dietary supplements, and even pharmaceuticals with the purpose of targeting specific disease. Recent research has also shown that peptides derived from milk proteins have alpha-glucosidase and DPP-IV inhibitory properties [33, 34].

Fig. 1
figure 1

Physiological functionality of milk protein derived peptides [26–32]

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The purpose of this non-systematic review is to look at the significance of milk protein-derived peptides as potential inhibitors of alpha-glucosidase and DPP-IV. However, other food protein-derived peptide inhibitors have also been taken into consideration for better understanding of the topic, but our primary focus is on milk protein-derived peptide inhibitors. The role of milk protein-derived alpha-glucosidase and DPP-IV inhibitory peptides will be considered in the context of a nutritional strategy for the management of T2D.

Milk protein and T2D

Milk proteins contain approximately 80 % casein and 20 % whey. The casein comprises α-s1, α-s2, β, and κ-casein, while whey comprises β-lactoglobulin, α-lactalbumin, lactoferrin, immunoglobulins, serum albumin, glycomacropeptide, enzymes, and growth factors. Whey proteins and caseins may help in diminishing the physiological effects of T2D and have been revealed to arouse insulin secretion and control blood glucose level in T2D patients [35, 36]. Recently, the role of protein in the diet as a physiological active ingredient has increasingly been acknowledged worldwide. Milk protein is an important source of amino acid is very well accepted, but in current times, it has been recognized that milk protein shows numerous functionalities in vivo by the action of bioactive peptides. Presently, milk proteins are considered as the most vital resource of a range of bioactive peptides [37]. For that reason, there is a growing interest for milk proteins or peptides as potential ingredients of health-promoting functional food targeted at metabolic syndrome, which linked to cardiovascular disease, T2D, and obesity. It seems that milk proteins or peptides may reduce the risk of metabolic syndrome through several mechanisms [32]. It was suggested that the antidiabetic properties of whey protein are primarily attributable to its content of bioactive peptides which, following their release during gastrointestinal digestion, could arouse the secretion of gut-derived hormones and/or inhibit enzymes involved in glucose homeostasis [38, 39].

Effect on insulin secretion

It was found that whey protein absorbed faster than casein. Therefore, plasma amino acid (AA) increased more rapidly after consumption of whey protein and that leads to more rapid secretion of insulin than micellar casein [40]. The absorption rate of casein in its native micellar form is lower because the acidic conditions in the stomach cause casein to clot and thus delay gastric emptying [41]. Insulin is sensitive to both the composition and concentration of plasma AAs, hence both whey and casein ingestion stimulate increased insulin secretion [42, 43]. However, the absorption of AAs and secretion of insulin speed up after the hydrolysis of casein relative to the micellar form of casein [44]. Therefore, different milk proteins may create a remarkable contribution to metabolic effects in insulin-sensitive tissues and in particular skeletal muscle anabolism by the stimulation of insulin secretion [45]. Extended, high fasting glucose is one of the main characteristics of T2D. Increased insulin resistance and defect in insulin secretion causes hyperglycemia [46].

Effect on postprandial glycemia

It is important to decrease the prolonged exposure to high blood glucose levels in individuals both with and without T2D for the management of postprandial glucose level [47]. Milk proteins, i.e., casein and whey stimulate the insulin secretion, have the potential to alter tissue glucose uptake, and suppress postprandial blood glucose excursions [48–50]. The insulinotropic effect of whey protein is mainly contributed by its AA profile. The numbers of insulinotropic AAs present in whey protein (e.g., leucine, isoleucine, valine, lysine, and threonine) are observed to show modified insulinemic and glycemic responses [43].

In a comparative study, the consumption of whey protein (18 g in lunch or breakfast) resulted in greater insulinotropic responses, circulating levels of the gut peptide glucose-dependent insulinotropic polypeptide (GIP), and suppression of postprandial glycemia than non-dairy protein (lean ham) in individual with T2D [35]. In healthy subjects, addition of whey protein supplement to a drink contacting 50 g glucose reduced the postprandial glycemia in a dose-dependent manner [51]. Similarly, whey protein in mice showed increased level of glucagon-like peptide 1 (GLP-1) and inhibit DPP-IV, a peptide that hydrolyze incretin hormones, resulting in an increased and prolonged insulin response [52]. Concerning about the casein, in an overweight subjects with T2D consumption of a casein hydrolysate (~30 g) and leucine (~10 g), beverage after food intake decreased prevalence of hyperglycemia over the period of 24 h [53]. Milk proteins are gaining considerable attention due to their beneficial effect on postprandial blood glucose, which are comparable to insulin secretagogues used for the treatment of hyperglycemia in T2D. Therefore, there is a rationale for regular whey protein consumption before or with meals to control postprandial glycemic responses in individuals with poor metabolic control or T2D [45].

Effect on satiety

It is very well examined that protein is the most satiating component of food [54]. In several clinical studies, dairy products showed a certain satiating effect. In a recent study with 49 overweight and obese adults, the weight loss between the high-dairy diet (1400 mg/day) group and low-dairy diet (750 mg/day) group was similar, but high-dairy diet group was found with slightly higher peptide YY concentrations in plasma and improved feelings of satisfaction [55]. In a comparative study, consumption of both pea protein hydrolysates and whey protein leads to greater satiety and fullness as compared to milk protein. A positive correlation was observed between insulin and both cholecystokinin (CCK) and GLP-1 for whey protein. However, both CCK and GLP-1 were increased by milk protein [56]. In addition, skim milk containing casein and whey protein showed more decreased in food intake than either protein alone in a study of isoenergetic preloads [57]. The mechanisms of action of different protein or peptides on satiety are still under investigation, but it has been proposed that it could be related to a delay of the gastric emptying, an increase in brain amino acids, or the presence of specific peptides or amino acids [58]. The satiating effect of whey protein is mainly due to a high concentration of branch chain amino acids, particularly l-leucine. Regarding the casein fraction of milk, it was proposed that peptides from casein hydrolysates activates the peripheral opioid and cholecystokinin receptors and blocks the antagonist receptors which reduces their effect on food intake [58, 59].

Effect on incretin system

The amino acids or peptides affect both the insulin secretion and release of incretin hormones, i.e., glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1 (GLP-1) from gut.

Glucose-dependent insulinotropic peptide

GIP, also known a gastric inhibitory peptide, is synthesized from K cells in the duodenum and jejunum after the food intake [60]. Recent studies show that GIP response was (+80 %) significantly enhanced by a whey drink in healthy subjects, while branched-chain amino acid mixtures did not [43]. Whey or casein hydrolysates elicited about 50 % more gastric secretion than whole protein solutions [44]. It is possible that bioactive peptides and/or other amino acids liberated during whey digestion are the key stimulators of GIP synthesis and secretion, as the same was found for insulin [61]. One more probability is that bioactive peptides released from whey protein may lead to increased half-life of GIP.

Glucagon-like peptide 1

GLP-1 secretion by intestinal L cells is dependent on the presence of nutrients in the lumen of the small intestine. It is a potent antihyperglycemic hormone, inducing glucose-dependent stimulation of insulin secretion while suppressing glucagon secretion. Whey generated the strongest response of active GLP-1 as compared to casein [62]. This stimulatory effect of whey protein on GLP-1 may result into many beneficial effects like increase in the glucose-induced insulin secretion and reduction in postprandial glycemia. Enhanced GLP-1 levels also boost the synthesis of pro-insulin and insulin stores in β-cells [63, 64].

Effect on appetite

Protein does not only increase energy expenditure, but also manifests reduction in energy intake through mechanisms that influence appetite control [65]. Many studies explained that dairy protein has a stronger effect on suppression of hunger compared to soy and egg albumin [62]. The appetite-suppressing effect of whey protein can be attributed to the high content in branched-chain amino acids (mainly, l-leucine), and the presence of certain peptides, such as CMP, or it can be mediated by the release of satiety hormones [66]. Recently, it has been found that whey protein may exert a central effect on appetite by suppression of food intake [67, 68]. The increase in levels of insulin by ingestion of whey protein not only helps in modifying the glycemic response, but is also strongly associated with satiety and decreased food intake by suppressing appetite [48, 49]. Directly or indirectly, other hormones are also involved in the regulation of food intake, such as ghrelin, CCK, and peptide YY (PYY) [38]. Figure 2 shows the metabolic effect of milk protein in T2D management.

Fig. 2
figure 2

Metabolic effect of milk protein in T2D management. Adapted from Jakubowicz and Froy 2013 [35]

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Bioactive food peptides and T2D

The significance of food proteins in the diet has been increasingly recognized because of the functionalities of biologically active peptides. In recent times, many studies reveal that antidiabetic properties of milk are mainly attributed to a variety of peptides derived from milk protein [21, 33, 69–71]. The two enzymes, i.e., dipeptidyl peptidase-IV and alpha-glucosidase play an important role in the development of hyperglycemia in T2D. Accordingly, inhibition of these two enzymes opens up new vistas for the treatment of T2D [33, 34].

Alpha-glucosidase enzyme inhibitory peptides

Enzyme alpha-glucosidase (EC 3.2.1.20) is located in the brush border of the enterocytes of the jejunum in the small intestine and happens to be a key enzyme in carbohydrate synthesis and breakdown [72]. It is an exo-type carbohydrolase, cleaving glycosidic bonds in complex carbohydrate to release absorbable monosaccharides (Fig. 3a) [73]. The hydrolyzed dietary carbohydrate is the main source of increased level of glucose in the blood. After the hydrolysis of dietary carbohydrate by pancreatic alpha-amylase, the subsequent absorption in the intestine is done by alpha-glucosidases [74]. One of the remedial strategies for managing T2D is to decrease postprandial hyperglycemia by retarding the absorption of glucose through inhibition of carbohydrate-hydrolyzing enzymes, e.g., alpha-glucosidase, in the digestive organs [75]. In addition, one interesting aspect of the alpha-glucosidase inhibitor is that, it has the ability to both enhance and extend GLP-1 secretion in normal individuals and patients with T2D [76, 77].

Fig. 3
figure 3

Mechanism of action of alpha-glucosidase inhibitors. Adapted from Arungarinathan et al. [89]. a Action of alpha-glucosidase on carbohydrate in the absence of alpha-glucosidase inhibitor. b Action of alpha-glucosidase on carbohydrate in the presence of alpha-glucosidase inhibitor. AG alpha-glucosidase, AGI alpha-glucosidase inhibitor

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Currently alpha-glucosidase inhibitors such as acarbose, miglitol, voglibose, and emiglitate are considered as therapeutic drugs for the treatment of diabetic individuals with postprandial hyperglycemia. However, the chronic use of these agents could result in side effects such as flatulence, abdominal cramping, vomiting, and diarrhea [78–80]. Therefore, a number of studies have been carried out to identify the natural sources of alpha-glucosidase inhibitors. A range of fruits, vegetables, and animals, including blueberry, strawberry, broccoli sprouts, green paper, peptides from sardine muscle, and more recently egg white protein, has been reported to display alpha-glucosidase inhibitory activity [81–85].

Alpha-glucosidase inhibitors act as competitive inhibitors of enzymes needed to digest carbohydrates, specifically alpha-glucosidase [86]. Inhibition of this enzyme in digestive tract delays carbohydrate digestion thus increases overall carbohydrate digestion time. As a result, less glucose gets absorbed, because the carbohydrates are not rapidly hydrolyzed down into glucose molecules and subsequently diminishing the postprandial blood glucose and insulin level (Fig. 3b) [87, 88]. The short time effect of these inhibitors is to decrease the current blood glucose level.

Alpha-glucosidase inhibitors also play a crucial role in secretion of GLP-1 in normal and diabetic patients. GLP-1 is secreted from intestinal L-cells in response to nutrient ingestion. GLP-1 is recognized to be involved in the regulation of insulin secretion [90], glucagon decreation [91], and β-cell turnover [92]. Since, alpha-glucosidase inhibitors slow down carbohydrate absorption, this in turn results in an elevated sugar absorption in the lower gut [93]. Taking into consideration that sugar absorption plays a significant role in GLP-1 secretion [94, 95] and that lower gut is plentiful in GLP-1 producing cells [96, 97], delayed carbohydrate absorption is considered a satisfactory contributing factor for stimulating GLP-1 secretion by alpha-glucosidase inhibition. Hence, alpha-glucosidase inhibitor is a promising therapeutic modality in T2D patients. The accurate mechanism by which peptides can inhibit alpha-glucosidase activity is unknown, but it has been proposed that non-saccharide compounds may apply their inhibitory activity by binding to the enzyme’s active site through hydrophobic interactions [98].

Food protein-derived bioactive peptides inhibiting alpha-glucosidase enzyme

Beyond their nutritional importance, proteins and peptides have a wide variety of physiological functions that may benefit the human health. Many food proteins showed the natural precursor of alpha-glucosidase inhibitory peptides.

Sardine muscle hydrolysates by alkaline protease at 0.3 wt%–17 h hydrolysis showed highest inhibitory activity against alpha-glucosidase with an IC50 value of 48.7 mg/mL. Various proteases-treated sardine muscle hydrolysates exhibited different alpha-glucosidase inhibition activity, which indicated that the main contributor to inhibitory activity was sardine protein hydrolysates. This indicated that differences in the inhibitory activity of hydrolysates by various proteases were due to the difference in the size of the hydrolytic product with their different substrate specificity. Further purification of hydrolysates with DEAE-Sephadex A-25 resin showed the two most potent alpha-glucosidase inhibitory fractions i.e., A and B with the IC50 value of 16.5 and 15.6 mg/mL, respectively [76]. Two peptides Val-Trp (IC50 = 22.6 mM) and Tyr–Tyr–Pro–Leu (IC50 = 3.7 mM) were identified from sardine muscle hydrolysates (Table 1) [99]. Lavigne et al. [100, 101] reported that cod proteins could improve glucose tolerance and insulin sensitivity in high fat-fed rats, and their studies showed that this might be attributed to certain amino acids. Huang and Wu [102] purified and characterized an antidiabetic peptide from shark liver which reduced the fasting plasma glucose level in diabetic mice.

Table 1 Food protein-derived peptides with alpha-glucosidase inhibitory activity
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Peptides from egg white protein hydrolysates displayed the potential alpha-glucosidase inhibitory activity. Among the eight synthetic peptides, two peptides, Arg–Val–Pro–Ser–Leu–Met and Thr–pro–Ser–Pro–Arg, demonstrate higher alpha-glucosidase inhibitory activity with an IC50 values at 23.07 and 40.02 µmol/L, respectively (Table 1). These results showed that the potential of bioactive peptides from the egg white protein exhibiting the alpha-glucosidase inhibitory activity could be considered as an ingredient for functional food product with the antidiabetic activity [85]. In another study, the peptides showed antidiabetic activity against alpha-glucosidase. Among the peptides, Lys–Leu–Pro–Gly–Phe displayed the highest inhibitory activity against alpha-glucosidase with the IC50 values of 59.5 µmol/L (Table 1) [103].

Milk protein-derived bioactive peptides inhibiting alpha-glucosidase enzyme

One of the studies conducted in vitro showed that milk and soy milk fermented with L. bulgaricus and L. acidophilus was used for the management of hyperglycemia linked to T2D. During the fermentation process, the alpha-glucosidase inhibition was increased, indicating that possible positive effect was due to the milk-derived bioactive peptides [74]. In another study, whey protein isolate (WPI), α-lactalbumin, β-lactoglobulin, serum albumin, and lactoferrin hydrolysates obtained by peptic digestion were investigated for their potential to serve as natural sources of alpha-glucosidase inhibitors. The peptides generated from whey proteins have dual beneficial effects on glycemic regulation and could be used as functional food ingredients for the management of T2D [33]. The exopolysaccharides and insulin-containing yogurt showed better alpha-glucosidase inhibitory activity vis-a-vis the control yogurt [104]. The alpha-glucosidase inhibitory activity was found in peptic treated whey protein hydrolysates at 2.50 mg/mL. Peptic digested β-lactoglobulin and WPI hydrolysates displayed the highest alpha-glucosidase inhibition, i.e., 33 and 36 %, respectively. The IC50 values for the β-lactoglobulin and WPI hydrolysates were 3.5 and 4.5 mg/mL, respectively (Table 2) [33]. In addition, the alpha-glucosidase inhibitory activity observed in the whey protein hydrolysates obtained by the use of serine protease was isolated from Asian pumpkins. The peptides from six fractions of WPC-80 hydrolysates with a molecular mass below 3 kDa, showed highest inhibitory activity with the IC50 values lower than 2.0 mg/mL (Table 2) [34].

Table 2 Alpha-glucosidase inhibitory activity of milk protein hydrolysates
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Dipeptidyl peptidase-IV (DPP-IV) enzyme inhibitory peptides

Dipeptidyl peptidase IV (DPP-IV/CD26; EC.3.4.14.5) is a multifunctional transmembrane glyco-protein, involved in different biological processes. It is a 766–amino acid serine protease that contains N-terminal dipeptidases activity, which selectively cleaves dipeptides after proline or alanine residues [105]. It is originally known as the lymphocyte cell surface marker CD26, or as the adenosine deaminase (ADA)-binding protein. The DPP-IV enzyme is widely distributed in human organs and tissue, including exocrine pancreas, sweat glands, salivary and mammary glands, thymus, lymph nodes, intestines and biliary tract, kidney, liver, placenta, uterus, prostate, brain, blood cells, and skin. DPP-IV is present in almost all organs in the body through the attachment to the plasma membrane of the endothelia [106]. The enzyme DPP-IV plays numerous roles in several physiological processes, including enzymatic incretin degradation, including immune and endocrine activity, cell adhesion, and dampening of cancer growth [107, 108]. DPP-IV is renowned for its inactivation of incretin hormones GLP-1 and GIP (Fig. 4).

Fig. 4
figure 4

Functions of the incretin active hormones and Relation between the incretin hormones and dipeptidyl peptidase IV (DPP-IV) activity

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Many studies reveal that hydrolysate and peptides from different proteins, including those from egg [109], fish [110, 111], amaranth [112], rice bran [113], corn [114], and milk [70, 71, 115–118] have been found to be the source of the DPP-IV inhibitors. Among those proteins, peptides derived from the milk protein i.e., whey and casein fractions of milk have been found to display in vitro DPP-IV inhibitory activity and in vivo reduction in blood glucose level [33]. The new strategy for enhancing incretin action consists in the administration of foods with the potential to inhibit intestinal DPP-IV to prevent incretin degradation [52, 119].

The incretin hormones, GLP-1 and GIP, are gut hormones released into plasma from K cells in the duodenum and L cells in the intestine mucosa, respectively, after the ingestion of food [120]. The incretin hormones enhance meal-induced insulin secretion from β-cells of the islets of langerhans into the bloodstream in glucose-dependent manner and play an important part in the maintenance of normal glucose homeostasis [121–123]. They also have a role in suppression of pancreatic glucagon release, delay gastric emptying, and modulate appetite [124, 125]. Released after the ingestion of the meal, the concentration of active endogenous GLP-1 and GIP increases from two- to threefold. These two hormones enhance approximately 60 % of the postprandial insulin release [126]. The DPP-IV enzyme is responsible for the degradation of both incretin hormones GIP and GLP-1, as these gut-derived hormones are endogenous physiological substrates of DPP-IV [127]. However, both GIP and GLP-1 have very short half-lives (<7 and 2 min, respectively) due to the action of DPP-IV enzyme [106].

A DPP-IV inhibitor enhances the exogenous and endogenous GLP-1 and GIP action by blocking their N-terminal degradation, and hence results in inactivation [128]. It was found that in patients with T2D, there is a decrease in the incretin response, resulting in decreased insulin secretion, increased postprandial glucagon levels, and elevated postprandial glucose [129]. DPP-IV inhibitors extend the half-life and increase the concentrations of circulating intact (active) incretins [130, 131]. As a result, the increased level of incretin causes the inhibition of glucagon, which in turn elevates insulin secretion, diminishes gastric emptying, and decreases blood glucose levels (Fig. 5). Therefore, a DPP-IV inhibitor can improve glucose tolerance, by augmenting the incretin effect in patients with T2D [120, 132]. It was found that canary seed peptides obtained by gastrointestinal digestion showed an inhibition of DPP-IV in a dose-dependent manner; the highest inhibition (43.4 %) was observed at peptide concentration of 1.4 mg/mL. However, the non-hydrolyzed proteins showed a low inhibition (9.3 %). This showed that peptides that are released during gastrointestinal digestion have an effect on blood glucose levels [133]. The presence of Leu and Pro in second and third position in the peptides sequence suggested a potential inhibitory role of these peptides; the flanking amino acids could affect the interaction between them and the enzyme [134]. Most of the DPP-IV inhibitors are competitive in nature with some exceptions. The peptides from amaranth 11S globulin interact with the active site pocket of DPP-IV, thereby blocking access to the substrate. Three peptides larger than 13 residues prevented the formation of the dimeric active form of DPP-IV, resulting in enzyme inhibition [112]. DPP-IV inhibitors signify a new class of oral antihyperglycemic agents to treat patients with T2D [135].

Fig. 5
figure 5

Mechanism of action of DPP-IV inhibitors

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Food protein-derived bioactive peptides inhibiting DPP-IV enzyme

The peptide derived from the Atlantic salmon skin gelatin showed DPP-IV inhibitory activity and the activity was dependent on the type of enzyme used to generate peptides [110]. The three proteases used in the study were alcalase, bromelain, and flavourzyme. At the concentration of 5 mg/mL, the Flavourzyme hydrolysates showed the greatest DPP-IV inhibition activity, i.e., 45.2 % as compared to alcalase and bromelain hydrolysates with the same E/S ratio. Further, hydrolysates fractionation by ultrafiltration gives peptides within the <1 kDa range. This UF fraction had the greatest (61.2 %) DPP-IV inhibitory activity and IC50 value of 1.35 mg/mL. The <1 kDa UF fraction was further separated by RP-HPLC, and most potent F-1 fraction had the highest DPP-IV inhibition rate of 68 % with IC50 value of 0.57 mg/mL. Two peptides Gly–Pro–Ala–Glu and Gly–Pro–Gly–Ala (Table 3) were identified in F-1 fraction, which had IC50 values of 49.6 and 41.9 μM, respectively. In another study, marine collagen peptides (MCPs) from fish hydrolysate in Chinese patients with T2D significantly reduced levels of fasting blood glucose, human glycated hemoglobin A1c (GHbA1c), and fasting blood insulin [136]. Nutripeptin™ a product containing cod hydrolysate and Fortidium Liquamen® a white fish (Molva molva) autolysate are commercialized as postprandial blood glucose-lowering food supplements [137].

Table 3 Food-derived peptides with dipeptidyl peptidase IV inhibitory activity
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Proteolytic or microbial enzymatic hydrolysis of East Asian azuki bean produces hydrolysates with DPP-IV inhibitory activity. The hydrolysate from 10 kDa UF permeate had 52 % DPP-IV inhibition at 1.0 mg/mL, which was generated using Umanizyme G® [138]. The glutelins, main protein fraction of amaranth seeds, contain the peptide with DPP-IV inhibitory activity [139]. Tryptic digested amaranth hydrolysates show the decreased DPP-IV inhibitory activity in a dose-dependent manner with an IC50 ranging from 1.2 to 2.0 mg/mL. The removal of fragments larger than 10 kDa was done by ultrafiltration, and the IC50 value of the ultrafiltrated samples ranged from 1.0 to 1.6 mg/mL. It indicated that peptides smaller than 10 kDa were able to inhibit the DPP-IV enzyme. In silico analysis identified four peptides in amaranth globulins [11S amaranth globulin f (1–13), f (18–39), f (69–81), f (92–143)] and which may be responsible for the DPP-IV inhibitory activity [112]. Hen’s egg hydrolysates also showed the inhibitory activity against the DPP-IV and the activity dependent on the specificity of the enzyme used [125]. The peptides from the rice bran showed the DPP-IV inhibitory activity [113]. It was found that peptides obtained by using the enzyme Umamizyme G were 11 times more effective in inhibiting DPP-IV than those obtained with Bioprase SP and had IC50 values of 2.3 and 26.4 mg/mL, respectively. Further purification of Umamizyme G hydrolysates found two DPP-IV inhibitory dipeptides (Leu–Pro and Ile–Pro) (Table 3).

DPP-IV inhibitory activity was found in tuna cooking juice hydrolysates, and it was produced using two fungal endoproteinases in order to generate DPP-IV inhibitory peptides. Hydrolysates showed the highest inhibition rate of 45.2 % at the enzyme concentration of 2 mg/mL and after 1 h hydrolysis. Further purified by gel filtration chromatography, the highest DPP-IV inhibitory activity was 40 % at 5 mg/mL, and after reversed-phase HPLC, the inhibition rate was ~60 % at 5 mg/mL in order to enrich the peptides with DPP-IV inhibitory activity. Three peptides (Pro–Gly–Val–Gly–Gly–Pro–Leu–Gly–Pro–Ile–Gly–Pro–Cys–Tyr–Glu, Cys–Ala–Tyr–Gln–Trp–Gln–Arg–Pro–Val–Asp–Arg–Ile–Arg and Pro–Ala–Cys–Gly–Gly–Phe–Tyr–Ile–Ser–Gly–Arg–Pro–Gly) isolated from a tuna cooking juice hydrolysate (Table 3). Three peptides showed the IC50 values between the 78 and 116 µM. However, peptides obtained in the study comprised 13–15 amino acid residues which were much longer than the preferable DPP-IV inhibitory peptides. The result reveals that the DPP-IV inhibitory activity is dependent on the composition and sequence of amino acids, but not the length [111].

Peptides derived from the porcine skin gelatin hydrolysates showed in vitro inhibitory activity against the DPP-IV. It was found that the inhibitory activity was increased with the E/S ratio and hydrolysis time. The results exhibited the greater DPP-IV inhibitory activity of hydrolysates with the smaller size of peptides due to the higher degree of hydrolysis. The hydrolysates obtained with the E/S ratio of 3 % and 4 h hydrolysis at the rate of 1 mg/mL were further fractionated by ultrafiltration with different cutoff membranes. The DPP-IV inhibition was highest (30.7 %) for the <1 kDa UF fraction as compared to other UF fractions. As the small-sized peptides passed through the digestive tract without degradation, therefore, <1 kDa UF fraction was selected for further study. The IC50 value of the <1 kDa fraction was 1.50 mg/mL. This fraction was further purified by HPLC, and the most potent F-3 fraction showed the highest DPP-IV inhibition rate of 64.6 % and the IC50 value was 62.9 μg/mL. Within these two peptides, i.e., Gly–Pro–Hyp and Gly–Pro–Ala–Gly were identified in F-3 fraction (Table 3), which inhibited DPP-IV. There were a range of structural resemblance between the two peptides, each peptide contained Pro as the second N-terminal residue, and Pro residue was flanked by Ala and Gly, and moreover, the peptides were composed of mostly hydrophobic amino acid residues [140].

Milk protein-derived bioactive peptides inhibiting DPP-IV enzyme

Casein contains a large amount of Pro residues, which increases the vulnerability to cleavage by DPP-IV and potential for the release of peptides with DPP-IV inhibitory activity. Lacroix and Li-Chan [117] examined the potential of dietary proteins from various food commodities to serve as precursors of DPP-IV inhibitors by using an in silico approach. Caseins from cow’s milk emerged to be the richest potential sources of DPP-IV inhibitors. The β-casein was found to have the greatest potential among milk proteins to serve as a source of DPP-IV inhibitors. A computer-aided analysis of milk proteins as sources of bioactive peptides [141] identified β-casein as the most promising milk protein precursor of DPP-IV inhibitory peptides. On the contrary, alpha-lactalbumin contained a limited number of peptides with DPP-IV inhibitory activity. Dairy protein hydrolysates examined for DPP-IV inhibitory activity. With the time, in vitro pepsin–pancreatin hydrolysis enhances the DPP-IV inhibitory activity of sodium caseinate, skim milk powder, and milk proteins concentrate hydrolysates. The maximum DPP-IV inhibitory activity (IC50 of 0.075 mg/mL) was found in Whey protein isolate (WPI) hydrolysate following peptic digestion. With the use of different proteases, hydrolysates generated from sodium caseinate showed the highest inhibitory activity than the majority WPI hydrolysates [69]. Milk protein-derived hydrolysates and dipeptides exhibited the inhibitory activity against the DPP-IV. Eight dipeptides showed the inhibitory activity, and the IC50 values for the dipeptides were ranging from 65.29 to 3216 µM (Table 5). Among all potent peptides, Trp–Val was found to be most potent, but paradoxically, the reverse peptide did not inhibit the DPP-IV. This indicated that N-terminal residue of peptide plays an important role in the inhibition of DPP-IV. Lactoferrin, casein, and WPH hydrolysates showed the inhibitory activity against DPP-IV. Of these, lactoferrin and casein hydrolysates were the most potent inhibitors (Table 4). The result indicated that both hydrolysates and dipeptides acted as competitive inhibitors of DPP-IV [21]. Antihyperglycemic effect was found in water-soluble extract of Gouda-type cheese, and several types of DPP-IV inhibitory peptides were identified (Table 5). Of all, cheese octapeptide Leu–Pro–Gln–Asn–Ile–Pro–Pro–Leu (β-CN f70-77) showed the highest inhibitory activity (IC50 of 46 µM), which increased during ripening period and played an important role in inhibiting DPP-IV in vivo [71]. Patent WO 2006/068480 [142] has demonstrated that the small peptides from casein hydrolysates possessed DPP-IV inhibitory activity.

Table 4 DPP-IV inhibitory activity of milk protein hydrolysates
Full size table
Table 5 Milk protein-derived peptides with dipeptidyl peptidase IV inhibitory activity
Full size table

Whey protein hydrolysates also showed peptides with DPP-IV inhibitory activity. The DPP-IV inhibitory activity of β-lactoglobulin hydrolysates from goat/sheep and bovine has been compared. Combined in silico examination and in vitro studies verified that caprine/ovine whey is abundant in the content of short peptides carrying weak DPP-4 inhibitory activity than bovine whey [115]. The bioactive peptide Ile-Pro-Ala (IPA) from bovine β-lactoglobulin showed inhibitory activity against the DPP-IV with IC50 value of 49 µM (Table 5) [70]. Similarly, the hexapeptide was generated from trypsin-treated β-lactoglobulin and identified as Val–Ala–Gly–Thr–Trp–Tyr (β-lactoglobulin f15–20) (Table 5). This hexapeptide showed a concentration-dependent DPP-IV inhibitory activity with IC50 value of 174 µM [116]. The most potent fractions from whey protein hydrolysates were the 2 kDa permeate with the lowest IC50 value of 0.48 mg/mL (Table 4). When WPH was subjected to stimulated gastrointestinal digestion, there was a significant decrease in the IC50 value i.e., 1.02 mg/mL [118]. The study demonstrated the release of DPP-IV substrate like peptide sequences by gastrointestinal enzymes using an in silico digestion of the milk proteins, and these peptides behave as a substrate or prodrug-type inhibitors to DPP-IV. The DPP-IV inhibitory activity was determined for the identified peptides (Table 5). The lowest IC50 value was found for the Ile–Pro–Ile (3.4 µM) [143]. In addition, peptides from trypsin-treated β-lactoglobulin showed DPP-IV inhibitory activity (Table 5). The notable DPP-IV inhibitory activity was shown by the peptide Ile–Pro–Ala–Val–Phe with IC50 value of 44.7 µM [144]. Whey protein hydrolysates obtained by peptic hydrolysis exhibited the inhibitory activity against the DPP-IV (Table 4). The maximum DPP-IV inhibitory activity was shown by the α-lactalbumin and WPI hydrolysates, with 91 and 82 % inhibition, whereas the IC50 value measured for both was found in the same order of magnitude, i.e., 0.036 mg/mL [33]. In another study, the DPP-IV inhibitory activity was shown by the peptides from the hydrolysates of β-lactoglobulin and WPC-80 obtained by serine protease isolated from C. ficifolia. The peptide fractions below 3 kDa from WPC-80 hydrolysates showed the highest inhibition with IC50 value <0.55 mg/mL (Table 4) [34].

Production of antidiabetic peptides from milk protein

Although consumption of low-fat milk and dairy products have a beneficial effect on the prevention or treatment of T2D [145], research has been focused on milk protein-derived peptides. Peptides may be released from their parent protein by following four ways [26, 146]:

  1. 1.

    Enzymatic hydrolysis during gastrointestinal digestion

  2. 2.

    Fermentation of milk with proteolytic starter cultures

  3. 3.

    Hydrolysis by enzymes obtained from microorganisms or plants

  4. 4.

    Combination of fermentation and hydrolysis

If the sequence of the peptide is known, it is also possible to synthesize peptides by chemical route, recombinant DNA technology, or enzymatic amide synthesis [147, 148]. After releasing from origin, bioactive peptides must reach the target receptor in the intestinal lumen or in other peripheral organs, passing via the systematic circulation [25].

Gastrointestinal digestion

During gastrointestinal digestion, physiologically active peptides are produced from several milk proteins. Hydrolysis may occur in various stages after ingestion of the protein. In the gastrointestinal tract, ingested proteins are hydrolyzed by gastrointestinal enzymes, usually pepsin, trypsin, chymotrypsin, and other membrane peptidases [23]. Other digestive enzymes and different enzyme combinations of proteinases, i.e., alcalase, chymotrypsin, pancreatin, pepsin, and thermolysin have also been utilized to produce bioactive peptides from various proteins [149, 150]. Absorption of peptides can be through the gastrointestinal wall by different mechanisms, such as by passive diffusion through the enterocytes, and para-cellularly through cytosis or through a carrier [151]. After absorption, bioactive peptides must reach their target sites at the luminal side of the intestinal tract or at specific peripheral organs to exert their physiological effects [152].

It has been hypothesized that peptides released from whey proteins during their transit through gastrointestinal tract might be responsible for the postmeal glycemic response produced after whey intake [48]. It has been suggested that the antidiabetic property of whey proteins is mainly due to its content of bioactive peptides which, following their release during gastrointestinal digestion, could stimulate the secretion of gut-derived hormones and/or inhibit enzymes involved in glycemic homeostasis [38, 39].

Microbial fermentation

Lactic acid bacteria utilize milk protein as their key source of essential and growth-stimulating amino acids [153]. Many industrially important dairy starter cultures are highly proteolytic in nature. Bioactive peptides can, thus, be generated by the proteolytic activities of the strains of starter and non-starter lactic acid bacteria (LAB) in fermented dairy products [154]. Besides, to live microorganisms, proteolytic enzymes isolated from LAB have also been successfully employed to release bioactive peptides from milk proteins. The production of a variety of bioactive peptides in fermented dairy products, e.g., yoghurt, sour milk, and dahi has been well documented in many studies [151]. Apostolidis et al. [74] observed that milk and soy milk fermented with L. bulgaricus and L. acidophilus generated α-glucosidase inhibitory activity. The activity suggested that peptides were responsible for the management of hyperglycemia linked to T2D. Uenishi et al. [71] identified several DPP-IV inhibitory peptides from Gouda cheese. The DPP-IV inhibitory activity was observed in the water-soluble fraction of a Gouda-type cheese, and this inhibitory activity increased during ripening. This inhibitory activity positively correlated with the blood glucose improving effect of Gouda cheese.

Consumption of water kefir in (10–30 %) concentrations for 5 weeks has shown beneficial effect on blood glucose level in animal model [155]. Similarly, the water-soluble fraction of Kefram–Kefir showed glucose uptake in skeletal muscle cells specifically in vitro [156]. In another study, the probiotic fermented milk (kefir) consumption causes the decline of fasting blood glucose and Glycated hemoglobin (HbA1C) in comparison with conventional fermented milk [157]. The results of Maeda et al. [158] showed that kefiran had hypoglycemic effects in KKAy mice. Fermented soymilk extract showed the suppression of postprandial blood glucose levels in diabetics through inhibition of alpha-glucosidase and alpha-amylase [159]. The probiotic dahi-supplemented diet significantly delayed the onset of glucose intolerance, hyperglycemia, and hyperinsulinemia in high-fructose-induced diabetic rats which indicates a lower risk of diabetes [160].

Enzymatic hydrolysis

The most common way to obtain bioactive peptides in vogue is by enzymatic hydrolysis of protein [23]. Proteases derived from microorganisms, animals, or plants have also been successfully employed in the proteolytic process to release peptides from milk proteins. In addition, enzyme combinations including alcalae, chymosin, pancreatin, trypsin, and thermolysin can be used to release bioactive peptides [146]. The commonly used enzymes of plant and animal origin are α-chymotrypsin, papain, neutrase, thermolysin, pepsin, alcalase, pronase, carboxypeptidase A, and trypsin [23, 161, 162]. Generation of DPP-IV inhibitory milk peptides by enzymatic hydrolysis from milk protein are more studied [21, 69, 70, 116]. Similarly, alpha-glucosidase inhibitory milk peptides have been produced from the milk protein by enzymatic hydrolysis [33, 34].

Clinical studies

So far, only some human studies have evaluated the role of milk protein hydrolysates or peptides in T2D. The impact of co-ingestion of intact or hydrolyzed protein with carbohydrate on postprandial plasma insulin and glucose responses was evaluated in T2D patients. Patients participated in a study received the single bolus of carbohydrate (0.7 g/kg: CHO) with or without an intact casein protein (0.3 g/kg: PRO) or its hydrolysate (0.3 g/kg: PROh). Results showed that protein co-ingestion strongly increased postprandial insulin release, with the insulin response +99 and +110 % greater in the CHO + PRO and CHO + PROh experiments when compared with the CHO experiment. The concomitant plasma glucose responses were 22 and 23 % lower in the CHO + PRO and CHO + PROh experiments, respectively [163]. Similarly, 11 long standing T2D patients and 11 healthy control subjects received a beverage containing casein hydrolysate (0.3 g/kg)/leucine (0.1 g/kg) mixture (PRO) or a placebo (PLA). In the PRO trial, glucose was significantly lower compared with PLA trial at 24 h. PRO resulted in a 11 % decline in the overall glucose response in diabetic patients [53].

Meric et al. [164] study the effect of four different beverages containing 6 % w/v whey protein isolate (WPI), whey protein hydrolysate (WPH), soy protein isolate (SPI), and 2.66 % WPI or a control (no protein added) on the glucose and insulin response in 25 healthy men. Result demonstrated that only beverages containing 6 % (w/v) of whey protein increased insulin response and decreased glucose level compared with control. Furthermore, in the Goudarzi and Madadlou [165] study, they demonstrated that whey protein hydrolysates showed distinctly better effectiveness in blood glucose control than intact whey protein. The effect of WPH on blood glucose level might be due to faster digestion and availability of its insulinotropic peptides and amino acids in the blood [166].

There are increasing evidences that milk protein, protein hydrolysates or bioactive peptides, and amino acids could be used as valuable nutritional strategies to improve blood glucose levels in T2D patients. Milk protein-derived peptides are commercially available as functional food, food supplements, and nutraceuticals with certain health claims. There is a need to validate the associated functional attributes of milk protein-derived alpha-glucosidase and DPP-IV inhibitors through well-designed clinical trials. At the same time, the challenge to the food industry remains to incorporate these bioactive peptides without adversely affecting the sensory profile, convenience, bioavailability, and safety.

Conclusions

Epidemiological evidences reveal that a diet rich in dairy products has been associated with the prevention and treatment of metabolic-related disorders, while evidence from observational studies points toward milk protein-derived peptides as dietetic ingredients which may aid prevention of T2D. One of the new promising therapeutic strategies for the treatment of T2D is the use of inhibitors of alpha-glucosidase and DPP-IV. However, synthetic drug inhibitors have been associated with some side effects and limitations. Therefore, there is an increasing attention toward natural, safe, food-derived peptide inhibitors as these are without any side effects. Milk protein-derived peptides with alpha-glucosidase and DPP-IV inhibitory traits potentially regulate the postprandial hyperglycemia in healthy and T2D subjects by inhibiting both the inactivation of the incretin hormones and the carbohydrate hydrolyzing enzymes. These antidiabetic peptides are liberated from milk protein by gastrointestinal digestion of milk, fermentation of milk with proteolytic starter cultures, or hydrolysis by proteolytic enzymes from plant or animal. Milk protein-derived alpha-glucosidase and DPP-IV inhibitory peptides could be used as ingredients for functional food, nutraceuticals, and pharmaceuticals applications for the management of T2D. However, there is a need to unravel the molecular mechanisms of action of these peptides on alpha-glucosidase and DPP-IV. Besides, clinical studies are necessary to validate the most in vitro and some in vivo data and to confirm the efficacy and bioavailability of milk protein-derived peptides in humans.