Metabolic Syndrome-Preventive Peptides Derived from Milk Proteins and Their Presence in Cheeses: A Review

: The metabolic syndrome (MetS) is deﬁned as the occurrence of diet-related diseases such as abdominal obesity, atherogenic dyslipidemia, hyperglycemia (insulin resistance) and hypertension. Milk-derived peptides are well-known agents acting against high blood pressure, blood glucose level, and lipoprotein disproportion. The aim of this review are metabolic syndrome-preventive peptides derived from milk proteins which were identiﬁed in cheeses. Special attention was paid to the sequences acting as angiotensin converting enzyme (ACE), dipeptidyl peptidase IV (DDP4), and α -glucosidase inhibitors, as well as antioxidative, hypocholesterolemic, antiobesity, and anti-inﬂammatory agents. Some results of meta-analyses concerning the consumption of cheese and the risk of MetS diseases were also presented.


Introduction
The metabolic syndrome (MetS) is defined as a clustering of four major cardiovascular risk factors, namely: abdominal obesity, atherogenic dyslipidemia, hyperglycemia (insulin resistance), and hypertension ( Figure 1) [1]. Moreover, the pathogenesis of MetS involves both genetic and acquired factors contributing to the inflammation [2]. MetS also represents a significant risk factor for the development of cardiovascular disease (CVD), type 2 diabetes mellitus (T2D), and all-cause mortality [1]. Moore et al. [3] analyzed data derived from the National Health and Nutrition Examination Survey (NHANES). Based on the analysis including the period from 1988 to 2012, MetS was defined as at least three conditions from these listed further that have to occur in a patient: elevated waist circumference, elevated level of triglycerides, reduced high-density lipoprotein cholesterol level, high blood pressure, and elevated fasting blood glucose level. According to the above-mentioned definition of MetS, in 2012 there were more than 66 million adults in the US with recognized MetS symptoms. They were observed in every sociodemographic group [3].  Huang [4].
A cohort study made in 2018 on 136,384 individuals from 21 countries on five continents demonstrated that dairy product consumption (especially of milk and yogurt) can be associated with a lower risk, of not only CVD, but also mortality. According to this study, even one serving of milk or yogurt significantly lowered the risks of the above-mentioned events. Additionally, the higher consumption of dairy products can be associated with significantly lower risk of stroke [10]. According to another study, every 200 mL increase of daily milk consumption reduced the risk of CVD by 6%. Based on the data from 2015 it was shown that three main milk constituents were responsible for the MetS-preventive activity, namely calcium, fat, and proteins [11]. One of the MetSpreventive potentials of milk and dairy products resulting from the presence of proteins might be related to the action of biologically active peptides [5]. It also reflects vast information found in. e.g., Scopus database [www.scopus.com]. Providing the following words as the input query "ALL (metabolic AND syndrome AND milk AND peptides)" revealed 7459 hits (accessed in September 2019) .
The first mentions about cheese manufacturing appeared around 5500 BC. Early adult humans were unable to digest lactose. Thus, making cheese was a good way to consume health beneficial nutrients derived from milk, without getting ill [11]. The casein, main milk protein, during cheese maturation is hydrolyzed into a variety of peptides. Their composition varies and depends on the activity of endogenous proteases and peptidases of milk, rennet, starter culture, and microbial flora [12]. Bovine casein has been shown to possess a variety of bioactivities, including MetS-preventive ones. In silico analyses and reviews conducted on bovine casein confirmed the casein-derived peptides to exhibit these activities [13,14]. Hence, considering the increasing role of biopeptides in the prevention of MetS, the aim of this paper was to describe MetS-preventive peptides derived mainly from bovine milk proteins which were also identified in the ripened cheeses. The main attention was paid to the peptides exerting antidiabetic, antihypertensive, antioxidative, cholesterolreducing, antiobesity, and anti-inflammatory effects.
Milk is the first functional food for every mammalian species. The contents of its nutrients, such as proteins (and their proportions), fat, sugar, vitamins, and minerals, vary between species as well as breeds. Moreover, milk composition is affected by feeding methods, age of an animal, and many other [9]. Milk and dairy products were also studied as anti-metabolic syndrome foods [10].
A cohort study made in 2018 on 136,384 individuals from 21 countries on five continents demonstrated that dairy product consumption (especially of milk and yogurt) can be associated with a lower risk, of not only CVD, but also mortality. According to this study, even one serving of milk or yogurt significantly lowered the risks of the above-mentioned events. Additionally, the higher consumption of dairy products can be associated with significantly lower risk of stroke [10]. According to another study, every 200 mL increase of daily milk consumption reduced the risk of CVD by 6%. Based on the data from 2015 it was shown that three main milk constituents were responsible for the MetS-preventive activity, namely calcium, fat, and proteins [11]. One of the MetS-preventive potentials of milk and dairy products resulting from the presence of proteins might be related to the action of biologically active peptides [5]. It also reflects vast information found in. e.g., Scopus database [www.scopus.com]. Providing the following words as the input query "ALL (metabolic AND syndrome AND milk AND peptides)" revealed 7459 hits (accessed in September 2019).
The first mentions about cheese manufacturing appeared around 5500 BC. Early adult humans were unable to digest lactose. Thus, making cheese was a good way to consume health beneficial nutrients derived from milk, without getting ill [11]. The casein, main milk protein, during cheese maturation is hydrolyzed into a variety of peptides. Their composition varies and depends on the activity of endogenous proteases and peptidases of milk, rennet, starter culture, and microbial flora [12]. Bovine casein has been shown to possess a variety of bioactivities, including MetS-preventive ones. In silico analyses and reviews conducted on bovine casein confirmed the casein-derived peptides to 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are hormones that decrease blood glucose levels by stimulating insulin secretion and inhibiting glucagon release. GIP serves several additional functions including promotion of growth and survival of the pancreatic beta-cell and stimulation of adipogenesis [15]. This effect is hindered by DPP4, therefore inhibition of DPP4 prolongs the incretin effect and lowers the postprandial blood glucose level ( Figure 2) [16].

Figure 2.
The involvement of the dipeptidyl peptidase IV inhibitors in prolonging the incretin effect according to Mkele [16].
Antidiabetic properties of peptides may also result from their ability to inhibit α-glucosidase (EC 3.2.1.20). α-Glucosidase is an enzyme located in the brush border of enterocytes of the jejunum in the small intestine and is a key enzyme in carbohydrate breakdown. It cleaves carbohydrate to easy absorbable glucose. Thus, one of the T2D preventive strategies is to decrease the postprandial hyperglycemia by delaying glucose absorption through the inhibition of α-glucosidase ( Figure 3). α-Glucosidase inhibitors also play a crucial role in the secretion of GLP-1 in healthy and diabetic patients, due to the prolonged incretin effect [18].
α-Amylase is another enzyme involved in the regulation of the blood sugar level [5]. This enzyme is responsible for the initial hydrolysis of the α-D-(1-4) glycosidic bonds of polysaccharides like glycogen and starch to oligosaccharides. Then, oligosaccharides are further hydrolyzed into monosaccharides by α-glucosidase (see above), which are next transferred into bloodstream [5].
Some milk proteins were reported as the sources of α-glucosidase inhibitors. Whey proteins: αlactalbumin, β-lactoglobulin, serum albumin, and lactoferrin hydrolysates obtained by peptic digestion were confirmed to exert such an inhibiting effect. β-Lactoglobulin and whey protein isolate hydrolysates were found to inhibit α-glucosidase (IC50 value of 3.5 and 4.5 mg x mL -1 , respectively) Antidiabetic properties of peptides may also result from their ability to inhibit α-glucosidase (EC 3.2.1.20). α-Glucosidase is an enzyme located in the brush border of enterocytes of the jejunum in the small intestine and is a key enzyme in carbohydrate breakdown. It cleaves carbohydrate to easy absorbable glucose. Thus, one of the T2D preventive strategies is to decrease the postprandial hyperglycemia by delaying glucose absorption through the inhibition of α-glucosidase ( Figure 3). α-Glucosidase inhibitors also play a crucial role in the secretion of GLP-1 in healthy and diabetic patients, due to the prolonged incretin effect [18].
α-Amylase is another enzyme involved in the regulation of the blood sugar level [5]. This enzyme is responsible for the initial hydrolysis of the α-D-(1-4) glycosidic bonds of polysaccharides like glycogen Appl. Sci. 2020, 10, 2772 4 of 36 and starch to oligosaccharides. Then, oligosaccharides are further hydrolyzed into monosaccharides by α-glucosidase (see above), which are next transferred into bloodstream [5].
Some milk proteins were reported as the sources of α-glucosidase inhibitors. Whey proteins: α-lactalbumin, β-lactoglobulin, serum albumin, and lactoferrin hydrolysates obtained by peptic digestion were confirmed to exert such an inhibiting effect. β-Lactoglobulin and whey protein isolate hydrolysates were found to inhibit α-glucosidase (IC 50 value of 3.5 and 4.5 mg × mL −1 , respectively) [18]. Dipeptide, like arginine-proline (RP), derived from whey proteins and being the component of commercial Pep2Dia ® product, affected the inhibition of α-glucosidase with an IC 50 value of 0.0025 mg × mL −1 [19]. To the best of our knowledge, there is no research papers about α-amylase inhibitors identified milk proteins and dairy products. Only one article concerned cheese as a source of α-glucosidase inhibitors. According to Grom et al. [20], Prato cheese inhibited α-glucosidase activity in vitro by 52.6%. Although, it was concluded that the consumption of probiotic Prato cheese can contribute to the reduction of postprandial glycemia in healthy individuals, but no peptide was identified [20].
Appl. Sci. 2020, 10, x FOR PEER REVIEW 4 of 39 vitro by 52.6%. Although, it was concluded that the consumption of probiotic Prato cheese can contribute to the reduction of postprandial glycemia in healthy individuals, but no peptide was identified [20]. It was evidenced on mice that peptides from cheese effectively decreased glucose level. Some epidemiological surveys showed a slightly higher risk of T2D development with increasing cheese consumption, but most of the meta-analyses concluded that increasing cheese consumption was correlated with a significant decreasing in T2D development risk [21]. Another epidemiological and cohort studies showed that dairy products can be beneficial in type 2 diabetes prevention. The latest research carried out on 34,224 men in the Health Professionals Follow-Up Study (1986 -2012), 76,531 women in the Nurses' Health Study (1986 -2012), and 81,597 women in the Nurses' Health Study II (1991 -2013) suggested that the decrease of total dairy intake by > 1.0 serving per day over a period of 4 years was associated with an 11% increase of T2D development risk [22]. However, there were some opposite remarks to these above. For example, it was stated that increasing cheese consumption by > 0.5 serving per day, caused a 9% increase of T2D development risk, when compared to the maintained, stable cheese intake [22]. In turn, these findings were not consistent with other studies. For example, the meta-analysis made by Gao et al. [23], concerning seven studies with a total of 178,429 subjects and 14,810 cases, led to opposite conclusions. They found an inverse linear correlation between the consumption of cheese and the risk of T2D development. The consumption of 30 g per day cheese decreased the relative risk (RR) of T2D development (RR 0.80 ± 0.12). The obtained results suggested that a modest increase in cheese consumption might prevent from T2D but further research is needed [23]. Another eight cohort studies concerning the meta-analysis of It was evidenced on mice that peptides from cheese effectively decreased glucose level. Some epidemiological surveys showed a slightly higher risk of T2D development with increasing cheese consumption, but most of the meta-analyses concluded that increasing cheese consumption was correlated with a significant decreasing in T2D development risk [21]. Another epidemiological and cohort studies showed that dairy products can be beneficial in type 2 diabetes prevention. The latest research carried out on 34,224 men in the Health Professionals Follow-Up Study (1986-2012), 76,531 women in the Nurses' Health Study , and 81,597 women in the Nurses' Health Study II  suggested that the decrease of total dairy intake by > 1.0 serving per day over a period of 4 years was associated with an 11% increase of T2D development risk [22]. However, there were some opposite remarks to these above. For example, it was stated that increasing cheese consumption by > 0.5 serving per day, caused a 9% increase of T2D development risk, when compared to the maintained, stable cheese intake [22]. In turn, these findings were not consistent with other studies. For example, the meta-analysis made by Gao et al. [23], concerning seven studies with a total of 178,429 subjects and 14,810 cases, led to opposite conclusions. They found an inverse linear correlation between the consumption of cheese and the risk of T2D development. The consumption of 30 g per day cheese decreased the relative risk (RR) of T2D development (RR 0.80 ± 0.12). The obtained results suggested that a modest increase in cheese consumption might prevent from T2D but further research is needed [23]. Another eight cohort studies concerning the meta-analysis of dairy food consumption and its effect on the T2D risk revealed that the consumption of 50 g cheese per day decreased the risk of T2D development (RR 0.92 ± 0.07) and a non-linear negative correlation was observed between cheese consumption and T2D risk [24]. The cohort study conducted on 2809 participants for 12 years showed a negative dose-response relation between cheese consumption and risk of T2D development. Out of the 1867 participants with no T2D symptoms, 902 were diagnosed as prediabetic. Consumption of ≥ 4 servings of cheese per week lowered the risk of T2D incident by 70%. Less than one cheese serving per week was associated with a 63% reduction of T2D incidents. None of the abovementioned meta-analyses, however, considered what type of cheese caused the risk-reducing effect [25].

Peptides with Antihypertensive Activity
The regulation of blood pressure that takes place in the organism involves several modes of ACE action. They include renin-angiotensin, kinin-nitric-oxide, and neutral endopeptidase systems. The first one (RAS; renin-angiotensin system), also called renin-angiotensin-aldosterone system (RAAS), is considered as the major in blood pressure regulation. Briefly, renin (EC 3.4.22.15) activates angiotensinogen and releases angiotensin I. Then, ACE ( Figure 4) transforms angiotensin I into angiotensin II (known as potent vasoconstrictor) and induces the release of aldosterone. Moreover, the increase of the blood pressure caused by angiotensin II causes some dysfunctions like damages of body organs leading to e.g., heart and kidney failure. Thus, the action of ACE receptor blockers or ACE-inhibiting peptides contributes to the blood pressure reduction [26].
Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 39 None of the abovementioned meta-analyses, however, considered what type of cheese caused the risk-reducing effect [25].

Peptides with Antihypertensive Activity
The regulation of blood pressure that takes place in the organism involves several modes of ACE action. They include renin-angiotensin, kinin-nitric-oxide, and neutral endopeptidase systems. The first one (RAS; renin-angiotensin system), also called renin-angiotensin-aldosterone system (RAAS), is considered as the major in blood pressure regulation. Briefly, renin (EC 3.4.22.15) activates angiotensinogen and releases angiotensin I. Then, ACE ( Figure 4) transforms angiotensin I into angiotensin II (known as potent vasoconstrictor) and induces the release of aldosterone. Moreover, the increase of the blood pressure caused by angiotensin II causes some dysfunctions like damages of body organs leading to e.g., heart and kidney failure. Thus, the action of ACE receptor blockers or ACE-inhibiting peptides contributes to the blood pressure reduction [26]. Milk proteins have been reported as the reservoir of ACE inhibitors for many years [27]. The most known sequences are VPP and IPP identified in β-and κ-casein, respectively. Due to their antihypertensive potential tested in animals and humans, they became the components of commercial milk drink nutraceuticals such as Calpis, Evolus, and Valio [28]. VPP and IPP sequences were also identified in 36 cheeses of Swiss origin (Table 1). Depending on the type of Swiss cheese, the content of these two sequences varied. For VPP, it ranged from 0 to 224 mg per kg of cheese, whereas for IPP from 0 to 95.4 mg. The highest content of both peptides, VPP and IPP, was identified in the hard cheese "Hobelkäse from the Bernese Oberland" (224.1 and 95.4 mg × kg -1 , respectively). That cheese sample had the highest ACE inhibitory activity of all analyzed chesses, with an IC50 value of 2.6 mg × mL -1 [29]. Different concentrations of these two peptides in cheeses were probably related to milk pretreatment, cultures, scalding conditions, and ripening time. Thus, it is recommended to develop a reproducible cheese-making process to produce higher concentrations of these peptides that could be used for clinical trials [30].
The potential of some cheeses containing the high concentrations of VPP and IPP to reduce blood pressure was tested in spontaneously hypertensive rats (SHR). Blood pressure was monitored after Milk proteins have been reported as the reservoir of ACE inhibitors for many years [27]. The most known sequences are VPP and IPP identified in βand κ-casein, respectively. Due to their antihypertensive potential tested in animals and humans, they became the components of commercial milk drink nutraceuticals such as Calpis, Evolus, and Valio [28]. VPP and IPP sequences were also identified in 36 cheeses of Swiss origin (Table 1). Depending on the type of Swiss cheese, the content of these two sequences varied. For VPP, it ranged from 0 to 224 mg per kg of cheese, whereas for IPP from 0 to 95.4 mg. The highest content of both peptides, VPP and IPP, was identified in the hard cheese "Hobelkäse from the Bernese Oberland" (224.1 and 95.4 mg × kg −1 , respectively). That cheese sample had the highest ACE inhibitory activity of all analyzed chesses, with an IC 50 value of 2.6 mg × mL −1 [29]. Different concentrations of these two peptides in cheeses were probably related to milk pretreatment, cultures, scalding conditions, and ripening time. Thus, it is recommended to develop a reproducible cheese-making process to produce higher concentrations of these peptides that could be used for clinical trials [30].
The potential of some cheeses containing the high concentrations of VPP and IPP to reduce blood pressure was tested in spontaneously hypertensive rats (SHR). Blood pressure was monitored after 6 h since oral administration. It was observed that the decrease of systolic blood pressure (SBP) differed depending on the cheese variety. The level of the SBP reduction (mmHg) in SHR was as follows: −24.7 and −17.  According to the scientific reports, sequential motif located between 60 and 70 residue of β-casein is considered as strategic zone due to its ACE-inhibiting, immunostimulating, and opioid potential [32]. The example of peptide derived from β-casein showing dual bioactivity is β-casomorphin-7 (YPFPGPI; fragment 60-66) acting as opioid and ACE inhibitory agent [26]. De Noni and Cattaneo [33] identified this peptide in different dairy products including commercial cheeses like e.g., Brie, Cheddar, Gouda, Fontina, and Gorgonzola (see Table 2). Some ACE inhibitors which matched the sequences of caseins were identified in Spanish cheeses. Those peptides which sequences fulfilled the structural requirements for being a potent ACE inhibitors were synthesized [34]. Their structural characteristics included the presence amino acids like lysine, arginine or residues possessing hydrophobic side chains as well as C-terminal proline [35]. Finally, it was reported that eight peptides (out of 11) inhibited ACE. Among them DKIHP peptide was found as the most potent (IC 50 = 113.1 µM) but the addition C-terminal phenylalanine (DKIHPF) caused the dramatic reduction of its ACE-inhibiting potential (IC 50 = 2419.4 µM) [36]. The ACE-inhibiting activity of synthesized peptide DKIHP (source Manchego cheese; β-casein) was also measured using different protecting groups, namely tert-butyloxycarbonyl (Boc) and fluorenyl-methyloxycarbonyl (Fmoc). The IC 50 values for DKIHP were 113.18 and 577.92 µM, respectively. It was concluded that the differences above were attributed to the presence of less active conformers in the structure of the peptide [37]. The other sequences of ACE inhibitors identified in some Spanish cheeses are given in Table 3.  [30]. Tonouchi et al. [38] isolated two peptides (LQP and MAP) from Danish skimmed milk cheese. LQP was originally known as ACE inhibitor identified in α-zein hydrolyzed with thermolysin. MAP sequence was identified in β-casein and exhibited ACE inhibition (IC 50 = 0.8 µM) and antihypertensive effect in SHR. Significant hypotensive effect of MAP tripeptide was observed at 8 h since oral administration (−17.0 mm Hg; dose 3 mg × kg −1 body weight) [38].
The potential of cheeses as diet components that may affect the reduction of blood pressure is also in the focus of interest of the scientists aiming at meta-analyses [39]. Nilsen et al. [39] carried out a cross-sectional study to analyze the impact of the consumption of Gamalost cheese (a traditional Norwegian cheese) on the reduction of the risk of hypertension development. Based on data obtained from the questionnaires of 168 participants, the average intake of Gamalost servings per week was 2 and the average value of systolic and diastolic blood pressures was 128 and 78 mmHg, respectively.
The increase of Gamalost servings reduced the systolic blood pressure by 0.72 mm Hg. Moreover, it was concluded that blood pressure reduction related to Gamalost consumption was associated with the presence of ACE inhibitory peptides [39]. Crippa et al. [40] investigated the antihypertensive effect resulting from the consumption of Grana Padano cheese and observed that supplementation of Grana Padano cheese at a dose 30 g per day significantly lowered the systolic and diastolic blood pressure in mild-hypertensive subjects (-3.5 and-2.4 mm Hg, respectively) [40].
One of the meta-analyses concerned studies evaluating the association of CVD, coronary heart disease (CHD), and stroke with high vs. low cheese consumption. This analysis revealed that high cheese consumption lowered the relative risk for: total CVD (RR 0.90; 7; 8076), CHD (RR 0.86; 8; 7631), and stroke (RR 0.90; 7; 10,449). Respective numbers in the brackets represent: relative risk, number of studies, and number of documented events provided. Finally, the results indicated a nonlinear relationship between cheese consumption and decreasing risks of CVD, CHD, and stroke. Consumption of approximately 40 g of cheese per day ensured the best results in reducing the risks of dysfunctions related to hypertension [41].
To conclude, epidemiological studies suggested that the antihypertensive potential of cheeses can be linked to the presence of biopeptides. According to above-mentioned analyses, the optimal daily cheese dose to reduce the risk of hypertensive dysfunctions was 30-40 g. These results suggest that cheese-as part of a diet-can prevent from the hypertension-related diseases.

Antioxidative Peptides
Free radicals are involved in many signaling processes, i.e., as defending the body from infections. Thus, the accumulation of free radicals in the organism can lead to unwanted cell damage. Oxidized cells can then initiate several body dysfunctions like e.g., rapid ageing, atherosclerosis, arthritis, diabetes, and cancer ( Figure 5). It is the so-called oxidative stress defined as the process of formation of highly reactive molecules (that is, reactive oxygen species-ROS). In order to act against the destructive activity of ROS, it is important to look for diet-derived compounds acting as antioxidants [42].
Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 39 EVLNENLLRF (source: αs1-casein, Fresco; IC50 = n.d.), FVAPFPEVFGK, YQEPVLGPVRGPF, YQEPVLGPVRGPFPI, YQEPVLGPVRGPFPIIV (source: β-casein; Fresco; IC50 = n.d.) [30]. Tonouchi et al. [38] isolated two peptides (LQP and MAP) from Danish skimmed milk cheese. LQP was originally known as ACE inhibitor identified in α-zein hydrolyzed with thermolysin. MAP sequence was identified in β-casein and exhibited ACE inhibition (IC50 = 0.8 µM) and antihypertensive effect in SHR. Significant hypotensive effect of MAP tripeptide was observed at 8 h since oral administration ( − 17.0 mm Hg; dose 3 mg × kg -1 body weight) [38]. The potential of cheeses as diet components that may affect the reduction of blood pressure is also in the focus of interest of the scientists aiming at meta-analyses [39]. Nilsen et al. [39] carried out a cross-sectional study to analyze the impact of the consumption of Gamalost cheese (a traditional Norwegian cheese) on the reduction of the risk of hypertension development. Based on data obtained from the questionnaires of 168 participants, the average intake of Gamalost servings per week was 2 and the average value of systolic and diastolic blood pressures was 128 and 78 mmHg, respectively. The increase of Gamalost servings reduced the systolic blood pressure by 0.72 mm Hg. Moreover, it was concluded that blood pressure reduction related to Gamalost consumption was associated with the presence of ACE inhibitory peptides [39]. Crippa et al. [40] investigated the antihypertensive effect resulting from the consumption of Grana Padano cheese and observed that supplementation of Grana Padano cheese at a dose 30 g per day significantly lowered the systolic and diastolic blood pressure in mild-hypertensive subjects ( -3.5 and -2.4 mm Hg, respectively) [40].
One of the meta-analyses concerned studies evaluating the association of CVD, coronary heart disease (CHD), and stroke with high vs. low cheese consumption. This analysis revealed that high cheese consumption lowered the relative risk for: total CVD (RR 0.90; 7; 8076), CHD (RR 0.86; 8; 7631), and stroke (RR 0.90; 7; 10,449). Respective numbers in the brackets represent: relative risk, number of studies, and number of documented events provided. Finally, the results indicated a nonlinear relationship between cheese consumption and decreasing risks of CVD, CHD, and stroke. Consumption of approximately 40 g of cheese per day ensured the best results in reducing the risks of dysfunctions related to hypertension [41].
To conclude, epidemiological studies suggested that the antihypertensive potential of cheeses can be linked to the presence of biopeptides. According to above-mentioned analyses, the optimal daily cheese dose to reduce the risk of hypertensive dysfunctions was 30 -40 g. These results suggest that cheese-as part of a diet-can prevent from the hypertension-related diseases. Gupta et al. [43] produced Cheddar cheese using different Lactobacillus casei cultures and screened for antioxidant activity. Two antioxidative peptides matching the fragments of bovine β-casein (VKEAMAPK) and α S1 -casein (HIQKEDVPSER) were identified in Cheddar. Both peptides exhibited 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity comparable to the commercial antioxidants such as butylated hydroxyanisole (BHA), tert-butyl hydroquinone (t-BHQ) or ferulic acid [44]. The results indicated that the antioxidant activity of the water-soluble cheese extracts (WSE) was dependent on the ripening period of cheese. The highest bioactivity was observed after 4 months of ripening. After that, the activity started to decrease, up until the 7th month of ripening and remained similar after 9 months [44]. Another research group evaluated the inhibition of DPPH radicals by the WSE of Australian Cheddar cheeses. They found a moderate antioxidant activity of the fractions containing peptides with a molecular weight > 10 kDa, although peptide identification was not carried out [45]. A study conducted to analyze the antioxidative potential of Mexican Cotija cheese revealed that the maximal antioxidant activity of this cheese was achieved after 24 weeks of ripening. Although no specific peptides were identified, it was concluded that the antioxidative potential of cheese was related to a significantly high content of bioactive sequences [46].
Timón et al. [47] produced hard cow cheese and analyzed the antioxidative potential of its WSE. After the fractionation of WSE, it was discovered that some of the extracts exhibited DPPH-radical scavenging and metal chelating bioactivity. Moreover, two peptides, namely EIVPN and DKIHPF which were known from their ACE-inhibiting function, were identified in WSE of the hard cheese. Another sequence i.e., VAPFPQ identified in cheese-derived extract was discovered as a new metal chelating peptide. To conclude, it was postulated that antioxidative potential of cheese water-soluble extracts may be the result of the presence of the above-mentioned peptides. However, the bioactivity of these sequences should be confirmed on synthetic peptides [47].
Some peptides with the antioxidative potential were identified in WSE of the Burgos-type cheese. It was shown that 22 fractions derived from Burgos-type cheese extracts exhibited radical scavenging activity (oxygen radical absorbance capacity with fluorescein (ORAC-FL) assay). Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identification revealed the presence of the aromatic amino acids like tyrosine, phenylalanine or tryptophan in some fractions of cheeses extracts. These amino acids itself possess the antioxidative potential [48]. It was also reported that some of the Burgos-type cheese-originating peptide fractions contained the sequences, namely: SDIPNPIGSENSEKTTMPLW (source: α s1 -casein), YQQPVLGPVRGPFPIIV (source: β-casein), and LLYQQPVLGPVRGPFPIIV (source: β-casein) [49]. According to Timón et al. [49], peptides matching the sequences of β-casein have already been known as antioxidative agents. The sequence SDIPNPIGSENSEKTTMPLW is probably found as the new peptidic antioxidant, however further tests to measure this activity need to be performed [49].

Hypocholesterolemic Peptides
Plasma lipoproteins are composed of the following elements: cholesterol, triglycerides, phospholipids, and apoproteins. Their varying composition affects the density, size, and electrophoretic mobility of each lipoprotein particle which enables for the classification of lipoprotein disorders [50]. According to Cox et al. [50], cholesterol is an essential precursor of some substances as adrenal and gonadal steroid hormones and bile acids. Moreover, it is an element of the animal cell membranes. Triglycerides are the major component of dietary fat as well as animal fat depots. Abnormal concentrations of cholesterol and triglycerides in blood indicate some dysfunctions in synthesis, degradation, and transport of lipoproteins and their increase levels lead to the different variants of hypercholesterolemia and hypertriglyceridemia [50]. In turn, phospholipids are the constituents of the natural membranes. They are polar lipids which are e.g., the components of the milk fat globule membrane involved in the circulation of the lipid droplets secreted by the mammary gland cells [51].
It was reported that some peptides/protein-derived hydrolysates exhibited activity against metabolic lipids' dysfunctions and their effect was tested in cellular systems and animal models [52]. To the best of our knowledge, no peptides acting as hypolipidemic were identified in cheeses. When thinking about milk, most of the studies concerning their "anticholesterolemic effect" conducted so far, have concerned the whey proteins. The ingestion of whey protein was observed to be correlated with a significant reduction in total cholesterol levels in rats fed cholesterol-free and cholesterol-enriched diets [53]. The cholesterol reducing activity of β-lactoglobulin was due to the presence of two major biopeptides, namely lactostatin (IIAEK) and β-lactotensin (HIRL) [54]. According to the literature, consumption of 300 mg IIAEK × kg −1 body weight per day reduced LDL level as well as increased HDL cholesterol level, and fecal steroid excretion. The lactostatin activity was linked to the degradation of cholesterol into bile acids in hepatic cells [55,56]. The HIRL sequence was shown to be involved in the reduction of total (22.7%), and LDL plus VLDL (32.0%) cholesterol levels in mice after 2 days of administration at the dose of 30 mg × kg −1 body weight [57]. The activity of both peptides was tested on mice fed a cholesterol-enriched diet. Despite the optimistic results, it should be noted that the exact mechanisms of the hypocholesterolemic activity of peptides are still unclear. It is speculated that it might be linked to the amino acid composition, but further research is needed in this respect [54].

Antiobesity and Anti-Inflammatory Peptides
The studies concerning the effectiveness of different types of diets and proportions of macronutrients on the weight loss is in the focus of the interest of the many scientists [58]. Some studies have shown that the high-protein content diet is the most effective when thinking about the reduction of body weight. It results from the fact that consumption of the protein contributes to the increased satiety and the reduction of the energy intake in addition to a greater dietary thermogenesis [59].
Although proteins accelerate caloric metabolism, some of them are more efficient on weight loss than others. It was observed that soybean-originating proteins more efficiently enhance weight loss than bovine milk proteins [60]. One of the explanations of this phenomenon results from the fact that peptides produced from proteins during gastrointestinal digestion initiate satiety signals from the gut and hence suppress food intake. Thus, scientists try to explain reliably the mechanisms of a protein-rich diet on the body's health [5]. Some of them include e.g., the impact of dietary protein on cholecystokinin (CKK) release, the activation of GLP-1 receptor to suppress appetite, and/or modulating the adipose genes affecting metabolic and vascular functions [60].
It was reported that opioid peptides may also contribute to the weight loss due to the reduction of food intake [61]. For example, casomorphins interact with gastric opioid receptors and slow down the motility of the gastrointestinal tract, which inhibits food intake [60]. The example of the opioid peptide identified both in bovine casein and several types of cheeses is β-casomorphin-7 (YPFPGPI) which was described above (see Section 2). It was indicated that dose of 179 mg × kg −1 body weight of modified β-casomorphins effectively impaired gastric emptying in pup rats. It was probably related to direct interaction of β-casomorphins with opioid receptors in the gut [62].
Some studies confirmed the causal direction of the relationship between adiposity and inflammation (the greater adiposity, the higher C-reactive protein level) [63]. Citing the words by Zielińska et al. [64], inflammation is defined as a "complex biological process, occurring through a variety of mechanisms and leading to changes in local blood flow and the release of several molecular mediators". Uncontrolled inflammation may lead to chronic dysfunctions such as rheumatoid arthritis, atherosclerosis, and it is linked to cancer [65]. It was reported that VPP and IPP milk-derived ACE-inhibiting tripeptides exhibited anti-inflammatory activity. First reduced leukocyte-endothelial cell interaction in vitro and this effect was related to that of inhibition of MAPK (mitogen-activated protein kinase) signaling pathway. The anti-inflammatory effect of both peptides was also tested in a model of intestinal enterocolitis. It was found that VPP and IPP were protective against atherosclerosis in mice with defect of endogenous apoprotein E (ApoE). These peptides suppressed the mRNA for inflammatory cytokines, oxidized low density lipoprotein receptor, and transcription regulators [65]. These two peptides were also identified in the different types of cheeses (see Section 2).
To the best of our knowledge, there is no more specific anti-inflammatory peptides that could be identified in cheeses. However, it was found that water-soluble extracts of Cheddar cheese possessed such an activity which was determined based on nitric oxide (NO) production in lipopolysaccharide-stimulated macrophage (RAW-264.7) cells [66].

Multi-Activity of Peptides in MetS Prevention: A Screening Using BIOPEP-UWM Database
Loads of publication data concern the analysis of the proteins as the sources of biopeptides exhibiting only one biological effect while the information about dual or multifunctional properties of peptides is rather poor [5]. Such statement may also refer to the MetS-preventive peptides. To the best of our knowledge, there is no a "universal" peptide sequence that would show all bioactivities described in this paper. Thus, databases of biological information like BIOPEP-UWM, available at: http://www.uwm.edu.pl/biochemia, may be a suitable option to acquire the knowledge about peptides exhibiting more than one biological function [67]. BIOPEP-UWM is a curated database providing comprehensive information on 3899 bioactive peptide sequences (accessed: March 2020).
We applied BIOPEP-UWM database to retrieve the profiles of potential biological activities of the four major casein fractions (see Table A1). Profile of the potential biological activity of the protein is defined as the type and the location of bioactive fragment in a protein chain [67]. Briefly, such analysis enables to answer the question: what bioactive fragments match the protein sequence? Then, each peptide sequence was pasted again to BIOPEP-UWM database of bioactive peptide sequences using the search option "sequence" and ticking "exact" [67]. Such searching was helpful to show what additional activities are exhibited by this peptide (the pasted one). This approach led us find that 66 peptides had dual i.e., ACE and DPP4 inhibitory function, and 17 were acting as ACE inhibitory and antioxidant agents. Some peptides exerted a triple bioactivity, i.e., eight dipeptides inhibited ACE and DPP4 as well as exerted an antioxidant activity. The example of the sequence with triple inhibitory bioactivity is YP peptide present in α S1 -, β-, and κ-casein sequence. It has proven ACE, α-glucosidase and DPP4 inhibitory function. The brief summary of peptides encrypted in the sequences of caseins and exerting e.g., more than one biological effect is shown in Table 4. For checking what particular sequences were found as dual or multiactive, see Table A1. Although, our bioinformatic approach is useful for searching multifunctions of peptides, it has some limitations. The important one is based on the idea of the positive selection assuming that sequence of interest matches the peptide sequences present in databases. Thus, it may not include peptides that are "new" and they are, at the time of screening, not displayed in the database. Thus, some authors postulate for the regular update of the databases with newly-reported bioactive peptides [68]. Nevertheless, such databases, if curated and reliable, may contribute to acquiring of some information about the peptides which multi-activity can be described as "MetS-preventive".

Conclusions
Some milk-derived peptides were identified in different types of cheeses. These biopeptides exhibited bioactivities playing an important role in the prevention from the dysfunctions related to metabolic syndrome. It needs to be noted that majority of studies concerned the identification of peptides in cheeses and did not included studies on animal and humans. Several epidemiological and cohort studies confirmed the positive effect of cheese consumption on the reduction of the risk of MetS diseases. A majority of studies concerning milk-derived peptides and their identification in cheeses focus on the determination of only one biological effect. Despite its limitations, bioinformatic-assisted approach can be helpful in the screening of multifunctional effect of peptides being the promising agents in the prevention of MetS.
Author Contributions: D.M. wrote Sections 3-7, and was involved in draft preparation and editing; A.I. was responsible for conceptualization, wrote Section 1, Section 2, Section 8, and reviewed the article. All authors have read and agreed to the published version of the manuscript.

Acknowledgments:
The authors wish to thank Małgorzata Darewicz for her valuable scientific suggestions when preparing this manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.