**2. Bioactive peptides and their functionalities**

The health potential of dairy protein is not only originating from the unique amino acid composition and their great bioavailability (**Figure 1**). Especially the high content of essential amino acids and their fast release as free amino acids during digestion [2], plus the high content of certain vitamins and minerals is important for the high nutritional value of bovine milk. However, there is a more hidden health potential of dairy products that is displayed by bioactive peptides. Bioactive peptides contain usually 3–20 amino acid residues and their composition and sequence determine their activity. They are encrypted in the primary sequence of proteins and get released via three different ways [3]:


**Figure 1.** Illustration of milk protein degradation by lactic acid bacteria (LAB) and during digestion via digestive enzyme action resulting in bioactive peptides.

These are the ways to produce bioactive peptides that can be afterwards purified and used as ingredients for manufacturers of functional foods. However, also the more natural way of processing via hydrolysis by proteolytic microorganisms can be an approach to enrich a specific bioactive function in a product. Bioactive peptides have been discovered not only in dairy products, but also in meat, eggs, fish, and other marine organisms and also in plant sources like certain grains, legumes, pulses, and oilseeds [4–6].

Interestingly, the processing method of the dairy products can influence the number and sequence of the resulting peptides after digestion and therefore also the content of bioactive peptides. Heat treatment, chemical and biochemical, and physical treatment can influence the bioactive functionality transmitted by the selected dairy product. In the future, it might be possible to target a wished bioactive function via processing. This book chapter just deals with the effects exhibited by bioactive peptides. However, also other milk components are affected by processing and can exhibit bioactive functions, e.g. effects on the lipids, minerals, and vitamins. Furthermore, the addition of certain bioactive ingredients to dairy products is also not discussed in this chapter. The focus is given to the possible effects transmitted via

The health potential of dairy protein is not only originating from the unique amino acid composition and their great bioavailability (**Figure 1**). Especially the high content of essential amino acids and their fast release as free amino acids during digestion [2], plus the high content of certain vitamins and minerals is important for the high nutritional value of bovine milk. However, there is a more hidden health potential of dairy products that is displayed by bioactive peptides. Bioactive peptides contain usually 3–20 amino acid residues and their composition and sequence determine their activity. They are encrypted in the primary

**Figure 1.** Illustration of milk protein degradation by lactic acid bacteria (LAB) and during digestion via digestive enzyme

bioactive peptides and the effect of processing on the peptide profile.

sequence of proteins and get released via three different ways [3]:

**3.** Action of proteolytic enzymes derived from microorganisms or plants

**1.** Hydrolysis by digestive enzymes

action resulting in bioactive peptides.

**2.** Hydrolysis by proteolytic microorganisms

**2. Bioactive peptides and their functionalities**

110 Technological Approaches for Novel Applications in Dairy Processing

The production of bioactive peptides for use as additives can be done by enzymatic hydrolysis or microbial fermentation [7]. Enzymatic hydrolysis applies digestion enzymes. Mostly trypsin, a pancreatic proteinase is used, but also chymotrypsin, pepsin thermolysin, pancreatin, elastase, carboxypeptidase or a proline-specific endopeptidase can deliver bioactive peptides. Additionally, proteases from bacteria, fungi, and plants also showed interesting properties [7]. Microbial fermentation uses bacteria or yeast that exhibit proteolytic activity to generate peptides. They are grown and added in their exponential phase to the protein of interest. The degree of hydrolysis is then dependent on the strain and its proteolytic activity. In both ways, a purification of the peptides is necessary. This can be for example reached by centrifugation methods, freeze drying, desalting, and membrane filtration techniques [8]. Examples are the production of caseinophosphopeptides from α-s-casein with an immobilized trypsin in a fluidized bed bioreactor [9] and a combination of diafiltration and anionexchange chromatography [10]. The peptide additives can be added to a product of interest to generate a functional food. For this purpose, also the stability of the peptides with regard to pH, temperature, and food matrix has to be considered. Furthermore, the more natural way to enhance dairy products with bioactive peptides is to directly add a bacterial culture to the dairy product and generate a fermented product containing bioactive peptides. This is the general processing method applied already for each fermented dairy product. If protein is not taken out, all dairy products result in a high quantity of bioactive peptides that might be absorbed in the small intestine. For the functionality of these peptides, the selection of bacteria strains is important to aim for a specific bioactive function via processing (see Section 3).

The possible, so far detected, functionalities of bioactive peptides are summarized in **Figure 2**.

**Figure 2.** Possible functionalities of bioactive peptides.

#### **2.1. Antihypertensive peptides**

Antihypertensive peptides can inhibit the angiotensin I-converting enzyme (ACE) (EC 3.4.15.1;ACE) that is involved in blood pressure regulation. ACE increases the blood pressure by converting angiotensin I into the vasoconstrictor angiotensin II and additionally degrades vasodilative bradykinin. ACE-inhibitory peptides were detected in different food proteins like bovine casein and human casein, whey protein, zein, gelatin, yeast, and corn [11]. The most ACE-inhibitory peptide in studies of [28], had an IC50 value of 77 μM and was originating from α-lactalbumin with the peptide sequence α-lactalbumin f(104–108). Different studies showed the bioavailability of the ACE-inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro in humans [12, 13]. These two tripeptides are the ones that are studied the most and show the highest evidence for their bioefficacy.

demonstrated promising results. Otani et al. showed for example that feeding mice a dietary casein phosphopeptide influenced the level of serum IgA and intestinal antigen-specific IgA [20]. The exact mechanism of the action exhibited by immune-modulatory peptides still has

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Peptides that inhibit dipeptidyl peptidase 4 (DPP-IV, EC 3.4.14.5) are known as satiety increasing peptides. DPP-IV degrades the satiety regulating glucagon-like-peptide 1 [21]. Kopf-Bolanz et al, monitored the fates of specific peptides with known satiety increasing action. They compared different dairy products and found the relative abundance of three potent DPP-IV inhibitory peptides deriving from β-casein. The best source for these peptides was Gruyere cheese. Two other peptides deriving from α-S1-casein were not detectable anymore after the pancreatic phase of the digestion [22]. Tryptophan seems to be an important amino acid in peptides that exhibit a DPP-IV inhibitory potential [23]. Three dipeptides containing tryptophan Trp-Arg, Trp-Lys, and Trp-Leu with half maximum inhibitory concentrations (IC50) < 45 μM could be detected in a study of Nongonierma and Fitzgerald that are potent

Another interesting peptide that has an influence on satiety is the glycomacropeptide (GMP) resulting from cheese production. It has demonstrated in several animals and human studies that it can stimulate the release of cholecystokinin and promote satiety. However, further studies would be necessary to demonstrate a clear bioefficacy [25, 26]. Peptides that increase

Peptides that have an affinity for the opioid receptor are categorized in this group. There are receptors that are responsible for specific physiological effects like emotional behavior and food intake. Opioid peptides have the same N-terminal sequence Tyr-Gly-Gly-Phe. There are also atypical opioid peptides with the ending of Tyr-X-Phe or Tyr-X1-X2-Phe. A tyrosine residue at the N-terminal and another aromatic amino acid at the third or fourth position are specific binding motifs of the opioid receptor. The first food-derived opioid peptides were β-casomorphins. Also, casoxins, lactorphins, and exorphins can bind to opioid receptors [28]. So far, a weak opioid activity for α-lactorphin (α-lactalbumin f(50–53)) and β-lactorphin (β-lactoglobulin f(102–105)) was detected in guinea pigs [11], but human data are still missing. Concentrations released from in vivo digestion of milk are quite low. The total amount of α-lactorphin and β-lactorphin in 1 L of bovine milk would be 32 mg (64 μmol), respectively 90 mg (162 μmol), but it might be difficult to obtain a full release of the possible peptide during in vivo digestion. It is so far not clear whether they can get liberated by in vivo digestion at all, but it was demonstrated that casomorphins are liber-

to be determined.

**2.6. Satiety peptides**

inhibitors of DPP-IV [24].

**2.7. Opioid peptides**

ated in vivo [11].

the satiety are also known as anti-obesity peptides [27].

#### **2.2. Anti-oxidative peptides**

Anti-oxidative peptides help against the oxidative damage caused by reactive oxygen species. The amino acids cysteine, lysine, histidine, methionine, tryptophan, and tyrosine can act as radical scavengers [14]. Therefore, they act as potential antioxidants.

#### **2.3. Antithrombotic peptides**

The formation of blood clots can be reduced by antithrombotic peptides. Especially known is the glycomacropeptide (GMP) originating from kappa-casein after enzymatic milk coagulation. GMP can inhibit the aggregation of blood platelets and binding of the human fibrinogen gamma-chain to platelet surface fibrinogen receptors [15]. Also, the absorption into the plasma could be observed in humans for 2 anti-thrombotic peptides [16].

#### **2.4. Anticancer peptides**

Anticancer peptides can inhibit cancer cell growth. In vitro experiments with HL-60 human leukemia cells showed for example, that after skimmed milk digestion with a proteolytic enzyme from yeast *Saccharomyces cerevisiae* apoptosis can be induced [17].

#### **2.5. Immune-modulatory peptides**

Immune-modulatory peptides are mostly found in dairy products. Enzymatic hydrolysis resulted in a number of biologically active peptides that can influence immune cells and release specific signals [18]. Some peptides can stimulate or inhibit immune responses and their positive health effects have been investigated mostly in vitro. These assays are performed with immune cells and target for example proliferation, phagocytosis, differentiation, and cytokine production. A survey of these assays can be found in the review of Maestri et al. [6]. The immunomodulatory potential of peptides originating from whey protein is discussed in the study of Gauthier et al. [19]. Interestingly, there are some in vivo studies that have demonstrated promising results. Otani et al. showed for example that feeding mice a dietary casein phosphopeptide influenced the level of serum IgA and intestinal antigen-specific IgA [20]. The exact mechanism of the action exhibited by immune-modulatory peptides still has to be determined.

#### **2.6. Satiety peptides**

**2.1. Antihypertensive peptides**

112 Technological Approaches for Novel Applications in Dairy Processing

their bioefficacy.

**2.2. Anti-oxidative peptides**

**2.3. Antithrombotic peptides**

**2.4. Anticancer peptides**

**2.5. Immune-modulatory peptides**

Antihypertensive peptides can inhibit the angiotensin I-converting enzyme (ACE) (EC 3.4.15.1;ACE) that is involved in blood pressure regulation. ACE increases the blood pressure by converting angiotensin I into the vasoconstrictor angiotensin II and additionally degrades vasodilative bradykinin. ACE-inhibitory peptides were detected in different food proteins like bovine casein and human casein, whey protein, zein, gelatin, yeast, and corn [11]. The most ACE-inhibitory peptide in studies of [28], had an IC50 value of 77 μM and was originating from α-lactalbumin with the peptide sequence α-lactalbumin f(104–108). Different studies showed the bioavailability of the ACE-inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro in humans [12, 13]. These two tripeptides are the ones that are studied the most and show the highest evidence for

Anti-oxidative peptides help against the oxidative damage caused by reactive oxygen species. The amino acids cysteine, lysine, histidine, methionine, tryptophan, and tyrosine can act as

The formation of blood clots can be reduced by antithrombotic peptides. Especially known is the glycomacropeptide (GMP) originating from kappa-casein after enzymatic milk coagulation. GMP can inhibit the aggregation of blood platelets and binding of the human fibrinogen gamma-chain to platelet surface fibrinogen receptors [15]. Also, the absorption into the

Anticancer peptides can inhibit cancer cell growth. In vitro experiments with HL-60 human leukemia cells showed for example, that after skimmed milk digestion with a proteolytic

Immune-modulatory peptides are mostly found in dairy products. Enzymatic hydrolysis resulted in a number of biologically active peptides that can influence immune cells and release specific signals [18]. Some peptides can stimulate or inhibit immune responses and their positive health effects have been investigated mostly in vitro. These assays are performed with immune cells and target for example proliferation, phagocytosis, differentiation, and cytokine production. A survey of these assays can be found in the review of Maestri et al. [6]. The immunomodulatory potential of peptides originating from whey protein is discussed in the study of Gauthier et al. [19]. Interestingly, there are some in vivo studies that have

radical scavengers [14]. Therefore, they act as potential antioxidants.

plasma could be observed in humans for 2 anti-thrombotic peptides [16].

enzyme from yeast *Saccharomyces cerevisiae* apoptosis can be induced [17].

Peptides that inhibit dipeptidyl peptidase 4 (DPP-IV, EC 3.4.14.5) are known as satiety increasing peptides. DPP-IV degrades the satiety regulating glucagon-like-peptide 1 [21]. Kopf-Bolanz et al, monitored the fates of specific peptides with known satiety increasing action. They compared different dairy products and found the relative abundance of three potent DPP-IV inhibitory peptides deriving from β-casein. The best source for these peptides was Gruyere cheese. Two other peptides deriving from α-S1-casein were not detectable anymore after the pancreatic phase of the digestion [22]. Tryptophan seems to be an important amino acid in peptides that exhibit a DPP-IV inhibitory potential [23]. Three dipeptides containing tryptophan Trp-Arg, Trp-Lys, and Trp-Leu with half maximum inhibitory concentrations (IC50) < 45 μM could be detected in a study of Nongonierma and Fitzgerald that are potent inhibitors of DPP-IV [24].

Another interesting peptide that has an influence on satiety is the glycomacropeptide (GMP) resulting from cheese production. It has demonstrated in several animals and human studies that it can stimulate the release of cholecystokinin and promote satiety. However, further studies would be necessary to demonstrate a clear bioefficacy [25, 26]. Peptides that increase the satiety are also known as anti-obesity peptides [27].

#### **2.7. Opioid peptides**

Peptides that have an affinity for the opioid receptor are categorized in this group. There are receptors that are responsible for specific physiological effects like emotional behavior and food intake. Opioid peptides have the same N-terminal sequence Tyr-Gly-Gly-Phe. There are also atypical opioid peptides with the ending of Tyr-X-Phe or Tyr-X1-X2-Phe. A tyrosine residue at the N-terminal and another aromatic amino acid at the third or fourth position are specific binding motifs of the opioid receptor. The first food-derived opioid peptides were β-casomorphins. Also, casoxins, lactorphins, and exorphins can bind to opioid receptors [28]. So far, a weak opioid activity for α-lactorphin (α-lactalbumin f(50–53)) and β-lactorphin (β-lactoglobulin f(102–105)) was detected in guinea pigs [11], but human data are still missing. Concentrations released from in vivo digestion of milk are quite low. The total amount of α-lactorphin and β-lactorphin in 1 L of bovine milk would be 32 mg (64 μmol), respectively 90 mg (162 μmol), but it might be difficult to obtain a full release of the possible peptide during in vivo digestion. It is so far not clear whether they can get liberated by in vivo digestion at all, but it was demonstrated that casomorphins are liberated in vivo [11].

#### **2.8. Antidiabetic peptides**

Diabetes is treated by synthetic antidiabetic drugs that can result in side effects like hypoglycemia or weight gain [8]. To overcome this issue, the application of antidiabetic peptides originating from food sources might be a solution. Antidiabetic peptides could be for example detected in sheep milk [29].

can occur over the whole dairy protein sequences, and there can be rare cases that people are allergic to a new peptide sequence arising from fermentation. However so far, mostly positive reports about the effect of fermentation are published [41, 42]. It is also important to mention that these functionalities were observed to a great extent with in vitro methods. Only very few

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To exhibit really a bioactive function in vivo, the peptides must be released during digestion from their originating protein or if they are already in the product as such, they have to be resistant to digestion enzymes. During digestion, the proteins get denatured by gastric acidification and subsequently degraded by pepsin and pancreatic peptidases like trypsin into peptides and amino acids. Furthermore, the final actions of the enzymes at the brush-border membrane in the small intestine have to be taken into account. There are peptidases that cleave amino acids or dipeptides from the N- or C-terminal of the interior bond of the oligopeptides. The mean size of the peptides in the jejunum considering the action of aminopeptidases and dipeptidases from the enterocytes is 3–6 amino acids. Di- and tripeptides can be transported actively by the peptide transporter PEPT1 [43]. Longer peptides can probably get absorbed ether via paracellular or transcellular pathways. The possible transport of a heptapeptide was shown using a cell culture model [44]. In the blood, the peptides must be able to reach their target site in the peripheral organs. In a human study of van Platerink et al., 17 ACE-inhibiting peptides with 5–6 amino acids length could be detected in the blood after consumption of drinks enriched with those peptides [13]. The first proof that the tripeptide Ile-Pro-Pro does not undergo intestinal degradation and can reach the circulation intact was shown from Foltz et al. [12]. Another human study showed the presence of a longer peptide after soybean consumption in the blood [45]. At the target cells, it is assumed that peptides can internalize via endocytosis and get digested in the lysosome. Peptides that do not enter target cells can accumulate in the liver and kidney and can be detected in urine or bile [6]. There is still the need to demonstrate a clear bioefficacy of the peptides and confirm the positive health effects in human studies. In the future possibly health claims for certain bioactive peptides could be developed. So far Japan declared certain antihypertensive peptides such as Val-Pro-Pro, Ile-Pro-Pro, Val-Tyr, and Cys-Pro-Pro as Food of Specific Health Use (FOSHU). In contrast, the European Food and Drug Association (EFSA) did

not authorize any claims regarding the effect of bioactive peptides in foods yet [46].

product is inserted into an in vitro digestion model that mimics human digestion.

Experiments concerning bioactive peptides are mainly done in vitro. Most of the time, a dairy

There are numerous in vitro digestion models that can be applied. It is important that a model close to human physiology and validated is applied. Recently, a harmonized digestion model was established during the COST digestion action. This model is very physiological and might be used for mimicking digestion [47]. The resulting peptides generated during the digestion process can be detected by peptidomic methods. Analytics of bioactive peptides aim toward

human studies have demonstrated an effect of bioactive peptides in vivo.

**2.13. Physiology of digestion**

**2.14. Detection of bioactive peptides**

three main directions [48]:

#### **2.9. Mineral-binding peptides**

Mineral-binding phosphopeptides can carry different minerals by forming soluble organophosphate salts [30]. Caseinophosphopeptides (CPP) can increase calcium absorption by limiting calcium precipitation in the ileum. Caseins are phosphorylated in the mammary gland at primary sequences rich in serine and glutamic acid forming triplet regions SerP-SerP-SerP-Glu-Glu that occur in α-S1-casein (66–70), α-S2-casein (8–12), (56–60), (129–133) and β-casein (15–19). The presence of CPPs has been shown in vivo. Several animal studies have demonstrated the effect of CPP to enhance calcium bioavailability. In contrast, convincing results from human are still missing [31]. A human study with CPP-enriched preparations (containing candidate functional food ingredients) on calcium absorption from a calcium lactate drink showed no significant results [32]. Another interesting peptide is lactoferricin consisting of 25 amino acid residues. The molecule is folded into two globular units, each capable of binding one ferric (Fe3+) ion [33].

#### **2.10. Cholesterol-lowering peptides**

So far mainly peptides derived from soy proteins have been shown to suppress cholesterol in the blood. Some can for example target the cholesterol receptor or suppress the presence of LDL. Important for the functions are mainly the hydrophobic residues [34]. A novel peptide (Ile-Ile-Ala-Glu-Lys) from a trypsin-treated hydrolysate of β-lactoglobulin showed a hypocholesterolemic effect in an animal study [35].

#### **2.11. Antimicrobial peptides**

Peptides that induce for example the lysis of bacterial membranes are antimicrobial peptides. They could be detected in α-lactalbumin, β-lactoglobulin, all casein fractions, and lactoferrin [36].

#### **2.12. Safety issues**

The safety and toxicity of bioactive peptides has to be considered. Different studies demonstrated that casein hydrolysates and Val-Pro-Pro from powdered fermented milk did not show any toxicological potential [37–39]. Processing can lead to Maillard reaction and result in the production of allergenic compounds [40]. Processing changes the protein structure and might influence the protein degradation and therefore also the response of the immune system. Therefore, it is important to determine the allergic potential that can arise from bioactive peptides. If fermentation takes place, for example, de novo peptides might originate and their allergenic potential has to be determined. First, a comparison with already known allergenic sequences can be done, followed by laboratory tests. The problem is that allergenic sequences can occur over the whole dairy protein sequences, and there can be rare cases that people are allergic to a new peptide sequence arising from fermentation. However so far, mostly positive reports about the effect of fermentation are published [41, 42]. It is also important to mention that these functionalities were observed to a great extent with in vitro methods. Only very few human studies have demonstrated an effect of bioactive peptides in vivo.

#### **2.13. Physiology of digestion**

**2.8. Antidiabetic peptides**

114 Technological Approaches for Novel Applications in Dairy Processing

detected in sheep milk [29].

**2.9. Mineral-binding peptides**

**2.10. Cholesterol-lowering peptides**

**2.11. Antimicrobial peptides**

**2.12. Safety issues**

cholesterolemic effect in an animal study [35].

Diabetes is treated by synthetic antidiabetic drugs that can result in side effects like hypoglycemia or weight gain [8]. To overcome this issue, the application of antidiabetic peptides originating from food sources might be a solution. Antidiabetic peptides could be for example

Mineral-binding phosphopeptides can carry different minerals by forming soluble organophosphate salts [30]. Caseinophosphopeptides (CPP) can increase calcium absorption by limiting calcium precipitation in the ileum. Caseins are phosphorylated in the mammary gland at primary sequences rich in serine and glutamic acid forming triplet regions SerP-SerP-SerP-Glu-Glu that occur in α-S1-casein (66–70), α-S2-casein (8–12), (56–60), (129–133) and β-casein (15–19). The presence of CPPs has been shown in vivo. Several animal studies have demonstrated the effect of CPP to enhance calcium bioavailability. In contrast, convincing results from human are still missing [31]. A human study with CPP-enriched preparations (containing candidate functional food ingredients) on calcium absorption from a calcium lactate drink showed no significant results [32]. Another interesting peptide is lactoferricin consisting of 25 amino acid residues. The molecule is folded into two globular units, each capable of binding one ferric (Fe3+) ion [33].

So far mainly peptides derived from soy proteins have been shown to suppress cholesterol in the blood. Some can for example target the cholesterol receptor or suppress the presence of LDL. Important for the functions are mainly the hydrophobic residues [34]. A novel peptide (Ile-Ile-Ala-Glu-Lys) from a trypsin-treated hydrolysate of β-lactoglobulin showed a hypo-

Peptides that induce for example the lysis of bacterial membranes are antimicrobial peptides. They could be detected in α-lactalbumin, β-lactoglobulin, all casein fractions, and lactoferrin [36].

The safety and toxicity of bioactive peptides has to be considered. Different studies demonstrated that casein hydrolysates and Val-Pro-Pro from powdered fermented milk did not show any toxicological potential [37–39]. Processing can lead to Maillard reaction and result in the production of allergenic compounds [40]. Processing changes the protein structure and might influence the protein degradation and therefore also the response of the immune system. Therefore, it is important to determine the allergic potential that can arise from bioactive peptides. If fermentation takes place, for example, de novo peptides might originate and their allergenic potential has to be determined. First, a comparison with already known allergenic sequences can be done, followed by laboratory tests. The problem is that allergenic sequences To exhibit really a bioactive function in vivo, the peptides must be released during digestion from their originating protein or if they are already in the product as such, they have to be resistant to digestion enzymes. During digestion, the proteins get denatured by gastric acidification and subsequently degraded by pepsin and pancreatic peptidases like trypsin into peptides and amino acids. Furthermore, the final actions of the enzymes at the brush-border membrane in the small intestine have to be taken into account. There are peptidases that cleave amino acids or dipeptides from the N- or C-terminal of the interior bond of the oligopeptides. The mean size of the peptides in the jejunum considering the action of aminopeptidases and dipeptidases from the enterocytes is 3–6 amino acids. Di- and tripeptides can be transported actively by the peptide transporter PEPT1 [43]. Longer peptides can probably get absorbed ether via paracellular or transcellular pathways. The possible transport of a heptapeptide was shown using a cell culture model [44]. In the blood, the peptides must be able to reach their target site in the peripheral organs. In a human study of van Platerink et al., 17 ACE-inhibiting peptides with 5–6 amino acids length could be detected in the blood after consumption of drinks enriched with those peptides [13]. The first proof that the tripeptide Ile-Pro-Pro does not undergo intestinal degradation and can reach the circulation intact was shown from Foltz et al. [12]. Another human study showed the presence of a longer peptide after soybean consumption in the blood [45]. At the target cells, it is assumed that peptides can internalize via endocytosis and get digested in the lysosome. Peptides that do not enter target cells can accumulate in the liver and kidney and can be detected in urine or bile [6]. There is still the need to demonstrate a clear bioefficacy of the peptides and confirm the positive health effects in human studies. In the future possibly health claims for certain bioactive peptides could be developed. So far Japan declared certain antihypertensive peptides such as Val-Pro-Pro, Ile-Pro-Pro, Val-Tyr, and Cys-Pro-Pro as Food of Specific Health Use (FOSHU). In contrast, the European Food and Drug Association (EFSA) did not authorize any claims regarding the effect of bioactive peptides in foods yet [46].

#### **2.14. Detection of bioactive peptides**

Experiments concerning bioactive peptides are mainly done in vitro. Most of the time, a dairy product is inserted into an in vitro digestion model that mimics human digestion.

There are numerous in vitro digestion models that can be applied. It is important that a model close to human physiology and validated is applied. Recently, a harmonized digestion model was established during the COST digestion action. This model is very physiological and might be used for mimicking digestion [47]. The resulting peptides generated during the digestion process can be detected by peptidomic methods. Analytics of bioactive peptides aim toward three main directions [48]:


asparagine, and desulphur cysteine and cysteine. Resulting end products might be lanthionine, lysine-alanine, iso-peptides and ornitho-alanine [52]. The digestibility of whey proteins increases after thermal treatment because the sites for enzymatic hydrolysis are easier to reach for the digestive enzymes. However, strong denaturation reduces digestibility [54]. Kopf-Bolanz et al. showed that heat treatment of dairy products led to an increased number of β-lactoglobulin peptides after in vitro digestion [22]. There is a greater susceptibility to hydrolysis following heat treatment [55]. Regarding the antidiabetic action of casein, there was a significant reduction observed after boiling compared to the raw casein [29]. The denaturation of whey protein via thermal processing led to an increase in the antibacterial activity of α-lactalbumin [56] and lysozyme [57]. The antioxidant action of whey proteins can be maintained by low-temperature processing. This results in high levels of specific dipeptides that can promote the synthesis of the antioxidant glutathione [58]. Extrusion cooking might also affect protein digestibility shown for example in a study of Onwulata et al. [59]. Data on the effect of ohmic heating are rare. Depending on the used temperatures, similar effects like with application of other heating methods might be expected [52]. It was also shown that spray drying or freeze drying did not exhibit negative effects on the immunomodulatory activity of a whey protein hydrolysate. The study also used whey protein concentrate (WPC) and sodium alginate as carriers for encapsulation to reduce bitter taste and resistance to hygroscopicity. They showed that spray drying of whey protein concentrate hydrolysate with the proper carriers did not affect the immunomodulatory activity and might therefore widen its application

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Hydrolysis by acid is applied which is known to improve their protein digestibility. It is used for example for enteral and hypoallergenic infant nutrition. For Mozzarella, the type of acid used is important for the protein yield obtained in the pre-cheeses [61] and might therefore also affect the profile of bioactive peptides. Treatment with alkali for hydrolysis is rarely applied in

Fermented dairy products like yoghurt and cheese result in a high number of bioactive peptides produced by the lactic acid bacteria. Especially the type of the starter culture, type of probiotic bacteria, and the fermentation parameters play an important role for the bioactive effect that the product might have. Furthermore, only via this way de novo peptides can be generated that do not occur after digestion of milk as such. *Streptococcus thermophilus* and *Lactobacillus bulgaricus* possess bacterial activity against Streptococci in vivo that probably derives from the antimicrobial peptides that they produce during fermentation [63]. It is very promising to test different lactic acid bacteria strains for their effect on a bioactive function. One study of Gobbetti et al. showed that a fermentation with *L. delbrueckii* ssp. *Bulgaricus SS1* versus a fermentation with *Lactobacillus lactis* subspecies *cremoris FT4* resulted in a higher ACE-inhibitory activity [64]. The most investigated ACE-inhibitory peptides were obtained after fermentations with *L. helveticus* and *L. helveticus CP790*. Also, the Finnish milk product Evolus contained *L. helveticus LBK-16H* strain as a starter and

the food industry. It would result in racemization and loss of protein digestibility [62].

in food systems [60].

**3.2. Chemical treatment**

**3.3. Biochemical treatment**

Peptidomics is the comprehensive qualitative and quantitative analysis of all peptides in a biological sample. In earlier days, protein digestion could be followed by HPLC or Edman sequencing [49]. Nowadays, MS-based techniques such as Liquid chromatography coupled to mass spectrometry (LC-MS) can be applied [22, 50]. Peptidomics of food hydrolysates, for example, led to the discovery of the exact sites of rennet cleavage on kappa-casein or the cleavage sites produced by bacteria during cheese ripening [49]. The detailed human study of Boutrou et al. was identified in the jejuna effluents of healthy adults, after consumption of 30 g milk casein and whey proteins, 356 and 146 peptides [50]. The in vitro model developed by Minekus et al., almost resulted in similar peptides [47]. The different analytical approaches that can be applied are summarized in the review of Dallas et al. [49]. Technology allows the prediction of the peptide sequence and can generate a peptide fingerprint. The peptides can be then compared to the known bioactive peptides from the literature in various databases. An example is the milk bioactive peptide database by Nielsen et al. [51]. This database comprises information on bioactive peptides from across hundreds of original research articles and is available to the public. Furthermore, whole in silico strategies for bioactive function generation including computational modeling might be applied, that still have limitations, but might be used in the future for the design of new products.
