The Contribution of Bioactive Peptides of Whey to Quality of Food Products

Marica Rakin^*

Maja Bulatovic^*

Danica Zaric^**
^* University of Belgrade, Belgrade, Serbia
^** IHIS Techno-experts D.o.o., Research Development Center, Belgrade, Serbia

Abstract

Whey proteins are sources of biologically active peptides that can be released by controlled enzymatic hydrolysis or bacterial activity. Whey peptides have a wide range of bioactivities (ACE-inhibitory activity, antioxidant activity, antiinflammatory, antimicrobial), so these peptides are more suitable as ingredients in functional foods than molecules with only one substantial characteristic.

Bioactive peptides derived from whey protein and added into probiotic-fermented products and confectionery products (as chocolate, biscuit, or cream) affect human health directly and indirectly. These peptides increase the viable number of probiotic bacteria, increase the stability of the probiotic products, and act in the human organism as antioxidants and ACE inhibitors, increase proliferation of intestinal epithelial cells, and so forth. Bioactive peptides are suitable for application in food because of the simple procedure to produce and separate it. In summary, this chapter describes the biological activity of peptides and their practical application in food products.

Keywords

whey

protein

peptide

ACE

antioxidant

bioactivity

immobilization

functional food

1. Introduction

Milk is a food that fulfills the nutritional needs of the mammal newborn and ensures development and growth during the first stages of its life. Milk is also recognized as a food that adults mammal do not consume. Humans are the only mammals that consume milk as adults, but many experts consider that this is not in accordance with “human nature.” This statement is helped by the knowledge that a large number of adults are lactose intolerant or have an allergic reaction to different component of milk [such as β-lactoglobulin (Lg)]. Milk has many components that contribute to human’s health, so it would be a waste to not consume it. Milk proteins are the most valuable components of milk. Normal bovine milk contains about 3.5% of protein, of which casein constitutes 80% and whey proteins 20%. The knowledge of bioactive peptides has steadily increased since 1979 with the “rediscovery” of whey.

Whey was discovered about 3000 years ago. In the 17th and 18th centuries, whey was considered a medicinal mostly because of biologically active proteins and peptides. Thereafter, whey was neglected in the 19th century but in the second half of the 20th century many techniques for protein isolation were developed in order to isolate whey protein as a very valuable component from whey. Before 1980, bioactive peptides were mostly fragments of α-, β-, and κ-casein, detected in milk. Bioactive peptides derived from whey proteins were rapidly discovered in the 80s and 90s. The activity is based on the amino acid composition and sequence. Bioactive peptides usually contain 3–20 amino acids per molecule. They have been used for recovery after exercise, weight management and satiety, infant nutrition, cardiovascular health, and wound care and repair. During the research it was observed that some peptides that do not show a particular bioactivity become bioactive after ingestion and passing through the gastrointestinal system. That was the stimulus for the development of research on the effect of enzymatic hydrolysis of the bioactive properties of proteins. Nowadays, science has mostly unraveled the secrets of whey proteins and achieved isolation of each component of whey and using it optionally.

Bioactive peptides, derived by enzymatic hydrolysis of whey protein, have demonstrated characteristics as health-promoting agents. Depending on these activities, bioactive peptides derived from whey protein can be used as ingredients for various food and medical preparations. A food can be considered as functional if, beyond its nutritional outcomes, it provides benefits for one or more physiological functions, thus improving health while reducing the risk of illness (Kailasapathy, 2009). The concept of functional food appears in Japan during the 1980s. The main idea was to improve the quality of life of the elderly population because it is been known that nutrition plays a key role in the prevention of various diseases. Whey protein is used in many different types of food as ice cream, pastry, and infant formula. It has also been used to replace fat in a number of products. With its high-protein quality score and branched-chain amino acid (BCAA) content, whey protein has also long been popular as a muscle-building supplement (Josse and Phillips, 2012; Lollo et al., 2011). Whey peptides are more useful molecules derived from whey protein. Produce of bioactive peptides is usually carried out as hydrolysis using mostly digestive enzyme, but also microbial, plant, or animal enzymes. Bioactive peptide can be produced by fermentation with some protolithic starter cultures. The functional food market abounds in fermented dairy products containing lactic acid bacteria with probiotic properties and produce secondary metabolites that deliver health benefits (Stanton et al., 2005). The global functional food and drink market increased 1.5-fold between 2003 and 2010, and was expected to grow a further 22.8% between 2010 and 2014 (Leatherhead, 2011). Other estimates predicting the market will reach €65 billion by the year 2016 (Marsh et al., 2014). Bioactive peptides are more likely to be used in formulation of functional food due to their wide scope of activity.

Bioactive peptides are a rapidly developing field of research. According to the current state of scientific knowledge, every protein can contain fragments that possess some of many bioactivities. This chapter reviews recent research about producing of bioactive peptides from whey and peptide applications in food and beverages to create functional food.

2. Bioactivity of Whey Protein and Peptide

Bovine milk contains about 3.0% of protein (Fox and McSweeney, 1998) of which 80% is caseins and 20% is whey proteins that remain in the supernatant after precipitation of caseins. The bovine whey protein contains six major proteins, β-Lg, α-lactalbumin (α-La), immunoglobulins (Ig), bovine serum albumin (BSA), proteose-peptone, and glycomacropeptide [caseinomacropeptide (CMP)], which together make up 85% of whey protein. Whey contains several proteins at very low levels as lactoferrin (Lf) and lactoperoxidase. These proteins are regarded as highly significant due to their bioactivities. Characteristics of major whey proteins are given in Table 9.1.

Table 9.1

Approximate composition and characteristics of major whey proteins (Sharma and Shah, 2010; Zydney, 1998).
Proteins	Concentrations (g/L)	MW (kDa)	Isoelectric Point (pI)
β-Lg	3.2–4.0	18.4	3.2
α-La	1.2–1.5	14.2	4.70–5.1
BSA	0.3–0.6	69	4.7–4.9
IgG, IgA, IgM	0.6–0.9	150–1000	5.5–8.3
Protease-peptone	0.5–1.1	4–20
Lactoferrin	0.05	78	8.0
Lactoperoxidase	0.006	89	9.6
Glycomacropeptide	1.2	7

Whey proteins have been reported to have utility in many different applications ranging from effects on muscles and bone, blood, immune system brain, cancer, and infection. Whey protein seems to be more effective in physiological systems than casein due to faster digestion and presence of bioactive components (Boirie et al., 1997). Casein is easily hydrolyzed by digestive enzymes in the gastrointestinal tract, but whey protein hydrolyzed at a slower rate and maintains its function in the gastrointestinal tract.

There is substantial evidence that whey proteins influence the release of some satiety hormones. Insulin and glucagon-like peptide-1 release are stimulated by the ingestion of whey protein (Brubaker and Anini, 2003; Samra et al., 2007). Whey protein increased of cholecystokinin secretion more than casein (Hall et al., 2003). The concentration of peptide YY in plasma increased after intragastric administration of whey protein or whey peptide hydrolysate fractionation (Calbet and Holst, 2004). Recent studies showed that a whey drink caused significantly enhanced glucose-dependent insulinotropic polypeptide response more than branched amino acid mixture. It is possible that bioactive peptides present in whey and formed during digestion are the primary stimulators of glucose-dependent insulinotropic polypeptide secretion (Nilsson et al., 2007). Whey proteins were found to inhibit the growth many types of tumors more effectively than other food proteins (Parodi, 2007). Attaallah et al. (2012) showed that a lesser number of tumor foci were observed when whey protein hydrolysate (WPH) was fed to colon cancer-bearing rats.

The therapeutic properties of milk and whey proteins have been investigated by many researchers in the recent past (Madureira et al., 2007; Mils et al., 2011; Parodi, 2007). The functions of bioactive proteins and peptides from whey in human body are shown in Fig. 9.1.

Figure 9.1 Function of Bioactive Proteins and Peptides From Whey.

2.1. β-Lactoglobulin

β-Lg is the major protein in bovine whey. β-Lg represents approximately 50% of the total whey protein (Creamer and Sawyer, 2003; Fox and McSweeney, 1998). It is also a rich source of amino acid (cysteine), which plays a key role in the synthesis of glutathione (GSH) (Anderson, 1998). Glutathione is important for human health because it is an intracellular antioxidant that protects the body from the onslaught of stressors. Yahya et al. (2013) have reported the nexus between GSH deficiency and IL-8-driven pathogenesis. β-Lg binds retinol (vitamin A) and vitamin D, and promotes uptake of retinol via gastrointestinal tract. It has been speculated that this protein plays a role in the absorption of many fatty acids (Chatterton et al., 2006). Also, β-Lg is a molecular carrier that increases the accessibility of linoleic acid (Le Maux et al., 2012).

β-Lg exhibits prebiotic effects on Bifidobacterium and Lactobacillus probiotics species.

Bovine β-Lg is quite resistant to gastric digestion. This resistance may contribute to the allergy to bovine milk and whey. Resistance to digestion by pepsin is not characteristic of all mammals. For example, ovine β-Lg is highly sensible to digestion by pepsin (El-Zahar et al., 2005). While β-Lg is resistant to the gastric digestion, it is likely to be digested in intestinal tract by trypsin and chymotrypsin.

β-Lg fragments obtained by intestinal digestion or limited hydrolysis by specific enzymes show many bioactivities as opioid, antihypertensive activity, antimicrobial activity, imunomodulatory activity, and so forth. More bioactivities of fragments are shown in the section “Bioactivity of Whey Protein Fragments.”

2.2. α-Lactalbumin

α-La appears quantitatively second in whey. It comprises about 20% of all proteins in bovine whey (Fox and McSweeney, 1998). α-La originates peptides with antimicrobial (Haque et al., 2009) and antistress properties. Antistress activity is mediated by a high content of amino acid tryptophan used in serotonin synthesis. Evening intake of α-La by human volunteers increased plasma tryptophan bioavailability and improved morning alertness and brain measures of attention (Markus et al., 2005).

α-La shows bactericidal effects in the upper respiratory system and protective effects on gastric mucosa and provides protective effects against induced gastric mucosal injury caused by intake of nonsteroid antiinflammatory drugs (NSAID) or a large dose of ethanol in animal experiments (Mezzaroba et al., 2006; Rosaneli et al., 2004). In an acidic environment, human α-La forms the complex with oleic acid named HAMLET (human α-La made lethal to tumor cells). HAMLETs positive effect on human health is shown as inhibiting proliferation of different tumor types by an apoptosis mechanism with high selectivity (Svanborg et al., 2003). The BAMLET complex is the bovine equivalent of HAMLET. The BAMLET shows a strong cytotoxic effect on eight lines of tumor cells by increasing the permeability of the lysosomal membrane (Rammer et al., 2010).

Some fragments of α-La showed ACE-inhibitory activity. α-La fragment known as α-lactophorin was given subcutaneously to conscious, unrestrained, spontaneously hypertensive (SHR) and normotensive rats in experiment. It is shown that blood pressure decreased in the SHR rat group. Antihypersensitive effects have been associated with bioactive peptides originating from α-La and other whey components (Camfield et al., 2011).

2.3. Bovine Serum Albumin

BSA is not synthesized in the mammary glands; it is derived from the blood and represents 0.7%–1.3% of all whey proteins (Fox and McSweeney, 1998). As BSA is poorly represented in whey, its bioactivity is mostly examined as part of whey protein bioactivity. BSA possesses ACE-inhibitory activity and opioid-like characteristic (Poltronieri et al., 2012).

2.4. Immunoglobulins

Ig are protein complexes synthesized by B lymphocytes, derived in milk from blood serum. Ig represents 10%–15% of the total whey proteins (Nakai and Modler, 1996). Ig protects the mucosa of gastrointestinal tract from pathogenic microorganisms. In colostrum Ig’s role is to confer passive immunity to the neonate while its own immune system is developing (Gapper et al., 2007). Bovine IgG at concentrations as low as 0.3 mg/mL suppressed the synthesis of human IgG, IgA, and IgM by up to 98%. On the basis of these findings, it is concluded that Ig from bovine whey have the potential to modulate the immune response in humans (Reitelseder et al., 2011; Tipton et al., 2004). Also, bovine milk contains specific antibodies to Salmonella typhimurium, Salmonella enteritidis, Escherichia coli, Shigella flexneri, and human rotavirus (Yolken et al., 1985).

2.5. Proteoso-Peptone (PP)

Proteoso-peptone (PP) fraction is a minor peptide fraction in bovine whey. This fraction is a mixture of heat-stable acid-soluble (at pH 4.6) 135 amino acid phosphorylated glycoprotein that does not derive from caseins. Lactophoricin is one of two domens of proteoso-peptone fraction. Lactophoricin is able to permeabilize planar lipid bilayers (Campagna et al., 2001, 2004). It is a peptide with bacteriostatic activity against Gram-positive and Gram-negative bacteria.

2.6. Glukosomacropeptide (GMP) and Caseinomacropeptide

Glukosomacropeptide (GMP) is a C-terminal glycopeptide f(106–169) released from the κ-casein during cheese manufacturing. The nonglycosylated form of GMP is often termed CMP. CMP comprises from 10% to 15% of whey proteins. It shows prebiotic effects on Bifidobacterium and Lactobacillus probiotics species. CMP has been shown to increase the solubility of calcium and enhance the absorption of calcium in colon. Also, this is characteristic for other phosphorylated peptides derived from casein (Martinez et al., 2009). Many studies have shown that CMP possesses antimicrobial activity to fight caries and the oral pathogens Streptococcus mutans and Porphyromonas gingivalis (Aimutis, 2004). Also, it favors the growth of Lactobacilli in the oral cavity. GMP has been reported to inhibit bacterial and viral epithelial adhesion and to modulate the immune system response (Brody, 2000; Manso and López-Fandino, 2004). CMP shows antiinflammatory activity on monocytes (Requena et al., 2009) and strong ACE-inhibitory activity (Gobbetti et al., 2007). Oral intake GMP stimulates cholesystokinin release, a satiety hormone (Yvon et al., 1994). Bovine, ovine, and caprine CMP have been shown to inhibit platelet aggregation and the formation of thrombi (Poltronieri et al., 2012).

2.7. Lactoferrin

Lf is a nonheme iron-binding glycoprotein, with antimicrobial, antioxidative, antiinflammatory, anticancer, and immune regulatory properties (Caccavo et al., 2002; Marnila and Korhonen, 2009; Wakabayashi et al., 2006). Lf has bacteriostatic and bactericidal activity against Gram-negative and Gram-positive bacteria, fungicidal activity against Candida species. Also, Lf is capable of inhibiting replication of viruses (Marnila and Korhonen, 2009; Wakabayashi et al., 2006). The N-terminal lobe includes the lactoferricin (Lfcin) sequence, which possesses antibacterial activity (Lopez-Exposito and Recio, 2006). Lf and its sequence lactoferricin inhibit the attachment of Streptococcus mutans to hydroxyapatite or purified saliva host ligands (Johansson and Holgerson, 2011). Bovine Lf inhibit the proliferative response and cytokine production of Th1, but not Th2 cell lines. Oral intake reduced the spontaneous production of IL-6 and TNF-α by cultured peripheral blood mononuclear cells (PBMCs) and enhanced chemotactic reactions by promoting the recruitment of leukocytes to the inflammatory site (Zimecki and Kruzel, 2007). Both bovine and human Lf are anabolic factors for the bone in a mouse model (Cornish, 2004). Lf protects against lethal endotoxin shock in germ-free piglets (Lee et al., 1998).

2.8. Lactoperoxidase

Lactoperoxidase is the most abundant enzyme in whey. It has demonstrated antibacterial effects against a range of species. It catalyzes an antimicrobial system consisting of the thiocyanate anion (SCN⁻) and hydrogen peroxide to generate short-lived oxidation products, primarily hypothiocyanate (OSCN⁻), which kill or inhibit the growth of a wide range of bacteria, viruses, fungi, and protozoa (Seifu et al., 2005).

3. Production of Bioactive Peptide From Whey

Besides intact proteins, increasing interest is focused on peptides obtained from proteins by enzymatic hydrolysis. Biofunctionality characteristics of peptides depend on the amino acid sequence and the number of residues. Peptides that contain the same amino acids may exhibit different bioactivity because of different steric forms or hydrophobicity. Also, hydrophobic amino acids contribute to the antioxidant activity in particular when they are located at the N terminal of a peptide (Li et al., 2011). Milk and especially whey proteins are currently the main source of biologically active components. Bioactive peptide fragments from whey protein have a wider scope of activity than intact proteins. They play regulatory roles, but also directly influence various developmental and metabolic processes. Moreover, some activities attributed to proteins are actually activities of peptide fractions released during digestion in gastrointestinal tract. For example, Lf during intestinal digestion released bioactive peptide called lactoferricin. Proteolysis of Lf by pepsin produces N-terminal arginine-rich fragments (lactoferricin), which have antimicrobial, antiviral, and antiparasitic activity (Marnila and Korhonen, 2009; Wakabayashi et al., 2006). Also, GMP has been detected in the plasma of volunteers after milk or yogurt ingestion (Chabance et al., 1998). Lactoferricin has many bioactivities but its releasing and stability in gastrointestinal tract is variable. The last step constant yields and constant quality of the bioactive peptides can be reached by controlled hydrolysis.

Bioactive peptides can be obtained in three ways:

1. hydrolysis by digestive enzymes
2. hydrolysis by enzymes derived from microorganisms or plants
3. fermentation of milk with proteolytic starter cultures

3.1. Hydrolysis of Whey Proteins During Fermentation Process With Proteolytic Starter Culture to Obtain Bioactive Peptides

A number of probiotic bacteria show proteolytic activity. These bacteria have been shown to hydrolyze whey proteins to increase the number of peptides available for their growth. The peptides and amino acids released during fermentation contribute to the typical flavor and texture of dairy products. That is the reason why many industrially used dairy starter cultures are highly proteolytic.

Fermented milk and peptides isolated from fermented milk products showed various bioactivities. Despite this, the fermentation is not an easy way to produced required bioactive peptide. Fermentation is a complex process and many factors have influence to the process and production of bioactive peptides. In order to understand the choice of the culture used, it is essential to be familiar with the peptide-releasing mechanism. The process starts when the cell-wall bound proteinases, which have a very broad specificity, initiate the proteolysis and release oligopeptides. Oligopeptides that cannot be transported into the cells can be further degraded after lysis of bacterial cell by released intracellular enzymes. Having this in mind, it becomes clear that is important to choose the strains or combination of strains with optimal proteolytic activity and lysis at the right time, as the number of bioactive peptides depends on a balance between formation and degradation of inactive peptides and amino acids (Korhonen and Pihlanto, 2007).

Many studies have demonstrated that Lactobacillus helveticus strains are capable of releasing ACE-inhibitory peptides as tri-peptides (VPP) and (IPP). Pihlanto-Leppala et al. (1998) studied the potential formation of ACE-inhibitory peptides from whey during fermentation with various lactic acid starters. ACE-inhibitory activity was not proved after fermentation, but further digestion with pepsin and trypsin was produced ACE-inhibitory peptides. It is identified peptide corresponding to β-lactorphin sequence in the hydrolysate after whey fermentation with Kluyvermyces marxinaus var marxianus (Belem et al., 1999). Elfahri (2012) studied the release of bioactive peptides from milk proteins by selected Lactobacillus species. Ten strains of Lactobacillus species (Lactobacillus helveticus 474, Lactobacillus helveticus 1188, Lactobacillus helveticus 1315, Lactobacillus helveticus 953, Lactobacillus delbrueckii ssp. bulgaricus 734, Lactobacillus delbrueckii ssp. bulgaricus 756, Lactobacillus delbrueckii ssp. bulgaricus 857, Lactobacillus delbrueckii ssp. lactis 1210, Lactobacillus delbrueckii ssp. lactis 1307, and Lactobacillus delbrueckii ssp. lactis 1372 were assessed for growth characteristics, proteolytic activity, and release of angiotensin-converting enzyme inhibitory peptides. The presence of selected LAB enhanced proteolysis significantly compared to the control (sample without LAB). Also, it has been shown that proteolytic activity varied with changes in pH.

All strains in Elfahri (2012) study released bioactive peptides with ACE-inhibitory activities between 1.26% and 48.69%. Three strains of L. helveticus (474, 1188, and 1315) showed high proteolytic and antihypertensive activity.

Pan and Guo (2010) identified and purified the new ACE-inhibitory peptides from whey protein hydrolyzed by crude proteinases (include cell-envelope proteinase and intracellular peptidases) of L. helveticus LB10. It was a new ACE-inhibitory peptide isolated from β-Lg f(148–153), with a peptide sequence of RLSFNP and an IC₅₀ value of 177.39 mM.

Fermentation is a complex process that includes the whole microorganisms, with all its enzymes. A large number of enzymes in the fermentation system require a complex control, with the large possibility of mistake. Despite the fact that the fermentation process is often an inexpensive way to produce the desired product, the fermentation is not an easy way to produced bioactive peptides. Controlled enzymatic hydrolysis better meets the requirement for constant and large yield.

3.2. Enzymatic Hydrolysis: The Most Common Way to Obtain Bioactive Peptides From Whey Proteins

Enzymatic hydrolysis converts protein into peptides of various sizes. Hydrolysis is stopped when the desired degree of hydrolysis (DH) is reached, usually by temperature inactivation of the enzyme. Incubation at 100°C for 10 min is adequate for inactivation of most of the enzymes, but some proteases, such as bromelain, retained about 20% of original activity after incubation at 105°C for 30 min (Poh and Abdul Majid, 2011). Hydrolysates properties are dependent on the type of enzyme used, the substrate pretreatment and the DH (Jeewanthi et al., 2014). Certain studies show that hydrolysis is effective after heat (Adjonu et al., 2013) or high-pressure pretreatment (Piccolomini et al., 2012). It has been shown that the DH increases significantly in the first 60 or 90 min and after that increases considerably more slowly (Dryakova et al., 2010; Herregods et al., 2015; Krunic et al., 2016a). Some papers report that the extension of the time of hydrolysis leads to stagnation or even decrease in some bioactivities, such as ACE–inhibitory activity and total reducing power of the hydrolysate (Corrêa et al., 2014; Herregods et al., 2015). DH is usually defined as the percentage of peptide bonds cleaved and it can be determined with the pH-stat method described by Adler-Nissen (1986). It is a method based on measuring the amount of acid or base needed to keep the pH constant during hydrolysis. Also, DH can be defined as the amount of nitrogen soluble in trichloroacetic acid after hydrolysis, thus not expressing the percentage of cleaved peptide bonds (Drago and González, 2000). Very common methods used for DH determination are based on quantify free amino groups with trinitrobenzenesulfonic acid, o-phthaldialdehyde (OPA), or formol titration. Silveira et al. (2013) compared OPA method, formol titration method, and freezing point measurement method for determination of DH during hydrolysis of whey proteins with pancreatin. Determination of DH is important because it is the most commonly used parameter to structurally differentiate between different hydrolysates. DH determination is not the topic in this chapter. The various methods of DH determination have been summarized in a review by Rutherfurd (2010).

Pancreatic enzymes, especially trypsin and chymotrypsin are commonly used for production of bioactive peptide. Also, proteinases, such as pepsin, thermolysin, alcalase, proteinase from bacterial and fungal sources or from plants and animals have been used. Many enhancements are reached in numerous studies with digestive enzymes. The hexapeptide VAGTWY, derived from β-Lg by trypsin hydrolysis was found to have all three examinated bioactivities (antioxidant, ACE-inhibitory activity. and DPP-IV inhibitory activity) (Power et al., 2014.). Also Power et al. (2014) detected two new bioactive peptides with ACE-inhibitory activity. Bioactivities of derived peptides are diverse. Alcalase-hydrolyzed whey protein liberated a pentapeptide (VHLKP), which demonstrated significant antioxidant activity and protection of human lung fibroblast MRC-5 cells from H₂O₂ activity (Kong et al., 2012). WPH derived by pepsin exhibited significant antimicrobial activity (Theolier et al., 2013). Quintieri et al. (2012) shows that pepsin-digested hydrolysate of bovine lactoferin possess antimicrobial activity against E. coli K12. Very active antimicrobial hydrolysates were obtained by hydrolysis of whey protein by pepsin and trypsin (Benkerroum, 2010). But also, fractions that contain proline are very resistant to the influence of digestive enzymes (Beermann and Hartunga, 2013).

Hydrolysis of α-La and β-Lg by pepsin and trypsin separately or in combination resulted in similar ACE-inhibitory activities. The order of enzyme additions affected types of derived peptides in final hydrolysates but not on ACE-inhibitory activity. A combination of different enzymes can alter digestion and peptides released, but will not necessarily result in greater bioactivity (Mullally et al., 1997).

In vitro digestibility of bioactive peptides derived from bovine β-Lg is not only influenced by pH, time of incubation, and enzyme/substrate ratio, but also by the length and the nature of the peptides (Roufik et al., 2006) Also, other peptides present in the medium can influence the hydrolysis by gastric enzymes. It was concluded that in a mixture, peptides may form complexes that interact more efficiently with the hydrolyzing enzymes compared with peptides alone. The thermal or pH denaturation of whey protein prior to enzymatic hydrolysis has been shown to affect the bioactive properties (Leeb et al., 2015; O’Loughlin et al., 2014).

Bertucci et al. (2015) showed that after 180 min of hydrolysis of bovine whey with peptidases from Maclura pomifera fruit, α-La and β-Lg were almost completely degraded. Peptides smaller than 3 kDa showed ACE inhibitory activity and antioxidant capacity. The authors concluded that the results support the conclusion that, by the presence of ACE-inhibitory and antioxidant peptides, it would be possible to use these WPHs for functional food manufacturing.

Tavares et al. (2011) showed that aqueous extracts from the plant C. cardunculus could bring about release of peptides from whey protein concentrate (WPC) that exhibit potent ACE-inhibitory effects.

Kim et al. (2010) showed that significant amounts of BSA, β-Lg, and α-La survived papain digestion of heated colostral whey. Alcalase completely eliminated BSA, β-Lg, and α-La, while pepsin completely removed BSA but not β-Lg.

Adriena et al. (2010) reported that on hydrolysis with microbial proteases (alcalase, flavorzyme, protamex, and neutrase) the antioxidant activity of whey protein increased from 7%–19.8% to 40%–54.2%. Naik et al. (2013) found increased antioxidant activity in WPHs compared to the intact proteins. Castro and Sato (2014) also found an increase in antioxidant activity up to 205.3% after hydrolysis of protein.

After hydrolysis, separation of bioactive peptides from other peptides in hydrolysates is an important step. Ultrafiltration (UF) is the most common way to separate small peptide.

3.3. Ultrafiltration as Method for Obtaining Bioactive Peptide

Various technologies have been applied for the separation of bioactive peptides from the hydrolysates. Membrane filtration shows the most potential to be used in food industry, especially UF with molecular weight cut-off membrane, due to its simplicity and low prices. UF membranes are traditionally produced using polymers, such as cellulose acetate, and regenerated cellulose. Many studies showed that ultrafiltration with MWCO membranes is useful to separate small bioactive peptides from the rest peptides (Krunić et al., 2015; Poste et al., 2012; Power et al., 2014). UF can be used in the cheese industry to fractionate the proteins from whey (Athira et al., 2015).

Power et al. (2014) concluded that the two most potent β-Lg derived ACE-inhibitory peptides were IPAVFK and IIAEK, which contain less than six amino acid residues. In contrast, the longer peptide TPEVDDEALEK showed very little ACE inhibitory activity. Recently, Demers-Mathieu et al. (2013) obtained a retentate rich in anionic peptides using a pilot scale nanofiltration from UF permeate of whey protein tryptic hydrolysate. It is determined the antibacterial activity of these peptides derived from β-Lg against L. monocytogenes and S. aureus. They also reported that obtained activity could be improved by process optimization.

Welderufael and Jauregi (2010) used UF for separation bioactive peptide from whey treated with proteolytic commercial mixtures. According to Welderufael and Jauregi (2010), incorporating 1 kDa UF membrane enables to separate the most active peptides from less active or inactive peptides.

Gupta et al. (2013) used UF for isolation bioactive peptide from water-soluble extracts of the peptides formed in cheddar cheese.

Athira et al. (2015) concluded that the hydrolysis of UF retentate of whey is an effective method for production of WPH directly from whey and it can be used as energy and cost-saving method compared to the industrial production of WPH from WPC.

Bordenave et al. (1999) showed that α-lactorphin (bioactive fragment) was generated with continuous hydrolysis of goat whey in an ultrafltration reactor.

Qian et al. (2011) used UF MWCO membrane (1, 3, 5, and 10 kDa) for separation peptide with the highest ACE-inhibitory and antioxidant activity from fermented skim milk.

Krunić et al. (2015) used MWCO membrane (3, 10, and 30 kDa) for separation peptide with the highest ACE-inhibitory and antioxidant activity from WPH obtained by proteinase K hydrolysis.

Uluko et al. (2014) used UF and nanofiltration to obtain fraction with the highest ACE-inhibitory activity and fraction with the highest antioxidant activity from milk protein hydrolysates.

Tavares et al. (2011) showed that the peptide mixture generated under optimal processing conditions exhibited an IC₅₀ value of 52.9 ± 2.9 μg/mL, but its fraction with MW below 3 kDa had an IC₅₀ of 23.6 ± 1.1 μg/mL.

Laboratory UF unit with MWCO membrane is shown in Fig. 9.2.

Figure 9.2 Photo of millipore ultrafiltration stirred cell unit (Model 8050 1 Unit, Millipore Corporation, Bedford, MA, USA) with whey protein hydrolysate (A), schematic of millipore ultrafiltration stirred cell unit (B).

The applications of membrane processes in food industry can be classified into three main areas, namely dairy industry, beverage industry, and fish and poultry industries. UF has found a major application in the dairy industry. It is used for production of WPC and whey protein isolate (WPI), but also for separation of small bioactive peptides with molecular weight less than 3 kDa. Pihlanto-Leppala et al. (2000) found that the ACE-inhibitory activity in the <1 kDa fraction was, in many cases, higher than in the other fractions.

These results indicate that ultrafltration is a great way to derive bioactive peptides from whey protein. This technique provides good possibilities for enriching peptides with a low molecular mass and it is easily upscaled.

3.4. Bioactivity of Whey Protein Fragments

As previously stated, some activities attributed to proteins are actually activities of peptide fractions released during digestion in gastrointestinal tract. Those activities and many other activities of fragment released from whey peptides by hydrolysis are shown in Table 9.2.

Table 9.2

Bioactive fragments obtained by hydrolysis of different whey proteins.
Proteins	Enzymes	Fragments		Bioactivities	References
α-Lactalbumin	—	f(97–104)	DKVGINYW	ACE-inhibitor	Tavares et al. (2011)
	—	—	KGYGGVSLPEW	ACE-inhibitor	Tavares et al. (2011)
	—	f(59–60)	IW	Antihypertensive	Martin et al. (2008)
	Trypsin	f(104–108) f(99–108)	WLAHK, VGINYWLAHK	ACE-inhibitor	Pihlanto-Leppala et al. (2000)
	Pepsin	f(117–121)	K7VGIN	Antimicrobial activity: mainly Gram (−) bacteria	Theolier et al. (2013)
	—	f(50–53)	YGLF	Opioid-like peptide, ACE inhibitor	Haque et al. (2009)
	—	—	YGGF	Opoid	Ijäes et al. (2004)
Lactoferin	Pepsin	—	—	Antimicrobial activity	Quintieri et al. (2012)
Serum albumin	—		ALKAWSVAR	ACE	Poltronieri et al. (2012)
Serum albumin	—	f(399–404)	YGFQDA	Opoid-like	Poltronieri et al. (2012)
GMP	Trypsin	f(106–112)	MAIPPKK	ACE-inhibitor	Gobbetti et al. (2007)
	Trypsin	f(106–111)	MAIPPK	ACE-inhibitor	Gobbetti et al. (2007)
		f(106–116)	—	Antithrombotic activity	Phelan and Kerins (2011)
	—	f(108–110), f(106–112), f(113–116), f(112–116)	—	Antithrombotic activity	Martinez et al. (2009)
β-Lactoglobulin	—	f(33–42)	DAQSAPLRVY	ACE-inhibitor	Tavares et al. (2011)
	—	f(40–42), f(122–124)	RVY, LVR	Antihypertensive	Hernández-Ledesma et al. (2004)
	—	f(46–48), f(142–145), f(15–20)	LKP, ALPM, VAGTWY	Antihypertensive	Català-Clariana et al. (2010)
	—	f(36–42)	SAPLRVY	ACE-inhibitor	Welderufael and Jauregi (2010)
	—	f(102–105)	YLLF	ACE-inhibitor, opoid	Sipola et al. (2002)
	—	—	TPEVDDEALEK	ACE-inhibitor	Picariello et al. (2010)
	—	f(78–80)	IPA	ACE-inhibitor	Meisel et al. (2009)
	—	f(102–103),	YL	ACE-inhibitor
	—	f(142–148)	ALPMHIR	ACE-inhibitor
	Trypsin	f(15–20), f(25–40), f(78–83), f(92–100)	VAGTWY, AASDISLLDAQSAPLR, IPAVFK, VLVLDTDYK	Antimicrobial activity Gram (+) bacteria	Pellegrini et al. (2001)
		f(71–75)	IIAEK	Hypocholesterolemic properties	Nagaoka (2006)
		f(22–25)	LAMA	ACE-inhibitor	Pihlanto-Leppala et al. (2000)
		—	VAGTWY	ACE-inhibitor, DPP-IV inhibitor antioxidant	Power et al. (2014)
		f(15–20)	VAGTWY	Antioxidant	Power et al. (2014)
		f(19–29), f(145–149), f(42–46), f(58–61), f(95–101)	— — — — —
		—	IPAVFK	DPP-IV inhibitor	Silveira et al. (2013); Uchida et al. (2011)
		—	VAGTWY
		—	TPEVDDEALEK
		f(15–20)	VAGTWY	ACE-inhibitor	Ijäes et al. (2004)
	Pepsin	f(50–54), f(117–121)	PEGDL, KVGIN	Antimicrobial activity: mainly Gram (−) bacteria	Theolier et al. (2013)
	Pepsin	f(14–18), f(123–125), f(147–149)	K14VAGT, VRT, IRL	Antimicrobial activity: mainly Gram (+) bacteria	Theolier et al. (2013)
	Pepsin + Trypsin + Chymotrypsin	f(94–100)	VLDTDYK	ACE-inhibitor	Pihlanto-Leppala et al. (2000)
	CorolasePP	f(19–29)	WYSLAMAASDI	Antioxidant	Hernández-Ledesma et al. (2005)
		f(42–46)	YVEE
		f(145–149)	MHIRL
	Chymotrypsin	f(146–149)	HIRL	Ileum-contracting activity	Sipola et al. (2002)

4. Functional Properties

4.1. Solubility

Of all functional properties of proteins, solubility is probably the most important one. It has the largest impact on their overall usefulness in food systems and insoluble proteins have very limited application potential in food. Other important functional properties, such as foaming and emulsifying or gelation properties also require the protein to be soluble in the relevant medium. Solubility of protein depends on hydrophobicity and the charge of the amino acid, as well as conformation and surface properties (Walstra, 2003). pH and ionic strength strongly influence a protein’s solubility in a defined medium. Larger proteins generally have lower solubility than small peptides. Also, proteins have low solubility on isoelectric point (pI) because protein–protein interaction is more favorable than protein–water interaction, so compatibility of protein and enzyme during hydrolysis is very important. pH value in which enzyme used for hydrolysis has the strongest activity should not be the same value as pI of protein. Whey proteins have very good solubility in comparison with plant proteins. That is also a reason why whey proteins are more suitable for application in functional foods. Cassiani et al. (2013) examined whey proteins solubility of the heated mixtures (whey protein, gelatin, starch, and sucrose) in media that disrupt different kinds of bonds. First, the system disrupts electrostatic bonds, whereas the second system disrupts electrostatic, hydrophobic, and hydrogen bonds. Solubility of whey protein was excellent in both systems in contrast to the other tested samples.

4.2. Water- and Fat-Holding Capacities

Water- and fat-holding capacities (WHC and FHC) of proteins are important for determining texture properties of food products, such as juiciness, mouthfeel, and tenderness (Zayas, 1997). WHC is an important criterion to evaluate the acceptability of food systems. Meat proteins have excellent WHC and FHC, which explains the enjoyable organoleptic properties of many meat products. Cassiani et al. (2013) showed that whey proteins strongly hold water. Whey proteins with good WHC and FHC can be used as meat protein alternatives. WHC of the hydrolysates is higher than WHC of the nonmodified protein. This may be due to increased availability of polar, ionizable groups liberated during hydrolysis.

4.3. Gel Formation

Gels are important for sensory and textural properties of many different food products. There is no simple definition of a gel. It consists of a three-dimensional polymer network in which the gaps are usually filled with water. This gel framework can be a polysaccharide, a protein, or a combined protein–polysaccharide network. To form a gel, the native protein has to unfold (by protein denaturation) to facilitate interaction between, and aggregation of, different protein strands (Wouters et al., 2016). Gel formation can be induced by a physical stimulant, usually heat or in some cases pressure, or by a biochemical stimulant, such as an enzyme. The most important interactions for gel forming are covalent bonds, such as disulfide bonds, hydrogen bonds, and hydrophobic interactions, but also properties of the food matrix, such as protein concentration, pH, temperature, pressure, and ionic strength (Foegeding and Davis, 2011). Gel quality can be evaluated by elasticity determination by compression or indentation tests with a texture analyzer (Nieto-Nieto et al., 2014), by determination of the storage modulus (G′) of gels (Zhao et al., 2011), and by determination of WHC of gels (Foegeding and Davis, 2011). Gel formation is important for whey protein application for probiotic encapsulation, which has been described below.

4.4. Foam Formation and Stability

Foams are dispersions of gas, usually air, in a continuous phase, usually a liquid. In products, such as chocolate mousse, coffee preparations with milk foams, meringue, or cakes foams play important textural and structural roles (Foegeding and Davis, 2011). The air–water interfaces are thermodynamically unstable and foams rapidly collapse. Some proteins act as surfactant and kinetically stabilize foam interfaces. The foaming potential of proteins is assessed based on foaming capacity, namely the initial amount of foam formed after stirring or whipping, and foam stability, which is the amount of foam remaining after a certain time.

Krunic et al. (2016a) showed that whey protein hydrolysated by thermolysin possess better foaming properties than nonhydrolysated whey protein.

4.5. Emulsification

Emulsions consist of two immiscible phases. The most common emulsion in food industry is mix of oil phase and water. Like foams, emulsions are thermodynamically unstable. The more important destabilizing factors are coalescence and creaming, which is the tendency of oil to rise to the top of an emulsified system. Hydrolysis is a common way to improve emulsification properties of protein dilution.

Krunic et al. (2016a) showed that whey protein hydrolysated by thermolysin possess better emulsification properties than nonhydrolysated whey protein. Authors conclude that well-controlled enzymatic hydrolysis is good way to improve technological characteristics of protein and peptide intended for application in food products.

Palatnik et al. (2015) asserted that the good emulsifying properties, high water and oil holding capacity, and the rheological behavior of the caprine WPC, suggested the application of it in the formulation of a dressing.

4.6. Flavor

Flavor and changes in texture and aroma are important factors for peptide application in food products. Bioactive peptides are usually tasteless or have a bitter taste, except glutamic acid and aspartic acid, which have a very sweet taste. The bitterness of peptide fraction increases with increasing hydrophobicity and the length of peptide sequences (Jakala and Vapaatalo, 2010). Four peptides identified in a WPH were shown to be responsible for 88% of the bitterness of hydrolysate. These four peptides were TGLF [α-La f(50–53)), IPAVF (β-Lg f(79–82)], LLF [β-Lg f(103–105), Lf f(298–300) and Lf f(659–641)] and YPFPGPIPN [β-CN f(60–68)] (Liu et al., 2014). Wang et al. (2013) showed a correlation between selected bioactivities (ACE inhibition and antioxidant activity) of di- and tri-peptides and their bitterness. Debittering of an ACE inhibitory WPH has successfully been achieved by utilization of commercially available exopeptidases while maintaining its bioactivity (Cheung et al., 2015) or by encapsulated peptides as shown in studies by Ma et al. (2014) and Subtil et al. (2014).

5. Bioactive Peptide in Food Industry

The global functional food market is growing because modern consumers show an increasing interest in foods that can improve health and reduce the risk of disease. In Western Europe and North America, fermented milk products, such as yogurt, are the most popular health beverages. Dairy-based produce accounts for approximately 43% of the functional beverage market, and is mainly comprised of fermented products (Ozer and Kirmaci, 2010). Some commercially available food and intermediate food products containing bioactive peptides derived from whey protein, such as BioPURE-GMP and BioZate, are mentioned in a review by Dziuba and Dziuba (2014).

5.1. Production of Bioactive Peptides During Food Processing

During processing, the proteins contained in food can modify. Bioactive peptides are often liberated during the manufacture of different dairy products. Proteases in food, such as plasmin in milk, can hydrolyze proteins during storage or food processing or in probiotic and fermented product intracellular peptidases of lactic acid bacteria will most likely contribute to further degradation after lysis. The strains should be able to produce bioactive peptides with desired bioactivities, but not to be that much proteolytic to destroy the product during storage time. The formation of bioactive peptides during the fermentation process was previously described in the section “Hydrolysis of Whey Proteins During Fermentation Process with Proteolytic Starter Culture to Obtain Bioactive Peptides.” The main goal is to find the best proteolytic culture and process conditions for certain medium.

Conti et al. (2012) studied the ability of mixture composed of L. helveticus, L. paracasei, L. fermentum, L. gasseri, L. parabuchneri, L. casei, L. panis, Pichia kudriavzevii, and S. cerevisiae to degrade whey proteins by fermentation to obtain a functional drink.

Pescuma et al. (2008) evaluated the potentiality of three lactic acid bacteria strains to design a starter culture for developing functional whey-based drinks. Lactobacillus delbrueckii subsp. bulgaricus CRL 454 was the most proteolytic (91 mg Leu/mL) strain and released the BCAAs Leu and Val. Lactobacillus acidophilus CRL 636 and S. thermophiles CRL 804 were able to degrade the major whey proteins, α-La and β-Lg. The amino acid release was higher for the starter Streptococcus thermophiles CRL 804 and Lactobacillus delbrueckii subsp. bulgaricus CRL 454 than for Lactobacillus acidophilus CRL 636 and S. thermophiles CRL 804.

Terzic-Vidojevic et al. (2014) investigated the composition of lactic acid bacteria in autochthonous young cheeses, sweet creams, and sweet kajmaks produced in central Bosnia and Herzegovina over a four-season period. These three products were made from bovine milk by a traditional method without the addition of a starter culture. Fifteen species were identified as follows: Lactococcus lactis, Lactococcus raffinolactis, Lactococcus garviae, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus helveticus, Enterococcus faecium, Enterococcus durans, Enterococcus faecalis, Enterococcus italicus, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc lactis, Streptococcus thermophiles and Streptococcus mitis. Proteolytic activity was exhibited by 45 % of the lactobacilli and 54.4% of the lactococci.

Table 9.3 shows proteolytic activity of different cultures at different temperatures during 4 h of fermentation. Fermentation medium was bovine whey obtained from domestic dairy plant Imlek a.d. (Belgrade, Serbia). The chemical composition of whey was: total solids 9.8% ± 0.03% (w/v); protein 2.6 ± 0.012% (w/v); fat 1.05% ± 0.08% (w/v); and lactose 5.6% ± 0.114% (w/v). Commercial lyophilized dairy starter culture “Lactoferm ABY 6,” supplied by Biochem s.r.l. (Monterotondo, Rome, Italy), and the strain Lactobacillus rhamnosus ATCC 7469, supplied by American Type Culture Collection (ATCC, Rockville, USA), were used for fermentation in this study. Starter culture “Lactoferm ABY 6” is mixture of Streptococcus salivarius ssp. thermophilus (80%), Lactobacillus acidophilus (13%), Bifidobacterium bifdum (6%), and Lactobacillus delbrueckii ssp. bulgaricus (1%).

Table 9.3

Viability and proteolytic activity of “Lactoferm ABY 6” and “Lactoferm ABY 6” with Lactobacillus rhamnosus ATCC 7469 after 4 h of whey-based substrate fermentation at 39, 42, and 45°C.
LAB Cultures	Fermentation Temperatures (°C)	Cell Growth Expressed as the Difference in the Viable Cell Count After and Before Fermentation (∆logCFU/mL)	Proteolytic Activity Expressed Over the Difference in the Content of Leucine After and Before Fermentation (∆mMLe)
“Lactoferm ABY 6”	39	1.45	0.46
	42	1.21	0.37
	45	1.82	0.39
“Lactoferm ABY 6” and L. rhamnosus ATCC 7469	39	0.87	0.11
	42	0.69	0.21
	45	0.72	0.13

Commercial yogurt culture “Lactoferm ABY 6” in combination with Lactobacillus rhamnosus ATCC 7469 showed the best proteolytic activity at 42°C, but the lowest cell growth. Commercial yogurt culture “Lactoferm ABY 6” showed even higher proteolytic activity at 39°C, the temperature with medium cell growth. Proteolytic activity is not proportional with cell growth but is correlated with it.

Miclo et al. (2012) showed that under nongrowth conditions, it is possible to obtain different digestion profiles of various casein hydrolysates. Agyei et al. (2012) reported that the higher production of cell envelope proteinase (CEP) of L. delbrueckii subsp. lactis can be achieved in the batch fermentation if the process is properly aerated with stable temperature at 45°C and initial pH between 5.5 and 6.5. Cell envelope proteinase is one or more cell wall protease(s) capable of hydrolyzing proteins to peptides containing 4–30 residues.

Elfahri (2012) investigated 10 strains of Lactobacillus species (Lactobacillus helveticus 474, Lactobacillus helveticus 1188, Lactobacillus helveticus 1315, Lactobacillus helveticus 953, Lactobacillus delbrueckii ssp. bulgaricus 734, Lactobacillus delbrueckii ssp. bulgaricus 756, Lactobacillus delbrueckii ssp. bulgaricus 857, Lactobacillus delbrueckii ssp. lactis 1210, Lactobacillus delbrueckii ssp. lactis 1307, and Lactobacillus delbrueckii ssp. lactis 1372) for proteolytic activity and release of angiotensin-converting enzyme inhibitory peptides using milk as substrate. The proteolytic activity varied with changes in pH, time of fermentation and of course it was strain dependent. Three strains of L. helveticus (474, 1188, and 1315) showed high proteolytic and antihypertensive activity. These strains were further investigated for aminopeptidase activity. Aminopeptidase activity was found in both extracellular and intracellular extract to various extent in all three selected L. helveticus strains, while only oligopeptidase activity was observed in extracellular extract. The highest antioxidant and ACE-inhibitory activity were observed in the soluble freeze dried peptides of crude proteinase extract of L. helveticus 1188 compared to the other strains used at 12 h of incubation time. The effects of soluble peptides produced by crude proteinase extracts of individually selected L. helveticus strains on cytokine production by human PBMCs were determine. Effects of soluble peptide samples on the stimulation of all tested cytokine Th2 Interleukin-10 and Th1 Interferon-γ production were detected in varied levels at 6 and 12 h. These bioactive peptides might have capability to drive immune responses in opposite directions in vitro and thus may bring about imbalance in the Th1/Th2 type cytokines. In a recent research it was found that L. lactis strains isolated from artisanal dairy starters or commercial starter cultures are potential for the production of fermented dairy products with ACE-inhibitory properties (Rodríguez-Figueroa et al., 2010).

Rodríguez-Figueroa et al. (2010) evaluated and compared ACE-inhibitory activity of water-soluble extracts isolated from milk fermented by wild and commercial starter culture Lactococcus lactis strains after 48 h of incubation. The highest ACE-inhibitory activities were found in water-soluble extracts of milk inoculated with wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures.

Cheese is a complex food containing a large number of different peptides, which change with the ripening time. Paul and van Hekken (2010) evaluated ACE-inhibitory activity of cheese extracts. Wang et al. (2011) evaluated the influence of certain probiotic strains on bioactivity of different kind of cheeses. In their studies, starter cultures with the addition of L. helveticus ND01 produced Gouda cheese with significantly more proteolysis than control cheeses. Gouda cheeses with L. helveticus ND01 adjunct revealed significant increase in ACE-inhibitory activity and GABA content.

5.2. Addition of Whey Protein and Peptide in Food (Enrichment of Food With Whey Proteins and Peptides)

Reports of therapeutic effects of functional foods and food ingredients have increased. A wide variety of dairy ingredients (especially milk proteins and peptides) is commercially available and used in common food products, such as confectioneries, cereal bars, beverages, and sports supplements. Up to now, whey proteins are usually used as dietary ingredients for bodybuilders and sportist. Many studies investigate the effect of whey protein on satiety. A mice-model study showed that filtered whey protein can prevent obesity. The beneficial effect was attributed to the α-La (Shi et al., 2012a) and Lf, which prevent fatty liver formation and slow down weight gain (Shi et al., 2012b). Also, a study in obese human subjects showed that WPC ingestion prior to main meal intake positively effects to body fat mass and lean muscle (Tahavorgar et al., 2014). Modern lifestyle requires dietary corrections and the wide range of application of bioactive components in the daily diet of human. A global survey has projected that 27 major cancers cause millions of deaths annually (Ferlay et al., 2015). Also, high blood pressure is a major risk factor for cardiac issues (Udenigwe and Mohan, 2014). Many studies provide strong evidence that free radicals from food play an important role in development of many diseases. The action of antioxidants may help to reduce the formation of oxidized molecules and prevent cellular deregulation and subsequent development of disease (Hernandez-Ledesma et al., 2011; Wojcik et al., 2010).

Lim et al. (2011) showed that commercial yogurt fortified with different whey hydrolysates possesses ACE-inhibitory activity. Also, physicochemical characteristics, such as pH (3.47–3.77), titratable acidity (0.81%–0.84%), coloration, viable cell count, and sensory qualities were not significantly different among the tested yogurt beverage samples during storage. These results showed that yogurt beverage fortified with WPHs maintained antihypertensive activity and underwent no unfavorable changes in physicochemical characteristics regardless of enzyme type.

Kim et al. (2010) showed that alcalase can be used to generate good iron-binding peptides in heated colostral whey. Also, Kim et al. (2010) concluded that iron-binding peptides could be suitable as a value-added food ingredient for food supplements.

Jisha and Padmaja (2011) used WPC to fortify protein in cassava flour. Flour was used for making two baked products, such as muffins and biscuits. WPC was found to be an excellent replacer for eggs for producing eggless muffins and biscuits, which also significantly elevated the protein content of these baked goods made from cassava. Baked goods having WPC could be promoted for patients suffering from egg allergy and also for vegetarians.

Wronkowska et al. (2015) used acid whey for fortified wheat and wheat-rye products. The results obtained in their study indicate that whey concentrated by UF could be used as a functional ingredient of wheat and wheat–rye baking products, especially due to high concentrations of elements significant for well-being.

Palatnik et al. (2015) investigated characteristics of ultrafiltrated caprine whey protein and its application in a food protein formulation. The high water and oil holding capacity, good emulsifying properties, and the rheological characteristics suggested the application of the WPC in the formulation of a dressing. Samples were similar to a commercial dressing in viscosity, texture, moisture, and ash content. Also, the sensory analysis demonstrated a good acceptance of the samples. The protein content of the product (0.97 ± 0.12 g/100 g) duplicated the value of the commercial sample incorporating higher added value to a product with high consumption.

If it is known that bioactive peptides can contribute a slight bitterness to food formulation; besides yogurt, chocolate and coffee can also be ideal flavor systems for application of those peptides.

Mann et al. (2015) used WPC and WPH (whey protein was hydrolyzed by using three commercial proteases; flavorzyme, alcalase, and corolase PP) for enrichment of milk. The strawberry- and chocolate-flavored milk was supplemented with WPC and WPHs. The addition of 2% of WPHs has shown an increase in antioxidant activity up to 42%. The results suggest that WPH could be used as natural biofunctional ingredients in enhancing antioxidant properties of food products.

Kumari et al. (2013) also found an increase in antioxidant activity after addition of 1% flavorzyme WPH and alcalase WPH up to 14.73% and 29.6% in chocolate ice cream.

Chatterjee et al. (2016) enriched Indian sweetened yogurt with 1%, 2%, and 3% (v/milk) of tryptic whey protein hydrolysate (TWPH). A level of 3%, v/v, of TWPH was most acceptable. Significant difference was observed in case of % ACE inhibition (33,11% for sample with 1% TWPH, 43.17% for sample with 2% TWPH and 57.19% for sample with 3% TWPH). Addition of 3% TWPH may not be beneficial to improve antioxidant properties of Indian sweetened yogurt but can enhance the % ACE inhibition.

Matumoto-Pintro et al. (2011) investigated the use of modified whey protein in yogurt formulations. Whey protein was modified to produce yogurt with acceptable texture properties. The study demonstrates that the modification of whey protein ingredient prior to addition to milk can improve the textural quality of both set-style and stirred-style yogurts.

Soukoulis et al. (2014) formulated a probiotic bread by applying a film-forming solution (edible film) based on a binary blend containing 0.5% (w/v) sodium alginate and 2% (w/v) WPC inoculated with the probiotic strain Lactobacillus rhamnosus GG. The addition of whey improved the survival of the L. rhamnosus strain during drying and storage.

Castro et al. (2013) studied a strawberry-flavored dairy beverage supplemented with whey. Beverages were prepared by mixing up to 80% of whey, strawberry pulp, and sugar and milk. The formulation was fermented using a mixed culture containing strains of S. thermophilus, L. delbrueckii subsp. Bulgaricus, and L. acidophilus until a pH of 4.7 was reached. The fermented beverage with best properties was the sample that contained 45% of whey.

Athira et al. (2015) ultrafiltrated mozzarella cheese whey to remove lactose and mineral. The retentate was hydrolyzed with food-grade enzyme alcalase and the hydrolysis conditions (pH, temperature, and time) were optimized for maximum antioxidant activity. WPH showed a high radical scavenging activity. Further, they used hydrolysate for enrichment of lemon-based beverage. The incorporation of WPH in lemon whey drink (5–10 g/L) increased its antioxidant activity from 76% to 90% compared to control. So, they concluded that WPH with good nutritional and biological properties can be used in health-promoting foods as biofunctional ingredients.

5.3. Whey Proteins and Peptides as Carrier for Immobilization

Whey proteins are widely used for probiotic encapsulation. As already mentioned fermented probiotic drinks are the majority of available functional foods. Probiotic beverages comprise between 60% and 70% of the total functional food market (Tripathy and Giri, 2014). That is not unexpected if it is known that probiotics have shown to play an important role in maintenance of the normal intestinal microflora, protection against gastrointestinal pathogens, lactose metabolism, reduction in the incidence of urogenital and respiratory diseases, prevention of some cancers and reduction of serum cholesterol level and blood pressure (Khani et al., 2012). Probiotics have been consumed in foods, such as yogurt for thousands of years and new application of probiotics has been proposed, such as bread, fermented vegetables, and chocolate (Zaric et al., 2016). The shelf life of probiotics should be controlled in order to manufacture products with a satisfactory number of live bacteria (at least 10⁷ CFU/g) to obtain health benefits of probiotic cultures (Oliveira et al., 2002). Unfortunately, many studies indicated that there is poor survival of probiotic bacteria in these products. Encapsulation of probiotics is a good way to improve viable cell number during manufacturing or in extreme conditions as refrigerator or gastrointestinal tract (Krunic et al., 2016b).

Soukoulis et al. (2014) developed probiotic pan bread by the inclusion of Lactobacillus rhamnosus GG cells in edible films applied onto baked bread surface. They did not encapsulate probiotic cells but delivered them through edible films, which were constituted by different combination of two biopolymers usually used as microencapsulating agents, sodium alginate, and whey proteins. Alginic acid is nontoxic and widely used in food applications.

The most common ways to encapsulate the probiotics with whey protein as carrier are spray-drying and extrusion (Fig. 9.3).

Figure 9.3 (A) Schematic presentation of the spray-drying procedure, (B) extrusion procedure.

Ying et al. (2013) encapsulated L. rhamnosus LGG by spray-drying in beads obtained from whey proteins isolate and resistant starch (RS) matrices mixed in different ratio to protect probiotic cells in apple juice stored at 4 and 25°C. Particles with higher WPI content favored the growth of the entrapped probiotic cells more efficiently than RS microcapsules.

Doherty et al. (2012) monitored L. rhamnosus LGG survival in juices. Probiotic cells were microencapsulated via extrusion in WPI matrices. It was followed by a secondary coating with different polysaccharides (apple pectin, citrus pectin, sodium alginate, kappa-carrageenan, iotacarrageenan, and inulin), and a final coating with an additional WPI layer to produce more mechanically stable capsules. WPI was efficient in protecting cells during a 28-day storage against low pH and high content of phenolic acids of pomegranate and cranberry juice. The combination of WPI and apple pectin provided the higher level of protection after gastrointestinal simulated passage in ex vivo porcine fluids.

Ribeiro et al. (2014) showed that pectin-whey proteins matrix was effective in protect cells during simulated gastrointestinal digestion of yogurt and presence of probiotic capsules was not negatively affect physiochemical parameters of yogurt.

Hernández-Rodríguez et al. (2014) investigated Lactobacillus plantarum immobilization in whey protein. Cell culture was entrapped in WPI and k-carrageenan complex coacervates at different pH values. Encapsulated of Lactobacillus plantarum significantly improve viability of cells in gastrointestinal condition (low pH value and bile salt present) compared to free cells. They also concluded that the complex coacervates made with a 16.7:1 WPI-k-carrageenan weight ratio at pH values of 4.0 and 4.5 could be used as structural elements and probiotic carriers in functional foods, such as creams, yogurts, fermented lactic beverages, and certain types of cheeses, such as Petit Suisse.

Krunić et al. (2016c) compared effect of probiotic immobilization with different carriers. It was investigated which carrier and which particle size is the most suitable for the fermentation of whey based substrate. WPC-alginate beads showed the highest increase in viable cell number within all examined particles. That can be explained with positive impact of WPC on growth of yogurt culture. Also, the percentage of inhibition of DPPH radicals significantly (P < 0.05) increased from an initial value of 15.1% to a variety of values recorded after the fermentation. The percentage of inhibition of DPPH radicals was approximately 34.9% ± 0.5% for all samples except samples with WPC-alginate beads. Samples with WPC-alginate beads had slightly higher inhibition of DPPH radicals, for both size of beads (36.5% ± 1.1% for beads with diameter 0.94 ± 0.07 mm and 38.8% ± 1.4% for bigger beads with diameter 2.55 ± 0.09 mm). It could be assumed that proteolysis and lactic acid production as the results of microbial activity during fermentation could be additional sources of antioxidant activity. Krunić et al. (2016c) concluded that alginate and chitosan-alginate beads are suitable for operation in systems where beads are subjected to a large number of washes, or systems that require as little as possible of free viable cells in the medium because these beads showed low cell leaking. WPC-alginate beads showed a high percentage of leaking, but also a high number of viable cells after fermentation. So, these beads are suitable for the production of fermented dairy products because they allow the best growth of yogurt culture and parameters of the product (pH and titratable acidity), as well as good antioxidant characteristics.

Further, Krunić et al. (2014) investigated viability of free and immobilized probiotic cells in simulated gastrointestinal conditions. In order to improve probiotic character of immobilized cells, the influence of beads wall material composition (alginate, WPC-alginate beads and chitosan–coated alginate beads) was examined. Bile tolerance showed that all samples with encapsulated culture have great viable cell number after 4 h incubation in MRS broth with 0.3% of bovine bile. Acid tolerance assay showed that free cells had a drastic reduction in the number of viable cells in 2 h (50%) and in the third hour there were no living cells at pH 2.5, samples with alginate beads had 36.18% surviving cells after 4 h. Samples with coated beads had 37.8% surviving cells after 4 h and samples with WPC-alginate beads had 40.0% surviving cells after 4 h.

Ilha et al. (2015) showed that whey used for encapsulation of Lactobacillus paracasei strain isolated from grape sourdough has protective effect on probiotic in simulated gastrointestinal condition (low pH and presence of bile).

Whey protein can also be used to encapsulate bioactive compounds due to its functional properties, such as surface activity, gelation and some protective properties (Livney, 2010).

Madadlou et al. (2014) obtained particles with whey protein via heat gelation of enzymatically reinforced, using caffeine as drug model and confirmed that this method was responsible for obtaining stable nanoparticles with 45 nm of diameter.

Zhang et al. (2016) investigate alginate-whey protein beads as carrier for carvacrol. They showed that alginate–whey protein beads could effectively minimize the absorption of carvacrol in the stomach and proximal intestine and increase the percentage of carvacrol being delivered to the distal small intestine. The microcapsules could completely release the encapsulated carvacrol during transition through the gastrointestinal tract of pigs.

Assadpour et al. (2016) encapsulated folic acid (vitamin B₉) in maltodextrin-whey protein double emulsions via spontaneous emulsification method.

Abbasi et al. (2014) used WPIs beads for encapsulating vitamin D₃. According to their results, beads had the higher content residual of vitamin D₃ compared to the control sample (water, native WPI, and denaturized WPI) during 7 days of investigation of its stability in presence of air. They concluded that these beads are applicable in the beverage industry.

Perez-Masia et al. (2015) successfully applied electrospraying technique to encapsulate folic acid, entrapped by WPC matrix and commercial RS. According to their results, WPC particles enhanced the bioavailability and stability of folic acid.

Chapeau et al. (2016) used β-Lg and Lf coassemblies to bind vitamin B₉. They showed that B₉-Lf–β-Lg coacervates displayed great performance in entrapping vitamin B₉ (10 mg B₉/g protein) and concluded that natural food components have great potential to be utilized as biocarriers in designing functional foods.

Wichchukit et al. (2013) showed that the combination of whey protein and alginate formed beads with good integrity and zero order rate kinetics for the release of riboflavin.

Rafe et al. (2016) showed that the gelling structure of the mixed gel of WPC–rice bran protein was improved by adding WPC. Also, the gel structure was produced by adding WPC to the nongelling rice bran protein. They concluded that it is compatible with whey and can be applied as a functional food for infants and adults.

6. Conclusions

Currently, consumers have a growing interest in foods that are not only a source of basic nutrition, but which can also promote health and quality of life. Whey is a rich source of biologically active protein and peptide that can be used in the production of functional food. Most whey peptides with biological activity are released by enzymatic hydrolysis, but microbial fermentation can also be used for this purpose. Bioactive peptides from whey are introduced to the food processing industry with the new technologies, such as UF. New manufacturing technologies, such as supplementation of fermented product with specific bioactive peptides concentrated from hydrolysates of whey proteins seem to provide a practical solution for the time being. Bioactive peptides derived from whey protein, added in probiotic-fermented products and confectionery products (such as chocolate, biscuits, or cream) have effects, directly and indirectly, to human health. These peptides increase the viable number of probiotic bacteria, increase the stability of the probiotic products, and act in the human organism as antioxidant, ACE-inhibitor, increase proliferation of intestinal epithelial cells, and so forth.

Immobilization is a proven method that improves peptide stability in foods and during digestion, but whey proteins and peptides have also proved to be good carriers for probiotic bacteria. Probiotic bacteria encapsulated with whey peptides as carrier are suitable for the production of fermented dairy products because they allow satisfactory growth of probiotic culture and parameters of the product, and also protect probiotic bacteria during their transit through the gastrointestinal tract. Using whey protein and peptide for probiotics encapsulation is a good practice for nondiary foods, too, since nondairy probiotic foods are becoming popular for people with lactose intolerance.

The potential of whey proteins and peptides for the formulation of functional foods has been long demonstrated, but they are still not mass-produced except as a dietary supplement for bodybuilders.

Bioactive peptides are suitable for application in food, due to a simple procedure to produce and separate them. Also, good technological properties of whey protein and peptides fractions are obtained by UF and contribute to their wide use in food manufacturing.