Extraction of Bioactive Peptides from Whey Proteins by Conventional and Novel Technologies

Abstract

Bioactive peptides show physiological properties in systems such as digestive, cardiovascular, and vascular. Bioactive compounds are found in animal and plant proteins. However, peptides obtained from milk proteins have better biological activity in terms of amino acid composition and sequence. It is important to industrially evaluate bioactive peptides after proper extraction, purification, and identification. There are conventional (enzymatic hydrolyzation and fermentation) and novel methods (ohmic heating, ultrasound and microwave) for the extraction of bioactive peptides. Novel extraction methods increase the degree of hydrolysis of peptides, making them more efficient, and peptides with high activity are obtained. The extraction method of bioactive peptides to be extracted from whey is important, and the method to be chosen must be evaluated in all its aspects. This chapter includes literature data on the importance of whey proteins, bioactive peptides, and extraction methods of bioactive peptides from whey.

1. Introduction

There is a growing scientific and industrial interest in natural and generally side-effect-free bioactive compounds. Reducing the risk of chronic diseases is possible through the bioactivity of peptides with physiological properties [1]. The functions of bioactive peptides (2–20 amino acids and ˂10 kDa) vary depending on amino acid composition, charge, sequence, and molecular weight [1, 2, 3]. Peptides can be obtained from animal and plant proteins. In milk, which is an important source of peptides, bioactivity varies depending on the joint effect of one and/or more peptides [4, 5]. The majority of milk-derived bioactive peptides are composed of casein (α, β, γ, κ-casein) and precursors of serum proteins [2, 4]. In addition to peptides derived from casein such as immunopeptides, cytokinins, casomorphins, and casoxins, there are peptides derived from serum proteins such as α-lactorphin, β-lactorphin, lactoferricin, lactoferroxins, phosphopeptides [2].

Whey (approximately 80–90% of milk), which is the remaining part of casein after coagulation in cheese production, is a yellowish-green liquid with a yellowish/waste product due to riboflavin [6, 7]. Whey becomes a risk for many living things due to the effect of microorganisms operating using organic matter and oxygen in the environment [7]. Due to its high oxygen demand, it is important to process whey and use the proteins it contains in various industries [8]. Many products (powder, protein, enzyme, and various acids) are obtained from whey by using physicochemical (spray drying, thermal/isoelectric precipitation, membrane filtration, coagulation) and biotechnological processes [9]. While the water content of whey is reduced, the protein content is increased, resulting in products with a longer shelf life and more nutritious products [10, 11]. The solubility (hydrogen bonds and surface hydrophobicity), gelling properties (sulfhydryl groups and disulfide bonds), and emulsifying properties (surface activity) of proteins vary depending on the different properties in their structures [9, 12]. Whey proteins are used in the production of foods thanks to their textural and sensory properties such as gelling, foaming, and fat binding, as well as improving nutritional quality with protein content [7]. Whey products are used in many products such as beverages, bakery, milk, meat, and sports foods [11]. Whey biofilms of fruits and vegetables reduce respiratory rate and microbial activities and extend their shelf life [13]. β-Lactoglobulin (β-LG), which is not found in human milk [14] but makes up a large proportion of whey proteins, is an allergen for infants [15]. For this reason, whey enriched with α-lactalbumin (α-LA) and lactoferrin (LF) is used in infant foods [13].

It is clear that whey is not made up of simple proteins, but is a complex structure made up of many bioactive peptides with their unique effects. The applied technologies are of great importance in the activity of these biopeptides [13]. The type, number, quantity, and properties of peptides obtained from whey proteins vary depending on the method used and its conditions. Conventional methods (fermentation, enzymatic hydrolysis, in vitro digestion, and chemical/thermal application) are used in the dairy industry to isolate peptides. Peptides in whey show individual bioactivity, but conventional methods used to separate each peptide are inadequate. Because peptides and their biological properties vary depending on the enzyme and microorganism or conditions used in the extraction method. Traditional methods also have disadvantages such as being costly and taking a long time to hydrolyze proteins. With the development of these methods or the use of a novel method, more peptides are obtained and the functional properties of these peptides are increased. With novel methods, costs are reduced, and faster and bioactive peptide production takes place [2, 3]. However, the inadequacy of some novel methods in obtaining peptides and their application before or at the same time as conventional methods contribute to peptide extraction. This book chapter will focus on extracting peptides from whey using old and novel methods.

2. Whey proteins/peptides

Whey, also known as serum proteins or soluble milk proteins, consists of water, lactose, whey proteins, minerals, milk fat, and vitamins [8, 16]. However, whey proteins are not correctly called serum proteins because they contain glycomacropeptide (GMP), which is both partially free in milk and formed by hydrolysis of casein [6]. Whey is effective in growth and development because it contains proteins and amino acids with high biological value [17]. The main proteins are β-LG (50–55%), α-LA (20–25%), GMP (10–15%), immunoglobulins (IGs, 10–15%), bovine serum albumin (5–10%), LF (1–2%), lactoperoxidase (LPO, 0.50%), and lysozyme (0.002%) [18]. Whey proteins contain many essential (such as cysteine and methionine) and nonessential amino acids [13, 19]. The content of whey varies depending on the quality of the milk, processing, and purification method [8]. The pH of the milk decreases with microbial growth or direct acid addition and the casein coagulates, thus producing sour/acid whey (max pH 5.1). The product formed as a result of coagulation of milk with the addition of enzymes is yeast/sweet (min pH 5.6) whey [10, 20, 21]. Sour/acid whey is nutritionally superior to yeast/sweet whey [10, 20].

Peptides can exert both synergistic and antagonistic effects. For example, Lf may have antimicrobial properties from the synergistic activity of defense proteins such as LPO and lysozyme [16]. Bioactive peptides can exhibit more than one different biological activity [22]. Whey proteins/peptides have antioxidant, anticarcinogenic, antihypertensive [7, 13], immunomodulating, mineral-binding, antiobesity [13], antimicrobial activity [13], antidiabetic [13, 16], opioid agonist, and antagonist effects in in vitro and in vivo conditions [23]. Whey proteins also have positive effects such as prevention of muscle degeneration, initiation of wound care and repair, benefits for infant nutrition, and healthy aging [13].

Peptides exhibit antioxidant properties due to their radical scavenging effects on the activity of enzymes in the cell [23] and the inhibition of enzymes such as xanthine oxidase [24] or by binding to prooxidant metal ions [1]. Some amino acids (cysteine, histidine) can bind free radicals by transferring hydrogen atoms or electrons [1]. Whey proteins also contain amino acids such as methionine and cysteine, which show antioxidant activity as a precursor to glutathione [25]. In cysteine, especially the sulfhydryl group is responsible for antioxidant activity [16]. LF, which binds iron, reduces the conversion of hydrogen peroxide to the hydroxyl group [13, 25]. Many peptides identified from whey have antioxidant activity [16, 22].

Opioid peptides have agonistic activity by binding to opioid receptors (μ-, δ-, and κ-type) and acting on antagonistic or N-terminal sequences. Opioid agonistic peptides such as α-lactorphine, β-lactorphine, and serorphine [16] have a drug-reducing and/or -inhibiting effect, while opioid antagonistic peptides such as lactotensin and lactofotoxin A and B have a reducing and/or inhibiting effect [5].

Bioactive peptides have an immune-boosting effect thanks to their effects such as antibody synthesis, cytokine regulation, and lymphocyte proliferation [1, 16]. However, the amino acid content, charge, length, sequence, and hydrophobicity of immune-acting peptides are important [1]. For example, LF with its positively charged parts [26], GMP with its effect on lymphocytes [13], and LF with lipoprotein-binding properties compete with viruses [27], β-LG containing sulfurous amino acids [6] and IGs [28] have immune system-supporting properties. Whey proteins contribute to immunity, especially in newborn babies, by reducing allergic reactions [16]. Glutathione deficiency in individuals with HIV was eliminated by the consumption of β-LG, α-LA, and LF, strengthening the immune system of these individuals [7].

Peptides in whey protein structures are effective on angiotensin I-converting enzyme (ACE, peptidyl dipeptide hydrolase, EC 3.4.15.1), which is a key antihypertensive [16, 29]. In addition, the ACE inhibition (ACEi) activity of peptides increases the mineral density in the bones and has a positive effect on bone health [30]. Whey peptides such as α-lactofores, α- and ß-lactorphins [31], albutensin [5], β-lactosin [32], and ß-lactokinins have ACEi effects [31]. Amino acid residues at the C (aromatic) and N terminal (hydrophobic) of peptides are effective in ACEi [1]. α-Lactorphine, whose N-terminal dipeptide is Tyr-Leu, has the highest ACEi activity among whey peptides [29].

Whey proteins, which increase the amount of glutathione and/or bind iron [7], have an anticarcinogenic effect by contributing to immunity [25]. In addition to preventing colorectal cancer by binding iron [33], LF has effects such as reducing anemia in chemotherapy patients [34]. α-LA and β-LG reduce the risk of developing colon and breast cancers [7]. Whey peptides are an important antiproliferative agent [16].

IGs, LF, and LPO have been shown to have antimicrobial properties from PAS proteins [35]. The positive charge of LF [36], its hydrophobic structure, and its secondary structure are thought to support its antimicrobial properties. LF contributes positively to the activity of antibiotics by increasing the permeability of the cell wall by binding microorganisms to its outer membrane [14]. LF, which has a stronger antimicrobial effect than LF, increases membrane permeability with the basic amino acids it contains [36]. LPO becomes a natural antimicrobial agent through the oxidation of thiocyanate with hydrogen peroxide [31].

Whey proteins have a positive effect on the control of insulin secretion and blood sugar. This effect is because peptides can induce insulin secretion [22].

Metal-chelating peptides improve divalent mineral bioavailability and do not bind cations such as calcium, iron, and zinc [16]. β-LG binds minerals and transports them along the intestinal walls [6]. Thanks to its iron-binding properties, LF plays an inhibiting role in microbial activities and oxidative reactions and supports bone development [13, 37]. Four peptides from β-LG and 2 peptides from α-LA have mineral-binding effects [16]. GMP [5] and lactotransferrin have antithrombotic effects by binding to receptors in platelets [36].

β-LG can bind vitamin A (retinol) and vitamin D due to its capacity to bind with a large number of hydrophobic and amphiphilic ligands due to the presence of calyx in its structure and hence can act as a carrier for them. It is also involved in the stimulation of lipase activity [6] and phosphorus production due to its ability to bind β-LG fatty acids [37]. α-LA, a good calcium carrier, is involved in synthesizing lactose [38]. α-LA reacts with β-1,4-galactosyl transferase-1 and increases its affinity for glucose by a factor of 1000, thus enabling more lactose to be produced [6]. Whey proteins provide long-term satiety with a low glycemic index and regulate glucose homeostasis levels. The leucine they contain provides fat loss and helps weight loss [7].

3. Extraction of bioactive peptides

Whey proteins, which are primary, secondary, tertiary, and quaternary under certain pH, ion strength, and temperature conditions, denatured with chemical, enzymatic, and physical processes, change their structure and physicochemical properties. Reactive amino acids and whey proteins, which emerge due to the changing structure, cluster depending on the pH. Since whey proteins have different molecular properties, structures, and denaturations, these factors change when they are found together. For example, when α-LA and β-LG are together, denaturation temperature and aggregate sizes are affected [39]. The quaternary structure of β-LG, consisting of 162 amino acids, varies with low concentration (<10 mg/mL) and high pH at temperature (<10°C). It is in the form of β-LG monomer at very low pHs (pH˂3.4) and high pH (pH˃8). β-LG exists as non-covalently linked dimers between pH 5.2–8.0 and as octomers at pH 3.5–5.2 [8]. With increasing temperature (65°C≤), β-LG denatures and aggregation of oligomers causes irreversible aggregation, releasing free sulfhydryl. However, in skimmed milk with a high water content, β-LG denatures to 75–100°C at higher temperatures [39]. α-LA (about 14 kDa), on the other hand, is less sensitive to pH changes [8].

Bioactive peptides are in inactive form but become active by the activity of digestion and commercial enzymes or by fermentation [16, 22, 36, 40]. The number, sequence, and type of amino acids in peptides and the carboxy or amino terminus are important in peptide activity [4]. In addition to microbial and enzyme activities [4], many different peptides are activated chemically by acid/base hydrolysis [20]. Additionally, when these are used in combination, shorter peptides are obtained [16]. Novel technologies include ohmic heating (OH), microwave-assisted extraction (MAE), pulsed electric fields (PEF), subcritical water hydrolysis extraction (SWE), ultrasound-assisted extraction (UAE), and high hydrostatic pressure (HHP) (Table 1). Conventional methods alone can be used to produce peptides. However, fermentation or enzymatic hydrolysis after some novel technologies (UAE, MAE, and HHP) can be used as pretreatment for peptide recovery [2, 3, 19].

		Advantages	Disadvantages
Conventional	EH	High specificity [41]; Elimination of organic solvents as per [41]; Non-toxicity of the resulting product [3]	A limited number of enzymes are available [41]; Costly [2, 41]; Low yield [41]; Bitterness [2]
	Fermentation	Elimination of multifunctional peptides [42]; Release of exopeptidases that hydrolyze N-terminal amino acids [43]; Natural and controlled conditions [2]; Relatively economical [41]	Depending on the microorganism strain, the degree of hydrolysis and functional properties vary [43]; Peptide production is low and specificity is low [2, 41]
	Chemical/thermal	Simple and cheap [41]	Difficult to control the process [41]; Low peptide purity [18]; Low functionality [41]; Solvent need [41]; Formation of undesirable compounds [41]
Novel	UAE	High energy efficiency [41]; Environmentally friendly [1]; Non-thermal [1, 2]; High extraction selectivity [2]; Acting on covalent bonds [13]	Temperature rise during application [41]; Insufficient for peptide extraction alone [41]
	MAE	Simple to implement [41]; Low cost [41]; Fast and uniform heating [1]; Easy to maintain [1]; Repeatability [2]
	HHP	Without damaging bioactive compounds [41]; When applied in combination with heat treatment, hidden peptides are also revealed [19]	No effect covalent bonds [41]; Denaturation may occur due to pressure [41]; High cost of infrastructure [41]; Failure to implement at large scale [41]
	PEF	Environmentally friendly [12]; Non-thermal [25]; Low energy needs [41]; Short implementation time [41]; Conformational effects on the secondary structures of proteins [41]	High investment cost [41]; Thermal aggregates may form due to temperature increase [13, 44]
	OH	Improvement in bioactive properties [45]; Uniform heating [41, 45]; Less time [41]; Energy conversion is efficient [41]	High investment cost [41]; Application only in liquids/solids with solid particles [41]; The rapid increase in temperature due to conductivity [41]
	SWE	Breaks peptide bonds when applied alone [41]; Elimination of low molecular weight peptides [46]; Green technology [41]; Non-toxic water and no solvent residue [41]; Improving efficiency with additives [47]; The resulting peptides can be used without purification [41]	No specificity in peptide modifications [41]; Industrial application limited [47]; Infrastructure costly [41]; Pre-treatment such as alkylation may be needed [41]; High energy requirement [46]; Toxic substances can be formed [46]
The novel/conventional technologies	UAE-EH	Change of peptide profile [48]; Shortening of hydrolysis time [48, 49]; Low molecular weight peptide extraction [48, 49]; Increase in bioactivity [48]; Reducing the amount of enzymes required [48]; Increasing substrate sensitivity [39, 41]
	HHP-EH	Increased number of bioactive peptides [50]; Enhancement of physiological properties [51]; Enzyme to improve substrate accessibility [41]	After HPP pretreatment, protein/peptide interaction may occur again [3]
	MAE-EH	Obtaining low molecular weight peptides [41]; Enables rapid and efficient hydrolysis [2, 52]
	OH-EH	Enhancing bioactive properties [48]

Table 1.

Extraction technology of whey bioactive peptides.

3.1 Conventional methods

3.1.1 Enzymatic hydrolysis (EH)

Biological catalyst enzymes are protein structures that show activity inside or outside the cell. The most commonly used method in protein hydrolysis is enzymatic [16]. EH contributes to the improvement of properties of proteins such as nutrition, functionality, emulsification, solubility, foaming, and gelling [53]. The degree of EH is determined as a percentage depending on the peptide bonds [13]. There is a direct relationship between the degree of hydrolysis and the activities of bioactive peptides [3]. EH takes place under controlled conditions (hydrolysis time, pH, temperature, enzyme/substrate ratio, and enzyme activity) and is an effective method of obtaining peptides [2]. The enzymes used for this purpose can be of animal, vegetable, microbial [2], and digestive (chymotrypsin, trypsin, alkalase, pancreatin, thermocillin, and pepsin) origin [16]. When these enzymes are used in combination, more peptides are formed and the activity varies according to the order of the enzyme used [16]. Plant-derived proteases are widely used in the production of peptides because they operate in wide temperature and pH ranges [54]. They can be of animal, plant, or microorganism origin with some enzymes. For example, transglutaminase has high activity at certain temperatures (≈50°C) and over a wide pH range (pH 5–8) [13].

To understand the hydrolysis of proteins during digestion, it has been shown that proteins are broken down by pancreatic proteinases (mostly trypsin), and many peptides are released in vitro. In addition, combinations of proline-specific endopeptidase, thermolysin, elastase, carboxypeptidase, pepsin, pancreatin, and chymotrypsin endoprotease can also produce bioactive peptides [2, 3]. Trypsin is a digestive enzyme used especially in obtaining peptides showing ACEi activity [16].

Pepsin is used to obtain peptides with bioactive properties different from α-LA (α-lactofophorin, α-lactorphine), β-LG (β-lactofophorin), serum albumin (albuntensin, serrorfin), and lactotransferrin (thrombin inhibitor peptide, casonplatelin). Trypsin produces peptides with different bioactive properties from α-LA, β-LG, β-lactoxine, and GMP. Enzymatic hydrolysis of bovine whey with α-LA with trypsin and alkalase formed the peptides LDQWLCEKL, ELKDLKGY, and ILDKVGINY, respectively [19].

Whey proteins are difficult to hydrolyze due to their spherical structure. Therefore, pretreatment (physical/chemical denaturation) is required, and the number of peptide bonds increases, making enzymatic hydrolysis easier. By pretreatment, the main proteins in the whey are broken down and the hydrophobic parts are collected on the outer surface so that the peptide bonds become suitable for enzymes. However, the functionality of the peptides varies depending on the pretreatment used. In addition, if sulfitolysis, which is a reversible reaction with the SS bonds of sulfite, is used as a pretreatment method for whey proteins, it increases the degree of enzymatic hydrolysis since heat and pressure are not applied [55].

3.1.2 Fermentation

In the production of dairy products, many different bioactive peptides are formed by biochemical changes during fermentation under natural or controlled conditions [16]. Lactic acid bacteria (such as Lactococcus lactis, and Lactobacillus helveticus) are usually used for whey, as well as nutrients, aroma, and physical healing [2] also occur in peptides [16]. The efficiency of fermentation, the degree of proteolysis of proteins, depends on the type of bacteria and fermentation conditions [2].

As a result of the fermentation of whey with 34 different lactic acid bacteria (48 h, 37°C), many peptides were obtained from Β-LG and Pediococcus acidilactici SDL1414 showed the strongest ACEi effect, especially in peptides with low molecular weight [43].

3.1.3 Chemical/thermal methods

The chemical hydrolysis of whey proteins is related to sulfides in the structures of proteins. The function and properties of proteins can be enhanced by lowering the pH with succinic acid or by influencing disulfide bonds by sulfitolysis [25]. When whey was brought to pH 3.0–4.6 with citric acid, high and soluble β-LG was obtained [18]. Chemicals such as phosphoric and hydrochloric acid can be used for hydrolysis. If acid hydrolysis and microwave are used simultaneously, the hydrolysis time is shortened [46].

When heat is applied to whey proteins, free thiol groups are released and denaturation occurs. Free thiol groups form aggregates as thiol-disulfide. Calcium facilitates the thermal precipitation of whey proteins. Generally, dehydrated whey is denatured at neutral pH at 90°C and then precipitated at pH 4.4–5.0 to obtain efficient but low solubility protein, which is important for functionality. For this purpose, it is recommended to first acidify with the addition of organic or inorganic acid (pH 2.5–3.5) and then apply heat treatment (90°C). The addition of iron contributes to solubility [56]. Seventeen peptides were obtained as a result of the treatment of β-LG with heat and lysine using different concentrations of calcium [57].

3.2 Novel extraction methods

Novel extraction techniques are based on physical processes used in the production of bioactive peptides to improve the degree of hydrolysis [2, 58].

3.2.1 Ultrasound-assisted extraction

The new UAE provides many benefits by using high frequency (≈20 kHz) waves that humans cannot perceive [1]. In the UAE method, ultrasound waves consist of mechanical waves propagating in an elastic medium. The cavities formed in a high-intensity sound wave are called cavitation bubbles [59]. At the cavitation point, temperatures increase locally and these cause chemical changes by producing radicals [44]. If the bubble is large, it will float and burst on a surface; if it is small, it explodes into the liquid [59]. This explosion can create the energy necessary for physical, chemical, and biochemical effects [13, 60]. Chemical effects can be seen at a frequency of 300–500 kHz, and physical effects can be seen at a frequency of 20 kHz [2]. Protein structures undergo conformational changes due to these effects; proteins are fragmented, and the free sulfhydryl content increases [2]. The viscosity and surface hydrophobicity of whey protein change in proportion to the applied energy density, and the particle size decreases [44]. Ultrasound can be applied using two equipment: an ultrasonic bath and a probe [61]. Ultrasonic baths generally operate at a frequency of around 40 kHz, but they get hot while operating [62]. Additionally, if the local temperature increases the temperature of the entire solution, proteins may denature. Since the rate of increase of this temperature depends on time, frequency, volume, and power, the UAE application needs to be optimized. Additionally, the temperature can be controlled during the process by adding ice, a cooling jacket, or by constantly changing the medium [44]. Depending on the frequency range, it can be applied as high (16–100 kHz, 10–1000 Wcm⁻²) and low intensity (100 kHz–1 MHz, 1 Wcm⁻²˃). Low-intensity ultrasound is used in the food industry to ensure high quality nondestructively, while high-intensity ultrasound is used for material modification such as emulsification, microbial inactivity, extraction of flavors, protein modification, gas, and defoaming [13].

The number of disulfide bonds increases with hydrophobic interaction with the heat treatments applied to obtain peptides from whey proteins and the proteins are denatured. It can be used for many purposes such as solubility, gelling, and foaming properties by ultrasound application in whey [13, 44]. Furthermore, cavitation bubbles induce induction and conformational changes in protein substrates. Thus, UAE reduces α-helical structure and increases β-sheet structures [48]. By reducing the size of fat particles coated with whey proteins, UAE increases the area of action of enzymes and allows more peptides to be obtained [2]. It has been shown that UAE administration of whey proteins before hydrolysis with alkalase shows ACEi and immunomodulatory activities depending on peptides. Whey proteins and peptides obtained after enzymatic hydrolysis with bromelain first (probe, 500 W, 20 kHz, 10 min) have low molecular weight (5 kDa˃) and high ACEi activity [48]. Similarly, the conditions of an application before enzymatic hydrolysis (pepsin and papain) with an ultrasonic sonicator (300–400 W, 20 kHz, 2–4 min) and the degrees of hydrolysis vary according to the enzyme used [49]. β-LG, high-intensity ultrasound (frequency of 20 kHz, 60 Wcm⁻²) has been reported to increase the sensitivity of pepsin and trypsin to proteolysis [39].

3.2.2 Microwave-assisted extraction

MAE uses microwaves with electromagnetic waves of different wavelengths (0.001–1 m) and frequencies (300 MHz–300 GHz) [1]. MAE extends the shelf life of foods and improves various functional properties [2]. The microwaves have positive effects on the taste and quality of products [1]. The power, frequency, time, and temperature to be applied in MAE are important [12]. Microwave energy causes molecular interactions involving ionic conduction and dipolar rotation mechanisms [2]. Changes in the structure of proteins occur by generating heat with the energy released by interaction at the molecular level [12].

It can be microwave, thermal, or noninductive, using nonionizing radiation. Thermal effects with microwaves arise from local heat generated by the friction of water molecules, while nonthermal effects arise from the rate of unfolding and rearrangement of proteins [2]. Because microwaves break down disulfide bonds in proteins, proteins open up [1]. This opening in milk proteins facilitates the application of enzymatic hydrolysis as in UAE. MAE (532 W, 40–50°C, 5 min) application of bovine serum albumin concentrate before proteolysis (such as pronase, chymotrypsin, papain, alcalase) increases enzymatic hydrolysis [2].

3.2.3 High hydrostatic pressure

HHP, also called “pascalization” or “cold pasteurization” [3], is a green new technology that covers the application of hydrostatic pressure (100–1000 MPa, max 30 min) with and without heat treatment. Depending on the HHP processing time and pressure, the temperature of the product increases optimally [2]. Proteins are denatured as the volume decreases with the applied pressure causing biochemical changes in the systems [44]. Unlike thermal treatments, HHP acts more on weak chemical bonds (hydrophobic, hydrogen, and ionic), activating only the secondary structures of proteins. The application of low pressure (˂400 MPa) increases the hydrogen bonds, while high pressure has the opposite effect on these bonds [3]. HHP can be applied intermittently, semi-continuously, and continuously [2]. Denaturation and/or aggregation occurs depending on the HHP conditions (pressure, temperature, and time) and the proteins (pH and composition) [13, 44]. At high pressures, β-LG aggregates through disulfide bond exchange, particle size increase, formation of aggregates, and conformational change. At low pressures (<120 MPa), partial opening of β-LG is observed with changes such as particle shrinkage and an increase in sulfhydryl groups [44].

When applied as a pretreatment, HPP can increase the efficiency of enzymatic hydrolysis (active site formation, chemical bonding, and interaction) by destabilizing whey proteins. For example, EH made using chymotrypsin, pepsin, and trypsin produced more hydrophobic peptides than β-LG pretreated with HHP [3]. Factors such as HHP conditions (pressure, temperature, and time), pH, ionic strength, substrate, and enzyme affect enzyme hydrolysis [2, 3]. In addition, hydrolysis should be performed without wasting time after HHP administration so that the susceptibility to proteolysis does not decrease. After HPP application, reaggregation of proteins may occur through protein and peptide interactions. When hydrolysis is performed simultaneously with HHP, protein agglomeration is prevented [3].

HHP contributes to the foaming properties as a result of increased opening and adsorption rates of proteins through intermolecular interactions at neutral pH. However, the application of pressure above 300 MPa reduces the stability of the foam by opening the protein [13]. In HHP application, it causes β-LG and α-LA denaturation of pressure higher than 100 MPa and makes it difficult to separate into fractions. β-LG has reversible effects on certain pressure (300 MPa>) applications [25]. Following the application of batch HPP (0.1, 400 and 600 MPa, 10 min, at room temperature) to bovine whey, QEAKDAFLGSF and WENGECAQKK peptides were obtained from β-lg by tryptic hydrolysis, and while the best yield was obtained with 400 MPa, 600 MPa pressure reduced their amount. Before β-LG chymotrypsin administration, HHP (400 MPa) showed longer and more hydrophobic peptide [2].

3.2.4 Pulsed electric field

PEF is used specifically for the inactivation of microorganisms [25] and enzymes [1]. PEF, which is a nonthermal process, generates a high-voltage field (≈50 kV/cm) with short pulsed electricity (20 μs˃) of different alternating currents (10–100 kV) [25]. Process factors such as electric field strength and flow rate are important in PEF application [13]. Factors such as proteins (such as solubility, composition, molecular weight, hydrophobic groups, and conductivity), pH, temperature, viscosity, and ionic strength are also important [12]. PEF improves the properties of proteins by optimizing these factors [13]. Generally, short-pulse electricity application is more advantageous [44] and free radicals are formed by the polarization of proteins and the energy released. With the interaction of these free radicals, the dielectric constant of the proteins increases and the proteins open [12]. If the duration of the electrical pulse is prolonged, the opposite effect occurs and protein aggregates can form. In addition, the increase in protein aggregation and surface hydrophobicity at high temperatures (≈70°C) in the PEF was mostly attributed to the thermal effect. The effect of β-LG application time is greater than the electric field intensity [13].

Although it is known that the effect of PEF on whey proteins varies depending on the electric field intensity, there are many contradictions in the literature regarding this. Some researchers have concluded that PEF therapy does not effect on proteins [12]. However, the functional and structural properties of peptides can be improved by optimizing electric field density and processing time in PEF application in whey proteins [63]. In addition, thermal pretreatment with PEF may have a positive effect on the opening of proteins [12].

3.2.5 Ohmic heating

OH, which is a thermal process, can be used for pasteurization. OH uses alternating electrical currents [1]. OH, which takes its name from Ohm’s Law (current, voltage, and resistance), is known by names such as joules, electrical resistance, direct resistance, and electro conductive heating. In this method, heat is generated by passing alternating current through the electrodes and resisting the conductive product against this current. The effectiveness of the method is related to factors such as voltage (transformers), current density, electrical resistance, and duration [46]. Electrical resistivity is an indicator of the composition and electrical conductivity of food [64].

OH differs from microwave and inductive heating methods in that it performs the heating process while in contact with food [65]. Compared to conventional heat treatment, it is effective in enzyme inactivation as well as guaranteeing the microbiological safety of the product [58]. OH, which has many advantages over fermentation and enzymatic hydrolysis, adjusts enzymatic activity and is seen as an important technique due to its positive effects on the properties of whey proteins (gelation, allergy). Electric field intensity affects the sensory and physicochemical properties of proteins and is also important in the production of bioactive peptides [66]. Alizadeh and Aliakbarlu [58] determined the antioxidant capacity of the fractions obtained by subjecting whey concentrate OH (50 Hz, 0–240 V, 5–15 seconds), UAE (24 kHz, 400 W, 25 mm titanium probe, 5–15 min), and enzymatic hydrolysis (pepsin). The degree of hydrolysis was increased by both pretreatments (excluding the combination of 5-min UAE and proteolysis), and the highest UAE was obtained with the treatment with a combination of 15 min, OH-15 seconds. It has been reported that this condition is caused by the breaking of covalent bonds and the denaturation of proteins by heat. It has been stated that antioxidant capacity increases depending on the degree of hydrolysis. In addition, UAE and OH showed similar effects when administered alone [58].

3.2.6 Subcritical water hydrolysis (SWE)

Subcritical water hydrolysis (SWE) (pressurized low-polarity compressed or pressurized hot water extraction) is a technology used for extraction or hydrolysis. In the method, it is possible to obtain liquid water with different polarities by decreasing the density and dielectric coefficient of water with the increase in pressure (0.10–22.1 MPa) and temperature (100–374°C) [67, 68]. With the increase of hydroxide and hydronium ions formed as a result of temperature and intermolecular vibrations, water behaves as an acid and/or base, becoming suitable for hydrolysis reactions [69]. The effectiveness of SWE depends on factors such as particle size, temperature, extraction time, flow rate, pressure, and solvent/sample ratio [67]. In addition, the type of atmosphere, raw material content, and reactor type can affect hydrolysis [70].

Unlike UAE, MAE, and HHP, which were applied before conventional hydrolysis methods, it is also effective alone and is a new alternative that has been successfully used in many substrates. In addition, SWE with additives (nitrogen, carbon dioxide, acetic acid, formic acid, sodium hydroxide, hydrochloric acid, and sodium chloride) can increase the yield and reduce the time and energy required. Although there are not many studies on whey SWE, promising results have been reported [46]. It has been reported that sodium bicarbonate increases the hydrolysis in SWE (291°C, 28 min) with five catalysts in whey and a low molecular weight peptide is obtained [47]. Briefly, with the optimization of the concentration, temperature, time, and pressure of the additive (acetic acid, lactic acid, etc.) used in SWE, the degree of hydrolysis of proteins increases and low molecular weight peptides are obtained [46].

4. Future perspective

The health-beneficial components of many foods are attracting attention day by day, and the methods used to isolate these components are gaining importance. However, factors that will affect the properties of individual bioactive peptides to be obtained by separating whey proteins into their fractions make it necessary to develop the method to be used. In addition, while whey proteins are used for many purposes in the industry, it may be possible to produce more valuable bioactive peptides industrially with appropriate methods. Activation and isolation of bioactive peptides by conventional methods have been shown in many studies. However, it has been shown by a limited number of studies that it is possible to obtain more and different peptides before the application of novel methods such as UAE and/or HHP before conventional methods. The synergistic effects of new and traditional methods also need to be demonstrated. In addition, there are very few studies on whey proteins of SWE and OH methods, which are used to obtain peptides in various protein sources. It is important to produce novel, different, safe, and functional peptides using faster and lower-cost methods. In the future, there is also a need for studies on the optimization of these novel technology conditions. In addition, after the application of new methods applied as pretreatment, the activities of peptides need to be investigated further.

5. Conclusions

Whey is a waste and/or by-product that is important to utilize because of both its high oxygen demand and its rich composition. Whey contains proteins of high biological value, including essential and branched-chain amino acids, and is a source of bioactive peptides. Whey peptides individually have better bioactivity than whey proteins. While bioactive peptides are obtained, their physiological properties vary depending on the protein composition, processing conditions (such as pH, and temperature), and the method used. However, existing methods such as enzymatic or fermentation and novel technologies such as USE, HHP, and MAE, which are not very effective when used alone, are used together to obtain better peptides with efficient, nutritional, and biological properties. In addition to being environmentally friendly and economical, novel methods also have positive effects such as reducing the amount of enzyme in enzyme hydrolysis and shortening the fermentation period. The novel methods applied as pretreatment make the bonds and structures more sensitive by unfolding/denaturation of proteins with effects such as surface hydrophobicity and sulfide bonds. Thus, it is possible to obtain peptides with lower molecular weight in the second step. In addition, SWE and OH methods applied alone can yield peptides with low molecular weight, but further studies with whey proteins are needed. In addition to the extraction method to be used in the isolation of whey peptides for industrial use, fractionation and purification methods are also effective in the activities of peptides. There is a need for the development of peptide production on an industrial scale.

Conflict of interest

The authors declare no conflict of interest.