**3. Influence of processing on protein digestion and peptide profile**

Dairy products are processed by the application of different physical and chemical methods. These methods change the protein structure irreversible or reversible depending on the impact of the treatment. The protein can be mainly denatured, hydrolysed, or glycosylated. This structural change can influence the access of the digestion enzymes to the protein and therefore changes the action of the digestion enzymes. An impact on the peptide profile that is generated before absorption into the blood takes place is the result. It is necessary to determine which processing methods and which processing variables are necessary to be able to reach or maintain a specific bioactivity.

#### **3.1. Thermal treatment**

Thermal processing is an important step to improve the microbial quality of milk. Additionally, enzyme activities are inactivated and some physicochemical changes can occur that might support processing. The nutritional value is greatly affected by thermal processing. Denaturation, β-elimination, racemization, or iso-peptide bond formation can occur that influence the nutritional value [52]. Denaturation is influenced by pH, protein concentration, ionic environment, genetic variant, and presence of ligands [53]. Heating might even particularly destroy tryptophan, can convert Arginine into citrulline and ornithine, can deamidate glutamine and asparagine, and desulphur cysteine and cysteine. Resulting end products might be lanthionine, lysine-alanine, iso-peptides and ornitho-alanine [52]. The digestibility of whey proteins increases after thermal treatment because the sites for enzymatic hydrolysis are easier to reach for the digestive enzymes. However, strong denaturation reduces digestibility [54]. Kopf-Bolanz et al. showed that heat treatment of dairy products led to an increased number of β-lactoglobulin peptides after in vitro digestion [22]. There is a greater susceptibility to hydrolysis following heat treatment [55]. Regarding the antidiabetic action of casein, there was a significant reduction observed after boiling compared to the raw casein [29]. The denaturation of whey protein via thermal processing led to an increase in the antibacterial activity of α-lactalbumin [56] and lysozyme [57]. The antioxidant action of whey proteins can be maintained by low-temperature processing. This results in high levels of specific dipeptides that can promote the synthesis of the antioxidant glutathione [58]. Extrusion cooking might also affect protein digestibility shown for example in a study of Onwulata et al. [59]. Data on the effect of ohmic heating are rare. Depending on the used temperatures, similar effects like with application of other heating methods might be expected [52]. It was also shown that spray drying or freeze drying did not exhibit negative effects on the immunomodulatory activity of a whey protein hydrolysate. The study also used whey protein concentrate (WPC) and sodium alginate as carriers for encapsulation to reduce bitter taste and resistance to hygroscopicity. They showed that spray drying of whey protein concentrate hydrolysate with the proper carriers did not affect the immunomodulatory activity and might therefore widen its application in food systems [60].

#### **3.2. Chemical treatment**

**1.** Tracing the pathways of formation of bioactive peptides from the parent proteins

**3.** Improving the "positive" properties discovered in natural peptides by design of synthetic

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

**2.** Identifying the biological properties

structural analogues or peptide mimetics

116 Technological Approaches for Novel Applications in Dairy Processing

but might be used in the future for the design of new products.

reach or maintain a specific bioactivity.

**3.1. Thermal treatment**

**3. Influence of processing on protein digestion and peptide profile**

Dairy products are processed by the application of different physical and chemical methods. These methods change the protein structure irreversible or reversible depending on the impact of the treatment. The protein can be mainly denatured, hydrolysed, or glycosylated. This structural change can influence the access of the digestion enzymes to the protein and therefore changes the action of the digestion enzymes. An impact on the peptide profile that is generated before absorption into the blood takes place is the result. It is necessary to determine which processing methods and which processing variables are necessary to be able to

Thermal processing is an important step to improve the microbial quality of milk. Additionally, enzyme activities are inactivated and some physicochemical changes can occur that might support processing. The nutritional value is greatly affected by thermal processing. Denaturation, β-elimination, racemization, or iso-peptide bond formation can occur that influence the nutritional value [52]. Denaturation is influenced by pH, protein concentration, ionic environment, genetic variant, and presence of ligands [53]. Heating might even particularly destroy tryptophan, can convert Arginine into citrulline and ornithine, can deamidate glutamine and Hydrolysis by acid is applied which is known to improve their protein digestibility. It is used for example for enteral and hypoallergenic infant nutrition. For Mozzarella, the type of acid used is important for the protein yield obtained in the pre-cheeses [61] and might therefore also affect the profile of bioactive peptides. Treatment with alkali for hydrolysis is rarely applied in the food industry. It would result in racemization and loss of protein digestibility [62].

#### **3.3. Biochemical treatment**

Fermented dairy products like yoghurt and cheese result in a high number of bioactive peptides produced by the lactic acid bacteria. Especially the type of the starter culture, type of probiotic bacteria, and the fermentation parameters play an important role for the bioactive effect that the product might have. Furthermore, only via this way de novo peptides can be generated that do not occur after digestion of milk as such. *Streptococcus thermophilus* and *Lactobacillus bulgaricus* possess bacterial activity against Streptococci in vivo that probably derives from the antimicrobial peptides that they produce during fermentation [63]. It is very promising to test different lactic acid bacteria strains for their effect on a bioactive function. One study of Gobbetti et al. showed that a fermentation with *L. delbrueckii* ssp. *Bulgaricus SS1* versus a fermentation with *Lactobacillus lactis* subspecies *cremoris FT4* resulted in a higher ACE-inhibitory activity [64]. The most investigated ACE-inhibitory peptides were obtained after fermentations with *L. helveticus* and *L. helveticus CP790*. Also, the Finnish milk product Evolus contained *L. helveticus LBK-16H* strain as a starter and they all contained the tripeptide IPP and exerted a hypertensive effect [65–67]. Another study demonstrated the effect of the time of cheese ripening on the ACE-inhibitory activity. Cheese was produced with a mixture of 12 different strains and showed an increase of the inhibitory effect during ripening as long as a certain level of proteolysis was not exceeded [68]. In 10 Swiss cheese types, the ACE-inhibiting peptides V-P-P and I-P-P were quantified. They detected contents of 19.1 mg/kg to 182.2 mg/kg depending on the cheese type that shows the huge effect of different processing ways probably via different lactic acid bacteria [69]. Also, the application of new techniques like next-generation sequencing that reveals the whole genome of bacteria strains might help to select promising strains with specific protease expressions. It was also demonstrated that fermentation reduced the allergenic potential of α-lactalbumin and β-lactoglobulin [41, 42]. The peptides that result after fermentation and enzyme hydrolysis might remain susceptible to further hydrolysis as long as the process goes on. This might lead to a decrease of bioactive function of these peptides. More important is also the stability of the generated peptides. They might be degraded by the digestive enzymes and result in zero activity in the body. The stability versus the action of gastric and pancreatic enzymes has to be tested beforehand. Another problematic point is that the microbial fermentations have to be reproducible [8]. Fermentation with known and established lactic acid bacteria cultures is a great strategy to enrich certain bioactive peptides with a special functionality. This would be a possibility to enhance a bioactive function in a natural way with a minimal processing approach that meets the interests of the consumer. The functionality and bioavailability of bioactive peptides generated via fermentation has to be more clarified.

**4. Influence of other factors on bioavailability of bioactive peptides**

all the factors that can affect peptide bioavailability in the target group of the product.

An enrichment of peptides that can inhibit DPP-IV could result in increasing satiety after consumption of a dairy product. The most promising approach to steer the peptide profile of the

**5. Possible approach for design of a dairy product with a satiety** 

**increasing effect**

**Figure 3.** Targeting bioactive function in product development.

Not only processing can influence the profile of bioactive peptides. Also, other external factors can influence protein digestion and therefore the bioavailability and generation of bioactive peptides. It is important to consider the effect of the food matrix and meal composition on digestion. For example, the addition of inulin to the dairy product can influence digestion and peptide bioavailability [75]. Also, proteins can form complexes with polyphenols, etc. that could lower protein bioavailability [76]. Furthermore, internal factors can influence the peptide profile. Children and the elderly have different enzyme activities and therefore the digestion enzymes will act slightly different and change the peptide profile [77, 78]. Genetic variations in people for example enzyme deficiencies or changes in the composition of the digestion juices due to different transporter expression can have an impact. The action of digestion enzymes depends on daytime, age and on *Helicobacter pylori* infection [79]. Also for a lot of other special physiological states, certain diseases and so on, the enzyme activity is affected and might therefore result in a different bioactive peptide bioavailability. It is very important to consider

Adjusting Bioactive Functions of Dairy Products via Processing

http://dx.doi.org/10.5772/intechopen.72927

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The use of milk-clotting enzymes and digestive enzymes to produce bioactive peptides is another processing approach. However, most of the resulting peptides had a bitter taste [7]. Membrane-separation technique is applied to enrich peptides with a specific molecular weight [3]. It was also shown that hydrolysed infant formulas show a different peptide profile compared to the standard formulas assuming that infants fed hydrolysed formulas might obtain bioactive peptides that promote other bioactive functions than the ones provided by the standard formulas [70].

#### **3.4. Physical treatment**

Homogenization applies pressure (14–18 MPa) and shear stress that alter the protein structure and improve the digestibility [52]. Use of ultra-high pressure homogenization with pressure around 400 MPa results in more severe protein denaturation [71]. Application of high hydrostatic pressure processing increased digestibility of β-lactoglobulin with pepsin with increasing pressures (400–800 MPa) [72]. Penas et al. also combined high hydrostatic pressure processing with selected food-grade proteases and demonstrated a reduction in antigenicity of the whey protein hydrolysates that can be used as ingredients of hypoallergenic infant formulae [73]. Ultrasound treatment is a non-conventional processing technique that can denature α-lactalbumin and β-lactoglobulin. In whole milk compared to skim milk, the denaturation was stronger and heat addition even increased this effect [74]. A very soft technology is membrane filtration that enables to separate proteins in their native state. This technology only enables a fractionation of different milk components and does not alter the protein structure as such, and it only influences the milk composition.
