Cellular and Molecular Milk Protein Synthesis

### **Chapter 3**

## Superior Stimulation of β-Casein mRNA Accumulation by Pseudophosphorylated Prolactin: Enhanced Transcription and Message Stabilization

*Wei Wu, Changhui Deng, Jennifer L. Brockman, Linda A. Schuler and Ameae M. Walker*

### **Abstract**

A proportion of secreted pituitary prolactin (PRL) is phosphorylated. However, because most commercial sources of PRL are recombinant proteins without posttranslational modification, the importance of PRL phosphorylation to the production of milk proteins is an understudied area. Here, we have examined the effect of PRL phosphorylation on expression of the milk protein, β-casein, using a phospho-stable mimic of the phosphorylated form (S179D-PRL) and analyzing promoter activation and mRNA stability over a 7-day treatment period in response to this and unmodified PRL. At equivalent concentrations, the phospho-mimic showed a superior ability to activate a −2300 → +490 region of the promoter, but not an artificial promoter consisting of three Stat5 consensus sites upstream of a minimal promoter. Unlike unmodified PRL, S179D-PRL was also able to stabilize β-casein mRNA. These effects of S179D-PRL were eliminated by incubation in the MAP kinase pathway inhibitor, U0126, bringing promoter activation down to the level seen with unmodified PRL and essentially eliminating the effect on mRNA stability. These results support an important role for the posttranslational phosphorylation of PRL and signaling through the MAP kinase pathway in the production of this milk protein.

**Keywords:** hormone regulation, posttranslational modification, phosphorylated prolactin, molecular mimicry, S179D prolactin, beta-casein, mRNA stabilization, length of promoter

### **1. Introduction**

PRL phosphorylation in the pituitary is regulated physiologically [1, 2]. Phospho-PRL has been demonstrated in rat [3], mouse [4], sheep, avian [5], bovine [6], and human [7] pituitary extracts. The phosphorylated form is very stable and cleared

from the circulation with similar kinetics to the unmodified hormone [6–8]. Differential function analysis of unmodified and phospho-PRL demonstrated different biological activities [9, 10]. Recent studies have utilized a molecular mimic of phospho-PRL to prevent conversion to the unmodified form during the course of an experiment. This mimic was made by substituting an aspartate residue for the normally phosphorylated serine [11, 12], thereby producing S179D-PRL.

Previous work from our laboratories has demonstrated different activities for unmodified PRL (U-PRL) and S179D-PRL in the control of proliferation in the mammary gland and mammary cells in culture, with U-PRL promoting cell proliferation and S179D-PRL antagonizing this effect [12–16]. Despite these demonstrations, which indicate that mammary cells recognize and respond to the two kinds of PRL differently, there has since been very limited investigation of the importance of PRL phosphorylation to the production of milk proteins. In large part, this is likely due to the lack of availability of purified phosphorylated PRL, another reason we developed the phospho-mimic, S179D-PRL.

Using S179D-PRL, we have previously shown [14] that compared to U-PRL this phospho-mimic had a superior ability to stimulate β-casein expression. This was surprising since S179D-PRL has an inferior ability to activate Stat5 [13], activation of which is crucial to milk protein production [17].

Here, we have investigated the molecular mechanisms resulting in superior β-casein expression in response to S179D-PRL by examining the relative roles of transcriptional and posttranscriptional activities and MAP kinase signaling to the accumulation of β-casein mRNA in response to each PRL.

### **2. Materials and methods**

### **2.1 Mammary cell culture and differentiation**

HC11 cells were a gift from Nancy Hynes (Friedrich Miescher Institute, Basel, Switzerland). Cells were grown in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) containing 10% FBS, 5 μg/ml insulin (Sigma, St. Louis, MO, USA), 10 ng/ml epidermal growth factor (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin. Once confluent, they were grown 3 more days with daily medium changes. The medium was then removed, and the cells were washed five times with RPMI 1640. Cells were then treated with priming medium-RPMI 1640 supplemented with 10% charcoalstripped horse serum (Cocalico Biologicals, Reamstown, PA, USA), antibiotics, 10 μg/ml insulin, 1 μg/ml hydrocortisone (Sigma) for 24 h followed by induction in priming medium plus U-PRL, or S179D-PRL (changed daily). This essentially follows Taverna et al. [18] except that 1 μg/ml, which maximally stimulates the endpoints measured here, instead of 5 μg/ml PRL, was used in this study. Potential differences in the uptake or degradation of U-PRL and S179D-PRL were monitored by ELISA and Western blot of media samples.

### **2.2 Transfection and β-casein luciferase assays**

Primed HC11 cells were transfected with −2300 → +490 of the proximal rat β-casein promoter [19] subcloned into pGL3Basic (Promega, Madison, WI, USA) and a CMV-β-galactosidase construct. Sub-confluent cultures in six-well plates were transfected with β-casein luciferase DNA (2 μg), β-gal DNA (0.5 μg), and 10 μl *Superior Stimulation of β-Casein mRNA Accumulation by Pseudophosphorylated Prolactin… DOI: http://dx.doi.org/10.5772/intechopen.101256*

lipofectamine/5 ml. After transfection, cells were treated with one or other form of PRL for 24 h in the absence or presence of U0126 (10 μM). For the 7-day experiment, cells were treated with the PRLs for 6 days, with daily medium changes, prior to transfection. For some experiments, cultures were transfected with an artificial promoter consisting of three Stat5 consensus sites upstream of a minimal promoter [20].

### **2.3 β-Casein mRNA stability**

HC11 cells were induced for 1–7 days with daily medium changes and RNA was isolated. To test the mRNA stability, the transcription inhibitor 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole (DRB) (Promega) was added (50 μg/ml) in the continued presence of the PRLs and in the absence and presence of the MEK1/2 inhibitor, U0126 (10 μM). Real-time PCR reactions were 12.5 μl SYBR Green PCR master mix (Applied Biosystems), 2.5 μl of 10 μM mouse β-casein forward primer (5′-CCC GTC CCA CAA AAC ATC CAG CC-3), and 2.5 μl reverse primer (5′-ATT AGC AAG ACT GGC AAG GCT G-3′), or 2.5 μl of mouse GAPDH forward primer (5′-CCA TGG AGA AGG CTG GGG-3′), and 2.5 μl reverse primer (5′-CAA AGT TGT CAT GGA TGA CC-3′), 1 μl diluted RT product and 6.5 μl ddH2O with 10 min 95°C followed by cycles of 95°C, 1 min; 55°C, 30 s; 72°C, 45 s; and 80°C, 10 s. Both annealing temperature and Tm of mouse β-casein and GAPDH primers are similar, allowing co-amplification and a comparative CT method for quantification of gene expression calculated by 2–ΔΔC T.

### **2.4 Statistical analysis**

Data were subjected to ANOVA with posttests comparing specific groups and Bonferroni corrections for multiple comparisons. Data are presented as mean ± SEM. The minimal number of experiments and replicates within each experiment was 3. Analysis of the real-time RT-PCR data was as per ABI PRISM 7700 Sequence Detection System User Bulletin #2.

### **3. Results**

Real-time RT-PCR allowed us to determine steady-state transcript levels as a function of time in response to U-PRL and S179D-PRL (**Figure 1**).

β-Casein mRNA increased over the 7-day period in response to each PRL. S179D-PRL was more efficacious than U-PRL, resulting in over twice the level of β-casein transcripts after 3 days, and 3–4 times higher levels after the full 7-day incubation. In order to determine whether these were effects on promoter activity, we utilized a β-casein promoter-luciferase construct. Importantly, this construct included the −2300 to +490 region of the β-casein promoter and not the usually employed, much smaller −344/−1 portion. As can be seen in **Figure 2A**, both PRLs stimulated reporter activity after 1 day of exposure. However, S179D-PRL was twice as effective as U-PRL. This was not the result of differential stability of these PRL forms since examination of the 24-h media from these incubations by ELISA and Western blot using an antibody, which recognizes both forms equally showed no evidence of different uptake or degradation of the PRLs (data not shown). Conduct of this experiment in the presence of U0126 demonstrated that the ERK pathway was important for the superior ability of S179D-PRL to activate the promoter (**Figure 2A**). In contrast, this inhibitor had no significant effect on U-PRL-stimulated activity and did not alter controls.

### **Figure 1.**

*Effect of U-PRL and S179D-PRL on β-casein mRNA levels as a function of days of stimulation. \*, p < 0.01 versus control (CON); #, p < 0.01 for S179D-PRL (S179D) versus U-PRL (U); \*\* p < 0.001 versus control; ##, p < 0.05 for S179D-PRL versus U-PRL.*

#### **Figure 2.**

*Effect of U-PRL and S179D-PRL on the −2300 → +490 β-casein promoter-luciferase. A, transfection at day 0 and assay at day 1 of treatment with and without inhibition by U0126; B, transfection at day 6 and assay at day 7 of treatment; C, ERK activation and β-casein-luciferase activity in response to S179D-PRL. \*, p < 0.01 for U-PRL and †, p < 0.001 versus control. #, p < 0.01 versus S179D-PRL.*

The relationship between both ERK activation and promoter activity and dose of S179D-PRL is shown in **Figure 2C**.

To determine the importance of Stat5 in the differential effects of the two PRLs at the promoter, we examined their relative activities at an isolated Stat5 enhancer.

Although both PRLs were able to significantly increase activity, S179D-PRL was slightly less effective (**Figure 3**). This result demonstrated that other regulatory

*Superior Stimulation of β-Casein mRNA Accumulation by Pseudophosphorylated Prolactin… DOI: http://dx.doi.org/10.5772/intechopen.101256*

sequences and mediators, in addition to Stat5, were important in the S179D-PRL response in the physiologic context of the β-casein promoter.

By 7 days of incubation, the difference in efficacies of the two PRLs in stimulating β-casein promoter activity was essentially eliminated (**Figure 2B**) and the degree of promoter stimulation in response to both ligands was increased. Based on the results in **Figure 1**, one would not have predicted the loss of the difference between U-PRL

#### **Figure 4.**

*Effect of U-PRL and S179D-PRL on mRNA half-life. Following a 6-day incubation in each PRL, the transcription inhibitor, DRB, was added. A, GAPDH mRNA; B and C, β-casein mRNA levels normalized to GAPDH as a function of time after addition of DRB in the absence (B) and presence (C) of U0126 (added at time 0). The ratio at time 0 in the control cells was set at 0.1 in B. \*, p < 0.001 for S179D-PRL versus control or U-PRL; #, p < 0.05 for U-PRL versus control; ##, p < 0.001 versus control.*

and S179D-PRL at day 7 if transcription was the most important regulator of steadystate mRNA levels. We therefore analyzed β-casein mRNA stability in response to both PRLs. Cells were incubated without or with the different PRLs for 6 days, and then, the transcription inhibitor, DRB, was added. **Figure 4A**, which plots GADPH mRNA as a function of time after inhibition of transcription, shows the expected decline in mRNA levels, with a half-life of about 4 h. This was not appreciably altered by either PRL, allowing us to use GAPDH to normalize the data for RT-PCR efficiency.

As seen in **Figure 4B**, β-casein transcripts had a half-life of about 4 h since when normalized to GAPDH, the ratio was unaltered by incubation in DRB in the controls or cells incubated in U-PRL. In contrast, incubation in DRB revealed a dramatic effect of S179D-PRL on β-casein mRNA stability. Since time 0 on this graph is after 6 days of U-PRL or S179D-PRL treatment, the effects of the two PRLs on overall mRNA levels are evident prior to DRB treatment. When U0126 was added along with DRB at 0 h, the elevated levels of β-casein mRNA in response to S179D-PRL were reduced by 2 and 4 h, indicating an important role for ERKs in S179D-PRL-induced mRNA stability (**Figure 4C**).

### **4. Discussion**

The results demonstrate that both U-PRL and S179D-PRL increase steady-state β-casein mRNA levels. However, S179D-PRL was more effective than U-PRL after shorter exposures and also elicited a later rise not found with U-PRL. This biphasic response to S179D-PRL suggested the possibility of different mechanisms of mRNA accumulation. A large body of literature has demonstrated rapid effects of PRL treatment on the β-casein promoter [21–23]. However, a second, much smaller number of papers, which have largely been forgotten in recent years, demonstrate that PRL increases β-casein mRNA stability and that this is quantitatively much more important in terms of steady-state mRNA levels than promotion of transcription [24–27]. However, these studies used pituitary extract preparations of PRL, which contained a mixture of both unmodified and phosphorylated PRL, and so the importance of PRL phosphorylation to these activities was unknown.

Regulatory sequences within the β-casein promoter are found over a fairly large region, but most investigators have limited their examination to the activity of the most proximal 344 nucleotides in reporter gene assays. However, this promoter may not detect all the responses to S179D-PRL. CREB, ATF1, and YY1 sites outside of this region can potentially be activated by ERK1/2 (reviewed in [28]). This signaling pathway is a major mediator of S179D-PRL, although U-PRL can also activate these kinases in HC11 cells to some extent [13]. In addition, increased β-casein gene expression can be achieved by removal of YY1 from the promoter [28]. We therefore utilized a −2300 → +490 fragment of the promoter, which includes the CREB, ATF1, and YY1 sites (in addition to the STAT5 site in the −344/−1 region) in an attempt to make the reporter assay more physiologically relevant. It should be noted, however, that even this larger piece does not constitute the whole promoter.

While both PRLs increased activity of this promoter, S179D-PRL was about twice as effective as U-PRL during the first-phase response. This was unexpected, since S179D-PRL is weaker than U-PRL in stimulating STAT5 tyrosine phosphorylation [13, 15], generally thought to be the most important regulator of β-casein expression. The MEK1/2 inhibitor, U0126, eliminated the difference between U-PRL and S179D-PRL on promoter activity, indicating that ERKs 1/2 are important for the superior stimulation with S179D-PRL, but not for the activity of U-PRL. With the construct

*Superior Stimulation of β-Casein mRNA Accumulation by Pseudophosphorylated Prolactin… DOI: http://dx.doi.org/10.5772/intechopen.101256*

containing only the Stat5 enhancer, there was no superior effect of S179D-PRL. Together, these results demonstrate the importance of other transcription factors in S179D-PRL signals to the β-casein promoter.

Increasing β-casein transcripts with duration of S179D-PRL exposure shown in **Figure 1** suggested increased promoter activation over time in culture. However, we have shown that the difference in β-casein promoter activation between S179D-PRL and U-PRL was reduced after 7 days, consistent with a mechanism other than promoter activity for the second phase of the response to S179D-PRL. Our data indicate that S179D-PRL markedly increased β-casein mRNA stability at this time, while U-PRL was without effect. Other investigators have used pituitary-derived PRL, which is a mixture of U-PRL and phosphorylated PRL [27] and hence have not been able to make this distinction.

There are a variety of mechanisms by which β-casein mRNA stability may be regulated, including the length of the poly A tail [29] and effects at the 5′ untranslated region (5′UTR) and other areas of the 3′UTR [26, 27]. ERK1/2 activation has previously been shown to be important for some proportion of the β-casein response to mixed PRL [23]. This is the first demonstration of effects on both transcriptional and posttranscriptional regulation.

Combining these results with previous work on the role of these two PRLs in cell proliferation, we suggest that optimal alveolar development and subsequent lactation are the result of well-orchestrated exposures to combinations of U-PRL, phospho-PRL, and other physiological stimuli including milk removal. U-PRL (or a lactogen acting like U-PRL) stimulates alveolar development [14] and some β-casein expression. Phospho-PRL, which has the capacity to inhibit alveolar development [14, 16], may slow alveolar development after parturition but robustly stimulate β-casein expression by virtue of its ability to increase both promoter activity and mRNA stabilization.

### **5. Conclusions**

These results support an important role for the posttranslational phosphorylation of PRL and signaling through the MAP kinase pathway in the production of the milk protein, β-casein. Furthermore, the results are an important reminder that use of bacterially derived recombinant prolactin that does not have normal secretory pathway posttranslational modifications may not fully represent normal physiology.

### **Acknowledgements**

This work was supported by CBCRP grant 10PB-0127 (AMW) and by NIH grants DK 61005 (AMW) and CA 78312 (LAS). The authors would like to thank Dr. Jeffrey Rosen (Baylor College of Medicine, Houston, TX) for provision of the -2300 → +490 promoter region of the β-casein gene.

### **Conflict of interest**

The authors declare no conflict of interest that would by any measure affect their impartiality in the presentation of results.

*Milk Protein - New Research Approaches*

### **Author details**

Wei Wu1 , Changhui Deng2 , Jennifer L. Brockman3 , Linda A. Schuler3 and Ameae M. Walker1 \*

1 Division of Biomedical Sciences, University of California, Riverside, CA, USA

2 Department of Biochemistry, University of California, Riverside, CA, USA

3 Department of Comparative Biosciences, University of Wisconsin, Madison, WI, USA

\*Address all correspondence to: ameae.walker@ucr.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Superior Stimulation of β-Casein mRNA Accumulation by Pseudophosphorylated Prolactin… DOI: http://dx.doi.org/10.5772/intechopen.101256*

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### **Chapter 4**

## Omics, the New Technological Approaches to the Milk Protein Researches

*Zitai Guo, Lu Ma and Dengpan Bu*

### **Abstract**

With the development of technological approaches, the perturbations of biological information in gene, mRNA, proteins, and metabolites have been gathered to broaden the cognition of synthesis processes during lactation. While omics, the series of application including genomics, transcriptomics, proteomics, and metabolomics, are mostly preferred and conducted in the investigation of lactation especially the milk protein. These new technological approaches provide a complete view of the molecular regulation pathways and make it possible to systematically investigate the lactation. The aim of this chapter is to comprehensively review the advances in knowledge regarding the great progress in milk protein synthesis as well as lactation physiology and pathology mainly in dairy cows obtained from omics technologies, meanwhile the milk proteins as well as their attributes are illustrated.

**Keywords:** milk protein, omics technologies, dairy cows, protein synthesis

### **1. Introduction**

Milk protein is one of the most important nutrients in milk. It contains a variety of essential amino acids required by body maintaining and is believed to have a variety of potential biological functions [1, 2]. In past few decades, plenty of research studies were conducted for improving the milk quality especially the milk protein production, while the morphological as well as physiological focused on mammary gland has been widely investigate [3–6]. However, since the synthesis of milk protein during lactation is a complex biological activity, only little perturbations in lactation can result in a certain difference in the composition and concentration of milk protein [7]. Furthermore, the interactions between proteins, genes, and factors are diversity and dynamics when performing physiological functions, which means the tradition approaches may no longer meet the requirement of current research studies. In recent years, new approaches including Omics in lactation-related research studies received extensive attention. While Omics was one of the most used approaches since it make the study possible to explain the synthesis comprehensively and systematically at the levels of DNA, RNA, proteins, and metabolites, which can assist the further

indentation of factors as well as processes regulating lactation [8, 9]. To present the use of omics approach in lactation especially milk protein synthesis research in dairy cows, the relevant introductions of omics and milk protein following the great progress in lactation physiology as well as pathology by these new approaches are illustrated in the current chapter.

### **2. Milk protein and its attributes**

Suppose milk was considered an important nutrient source due to its perspective of molecular composition, milk proteins should be the most important source of bioactive peptides. Milk proteins were proved to have higher digestibility and more suitable content of amino acids for human being [10], which contribute to more than 3% of content in milk [11]. Based on the properties of structure, function, and solubility, milk proteins are normally divided into three categories including caseins, whey proteins, and milk fat globular membrane proteins, while these complex components make the milk proteins to vary widely [12, 13]. In short, caseins account for about 80% of total milk proteins and were mainly classified by four basic types of molecules: the αs1-, αs2-, β-, and κ-caseins [14, 15]. Whey proteins are the major component of milk whey, their components mainly include β-lactoglobulin (β-LG), α-lactalbumin (α-LA), serum albumin (SA), immunoglobulin G (IgG), and also include several small molecule proteins especially low-abundance proteins, such as enzymes and metal-binding proteins [16]. Milk fat globular membrane proteins only contribute to 1–4% of milk proteins, but play the important roles in the defense mechanisms and process of cell growth in newborns [17]. So far, plenty of research studies have investigated the types, structures as well as synthesis of those components in milk proteins, which would be reviewed in later paragraphs.

### **2.1 Caseins**

Casein is a kind of phosphorous-containing protein [18]. The serine hydroxyl group inside casein forms an ester bond to the phosphate group, which gives it the common feature of amphiphilic. Caseins classified by different types may contain different amount of phosphate groups [19]. However, all kinds of caseins keep at least one phosphate with ester bonded. Since the caseins contain amount of phosphorylated serine groups, which bind in the form of covalent bonds [20], these groups keep the form of clusters on the surface of molecules and provide conditions for its binding to calcium ions [21], which was considered to be the most important nutritional function of casein.

The αs1-, αs2-, β-, and κ-caseins are four basic types of casein molecules as mentioned above, which are also the types synthesized by the mammary gland of dairy cows. Except for major installations, there are numerous minor fractions of caseins such as γ-casein. While none of those were found in the composition of milk [22]. Though the amino acid sequence, number, and total charge of casein are not completely the same among different variant individuals, the composition of amino acids still has the following features [23–25]:

1.The amino acids with polar and nonpolar are unevenly distributed, forming obvious hydrophilic and hydrophobic regions.


Though the synthesis of milk protein is closely related to animal science, the research on its structures was mostly investigated in field of food science. For caseins, the research on those monomers has become the hotspot in recent years. Previous study has predicted the structure of αs-casein by molecular simulation, and its quaternary structures of caseins were investigated by Fourier transform infrared spectroscopy as well [26]. Specifically, αs1-casein has the ability of self- aggregation, but is affected by factors including condition pH as well as the ionic strength [27]. The increase of pH value will weaken its self-aggregation, to the opposite, the increase of ionic strength will enhance self-aggregation [28]. The ability of self-aggregation in αs2-casein is similar to that in αs1-casein, which mostly depends on the ionic strength, but the aggregation begins to be weakened once the strength arrives at 0.2 mol/L [29].

The research related to the structures of β-casein is contradicted and still unclear. Noelken et al. firstly predicted the form of β-casein in random coils in aqueous solution with only a small amount of regular structure [30], However, the results cannot be consistent due to the difference in temperature, ionic strength, protein concentration, etc., and these factors all contribute to affecting the ability of its self-aggregation [31]. β-casein exists as a monomer at low temperature and begins to aggregate as the temperature rises. While self-aggregation gradually increased and reached the maximum, once the protein concentration surpasses the threshold [32]. However, the effect of pH on the self-aggregation ability of β-casein is still inconclusive.

κ-casein is located on the surface of the casein micelle structure. Its most important role is to maintain the stability of the micelle structure and act as transition in the hydrophobic casein and water [33]. The unique disulfide bonds give κ-casein special properties different from the rest three types [34]. Though the self-aggregation of κ-casein consists of a core surrounded by multiple layers of variable polypeptide regions [35], which is similar to that of β-casein, it's not affected by temperature and ionic strength within a certain range and forms a fixed-size polymer.

In addition to nutritional functions, the potential biological activity of peptides formed as a result of casein proteolysis in the gastroenteric tract has been widely valued [36]. These peptides may affect the cardiovascular, nervous, immune, and digestive systems [37, 38]. However, it has been established that peptides are distributed unevenly in the composition of primary structure of caseins, which has attracted more attention to related research studies.

### **2.2 Whey proteins**

Whey is a by-product during casein production, it can remain the form of liquid after coagulation by rennet, while the proteins left are the whey proteins [39]. Whey proteins are in full value and contain all kinds of amino acids that make up proteins [40]. And except for a little bit lower content in sulfur-containing amino acids, whey proteins have higher contents of the rest of essential amino acids compared with other proteins [41]. Therefore, whey contributes to nearly half of the nutrients in milk even though its total solids are only around 6.0 ~ 6.5% [16]. The high quality of whey proteins with complete and appropriate proportion of essential amino acids meets the

requirement of human being, which also determines the functions of whey proteins. As mentioned above, whey proteins consist of a variety of biologically active ingredients including β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulin, and a variety of growth factors and biologically active peptides.

It's clear that the composition of amino acids determines the biological activity of each component in whey proteins. The various amino acids including threonine, cysteine, methionine are important to the intestine, muscle, and antioxidant systems [42]. While the physiological functions of whey proteins as well as the main amino acid related are reviewed below.

The β-lactoglobulin is the most prevalent one and comprises more than half of the whole proteins, while its prevalence affects the attributes of whey, β-lactoglobulin contains 162 amino acids with two variants differ in one amino acid, the disulfide and free sulfhydryl groups in its molecules forms let it become the major source of sulfurcontained amino acid [43]. β-lactoglobulin is manufactured in the ruminants while almost all of non-ruminants cannot synthesis it in the mammary gland [44]. The hydrophobic area on molecule of β-lactoglobulin is a quite effective in binding retinol, which may contribute to regulating the mammary gland by vitamin A [45]. However, the related biological functions of β-lactoglobulin are not commonly accepted now.

α-lactalbumin comprises around 13% of the whole whey proteins, with four disulfide linkages and no phosphate group molecule. The function of modifying the activity of the enzyme galactosyl transferase was proved in former studies, which promoting the transfer of UDP galactose to glucose [46]. α-lactalbumin is closely related to lysozyme evidenced by the similar synthesize of linkage but does not work on the same substrates, nor antigenically [47]. In addition, the α-lactalbumin is more heat-stable in the presence of calcium rather than in the absence of calcium [48], which is unusual compared with other proteins.

Different from the former two components, the serum albumin and immunoglobulins are not synthesized in the mammary gland. The serum albumins isolated from milk have the same molecule to those serum proteins since they are leaked into milk from the bloodstreams [49]. Therefore, serum albumins are identical to be the same molecule with serum proteins. Serum albumins contain no phosphorous with only one free sulfhydryl group, which gives those a specific binding sites for hydrophobic [50]. While the immunoglobulins in milk proteins comprise more than 2% of the total content and are classified by four types including IgG1, IgG2, IgA, and IgM. Immunoglobulins in colostrum can provide the passive immunity to the calf until the synthesis of antibodies activates in their body [51].

### **2.3 Milk fat globular membrane proteins**

Milk fat globule membrane (MFGM) is the layer of film wrapped on the surface of milk fat, the function of which is to protect fat globule from polymerization or enzymatic degradation [52]. Milk fat globular membrane proteins (MFGMPs) are protein component in MFGM and contribute to the 25% ~ 70% of total contents [53]. MFGMPs have the most diverse biological functions and play an important role in the cell growth process and defense functions of newborns [54, 55].

The three-layer structure theory of milk fat globule membrane has been widely accepted [56]. While the main components of MFGMs including xanthine oxidoreductase, butyrophilin, and lactadherin play the most important role [57]. The innermost layer of the milk fat globule membrane is a single-layer membrane composed of polar lipids and proteins synthesized by the endoplasmic reticulum, which wraps

### *Omics, the New Technological Approaches to the Milk Protein Researches DOI: http://dx.doi.org/10.5772/intechopen.102490*

the fat droplets in the core of the fat globule, followed by a high-density protein layer attached to the inner surface of the double-layer membrane; The last is the lipid bilayer that comes from the apical membrane of breast epithelial cells [58]. The cytoplasm forms a cytoplasmic crescent between the high-density protein layer and the outer double-membrane layer.

Xanthine oxidoreductase (XO/XDH) is the main component of milk fat globular membrane protein. It has been confirmed that it has a certain role in breast development, intestinal antibacterial and tissue damage [59]. XO/XDH is a member of the flavoprotein family of molybdenum dehydrogenase, which is a key enzyme for purine metabolism in the organism. However, the role of XO/XDH in the process of milk fat droplet wrapping and secretion may not express enzymes [60]. In addition, xanthine oxidoreductase has been confirmed to have a certain role in mammary gland development, intestinal antibacterial and tissue damage [61].

Butyrophilins (BTNs) are proteins related to fat droplets and a member of the immunoglobulin family. Many members of the BTN family have been confirmed to have immunomodulatory effects [62]. For example, the BTN3A1 and BTN3 families can inhibit T cell activation [63]. Milk-derived BTN has cross-reactivity with specific neuronal antibodies, which may be related to autoimmune regulation of diseases such as autism and multiple sclerosis [64].

Lactadherins are immunogenic lipophilic glycoproteins and are also known as milk fat globule epidermal growth factor 8 [65]. They are mainly distributed in secretory cells at the top of the milk tubules. In recent years, research on MFG-E8 has mainly focused on the phagocytosis of apoptotic cells, immune regulation, coagulation, and thrombosis [66]. Nakatani et al. found that MFG-E8 can recognize apoptotic cells in breast recession and activate phagocytes to phagocytose apoptotic epithelial cells. MFG-E8 can also resist rotavirus infection [67]. The protective effect of MFG-E8 on the intestinal tract has also become a research hotspot, which is mainly reflected in its antiinflammatory, anti-apoptotic, and promoting intestinal mucosal repair effects [68].

### **3. Omics, a series of novel approaches to study milk protein**

With the advent of the post-genomic era, the interactions between proteins, genes, and factors are followed by researchers, the diversity and dynamics of physiological functions cause the tradition approaches unable meet the requirement of current research studies. In recent years, a large number of research approaches including Omics have emerged in the research fields of biology [69, 70]. The emergence of these new applications can provide a complete view of the molecular regulation pathways of cells and organisms and make it possible to systematically investigate the lactation at the levels of genes, proteins, and even the metabolites, which is much helpful for the investigation of lactation especially the milk protein.

Omics are series of applications including genomics, transcriptomics, proteomics, and metabolomics in biological research. Genomics focus on the heterogeneity of coding genes, to investigate the sequence and expression of DNA, it provides the insight into the genetic structures by mapping as well as the performing the sequence analysis [71]. Transcriptomics profile the expression of mRNA in cells at specific time or state, while it can simultaneously work on more than thousands of changes in mRNA expression. Proteomics are used to determine the perturbations of expression patterns, abundance, and posttranslational modifications in proteins, and specialized in the differences caused by these factors [72]. While metabolomics monitor the

changes in large groups of metabolites in biological samples, during which the further integration is conducted to reflect the physicochemical properties [73]. By applying these new approaches, the knowledge related to dairy science especially milk synthesis has been pushed forward tremendously in recent years, meanwhile the determination and analysis methods applied were developing as well, which were specifically overviewed in later section.

### **4. Genomics in milk protein research**

Genomics involves genome mapping by genetic, physical, and transcript, gene sequence analysis and gene functional analysis, and was used in breeding selection. Since the DNA sequencing as well as high -density microarray analysis (gene chips) was commonly used in inferring genomics, the widespread of new next sequencing technology pushes the application of these two technologies in genomic studies related to lactation [74]. To improve the performance, dairy cows were already fully sequenced, and former studies of genomics in lactation research studies focused on the nutritional strategies and specific genes linked to the milk quality. However, the available data of genomics research on milk protein synthesis were still scarce in recent years. Since most of related studies concern more on the association of milk proteins with milk production traits instead of milk proteins variants with milk proteins composition [75]. The applications of genomics are mainly focused on the strong candidate of QTL, the variants of milk proteins, and the diseases that may affect the milk protein synthesis (i.e., Mastitis) [76]. Here we summarized those from mapping approaches to genome-wide association studies.

Lactation is usually affected by the factors such as environment, nutritional manages, breed, etc. The combination of the genetic data with the nutrition management including dry matter intake, body condition (in different periods) contributes to the efficient prediction of traits related to lactation as well as milk protein synthesis [77]. Former studies focused on the detection of relationships between lactation and the candidate genes related to milk proteins. And it has been shown that genes on chromosome 3 of bovine including the insulin-like growth factor 2 (IGF-2) and rap-1A are the strong candidate for the quantitative trait locus (QTL) and may affect the milk performance [78, 79], IGF-2 was found to have the functions of muscle mass and fat deposition in swine and was widely investigated in human medical. A parametric bootstrapping procedure found by Veerkamp et al. makes the estimate of heritability and genetic correlations between traits possible [80]. In addition, the development of genome-wide analysis makes it become a better solution to explain key genes and pathways. Berkowicz et al. indicated that a single genotyped single-nucleotide polymorphism (SNP) and traits related to animal growth also support the locus as harboring a potentially important quantitative trait nucleotides (QTNs) [79], suggesting that reprinter genes together with those documented biological roles represent important reservoirs for genetic improvement of dairy cows.

The genomics studies of milk proteins mainly focus on the amino acid changes caused by polymorphisms in the corresponding genes, while previous research studies focus on the variants on the components of caseins as well as whey proteins. To be specific, the A and B variants of β-LG and κ-CN on their concentration and total proteins in milk, which indicated that the A variant of β-LG is associated with a greater concentration of β-LG and a lesser concentration of casein, and that the B variant of κ-CN is associated with a greater concentration of κ-CN in milk [81].

### *Omics, the New Technological Approaches to the Milk Protein Researches DOI: http://dx.doi.org/10.5772/intechopen.102490*

But the research on the genotypes of all major components in milk proteins was not widely investigated in different kinds of animals. While in dairy cows, the A and B variants of β-LG and κ-CN, the E variant of κ-CN, and the A1, A2, and B variants of β-CN frequently occurred [82]. The genes of four different caseins have been found to be located at chromosome 6 of bovine and are closely linked meanwhile organized in a casein locus [83]. Heck et al. indicated that the selection for both the β-LG genotype B and the β-κ-CN haplotype A2B will result in cows that produce milk that is more suitable for production [84]. While related studies have also found the specific genes linked to milk proteins synthesis were also affected by the factors including seasons, lactation stages [81]. Therefore, the advance investigation of genes as well as variants will greatly enable the assessment of breeding to milk protein composition.

Mastitis is the most common disease in dairy cows, which is hallmarked by high somatic cell count (SCC). Mastitis negatively affects the dairy industry and enormously causes losses in milk performance and management costs [85]. Related research studies have shown that the stromal fibroblasts derived from cows that suffered mastitis were upregulated in the expression of inflammatory-related cytokines TNF-α and IL-8 [86], which contribute to the inhabitation the synthesis of milk proteins components including β-casein. Therefore, genomics research studies on mastitis have been conducted and SNPs on Bos Taurus autosome 4 (BTA4) and BTA18 were found to be significantly associated with mastitis [87]. Furthermore, 665 interactions in more than 140 genes were found in a co-expression network via the Genemania gene network analysis, which were recognized as candidate QTL for mastitis in the Holstein cows [88].

### **5. Transcriptomics in milk protein research**

The transcriptome broadly refers to the sum of all RNAs transcribed by organism, tissues, or cells in a specific state or period. And its field can be classified by different RNA types or methodology [89]. Since gene expression is complex and involves a variety of regulatory mechanisms, such as histone modification, promoter region, variable shearing, all of which affect the intracellular genes expression and play a regulatory role [90]. The expression level of genes among different tissues or cells is not consistent; hence, the complex and variable functions can be performed during different condition of physiological and pathological [91].

Methods of transcriptomics are similar to genomics, including microarray and RNA sequencing, while the latter one overcomes the weakness of Sanger sequencing due to the higher accuracy as well as sensitivity. Nowadays, the next-generation platforms such as Illumia, SOLID, and 454 are commonly used in related research studies to obtain reads and the transcript assembled, which revolutionize the analysis of eukaryotic transcriptomic [92]. In research studies related to lactating dairy cows, the expression profiles of mRNA were more focused especially in mammary glands, to reveal the candidate genes related to lactation. While the long noncoding RNAs also contribute to the pathway regulations in mammary gland and are essential for breeding [93]. However, the quality as well as quantity of RNA determines the reliability of sequencing results, so the extraction of total RNA from mammary gland significantly affects the analysis of the transcriptome. Li et al. reviewed the RNA sources used in transcriptome of lactating bovine mammary gland [9], which were used to the investigate the mammary gland development, effects of nutritional management on synthesis of milk composition, etc.

To specifically investigate the synthesis of milk protein, former studies revealed that the insulin signaling and mammalian target of rapamycin (mTOR) pathway participate in the regulation of milk protein synthesis by RNA-seq [94]. MenziesK et al. demonstrated that insulin plays a pivotal role in regulating milk proteins in dairy cows, while the recent works also report the similar results in other mammal animals [95]. Insulin was found to both directly and indirectly regulate the milk protein synthesis that is the involvement of control in gene expression and the regulation in translation [96]. Specifically, insulin can strongly activate the STAT5 pathway by increasing phosphorylation of the transcription factor. While the expression of ELF5 is induced by insulin in mammary tissue of dairy cows, which can co-activate and amplify the STAT5 [97]. In addition, insulin can regulate amount of translation via the mTOR hence indirectly control milk protein synthesis. mTOR particularly MTORC1 among the two mTOR complexes was well defined to play an important role in regulate protein synthesis by affecting the translation in all tissues of mammals [94]. Protein synthesis in mammal is inhibited by the association of the unphosphorylated eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) with the eukaryotic translation initiation factor 4E (eIF4E), the insulin can increase the specific phosphorylation in mTOR, which in turn phosphorylates 4EBP1 and results in releasing of eiF4E. eiF4E participates in the formation of the translation initiation complex and hence initiates the translation of mRNA into protein [98]. Furthermore, there are also additional mechanisms of activated mTOR complex to enhance the translation process through phosphorylation of the ribosomal protein S6 kinase (S6K1) and ukaryotic elongation factor-2 kinase (EEF2K) [99].

Except for the synthesis mechanism, transcriptomics conducted in investigation of nutritional management. The different expression of genes in milk synthesis was found in factors including short-term feeding restriction, low-quality total mixed ration, inducing the reduction in milk protein associated with the downregulation of protein synthesis [100, 101]. While the management such as the frequency of milking also causes the perturbation in lactation, former study reported that the increased milking frequency affects the expression of genes involved in reconstruction of extracellular matrix, κ-CN, α-lactalbumin, which may affect the milk protein components [102, 103]. Moreover, the related research studies also reported the perturbation of expression on milk protein synthesis induced by condition changes. For example, Gao et al. found that dairy cows suffered heat stress upregulated the inflammation-related genes, which interfered and downregulated the milk protein synthesis [104].

### **6. Proteomics in milk protein research**

Proteomics is defined as the technology of protein expressions from transcription and translation. By analyzing the posttranslational modification meanwhile identifying the differential proteins [105], the interaction among proteins, proteomics reflects the body metabolic changes to internal and external environmental changes and reveals biological functions inside. The advancement of analytical techniques pushes the development of proteomics rapidly. Two-dimensional electrophoresis (2-DE) is a well-developed technology and was used to separate proteins in the past decade [106]. As early as 1977, O'Farrel used 2-DE technology to separate about 1100 proteins from *E. coli* [107], which proved that 2-DE technology has the characteristics of high resolution and high sensitivity and is effective for analyzing complex biological samples and separating proteins.

### *Omics, the New Technological Approaches to the Milk Protein Researches DOI: http://dx.doi.org/10.5772/intechopen.102490*

The 2-DE technology performs one-dimensional isoelectric focusing according to different isoelectric points of proteins and then separates proteins of different molecular weights by polyacrylamide gel electrophoresis [108]. The 2-DE formed by the combination of these two technologies effectively separates protein vesicles in both directions of charge and relative molecular mass. Therefore, the information of the differential protein can be obtained through subsequent mass spectrometry identification and combined with bioinformatics analysis to analyze its biological function. The application of 2-DE combined with MS was usually used to identify the biomarkers related to treatment [109]. However, quantitative proteomics is challenging the 2-DE and MS methods as it allows for the massive multiplexing of primary data with better quality than established methods [110]. The method of quantitative proteomics, the isobaric tags for absolute and relative quantification (iTRAQ ) combined with MS, is commonly employed to analyze the multiplicity of different samples; however, the accuracy of iTRAQ might be compromised due to the influence of near isobaric ions contamination in a sample [111]. In fact, the methods of 2-DE combined with MS and iTRAQ have been widely selected in research related to lactating cows, particularly in the investigations of mastitis and effects of nutritional management.

As a novel research approach, proteomics was conducted to investigate the milk protein including the milk protein profile and the MFGM components. The investigation of lactation periods found the alternation of whey proteome as evidenced by the significantly decreased content of immunoglobulins and caseins and in particular the colostrum at 48 h postpartum [112]. While in mid-lactation, Reinhardt et al. found that the proteins associated with lipid transportation, synthesis, and secretion are upregulated, and 120 proteins were identified to associate with cell signaling and membrane/protein trafficking [113]. Except for the milk from different periods, the proteomic studies also focus on the milk components different among different species. For example, β-LG lacks in camel milk, while it is the main whey protein in the caprine and bovine [114]. This technology can also be used to identify the adulterated milk products meanwhile getting the information of the sources of hypoallergenic replacements. By the application of iTRAQ and MS, specific proteins from different species can be classified and be used for characterizing. For example, primary amine oxidase is unique in cows, while biglycan is source of goat [115, 116]. Furthermore, the investigation of MFGM profiles meanwhile provides the overview of MFGM proteome among species [117], which indicated that these differences of protein components may be related to differences in heredity.

The application of proteomics in pathology such as mastitis has been conducted to find biomarkers to overcome the challenges of quantification in mastitis diagnosing. While proteins including hemoglobin β, cytochrome C oxidase, annexin V, and α-1-acid glycoprotein as well as collagen type I α 1 and inter-α (Globulin) inhibitor H4 show more abundance in dairy cows that suffered mastitis [118, 119], which may participate in the repairment of tissue damage.

### **7. Metabolomics in milk protein research**

Metabolomics focus on the quantitatively analyzing of all metabolites in body to find the relative relationship between metabolites and physiological changes. Most of its research objects are small molecular substances with a relative molecular mass of less than 1000. In recent years, studies have reported the effects of different environmental factors on small molecular metabolites in animals and plants. While metabolic map was drawn through in-depth research on the metabolites of the body to seek the biomarkers [120, 121]. Moreover, analysis of NMR and MS indicated that biomarkers such as phosphorylated saccharides, acetone, and β-hydroxybutyrate (high levels) are closely correlated with the metabolic status in dairy cows during early lactation [9], which may contribute to the breeding selection to alleviate the metabolic stress in dairy cows during early lactation.

Similar to the application of proteomics in searching unique biomarkers, the investigation using NMR metabolomics approach found that acetate and novel metabolites including hippurate, isoleucine, butyrate, fumarate, and β-hydroxybutyrate are associated with milk composition [122]. While improving abundance of volatile fatty acid (VFA) as well as β-hydroxybutyrate and low abundance of hippurate and fumarate in milk are coupled with high levels of somatic cells [123], which contribute to the potential biomarkers for milk quality when dairy cows in high SCC conditions. Furthermore, metabolomics also provides new approach on highlighting interspecies differences from analyzing the metabolites. By using NMR and LC–MS or GC–MS method, the unique metabolites in milk among horse, Jersey cow, camel, yak, goat, caprine, buffalo, and dairy cow were found respectively, and results validated that metabolomics is a feasible approach for milk composition analysis [124, 125].

Although metabolomics has a higher accuracy and the high-throughput abilities, its application is still in the junior stages in research studies related to lactation especially the milk protein. With the advantage of combination with multivariate data analysis tool, metabolomics will obviously push the process of lactation research in the future. While the unique metabolites identified by these technologies will also provide much better perspective for the investigation of milk protein.

### **8. Conclusions**

The synthesis of milk protein is complex in mammary gland and is regulated by multiple factors, the protein components in milk are specific with the mammary tissue since they are minor or no expressions in the rest organs. Several hormones including insulin participate in the regulation of milk protein synthesis with a pivotal role of perturbation pathway such as mTOR. However, the control of milk synthesis is still need to be completely understood, to open new insight of research not previously considered. The advent of Omics technologies provides the possibilities to further investigate related complex mechanisms. These novel research approaches combined with bioinformatics constitute to be useful and powerful to generate large datasets for lactation sciences, which contribute to reveal the mechanism of milk protein synthesis and the novel biomarkers in milk affected by some factors. However, considering the limitation of cost, reproducibility, and throughput, it should be well arranged and prepared when choosing these new research approaches.

### **Funding**

The present study was financially supported by the National Natural Science Foundation of China (31872383), the Agriculture Science and Technology Innovation Program (ASTIP-IAS07-1) and the Key Research and Development Program of the Ningxia Hui Autonomous Region (2021BEF02018).

*Omics, the New Technological Approaches to the Milk Protein Researches DOI: http://dx.doi.org/10.5772/intechopen.102490*

### **Author details**

Zitai Guo, Lu Ma and Dengpan Bu\* State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China

\*Address all correspondence to: budengpan@126.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 4

## Milk Protein and Food Allergens

### **Chapter 5**

## Characteristics of Cow Milk Proteins and the Effect of Processing on Their Allergenicity

*Roua Lajnaf, Sawsan Feki, Hamadi Attia, Mohamed Ali Ayadi and Hatem Masmoudi*

### **Abstract**

Milk proteins are well known for their nutritional and functional properties. However, they are also members of the Big-8 food allergens including egg, fish, shellfish, soy, peanuts, wheat and tree nuts, in terms of prevalence. The most common milk allergens are casein fractions and β-lactoglobulin naturally not present in human breast milk. Thus, the examination of cow's milk proteins as potential allergens that may cause food allergies and the identification of methods of reducing their immunogenicity are of great interest. The main objective of this chapter is to review the physico-chemical characteristics cow milk proteins as well as their studied allergenicity and immunogenicity as a function of some denatured dairy processes such as heating, high pressure, enzymatic hydrolysis and lactic acid fermentation.

**Keywords:** cow's milk proteins allergy, protein allergenicity, immunoreactivity, milk processing, β-lactoglobulin, caseins

### **1. Introduction**

Food allergy is a major public health which has been estimated to affect around 1–2% of the adult population and 5–8% of pediatric population at the age below 3 years [1–3]. It is thought to result from disorders of the immune response to food allergens proteins and develop due to the defect in oral tolerance. Food allergens are contained in eight common foods including animal-based foods (cow's milk, eggs and fish) and plant-based foods (crustacean/shellfish, peanuts, soy, nuts and wheat). These allergens account for over 90% of the occurrence of all serious allergic reactions to foods worldwide [4]. Epidemiological studies have reported that animal food allergens, especially cow milk proteins allergy, was the most prevalent allergy for infants or young children, meanwhile, plant based food allergens was more encountered for adults [5].

Thus, Cow's Milk Allergy (CMA) represents 10–40% of the total food allergies. As an animal proteins allergy, it concerns mostly young children and less frequently adults. CMA is reported to affect approximately 3–8% of the total

pediatric population worldwide with symptoms at different levels of severity, which can endanger the patient's life [6]. Indeed, CMA is considered as the most common food allergy responsible for anaphylaxis reactions in young children as it is the first food eaten since birth. CMA is ranked third among all food allergies responsible for serious anaphylactic reactions to adults representing 15% from all allergic cases [7].

CMA disappears spontaneously at the age of 5 years in the majority of patients representing approximately 80% [8]. However, it seems that a minority of patients remains allergic in adulthood [9]. In most cases, the food allergy manifests itself as an immediate hypersensitivity reaction induced after recognition of food antigens by specific immunoglobulins type E (IgE). Other forms of allergy can also involve mechanisms not mediated by IgE. Their frequency is increasing but the immune responses involved are still poorly defined.

Like all food allergies, CMA involves both immunological reactions: immunoglobulin E (IgE) which is encountered in most allergic cases and non-IgE mediated reactions [10, 11]. The immunological reactions of IgE-mediated reactions occur immediately after proteins ingestion because of the interaction between allergens and immune mechanisms. This allergenic reaction is characterized by the production of IgE antibodies in allergic patients resulting in the degranulation phenomena of mast cells, the release of inflammatory mediators including histamine, 5-hydroxytryptamine (5HT) and prostaglandin E2 (PGE2) (**Figure 1**). These mediators induced the resulting allergy symptoms (hives, diarrhea, vomiting, and breathing difficulty). On the other hand, non-IgE mediated immunological reactions take up between 1hour and several days after ingestion of milk to develop involving the immune system as the IgE-mediated reactions [12–14].

The complete exclusion of cow's milk protein from the diet is still the only safe treatment that can be offered to patients today. In this case, infant formulations

### **Figure 1.**

*A schematic representation of allergenicity mechanism of bovine milk in the body (abbreviations: β-Lg: β-lactoglobulin; IL-4, IL-5 and IL-13, inflammatory cytokines; IgE, immunoglobulin E; 5HT, 5-hydroxytryptamine; PGE2, prostaglandin E2) [12].*

*Characteristics of Cow Milk Proteins and the Effect of Processing on Their Allergenicity DOI: http://dx.doi.org/10.5772/intechopen.102494*

containing cow's milk proteins are replaced by milks which are designed from more or less extensive hydrolysates of bovine proteins from whey or from the casein fraction in order to limit allergenicity as much as possible residual product. On the hand, researchers, scientists and industrials keep searching for new potential milk alternatives including hydrolyzed milk formulae, plant-based formulae and other milks from different mammalian species such as goat, sheep, donkey, mare and camel milks [11, 12, 15, 16].

### **2. Characteristics of cow's milk proteins**

Milk proteins represent an important nutritional source due to their high biological value and the presence of essential amino acids. They are also the source of various dairy products due to the important techno-functional properties of its proteins. Cow milk is a heterogeneous mixture of proteins with different structural and physicochemical properties. As milks from all mammalian species, cow milk proteins are divided according to their solubility into two fractions: caseins (insoluble in acidic conditions) and whey proteins (soluble proteins). Indeed, caseins precipitate at their isoelectric pH which is located at 4.6, while whey proteins remain soluble in this pH level [17].

### **2.1 Caseins**

Caseins are phosphoproteins which represent the most abundant protein fraction in milk. They represent approximately 80% of the total milk protein.

Caseins consist of 4 proteins which differ in contents of phosphorus, concentration, amino acid composition, isoelectric point (pI) and molecular weight (MW): alpha S1, alpha S2, beta, and kappa (αS1, αS2, β and ĸ). The α and β caseins are calcium sensitive caseins as they precipitate at a calcium concentration at 30 mM while the ĸ-casein remains in solution under these conditions. The β-casein represent 39% of total caseins, followed by αS1, αS2 and κ caseins which represent 38%, 10% and 13% of total amounts of caseins, respectively (**Figure 2**) [19].

#### **Figure 2.**

*Proportions of the different caseins (a) and whey proteins (b) in cow's milk (abbreviations: β-CN: β-casein; αS1-CN: αS1-casein; αS2-CN: αS2-casein; ĸ-CN: ĸ-casein, β-Lg: β-lactoglobulin; α-La: α-lactalbumin; SA: serum albumin; Ig: immunoglobulins; Lf: lactoferrin [18].*

The 4 different caseins are associated with minerals forming colloids called casein micelles (concentration of minerals 80mg/g of caseins) with a diameter ranging between 100 and 140 nm. Bovine casein micelles and their characteristics have been the subject of much research and different micellar models have followed one another over the years (models by Horne, Holt, Bouchoux, etc.) [20, 21].


### **2.2 Whey proteins**

Soluble protein fraction or whey protein, is the second main protein fraction in milk (20–25% (w/w) of total protein). Overall, the protein composition of whey varies depending on the mammalian species. For cow's milk whey, the protein composition is as follows: β-lactoglobulin is the main protein (~56%), followed by α-lactalbumin (~21%), immunoglobulins (14%), bovine serum albumin (BSA) (7%) and lactoferrin (2%) (**Table 1**, **Figure 2**).

• **β-Lactoglobulin (β-Lg)** is a globular protein, present in the milk of all mammalian species except camelids, rodents and humans. The biological function of this *Characteristics of Cow Milk Proteins and the Effect of Processing on Their Allergenicity DOI: http://dx.doi.org/10.5772/intechopen.102494*


*Abbreviations: pI: isoelectric point, α-La: α-lactalbumin, β-Lg: β-lactoglobulin, BSA: bovine serum albumin, Lf: lactoferrin, Ig: immunoglobulins.*

*a Proportion of individual caseins in the whole casein fraction of milk.*

*b Percentage of globular whey protein in the soluble fraction of milk.*

*c The allergenicity of the main proteins as reported by El-Agamy and Peñas et al. [13, 27].*

### **Table 1.**

*Physico-chemical characteristics of the main cow's milk proteins and their allergenic activity (% of patients) [18].*

protein is to transport the fatty acids, retinol and vitamins (A, D), binding Cu2+ and Fe2+ ions and inhibiting autooxidation of fats during digestion [28]. The β-Lg is the major protein in the soluble fraction of cow's milk with a concentration ranging between 2 and 4 g/L representing approximately 56% of the total bovine whey proteins [29]. The secondary structure of β-Lg consists of 10% α-helices, 45% β-sheets. It has two disulfide bonds at the cysteine residues (Cys106-Cys119 and Cys66-Cys160) and one free cysteine (Cys121) [30]. This protein is characterized by different quaternary structures depending on the environmental conditions of the protein (pH, temperature, ionic strength). The β-Lg, the primary structure comprises 162 amino acid residues with a MW of 18.281 kDa and a pI of 5.2. The β-Lg is an allergenic protein present due to its highest proportion among whey proteins (56% of total whey proteins) and due to the fact that this protein is totally absent in human milk [11, 31]. Food allergies associated with this allergenic protein may be present even in 80% of the total population [32].

• **α-Lactalbumin** (α-La): The α-La is a less allergenic protein than β-Lg and constitutes 21% of total whey protein [25]. Furthermore, chemical composition of bovine and human *α*-La bears a strong resemblance. This protein is a small protein of 123 amino acid residues (14.186 kDa, pI 4.65) known for its high content in the essential amino acids and for its important role in the biosynthesis of lactose with lactose synthetase and UDP galactosyl-transferase [33]. The α-La is a metalloprotein that contains one Ca2+ atom per mole of protein molecule, a divalent cation that plays an important role in stabilizing its spatial structure. The binding of this calcium ion is affected by the acid functions of the aspartic acid residues located

in position 82, 87 and 88. There is also a second calcium binding site occupied by the zinc, but which has an affinity 105 times lower than that of calcium. The α-La contains 4 disulfide bridges (Cys6/Cys120, Cys28/Cys111, Cys61/Cys77 and Cys73/ Cys91) but no free thiol groups. This configuration makes it more resistant to the phenomenon of protein aggregations caused by heat treatment, even though its denaturation temperature is relatively low (~64°C) [34].

The tertiary structure of this protein contains


### **3. Allergenicity of cow's milk proteins**

Cow's milk contains approximately 30–35 g/L of proteins divided into 30 proteins, some of them are potentially allergenic and called "Bos d" and numbered according to the protein type [39].

### *Characteristics of Cow Milk Proteins and the Effect of Processing on Their Allergenicity DOI: http://dx.doi.org/10.5772/intechopen.102494*

The main cow milk allergens in are caseins (Bos d8) including β-casein (Bos d11), αS1 casein (Bos d9), αS2-casein (Bos d10) and κ-casein (Bos d12). On the other hand, whey consists of high allergenic proteins including α-La (Bos d 4), β-Lg (Bos d 5), immunoglobulins (Bos d7), BSA (Bos d6) and traces of Lf (Bos d Lf) [31, 40].

Scientists confirmed that the most commonly allergens which are usually detected in cow milk allergic patients are whole caseins especially the αS1-casein (Bos d9), β-Lg (Bos d5) and α-La (Bos d4). Indeed, 66% of CMA is caused by the main cow milk allergen which is the β-Lg, followed by caseins (Bos d8) and significantly less by α-La and BSA (18%) [13, 25]. The high allergenicity of the β-Lg is attributed to the fact that this protein is totally deficient in human milk. Indeed, IgE response against β-Lg precedes those against the other allergens including caseins and α-La since birth. Afterwards, before the age of 1 year, IgE response toward caseins becomes predominant, whereas, the IgE response to α-La appears later after the age of 1 year [41].

However, the major problem of CMA is the fact that that only 27% of total patients with CMA are allergenic to only one allergen, meanwhile, the other patients present sensitization to two and more cow milk allergens leading to conclude that none of the main milk proteins allergens can be considered as the only responsible for the allergenicity of this food [11]. IgE do not react entirely with the antigenic protein but only with its allergenic part which is called epitope. Hence, one allergenic protein may have several epitopes, which might be the same or different depending on its quaternary structure and its exposed allergenic peptides. Epitopes of proteins molecules include immunodominant epitopes, which are the high allergenic epitopes and the main targets of immune response system. Allergy can not only be caused through bloodstream by the absorption of allergen but also by direct skin contact with the allergen [13].

The allergenicity of proteins, as well as the IgE epitopes of milk proteins, can be mapped and carried out using various bioinformatic tools through an *in silico* analysis. Overall, the primary protein sequences were taken from the UniProtKB protein Blast database, while the three-dimensional structures (downloadable as a pdb file) are listed in the PDB Protein Data base.

PD index, Bepipred, AlgPred, Discotope-2.0, Ellipro (prediction of linear and discontinuous epitopes) are some bioinformatic tools to study the allergenicity of proteins:


 **Figure 3.**

*An example of discontinuous B-cell epitopes predicted by the ElliPro. (a–e) Three-dimensional representation of conformational or discontinuous epitopes of bovine β-Lg. The epitopes are represented by yellow surface, and the bulk of the protein is represented in gray sticks.* 

machine) method of AlgPred calculates the allergenicity score of the protein that qualifies as "Allergen" for a score ≥ −0.5 [ 44 ].


### **4. The effect of different processes on the allergenicity of cow's milk proteins**

 Food processing and additional ingredients cause changes in immunodominant epitopes and hence, the allergenic properties of proteins. Food processing may lead to the destruction of epitopes structures and/or the formation of new epitopes which are called neo-allergens. On the other hand, food processing can be associated with the reduction of allergenic properties of proteins or/and can have no influence on their allergenicity, it can even increase the immunogenicity of the treated proteins by the appearance of new epitopes [ 47 ].

**Type of technological process Operating conditions The effect of process of the allergenicity of cow milk protein The reference** Heat treatment Pasteurization 90°C during 15 s Low decrease on the immunoreactivity of whey proteins such as α-La and β-Lg Wróblewska and Jędrychowski [50] Pasteurization 90°C during 15 min Ultrasound at 52°C during 60°C A significant reduction of the immunorectivity of α-La and β-Lg Heat treatment at 80°C and 90°C during 30 min The reduction of the allergenicity of immunoglobulins in milk Jost et al. [51] Heat treatment at 120°C for 20 min The reduction of the allergenicity of α-La by 25% compared to the native protein Kleber and Hinrichs [52] Heat treatment of the cow milk proteins powder at 500°F (260°C) for 3 min • 68% of children (n = 100, mean age, 7.5 years; range, 2.1–17.3 years) tolerated heated milk. • Smaller skin prick test wheals for heated milktolerant subjects • Lower milk-specific and casein specific IgE and lower IgE/IgG4 ratios to both of caseins and β-Lg (compared subjects with allergy to heated milk) Nowak-Wegrzyn et al. [53] High-pressureprocessing High-pressure treatment at 200–600 MPa and (temperature between 30°C and 68°C) An increase of the antigenicity of the treated β-Lg in the WPI solution, sweet whey and skim milk Kleber and Hinrichs [52] High-pressure treatment at 200 and 400 MPa • The increase of the binding to β-Lg specific IgG from rabbit, • No effects on the IgE from allergic patients Chicón et al. [54] High-pressure treatment at 600 MPa The distribution of the structure of casein micelles and the decrease of the immunogenic capacity of milk proteins Bogahawaththa et al. [55] High-pressure treatment at 400 MPa during 50 min The loss of the allergenicity of the β-Lg hydrolysates with chymotrypsin by the absence of anaphylactic symptoms López-Expósito et al. [56]

*Characteristics of Cow Milk Proteins and the Effect of Processing on Their Allergenicity DOI: http://dx.doi.org/10.5772/intechopen.102494*


### **Table 2.**

*The effect of food processes (heating, high pressure, enzymatic hydrolysis and lactic acid fermentation) on the allergenicity of cow milk proteins.*

### **4.1 The effect of heat treatments**

Heating is an important process in the manufacturing of dairy products in order to obtain bacteriologically safe products leading to extend their shelf life. During the heating process, various structural modifications occur in the milk proteins depending on temperature, heating time, and heating exchanger. The structural and chemical changes in heating milk proteins such as denaturation, aggregation and "Maillard reaction" may have significant impacts on the antigenicity level of milk proteins [3, 48]. Among cow's milk proteins, caseins are the most heat stable proteins contrary to globular whey proteins which are sensitive to heat treatment and start to denaturate at temperatures above 60°C in the following order: BSA (denaturation temperature 94.9°C) < β-Lg (denaturation temperature 79.6°C) < α-La (denaturation temperature 70.5°C) [49].

Sterilization and pasteurization, which are the major categories of thermal processes have a significant impact on structural and functional properties of milk proteins leading to the increased, reduced or similar allergenicity. of allergenicity [50]. Wróblewska and Jędrychowski [50] noted that pasteurization of milk at 90°C resulted in a low decrease on the immunoreactivity of whey proteins such as α-La and β-Lg, while ultrasound treatments at 52°C during 60 min reduced greatly the immunorectivity of these proteins (**Table 2**). On the other hand, Jost et al. [51] reported that heating whey proteins at a temperature ranging between 80°C and 90°C during 30 minutes reduces the immunoglobulins contents as well as their immunogenicity.

Other researches carried out with bovine whey proteins confirmed that the antigenicity of β-Lg and α-La increases when heating temperature rose from 50 to 90°C because of the exposure of allergenic epitopes buried inside the native molecule due to the unfolding of conformational structure during heat denaturation. However, the antigenicity of these proteins decreased significantly above 90°C. Furthermore, the antigenicity of α-La decreased by 25% compared with its native state when it is treated at 120°C for 20 min [52, 63].

Other researches have evaluated whether children (n = 100) with CMA can tolerate extensively heated milk proteins and they found that approximately 68% tolerated the extensively heated milk. Furthermore, Heated milk-tolerant subjects showed significantly smaller skin prick test wheals, lower milk-specific and casein specific IgE as well as lower IgE/IgG4 ratios to both of caseins and β-Lg when compared subjects with allergy to heated milk [53]. Hence, some manufacturers use denatured whey proteins for the production of hypoallergenic infant formulae [25].

### **4.2 The effect of high-pressure processing**

High-pressure processing is considered as a suitable nonthermal alternative method for milk pasteurization when it is in the range of 300–600 MPa [64]. Highpressure processing can even preserve the organoleptic and nutritional properties of the treated foods. However, this process can also alter structural and physico-chemical characteristics of proteins and result in their denaturation of native milk proteins. Indeed, high-pressure leads to the denaturation of whey proteins as the β-Lg and the changes of the casein micelles structures by their disassociation [55]. These changes may also influence the allergenicity of milk proteins. For instance, high-pressure treatment (200–600 MPa) at a temperature ranging between 30 and 68°C increased the antigenicity of β-Lg in the WPI (whey protein isolate) solution, sweet whey and skim milk [52]. Another study indicated that the high-pressure processing caused a

severe whey protein denaturation especially the β-Lg and the minor whey proteins (Immunoglobulins) but no effect was observed for the α-La. Indeed, a high-pressure processing at 600 MPa induced the formation of large protein aggregates involving both of β-Lg and ĸ-casein through the thiol/disulphide interchange reactions. Furthermore, this treatment can disturb the structure of casein micelles leading the alteration of the immunogenic capacity of milk proteins diminished at 600 MPa [55]. Chicón et al. [54] found that the high pressure treatment of the pure β-Lg and whey protein isolate solution at 200 and 400 MPa resulted in an increase of the binding to β-Lg specific IgG from rabbit, without any effect on the IgE from allergic patients (**Table 2**). This behavior can be explained by the exposure of the buried epitopes in the unfolded protein molecules becoming more accessible for the antibodies.

Several researches focused on the effect of high-pressure on milk proteins hydrolysates. For instance, it was reported that a significant high degree of hydrolysis was levels obtained in high pressure (600 MPa), in comparison to atmospheric pressure depending upon the used enzyme. This behavior is attributed to the increased enzyme availability of immunogenic hydrophobic areas which, as a result, intensifies hydrolysis [25, 57].

On the other hand, hydrolysates obtained via the enzymatic treatment of main allergen in cow milk: β-Lg under high-pressure may result in a lower antigenicity and IgE binding ability [3, 57]. Indeed, the evaluated *in vivo* allergenicity of the β-Lg hydrolysates with chymotrypsin indicated that the tested hydrolysates with highpressure treatment at 400 MPa during 50 min resulted in the loss of the allergenicity of the studied protein by the absence of anaphylactic symptoms. These results demonstrate the safety of hydrolysates produced under high-pressure conditions for manufacturing of novel milk formulae [56].

Other studies carried out with milk protein hydrolysates have also reported that the application of high-pressure treatment during enzymatic hydrolysis can significantly reduce the antigenicity of the treated proteins due to the increase of accessibility of the potentially immunogenic regions to the enzyme [3, 27, 54, 57].

### **4.3 The effect of enzymatic hydrolysis**

Proteolysis have been usually considered as an efficient process to reduce allergenicity of milk proteins by destroying their allergenic epitopes [65]. The enzymatic hydrolysates were prepared with the use of digestive enzymes including pepsin, trypsin and chymotrypsin in order to imitate potential digestion processes and to reduce intestinal activity and the activity of enzymatic system in children [58]. However, the differences in the types of enzymes in this process as well as hydrolysis model and the hydrolysis degree may result in some discrepancies in the composition of the resulted peptide and a residual antigenicity of the hydrolysates as well as their taste [3]. Previous researches showed that the overall antigenicity of whey protein can be reduced by hydrolysis with trypsin alone. Caseins including *α*-casein and β-casein also show sensitivity to trypsin (unlike immunoglobulins and BSA). However, Nakamura et al. [66] noted that using many enzymes at the same hydrolysis process including papain, neutrase, alcalase and protease is more efficient in reducing the allergenicity of whey proteins when compared to those treated with a single enzyme. Thus, the hydrolysis of β-Lg by trypsin alone or in combination with chymotrypsin and pepsin.

It was proved that hydrolysis of β-Lg (Bos d5) by trypsin alone or in combination with both of chymotrypsin and pepsin reduces its allergenicity without eliminating

*Characteristics of Cow Milk Proteins and the Effect of Processing on Their Allergenicity DOI: http://dx.doi.org/10.5772/intechopen.102494*

it, while the combination of both of enzymatic hydrolysis and heat treatment was reported to reduce greatly the allergenicity of β-Lg [57, 58].

The combination of pepsin and *α*-chymotrypsin is considered as the most effective combination of enzymes used for the reduction of allergenicity and act by a selective proteolysis of both allergens *α*-La (Bos d4) and β-Lg (Bos d5) with a degree of hydrolysis of 1–20% and depending and incubation time [58].

An innovative technique of preparing hypoallergenic formulae for newborns involves the combination of hydrolysates and probiotics, which reduces allergic symptoms. Probiotics, including *Lactococcus lactis*, *Lactobacillus rhamnosus* and *Bifidobacterium lactis* significantly reduced the severity of atopic dermatitis in breastfed infants after 2 months of treatment. Indeed, probiotics probably participate in mucosal degradation of macromolecules, leading to reduced allergenicity of milk proteins [25, 67–69].

Despite all these advantages of the hydrolysis of milk proteins for the reduction of their allergenicity, some researches confirmed the increase of the allergenicity of proteins by the exposure of new epitopes that appeared upon hydrolysis treatment. For instance, Haddad et al. [59] detected serum IgE from allergic patients using radioallergosorbent tests with a total tryptic hydrolysate of β-Lg (Bos d5) even when no IgE response was detected with the native protein of β-Lg (**Table 2**). Schmidt et al. [61] reported that no differences were found in the antigenic properties of the whey protein hydrolysates including α-La, β-Lg, BSA and bovine immunoglobulin G at pH 2 or 3, whereas, at pH 4 a further decrease in pepsin hydrolysis resulted in enhancement of antigenicity of all these proteins except the β-Lg. In the same way, the enzymatic proteolysis with Corolase 7092 was reported to increase the antigenicity of proteins including BSA and immunoglobulin G by exposing more antigenic sites during hydrolysis [62]. *In vitro* tests of Selo et al. [60] showed that that tryptic hydrolysis retained and even enhanced the allergenicity of β-Lg. In fact, the derived peptides showed a specificity to bind human IgE by ELISA assays. These authors also noted that numerous epitopes are widely scattered all along the β-LG molecule. They may be located in hydrophobic parts of the protein molecule, inaccessible for IgE antibodies in the native conformation of the protein but become bio-available after hydrolysis processes [60].

### **4.4 The effect of fermentation**

The lactic acid fermentation process may have a potential influence on the allergenicity of cow milk proteins. Thus, researches were conducted with the use of several mesophilic and thermophilic bacterial strains which are already used in the production of fermented dairy products [25]. This process did not show a significant influence on the allergenic properties; indeed, the *in vitro* studies were not consistent with those *in vivo*.

Many studies have reported that Lactobacillus fermentation can induce degradation of milk allergens. For instance, lactic acid bacteria *Lactobacillus casei*, which are defined as probiotics [58]. In the same way, *Lactonacillus rhamnosus* GG has the ability to reduce phagocytosis which is stimulated by milk allergens by blocking receptors involved in phagocytosis on neutrophils and monocytes. It can even modify clinical symptoms in children with dermatitis and eczema [25, 70].

Clinical investigations have noted that dietary consumption of fermented foods, such as yogurt, can alleviate some of the symptoms of atopy and might also reduce the development of allergies through a mechanism of immune regulation.

The consumption of fermented milk cultures containing lactic acid bacteria can enhance the production of both Type I and Type II interferons at the systemic level [71]. However, changes of cow milk protein antigenicity and allergenicity depend on the species of lactic bacteria as well as the conditions of fermentation (**Table 2**). Lactic acid fermentation can reduce 90% of the antigenicity of the β-Lg in skim milk and 70% of this protein in sweet whey compared with untreated samples [52].

Finally, the reduction in antigenicity suggested that during the fermentation process with *Lactobacillus*, some epitopes of proteins were destroyed. These results are very useful for the preparation of new fermented milk products with reduced antigenic properties [3].

### **5. Conclusion**

Cow's milk is a high nutritious food. However, it should be noticed that it contains many proteins which are considered as major food allergens leading to induce allergic reactions especially in infants.

The challenge for the food scientist, nutritionists and physicians is to resolve the problem of the CMA by searching new cow milk alternatives and/or new dairy processes that may reduce the allergenicity of cow milk proteins. Some processing technologies (heating, high pressure, enzymatic hydrolysis and lactic acid fermentation) can be used to effectively reduce the allergenicity of milk proteins by optimizing and controlling the processing conditions. However, attention should be paid during modification of milk proteins upon the used processes in order to prevent the appearance of some new epitopes during processing which are buried inside the native molecule. On the other hand, *in vitro* tests should be carried out to further detect the residual allergenicity of proteins and ensure the edible safety of milk products obtained by processing technologies. These strategies should provide valuable support for the development of the hypoallergenic milk formulae especially to infants.

### **Author details**

Roua Lajnaf1,2\*, Sawsan Feki2 , Hamadi Attia1 , Mohamed Ali Ayadi1 and Hatem Masmoudi2

1 Alimentary Analysis Unit, National Engineering School of Sfax, Sfax, Tunisia

2 Immunology Department, Habib Bourguiba University Hospital, Sfax, Tunisia

\*Address all correspondence to: roua\_lajnaf@yahoo.fr

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Characteristics of Cow Milk Proteins and the Effect of Processing on Their Allergenicity DOI: http://dx.doi.org/10.5772/intechopen.102494*

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Section 5
