Development of a method for obtaining the complex "casein polyphenolic compounds" enriched with chlorogenic acid scientific research group

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Development of a method for obtaining the complex "casein polyphenolic compounds" enriched with chlorogenic acid scientific research group

Ivanov Evgen Gennadievich

Candidate of biological sciences,

Associate Professor of the Department of Molecular Biology and Biotechnology, Faculty of Biology

V. N. Karazin Kharkiv National University, Ukraine

Ganin Vladimir Yuriyovych

PhD student of the Department of Molecular Biology and Biotechnology Faculty of Biology ,

V. N. Karazin Kharkiv National University, Ukraine

Summary. Milk casein micelles can serve as a form of stabilisation and delivery of various biologically active substances, in particular polyphenolic compounds ("PPs"), to the body. The work is dedicated to the production of casein micelles in which PPs are incorporated. The idea of the work was to introduce isolated polyphenolic compounds from sunflower meal enriched with chlorogenic acid in certain concentrations (1.5 g or 0.75 g per 30 g of casein) into skimmed milk and, after incubation, to induce the formation of casein micelles in which "PP" can be incorporated. It was shown that 'PP' was incorporated into the protein micelles of colostrum casein with the optimum ratio between 'PP' and casein being 0.75 g to 1.5 g per 30 g of casein on a dry matter basis. It was found that the binding of PP to casein micelles was accompanied by an increase in the extractability of some proteins from the micelles. It is suggested that PPs are held in the micelles by weak intermolecular bonds, as evidenced by the transition of PPs to the aqueous phase after extraction.

Keywords: casein, colostrum, polyphenolic compounds, chlorogenic acid, delivery methods. casein colostrum polyphenolic compound

Introduction

Currently, the development and use of functional foods is becoming increasingly important [1,2]. They occupy an intermediate position between traditional foods and medicinal products, but they have no toxic properties, are preventive and, in some cases, can have a therapeutic effect in the presence of chronic pathologies that traditional medicinal products cannot offer. Functional foods usually combine several biologically active substances of natural origin. In this context, casein is of particular interest as a basic ingredient for functional foods. Casein is known to be the main protein in milk and colostrum. However, first milk colostrum contains 2-3 times more casein than mature milk [3]; moreover, bovine casein is represented by four isoforms: aS1, aS2, b and k, which differ in amino acid composition, phosphorus and carbohydrate content, but are similar in their amphiphilic character [4, 5]. Milk contains significantly more k-casein than other casein isoforms. For example, the ratio of b-casein to k-casein in colostrum is 0.61, compared to 0.30 in mature milk [6]. Because of these properties, colostrum casein is of great interest as a potential pharmaceutical substance and as a basis for functional foods.

Of particular interest is the ability of casein isoforms to form nanoclusters, i.e. they are capable of self-assembly into micelle nanostructures [7, 8, 9]. During the formation of nanostructures, a variety of biologically active compounds can be incorporated. This knowledge has stimulated research into the structural features of casein isoforms and the possibility of using them to deliver bioactive compounds into the body. However, there have been no studies on the use of colostrum casein as a carrier of bioactive compounds.

The use of casein micelles as a biological matrix for bioactive compounds has another important feature. It allows the stabilisation of such compounds and this is particularly relevant for rapidly oxidising and unstable compounds. This is particularly true for polyphenolic compounds, and in particular chlorogenic acid, which is under active investigation.

Furthermore, the influence of polyphenolic compounds contained in casein micelles on the structure and digestibility of casein cannot be excluded.

In particular, it has been shown that polyphenolic compounds are able to regulate signalling pathways, have anticancer effects, participate in the regulation of inflammatory responses [10-12]. It has been shown that polyphenols can interact with proteins through covalent and non-covalent interactions [13, 14]. Covalent binding of polyphenols to proteins is an irreversible interaction that usually occurs when the phenolic compound is oxidised to a quinone in the presence of polyphenol oxidase or in an alkaline environment [15, 1 6].

However, it is much more common for polyphenolic compounds to form non- covalent, weak bonds with proteins. These bonds are the result of hydrophobic association, hydrogen bonding, electrostatic attraction and van der Waals forces. Hydrophobic interaction and hydrogen bonding are considered to be the most important non-covalent bonds in polyphenol-protein complexation [16]. This is due to the presence of hydroxyl groups in polyphenols and carbonyl groups in proteins which can form hydrogen bonds, while the hydrophobic regions of protein amino acids (benzene and aliphatic side chains) and aromatic cores of polyphenols play a key role in stabilising polyphenol-protein complexes [17]. The presence of weak intermolecular interactions in polyphenol-protein complexes can, on the one hand, provide stabilisation and preservation of polyphenols in such associations and, on the other hand, polyphenols can be relatively easily released from such associations under changing conditions.

Methods

Extraction of casein from colostrum. The milk of the second lactation was obtained at the farm "Alpha", located in Zolochevsky district, Kharkov region, from cows of the breed " Ukrainian Black-and-White ". After cooling it was stored at - 15 °C for no longer than one month. Before casein extraction, the milk was thawed at room temperature, diluted with sterile distilled water in the ratio of 1:3 by volume. The resulting samples were centrifuged at 3,000 g for 15 min at room temperature to remove lipids. The lipid removal procedure was repeated twice for complete skimming of the colostrum.

Polyphenolic compounds enriched in chlorogenic acid were extracted from sunflower meal as described in [1 8].

Three variants of at least three parallel samples were prepared from defatted colostrum: experimental variant №1, to which 10 ml of aqueous solution containing 1.5 g PP was added; experimental variant №2, to which 10 ml of aqueous solution of PP containing 0.75 g PP was added; control variant, to which 10 ml of sterile distilled water was added.

To form casein micelles and incorporate PP into them, 1 n acetic acid was added to the samples at pH -4.6 at 37 °C for 30 min.

The resulting samples were slowly cooled and centrifuged at 3000 g for 15 min.

The protein content and PP compounds in the supernatant were monitored by spectral characteristics on a Shimadzu UV-2600 spectrophotometer.

To evaluate the "degree" of PP binding to casein micelles, samples were incubated at 37°C for 10 min with vigorous stirring and casein was precipitated again by centrifugation. All procedures were repeated at least three times.

Results and discussion

Sufficiently diverse polyphenolic compounds ("PPs") were obtained from sunflower meal, absorbing light in the range of 310-360 nm, with chlorogenic acid isoforms accounting for 40 to 70% (Fig. 1).

Fig. 1. Absorption spectra of polyphenolic compounds in aqueous solution in the range of 310 to 360 nm at high concentration - 1.5 g in 10 ml solution - (A) and at lower concentration - 0.75 g in 10 ml solution (B). Typical absorption spectra of polyphenolic compounds are shown.

It was observed that in the control variant, without application of "PP" and after the formation of casein micelles and their subsequent removal in the aqueous solution, a small amount of amino acids and proteins was absorbed in the ultraviolet part of the spectrum (220 to 300 nm) (Fig. 2 A).

After application of "PP" in the amount of 1.5 g per 30 g of casein, prior to the formation of casein micelles and subsequent induction of micelle formation, the amount of "PP" compounds in the supernatant, i.e. not bound to casein micelles in the high "PP" concentration variant, was reduced by only 20% in comparison with the initial amount of "PP" (the amount of "PP" was determined by the area of absorption peaks). (Fig. 2 B and 1 A). At the same time, when 2 times less "PP" compounds were added to the casein samples, 3 times less "PP" compounds remained in the aqueous solution compared to the initial amount of "PP" (Fig.2C and 1C).

Fig. 2. Absorption spectra of the post-casein fraction without the application of "PP", in which small amounts of milk proteins are detected (A), and absorption spectra of "PP" not associated with casein micelles in the case of the application of a large amount of "PP" (1.5 g per 30 g of casein) (B) and also after the application of "PP" in the amount of 0.75 g per 30 g of casein (C). Typical spectra from three series of determinations are shown.

Consequently, the introduction of chlorogenic acid-enriched polyphenolic compounds into casein solution, followed by the induction of micelle formation, was accompanied by the incorporation of "PP" into the micelles. The efficiency of "PP" incorporation into casein micelles (the ratio of "PP" incorporated into the solution to the amount of "PP" incorporated into the micelles) depends on the ratio between the amount of casein and polyphenolic compounds.

As mentioned above, polyphenolic compounds can form weak intermolecular interactions with proteins, including hydrogen bonds. In order to determine the degree of binding of PP to casein micelles, we carried out a series of "washes" - aqueous extraction of the formed "casein-"PP" complexes.

It was found that during additional aqueous washing of casein micelles, which did not contain "PP", a small amount of compounds absorbing in the range of 220240 (amino acids) and 260-280 (proteins) was transferred to the aqueous phase, while in the aqueous solution there were no compounds absorbing at 250 nm (Fig. 3 A).

It is very interesting that, on the contrary, after aqueous washing of casein containing "PP" compounds, the transfer of peptides absorbing at 250 nm into the aqueous phase increased significantly (Fig. 3 B).

Consequently, the inclusion of "PPs" in casein micelles was accompanied by such rearrangements of the protein part of the micelles, which led to a decrease in the degree of their "retention" in the composition of these complexes, and this may serve as indirect evidence of the interaction of polyphenolic compounds with casein micelles. Further evidence for the incorporation of PPs into the micelles is the decrease in the amount of polyphenolic compounds extractable from casein micelles compared to the amount of PPs remaining in the micelles (Fig. 3 B).

Fig. 3. Absorbance spectra of substances released from casein without PP after washing 30 g of casein with 45 ml of water and intensive stirring for 15 min, followed by precipitation of the casein by centrifugation at 4 000 g for 15 min (A) and absorbance spectra of substances released under the same conditions but from casein with PP after inclusion of a high concentration of PP (1.5 g of PP per 30 g of casein) (B). Typical spectra from a series of determinations are shown.

After the second washing of the "casein-PP" complex, which included the highest concentration of PP (1.5 g PF per 30 g casein), it was observed that the release of peptides into the aqueous phase after washing of the casein without PP decreased 2-times compared to the first washing (Fig. 4 A).

At the same time, the release of PP from the casein-PP complex decreased insignificantly and the peak that appeared at 250 after the first wash was detected at 240 nm (Fig. 4 B).

Fig. 4. Absorption spectra of substances released from non-PP casein micelles after washing 30 g of casein with 45 ml of water after 15 min of intensive stirring at 37°C and subsequent precipitation of the casein by centrifugation at 4 000 g for 15 min (A) and absorption spectra of substances released under the same conditions but from casein containing PP after inclusion of a high concentration of PF (1.5 g/30 g of casein) (B). Typical spectra from a series of determinations are shown.

These results confirm that the isoforms of casein proteins that form micelles are held together by weak intermolecular bonds and are easily disrupted by intensive stirring in the physiological solution. The introduction of polyphenolic compounds into casein micelles ensured their inclusion in the micelles, and polyphenolic compounds modified the interactions between casein protein isoforms in the micelles to such an extent that they were relatively easily extracted by water and physiological solution.

Further studies of the mechanisms of interaction of polyphenolic compounds and, first of all, chlorogenic acid with protein components of casein micelles will allow to substantiate the use of such complexes in the prevention and treatment of such pathologies as liver diseases and a number of other pathologies.

Conclusion

A method for the incorporation of polyphenolic compounds into casein micelles of colostrum has been developed. The efficiency of incorporation of polyphenolic compounds depends on the ratio between the amount of casein and "PP", the optimum being not less than 0.75 but not more than 1.5 g to 30 g of casein (dry residue). Polyphenolic compounds in the composition of casein micelles influence the extractability of casein proteins, which proves their participation in changing the structural properties of proteins in the composition of micelles.

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