THE PHYSICAL PROPERTIES OF THE CASEIN IN SOLUTION: EFFECT OF ULTRA-HIGH PRESSURE

КЛЮ ЧЕВЫ Е СЛО ВА: казеин, обр абот к а высоким давлением , гидродинамический радиус, ф люоресценция т рипт оф ана и т ирозина, И К -спект роскопия казеи н а Целью данного исследования было изучение влияния воздействия давления (50; 90 ; 160; 250; 350 МПа) на ряд физических свойств мицеллы казеина: гидродинамический радиус, флюоресценцию тирозина и триптофана и характеристику ИК-спектров. По данным фотонно-корреляционной спектроскопии сред­ ний гидродинамический радиус мицеллы казеина составил 128 нм, увеличиваясь при 50 МПа до 467 нм с образованием конгломератов. Дальнейшее увеличение давления привело к формированию двух фрак­ ций частиц, различающихся по величине гидродинамического радиуса. При давлении в 350 МПа основную часть (75 %) составляли частицы со средним радиусом 121 нм. Сопоставление гистограмм гидродинамиче­ ского радиуса и флуоресценции тирозина обнаружило снижение интенсивности свечения при увеличении доли частиц больших размеров и рост излучения в растворе при уменьшении размера мицелл. Рост флу­ оресценции казеина по триптофану и её снижение по тирозину указываю т на изменение конформации белковых молекул при обработке давлением. ИК-Фурье спектроскопия выявила изменение интенсивности оптической плотности в диапазоне амид I, амид II и валентных связей тирозина, подтверждая отсутствие появления новых связей. Полученные физические данные указываю т на изменение структуры казеиновых мицелл с увеличением доли (25 %) крупных частиц после действия высокого давления (350МПа), что следует учитывать в перера­ ботке молока. Флюоресценция казеина при обработке давлением является слабо исследованным физиче­ ским показателем и может нести прикладное значение для технологической обработки молочного сырья.

The aim o f th is work was to study th e effect o f pressure (50; 9 0 ; 160; 250; 350 MPa) on a physical property of casein m icelle: hydrodynam ic radius, tyrosine and tryptophan fluorescence and IR spectra ch aracteristics. Ac

Introduction
High-pressure processing is a promising and relatively new approach of non-thermal milk processing technology, capable of inactivating microorganisms in food products, as well as modi fying milk proteins. Physical and chemical changes in dairy sys tems caused by high pressure include change of particle size and milk color (turbidity reduction) [1,2].
Casein micelles are the predominant protein components of milk, polydisperse, roughly spherical aggregates consisting of several phosphoproteins: AS1 -, AS2 -, p -, and k-casein [3]. The structure of casein micelles is poorly studied, but a number of models with their consideration as amphiphilic proteins are proposed [4]. Caseins in milk can be considered as nanocapsules providing the supply of nutrients, their size ranges from 50 to 500 nm [5]. According to the Horne model [6], the casein micelle is stable due to the balance between electrostatic repulsion and hydrophobic attraction. The hydrophobic interaction in the mi celle is based on the interaction between casein and calcium phosphate. The action of high pressure leads to the dissolution of colloidal calcium phosphate and dissociation of hydrophobic and electrostatic interactions. It is known that casein micelles under pressure can irreversibly decompose or to form larger particles [7,8]. Protein structuring in high-pressure processing can be used in high-tech food production of protein nanopar ticles, which requires the search for new physical methods of research [9].
The aim of this study was to investigate the high pressure effect on a physical properties of casein micelle: hydrodynamic radius, fluorescence of tyrosine and tryptophan and IR spectra characteristics.

M aterials and R eagents
The study was carried out in the research laboratory «Nano technology and Biophysics» (Center for biomedical engineering, NCFU). For the preparation of solutions used deionized water (Smart2Pure, Thermo Sc ientific, USA.) and reagents qualification «p.a». (AppliChem Inc., USA). Casein was obtained from skim milk by its acidification. In 100 ml of milk, 1 ml of 10% acetic acid is added to achieve pH = 4.6. Then the samples were centri fuged for 10 min at 3000 rpm, the precipitate of casein washed three times with distilled water and centrifuged at 3000 rpm for 15 min, followed by drying at 40 °C.
Dry casein powder was dissolved in deionized water at a con centration nf 1 mg/ml using; a magnetic stireer at room tempeeature; to standardize the conditions, the solution was brought to neutral pH ■values usingNaOH and HCl solutions. To /emove undissolved protein particles, the solution was centrifuged at 10,000 rpm (MPW-352r, MPW MED INSTRUMENTS, Poland).

R esearch M ethods
The solution of casein was treated with a laboratory highpressure homogenizer (Stansted Fluid Power Ltd., UK). The prepared solution was placed into the cylinder of the syringe a volume of 20 ml and was inserted into the inlet valve, the de vice automatically selected the right amount to fill the cell and began processing. Processing was carried out in the range from 50 MPa to 350 MPa. The treated sample was drained from the exhaust valve into a separate tube and stored at plus 4 O C. The hydrodynamic radius was determined using photon-correlation spectroscopy on the spectrometer Photocor Complex (Fotokor, Russia). Casein fluorescence was evaluated on a multi-modal reader Varioskan Flash (Thermo Scientific, USA) by tryptophan and tyrosine at the following wavelengths, respectively: exci tation 180 nm n leading; 3 2 8 -3 5 0 nm and excitation 275 nm / reading 303 nm. IR spectra of casein solutions were studied by ATR-FTIR spectrometer Nicnlet iS50 -rharmo Fisher, USA). Sta tistical analysis of the data was carried out using the descriptive statistics unit, Excel (Microsoft Office, USA).

The change in the hydrodynam ic radius o f casein after exposure 1o high pressure hom ogenization
As a sample for comparison, casein without high pressure procnssmg cnntaming one particle fraction was used (Fig.1). The pressure of 50 MPa led to a hydrodynamic radius fourfold in crease, and indicated the formation of nonglomerates based on  ing of as1 -and K-casein, which is responsible for aggregation of casein submicellae. This confirms the data that after pressure treatment of skim milk, an increase in the number of clusters derived from casein submicellae and monomers [10]. Thus, it is believed that the increase in the size of the particles is preceded by the disintegration of the casein micelles [11]. Treatment of a casein solution at 90 MPa caused the sepa ration of the particles into two fractions. The smaller fraction, which made up 40 % of the total, included large structures, and the remaining 60 % of the particles slightly changed relative to the original values of the micelles size, which can also be consid ered as a further destruction of the «hair» layer [9].
After increasing the pressure to 160 MPa and 250 MPa, an increase in the fraction of larger particles to 56 % and 68 %, re spectively, was observed, with a tendency to reduce the size of the average hydrodynamic radius in both fractions.
After treatm ent with a pressure o f 350 MPa aggregated conglomerates greatly increase in size, but their share was re duced to 25 %, and the bulk (75 %) was particles close in size to the casein micelles without pressure processing. The results obtained are confirmed by the data on the decay of the casein micelle at a pressure between 2 0 0 -2 5 0 MPa (room tem pera ture), and the average radius of the micelles after pressure processing gradually increases and then, when it reaches at 350 MPa, decreases [12].

Tyrosine and tryptophan fluorescence o f casein
Samples of casein were characterized by an expressed inten sity of tyrosine fluorescence prior to pressure treatment. The pattern of tyrosine fluorescence had a tendency to increase with increasing values of the pressure. The lowest value of fluores cence was recorded at 50 MPa. A significant spikes were regis tered at 90 and 250 MPa, but the fluorescence did not reach the level of the control sample. (Fig. 2 A).
The fluorescence of tryptophan in the control sample was significantly lower than after pressure processing. The pressure rise was accompanied by fluorescence increase in two stages: the first at pressure of 50 to 160 MPa and the second at 2 50-3 5 0 MPa (Fig.2 B). Fluorescence was estimated at the time of maximum emission [13] and depends on a number of factors, including the indole component fluorescence, the presence of several tryptophan molecules in most proteins and the environment of the molecule [14]. The fluorescence intensity can be due to the different number of amino acids, so the casein micelle contains 41 tyrosine residues and 7 tryptophan residues. [15].
Comparison of histograms and fluorescence of tyrosine indi cates a decrease in the intensity of the glow with an increase in the proportion of large particles and the increase in radiation in the solution with a decrease in the hydrodynamic radius. Pecu liarities of casein amino acids glow are associated with structur al rearrangements during the processing pressure. The growth of tryptophan and decrease of tyrosine fluorescence under the influence of high pressure indicate a change in the conformation of protein molecules of casein with the reallocation provisions of amino acids [16]. Unidirectional changes in the amino acids glow, after treatment, can be due to the energy transfer from ty rosine to tryptophan residues in proteins. [15].

C haracterization o f IR spectra o f casein micelle
In the carried out studies of casein, stable peaks of 1638 1649 cm-1 belonging to a characteristic region of the spectrum for amide I, 1600-1700 cm-1 [17,18,19] were revealed. Spectral range 1700-1550 cm-1 mid-IR includes all of the protein second ary structure (a-helix, p-sheets and p-turns), as well as the dy namic conformations of proteins, characterized by one or more (major and minor) of characteristic bands and the respective ex tinction coefficients in the amide I region [18,19].
At 50 MPa, the IR spectrum shows an increase in the to tal intensity and smoothing of all peaks, which is accompanied by the described above decrease of tryptophan fluorescence in tensity.
Probably, when protein particles stick together, the transi tion of the photon taken by the molecule to other bonds oc curs and the value of emission changes at fluorescence, which is consistent with the previously described data [20]. With fur ther pressure increase, there is a change in the IR spectra optical density, with an increase in intensity in various zones, namely the protein groups of amide I (1638-1649 cm-1), amide II (1560 1567 cm-1) and the bonds characteristic of the tyrosine ring (1514-1519 cm-1) and may indicate an increase in the number of this type of bonds. In studies [21], similar changes in the optical density of the amide I region and dissociation of casein micelles due to electrostatic repulsion to smaller negatively charged fragments are shown. Bands centered near 1647 cm-1 can be theoretically referred to elements of a-spiral. The bands located near 1669 cm-1 and 1680 cm-1 refer to p-coils and p-sheets, re spectively [22]. Consequently, the absence of displacements of specific IR spectra indicates the stability of the p-structure of casein.

Conclusion
In accordance with the purpose of this study, the effect of high pressure on a number of physical properties of the casein micelle was studied: hydrodynamic radius, fluorescence of tyro sine and tryptophan and the characteristic of IR spectra. Pres sure processing of casein micelles leads to transformation of their size, which is due to the adhesion of particles (50 MPa) and stepwise crushing with pressure increasing. This is accompanied Figure 3. IR spectra of casein before and after high pressure treatment by fluctuations in the fluorescence level of tyrosine and trypto phan, indicating a rearrangement of the casein molecular struc ture. IR-Fourier spectroscopy revealed a change in the intensity of the optical density in the range of amide I, amide II and va lence bonds of tyrosine, confirming the absence of new bonds. The obtained physical data indicate a change in the structure of casein micelles with an increase in the proportion (25 %) of large particles after the action of high pressure (350 mpa), which should be taken into account in milk processing. The fluores cence of pressure-processed casein is a poorly investigated physical indicator and can have an applied value in the process ing of raw milk.

Acknowledgements
The authors are grateful to Kirpa I.V., kay accaunt manager of GK Arma, for the possibility to perform research on labo ratory high-pressure homogenizer, and Andrey Blinov, post graduate student of the applied biotechnology department (NCFU) for the assistance in carrying out of photon-correlation spectroscopy.