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2 Chapter 2 Measurement of Casein Mielle Size in Raw Dairy Cattle Milk by Dynami Light Sattering Peter Hristov, Ivan Mitkov, Daniela Sirakova, Ivan Mehandgiiski and Georgi Radoslavov Additional information is available at the end of the hapter Abstrat The partile size of milk influenes its mirostruture and defines the qualities of dairy produts, suh as olloidal stability and texture. Moreover, differenes in asein mielle size may affet milk proessing, espeially heese making. Hene, the size of asein mielle is an important harateristi of raw milk and determines the yield of dairy produts. The aim of the present researh is to estimate the asein mielle size in the raw milk of dairy attle by dynami light sattering. The obtained results may be used for geneti elaboration of the breed, as well as to inrease the ompetitiveness of the milk industry by seletion of animals with higher asein mielle size. Keywords: dairy attle, asein mielle size, dynami light sattering, DLS, ow milk 1. Introdution It is well known that the asein fration of bovine milk exists as polydisperse, large, roughly spherial olloidal partiles, nm in diameter (average ~150 nm), alled asein mielles [1]. The size, form, and struture of the asein mielle are of great importane for the milk industry espeially for heese making, yellow heese, et. [2]. Casein mielle ontains an average of 3.4 g H 2 O per gram dry matter, whih onsists approximately of 93% protein and about 7% of inorgani omponent (Ca 2+ ), formed phosphoprotein omplexes. The asein mielle in milk onsists of four aseins: αs1- (CSN1S1), αs2- (CSNS2), β- (CSN2), and k-asein (CSN3). The latter is important for the stability and properties of the asein mielle. Although k-asein is in a relatively low amount of the asein system (12 15% of whole asein), it is soluble in the presene 2016 The Author(s). Liensee InTeh. This hapter is distributed under the terms of the Creative Commons Attribution Liense ( whih permits unrestrited use, distribution, and reprodution in any medium, provided the original work is properly ited.

3 20 Milk Proteins - From Struture to Biologial Properties and Health Aspets of Ca2+, whereas the remaining 85% of asein are preipitated by Ca2+. CSN3 stabilizes up to 10 times its weight of the Ca2+-sensitive aseins via the formation of mielles [3] (Figure 1). Figure 1. Eletron mirograph of an individual asein miell [4]. One of the most important issues onerning the asein mielle is how the asein partiles (mielles) in milk are stabilized and destabilized by the ation of various agents and onditions. Thus, various hypotheses of asein mielle struture are proposed. 2. Casein mielle struture 2.1. Coat-ore models The first model of asein mielle struture was proposed by Waugh and Nobel in 1965 [5]. This model is based on the asein solubility in Ca2+ solutions. The model desribes the formation of low-weight-ratio omplexes of αs1- and k-aseins in the absene of alium. The monomers of αs1- or β-aseins interat with a harged phosphate loop (Figure 2), then begin to aggregate Figure 2. Struture of asein mielle proposed by Waugh [6]. (A) Monomer model for αs1- or β-aseins with harged loop. (B) A tetramer of αs1-asein monomers. (C) Planar model for a ore polymer of αs1- and β-aseins [7].

4 Measurement of Casein Mielle Size in Raw Dairy Cattle Milk by Dynami Light Sattering 21 to a limiting size (the aseinate ore) while the alium ions are added. Preipitation of the aseinate is stopped after a monolayer of the low-weight αs1-k-asein omplexes is formed. This oat omplex has the k-asein monomers spread out ompletely on the surfae [8] Sub-mielle (subunit) models The first sub-mielle model was proposed by Morr in 1967 [9]. Morr onsidered that αs1-, β-, and k-asein monomers form small uniform submielles. The submielles, estimated by sedimentation veloity studies, are stabilized by hydrophobi bonding and alium aseinate bridges, and the submielles are aggregated and held together by olloidal alium phosphate linkages with a mielle struture overed by αs1- and k-asein [7, 10]. The model, desribed by Slattery and Evard [11] and Slattery [12], falls in the last ategory. In this model, the aseins first aggregate via hydrophobi interation into subunits of moleules eah. The pattern of interation is suh that it brings about a variation in the k-asein ontent of these submielles. The k-asein ongregate on the mielle surfae, those submielles poor (like αs and β-asein) or totally defiient in k-asein are loated in the interior of the mielle (Figure 2). This model does not explain what provokes the segregation of the k-asein or why k-asein moleules, having preferred to assoiate with their own kind to form aggregate pathes in the k-rih submielles, should then assoiate with the other aseins to omplete the building of the submielle. Another ritiism of this model is the late entry of alium phosphate into the assembly proess by Slattery and Evard [11]. Separating the aseins from alium and phosphate until this point in the assembly proess is not really possible, sine both alium and phosphate are involved in the phosphorylation of the protein hain whih ours posttranslation [13] immediately and presumably before the assoiation of the hains into submielles. Figure 3. Representation of the asein mielle dual binding model proposed by Horne [16].

5 22 Milk Proteins - From Struture to Biologial Properties and Health Aspets 2.3. Internal struture models The last ategory of models is based on the properties of the isolated asein onstituents, ausing or direting the formation of the internal struture of the asein mielle [7]. The first internal struture model was suggested by Rose in 1969 [14]. He assumed that β-asein monomers begin to self-assoiate into hain-like polymers. Subsequently, αs1-aseins mole ules are attahed to the β-asein polymers, while k-aseins interat with αs1-aseins, forming aggregates of limited size. Upon forming the mielle struture, olloidal alium phosphate ats as a stabilizing agent and ross-links the network. In addition, these mielle networks are oriented in suh a way that β-asein is direted internal, while the k-asein is direted external [8]. An alternative internal strutural model is proposed by Holt [15] with the help of transmission eletron mirographs. This model emphasized the role of hydrophobi interations in giving rise to submielles, the Holt model relies solely on the interations between the aseins and alium phosphate to hold the mielle together. In this model, the alium phosphate is in the form of nanolusters and the interation sites on the aseins are the phosphoseryl lusters of the alium-sensitive aseins (Figure 3). Beause as1- and as2-aseins have more than two suh lusters, arguably in the ase of as1-asein, they are able to ross-link the nanolusters into extended 3-dimensional network strutures. The dual-binding in Holt models is in the size of the nanoluster and in the number of phosphate lusters (or asein moleules) that the surfae of the nanoluster an aommodate (Figure 4). Figure 4. Shemati representation of Holt s model. The alium phosphate formed nanoluster as interat with phos phoseryl residue of aseins [16].

6 Measurement of Casein Mielle Size in Raw Dairy Cattle Milk by Dynami Light Sattering Dynami light sattering Dynami light sattering (DLS), sometimes referred to as quasi-elasti light sattering (QELS) or as photon orrelation spetrosopy (PCS), is a non-invasive, well-established tehnique for measuring the size and size distribution of moleules and partiles typially in the submiron region, and with the latest tehnology lower than 1 nm. A laser or any other monohromari light soure is shot through a polarizer and into a sample. The sattered light is then sent through a seond polarizer where it is olleted by a photomultiplier. The resulting image is then projeted onto a sreen and this phenomenon is known as spekle pattern (Figure 5) [17]. Figure 5. Priniple of dynami light sattering. All of the moleules in the solution are being hit with the light and all of the moleules diffrat the light in all diretions [18]. The diffrated light from all of the moleules an either interfere onstrutively (shown as light regions) or destrutively (shown as dark regions). The proess is repeated at short time intervals. The resulting set of spekle patterns is then analyzed by an autoorrelator in order to ompare the intensity of light at eah spot over time. The polarizers that are being used an be set up in two geometrial onfigurations. One of the polarizers is set in a vertial/vertial (VV) geometry, while the seond polarizer allows light through whih is in the same diretion as the primary polarizer. On the ontrary, in vertial/horizontal (VH) geometry, the seond polarizer allows light whih is not in same diretion as the inident light Pratial realization DLS is used for haraterization of the various partile sizes (PSs), inluding proteins, arbohydrates, mielles, polymers, and nanopartiles. If the system is not dispersing in size, the average effetive diameter of the partiles an be determined [19]. This measurement depends on many fators as size of the partile ore, size of surfae strutures, partile onentration, and the type of ions present in the medium. The diffusion oeffiient of the partiles an be determined sine DLS typially measures flutuations in sattered light intensity due to diffusing partiles. DLS software of ommerial instruments displays the distribution of partile population at different diameters [20]. In a monodisperse system, there should be only one population, while a polydisperse system would exhibit multiple partile populations. If a sample ontains more than one size population, then either the CONTIN analysis should be

7 24 Milk Proteins - From Struture to Biologial Properties and Health Aspets applied for PCS instruments or the power spetrum method should be applied for Doppler shift instruments [21]. DLS an be used for onvenient stability studies. Conduting periodial DLS measurements of a sample an demonstrate whether the partiles aggregate over time. This an be ahieved by heking for an inrease in the partile hydrodynami radius. If partiles atually aggregate, a larger population of partiles with a larger radius should be observed. In addition, some DLS mahines an analyze stability depending on temperature by ontrolling the temperature in situ. 4. Measurement of asein mielle size by dynami light sattering (DLS) The average asein mielle size varies widely between milk samples of individual ows. Casein mielle size is also variable and an range between 154 and 230 nm in milks from individual ows [22]. Moreover, mielles as small as 125 nm and as large as 487 nm have been reported in frationated bulk milk [23]. Casein mielle size may be influened by ow genetis, e.g., asein protein variants [24 26], protein post-translational modifiation involving phosphorylation and glyosylation of asein moleules, and the levels of mineral ompounds, suh as alium, in the milk [27]. Mielle size may also be influened by farming and environmental fators suh as feed [28] and season [29]. On the other hand, asein mielle size is of an important signifiane onerning influene the renneting behavior and the texture of manufatured dairy produts [30]. The main used tehniques for the analysis of PS and partile size distribution (PSD) are DLS, nanopartile traking analysis (NTA), sanning eletron mirosopy (SEM), size exlusion hromatography (SEC), ell eletrophoresis, analytial ultraentrifugation (AUC), et. Various analytial tehniques may give different results [31 34]. DLS is the most user-friendly and it gives relatively aurate and onsistent results of protein samples whih an be obtained in a short period of time [35]. The size of asein mielle is estimated in raw milk samples, pasteurized milk, bulk milk, milk powder, et. Here we will highlight determining of asein mielle size in raw milk by the DLS method. Researh of Bijl et al. [25] have revealed the asein mielle size in raw milk in 50 Holstein- Friesian ows and 54 Montbéliarde ows. Initially, all animals were genotyped for the k-asein gene. On the basis of average asein mielle size, the authors defined two types of mielles: small (170.6 ± 9.1 nm) and large (206.7 ± 5.0 nm). The results showed no signifiant differenes between the two types of mielles and aseins onentration in milk. On the other hand, there was a positive influene between k-asein and asein mielle size. The mean asein mielle size in CSN3 AA ows was ± 14.6 nm), while in ow with AB genotype, it was ± 5.4 nm. This study was the first to report a orrelation between asein mielle size and glyosylation of k-cn. The authors onluded that hanges in the struture of k-asein lusters aused by glyosylation an influene miellar stabilization during or after asein mielle formation in the mammary gland, and thereby influene asein mielle size. Another study by de Kruif and Huppertz [22] has revealed the asein mielle size in individual milk samples in 18 Holstein-Friesian ows by DLS measurement. Data from Figure 4 showed lear differenes between hydrodynami radius (Rh) between ows but do not vary as a

8 Measurement of Casein Mielle Size in Raw Dairy Cattle Milk by Dynami Light Sattering 25 funtion of stage of latation. To study the effet of different latations on asein mielle size, the authors have olleted the individual milk samples from 68 ows. The results revealed that Rh did not hange during milking, latation, or even over a period of 3 years. This suggests that asein mielle size is strongly genetially determined and is extremely onstant in the milks of individual ows. In this study, asein omposition of eah individual milk samples was determined by RP-HPLC. The results showed that first there were no signifiant differenes between asein omposition, as a funtion of stage of latation, and seond, asein omposition did not influene on the asein mielle size (Figure 6). Figure 6. Hydrodynami radius at a sattering angle of 93 in the milk of 15 individual ows taken at 4 different time points during a single latation [22]. The study of Hristov et al. [26] aimed to ompare the size of asein mielle in individual milk samples in dependene of kappa asein (CSN3) geneti polymorphism in 16 ows of Bulgarian Rhodopean attle breed. The three defined kappa asein genotypes AA, AB, and BB were used for determining the asein mielle size by DLS. On the other hand, individual milk samples of eah ow were assessed for protein and fat ontent. The results showed differenes in the size and polydispersity of the asein mielles in milk from ows with different genotypes. The hydrodynami radii of mielles at a sattering angle of 90 C varied from 80 to 120 nm and polydispersity varied from 0.15 to The authors onluded that the asein mielle size of CSN3 AA ows (~120 nm) exeeds in about 60% of ows with AB (~80 nm) and BB genotype (~70 nm). In ontrast, protein and fat ontent in milk annot be orrelated to asein mielle size. The obtained results ould be useful for improving tehnologial properties of milk and the yield of milk produts (Figure 7). Beliiu and Moraru [36] have analyzed the effet of the solvent on the auray of asein mielle PS determination by DLS at different temperatures and how to establish a lear protool for these measurements. DLS analyses were performed at 6, 20, and 50 C in raw and pasteurized skimmed milk as soures of asein mielles. The ph, ioni onentration, refrative index, and visosity of all solvents were determined. The solvents were evaluated by DLS to ensure that they did not have a signifiant influene on the results of the PS measurements. The authors

9 26 Milk Proteins - From Struture to Biologial Properties and Health Aspets onluded that when an ultrafiltered permeate was used as a solvent, the PS and polydispersity of asein mielles dereased as temperature inreased. The effetive diameter of asein mielles from raw skimmed milk diluted with ultrafiltered permeate was ± 5.3 nm at 6 C, ± 1.9 nm at 20 C, and ± 2.7 nm at 50 C. Overall, the results of this study suggest that the most suitable solvent for the DLS analyses of asein mielles was asein-depleted ultrafiltered permeate. Dilution with water led to mielle dissoiation, whih signifiantly affeted the DLS measurements, espeially at 6 and 20 C. A similar investigation was arried out by Mootse et al. [37]. The authors aimed to study asein mielle size in individual Estonian Holstein dairy ows during 1-year period by DLS. The main results of this study an be summarized as follows: 1) The average mean intensity (mode) of asein mielle partile size distribution (CM PSD) in raw milk of Estonian Holstein dairy ows was nm and its variation (range nm) resembled statistially normal distribution. 2) There was a weak orrelation between average mode and its variation in milk samples of individual ows whih may refer to the possible influene of ows' physiologial status, disease inidenes, and stages of latation, et., whih will be studied in a further researh. Devold et al. [28] have sreened the influene of geneti milk protein variants on mean size of native and heated asein mielles in 58 ows of Norwegian red attle breed. The results showed that the mean size of native and heated asein mielles was signifiantly influened by the following parameters: group of ows (different feeding regime), genotype of αs1-asein (native mean size only) and k-asein, ph and the ontent of asein, whey protein, and asein number (Table 1). Figure 7. Representative DLS distributions in raw milk sample from heterozygous AB (CSN3) ow: a) orrelation funtion, b) intensity partile size distribution, and ) multimodal number partile size distribution [26].

10 Measurement of Casein Mielle Size in Raw Dairy Cattle Milk by Dynami Light Sattering Variable Mean size of asein mielles Native Heated Change αs1-cn (BB > BC)a β-cn α-cn (AA, AE > AB)b (AA, AE > AB)a β-lg Fed. reg. (Gr.3E > Gr.1)a (Gr.3E > Gr.1)a W.O.Lat. Casein (%) b b WP (%) b b Casein no. b a ph a a Ca2+ a Ca Mg Citrate p < p < N.S. = non-signifant [28]. a b Table 1. Variation for mean size of native and heated asein mielles and for heat-indued hange in size. Figure 8. Sanning eletron mirosopy (SEM) imaging of the asein gel network produed from left, small asein mi elle, and right, b large asein mielle milks. Images are at 10,000 magnifiation, sale bar = 1 μm [30]. Logan et al. [30] investigated the ombined effets of milk fat globule size (MFG) and asein mielle size in bulk milk on the onset of gelation and the maximum rate of gelation. The results showed that asein mielle size is the major determinant of rennet gel strength. Milk fat globules ould oneivably enhane the rheologial properties of the rennet gel depending on their size in relation to the pore size of the asein mielle network. When the gels were formed with milks of large asein mielle, the size of MFG did not affet the gel properties, probably 27

11 28 Milk Proteins - From Struture to Biologial Properties and Health Aspets beause both large and small MFG were smaller than the pore size of the large asein mielle network. On the other hand, when the gels were formed with milks of small asein, the large MFG enhaned the gel firmness more than the small MFG. It is possible that when the size of large MFG fits well with the pore size of the asein mielle network, a synergisti effet on rennet elasti gel network is provided (Figure 8). Finally, the effet of gross omposition, protein omposition, total and ioni alium ontent, phosphorous ontent, and asein mielle size on hymosin-indued gelation was determined in milk from 98 Swedish Red ows by Gustavsson et al. [36]. The obtained results showed that protein ontent, ioni alium onentration, total alium ontent, and asein mielle size were the most important fators explaining the variation of gelation properties in this sample set of Swedish red ows. Furthermore, it was shown that omposite effet of beta and kappa asein geneti variants have an effet on asein mielle size and it was suggested that this ould be the reason for previously published differenes in gelation properties between the omposite genotypes in the present data set. Non-oagulating milk is a problem in Swedish red ows and the present study showed that non-oagulating milk is more ommon in ows in their first parity than in ows in their seond parity. 5. Conlusion Many produts in the food industry, inluding milk, ie ream, mayonnaise, jam, et., are olloids or have been produed via olloids. Dispersed partiles determine, through their onentration, distribution, size, and struture, the physiohemial and organolepti quality properties of a produt. Additionally, the omplex interations between olloidal partiles and with other ingredients will ontribute to the rheology, texture, stability, appearane, and many other food harateristis. Tehniques to measure the partile properties of the olloid systems allow for better understanding of these omplex relations and further improvement food proessing tehniques and reipes. Furthermore, the implementation of suh sensing-tehnologies in a food plant ould enable online monitoring of the olloids or the derived produts during the prodution proess and promote early detetion of an altering produt quality. Author details Peter Hristov 1*, Ivan Mitkov 1, Daniela Sirakova 1, Ivan Mehandgiiski 2 and Georgi Radoslavov 1 *Address all orrespondene to: peter_hristoff@abv.bg 1 Department of Animal Diversity and Resoures, Institute of Biodiversity and Eosystem Researh, Bulgarian Aademy of Sienes, Sofia, Bulgaria 2 Experimental and stokbreeding station, Agriultural Aademy, Smolyan, Bulgaria

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14 Measurement of Casein Mielle Size in Raw Dairy Cattle Milk by Dynami Light Sattering 31 attle breed. Biotehnology in Animal Husbandry. 2014;30(4): DOI: / BAH H [27] Glantz M, Devold TG, Vegarud GE, Lindmark Månsson H, Stålhammar H, Paulsson M. Importane of asein mielle size and milk omposition for milk gelation. Journal of Dairy Siene. 2010;93(4): DOI: /jds [28] Devold TG, Brovold MJ, Langsrud T, Vegarud GE. Size of native and heated asein mielles, ontent of protein and minerals in milk from Norwegian Red Cattle effet of milk protein polymorphism and different feeding regimes. International Dairy Journal. 2000;10(5): DOI: /S (00)00073-X. [29] Holt C, Baird L. Natural variations in the average size of bovine asein mielles: I. Milks from individual Ayrshire ows. Journal of Dairy Researh. 1978;45(03): DOI: /S [30] Logan A, Day L, Pin A, Auldist M, Leis A, Puvanenthiran A, Augustin MA. Interative effets of milk fat globule and asein mielle size on the renneting properties of milk. Food and Bioproess Tehnology. 2014;7(11): DOI: / s [31] Anema SG, Lowe EK, Stokmann R. Partile size hanges and asein solubilisation in high-pressure-treated skim milk. Food Hydroolloids. 2005;19: DOI: / j.foodhyd [32] Tran Le T, Saveyn P, Hoa HD, Van der Meeren P. Determination of heat-indued effets on the partile size distribution of asein mielles by dynami light sattering and nanopartile traking analysis. International Dairy Journal. 2008;18: DOI: /j.idairyj [33] Dejan A. Dynami Light Sattering and Appliation to Proteins in Solutions. Ljubljana: University of Ljubljana Faulty of Mathematis and Physis, Department of Physis; 2010, p. 19. [34] Raza H, Claire G, Joël S. Revealing asein mielle dispersion under various ranges of NaCl: Evolution of artiles size and struture. World Aademy of Siene, Engineering Tehnology. 2011;51: [35] Vaso F, Andrea H, Wim J. Critial evaluation of nanopartile traking analysis (NTA) by NanoSight for the measurement of nanopartiles and protein aggregates. Pharmaology Researh. 2010;27(5): DOI: /s [36] Gustavsson F, Glantz M, Buitenhuis AJ, Lindmark-Månsson H, Stålhammar H, Andrén A, Paulsson M. Fators influening hymosin-indued gelation of milk from individual dairy ows: Major effets of asein mielle size and alium. International Dairy Journal. 2014;39: DOI: /j.idairyj

15 32 Milk Proteins - From Struture to Biologial Properties and Health Aspets [37] Mootse H, Pisponen A, Pajumägi S, Polikarpus A, Tatar V, Sats A, Poikalainen V. Investigation of asein mielle partile size distribution in raw milk of estonian holstein dairy ows. Agronomy Researh. 2014;12(3): [38] Beliiu CM, Moraru CI. Effet of solvent and temperature on the size distribution of asein mielles measured by dynami light sattering. Journal of Dairy Siene. 2009;92(5): DOI: /jds