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ÖgeProduction of a casinomacropeptide concentrate from sweet whey and usage in a milk-like drink(Fen Bilimleri Enstitüsü, 2020) Karimidastjerd, Atefeh ; Kılıç Akyılmaz, Meral ; 629685 ; Gıda Mühendisliği Ana Bilim DalıCaseinomacropeptide (CMP) is a valuable polypeptide with structural and health-improving functional properties. CMP is part of κ-casein that is transferred into sweet whey after rennet coagulation of milk in cheese manufacturing. CMP is composed of fractions including glycosylated gCMP and its aglycon aCMP at almost equal proportions in whey. gCMP has a sugar moiety mostly sialic acid bound which was found to be responsible for some of its activities. CMP does not contain any aromatic amino acids such as phelnylalanine and tyrosine which makes it an alternative protein source for phenylketonuria patients. CMP has structural functionality including foaming, gelling and emulsifying. Bioactive properties such as antimicrobial, antioxidant, immune-modulating, satiety providing and prebiotic activities were also reported for CMP. These properties make CMP a special protein source to be utilized as a food ingredient. CMP is present about 1.2-1.5 gL-1 in sweet whey that makes up 15-20% of whey proteins. There are various methods to separate CMP from other sweet whey components for its isolation which are combinations of heat treatment, acid treatment, ultrafiltration (UF) and ion-exchange chromatography. In this study, CMP was isolated from sweet whey by a two-stage process where it was separated from other whey proteins by a heat-acid treatment firstly and then further purified by UF. Effects of process parameters in both stages on recovery and purity of CMP were determined to select best conditions. Final CMP concentrate was utilized as an ingredient in development of a rice drink that can be consumed for phenylketonuria patients. In the first part of this study, separation of CMP from other whey proteins by heat and acid treatments was investigated. Especially, the impact of order of the heat and acid treatments on isolation of CMP from sweet whey and its composition was explored. Acidification of sweet whey to pH values in the range of 3.0-5.0 and heat treatment at 90oC for 1 h in different order were applied to precipitate and separate whey proteins. The aim was to obtain the highest amount of CMP the lowest amounts of other whey proteins in the supernatant. Dry matter, crude protein, non-nitrogen protein, ash, lactose, bound sialic acid, phenylalanine and tyrosine contents of the supernatants from treated whey samples were determined. CMP, α-lactalbumin and β-lactoglobulin in the supernatants were quantified by RP-HPLC. Order of heat and acid treatments and pH had no effect on amounts of lactose and ash in treated samples. The lowest crude protein and dry matter contents in treated samples were found at pH values 4.0, 4.5 and 5.0 regardless of the order of heat and acid treatments. Heat-acid treatment at pH 4.5 and 5.0 were found to yield the highest CMP content and the lowest concentration of α-lactalbumin and β-lactoglobulin, respectively. Concentrations of Phe and Tyr were the lowest at pH values of 4.0, 4.5 and 5.0 in both treatments. Bound sialic acid was recovered at the highest extent in the treated sample that was heated and then acidified to pH 5.0. In the second part of the study, the pre-treated whey which was heated and then acidified to pH 5.0 that yielded the highest CMP and bound sialic acid contents was further purified by ultrafiltration and then diafiltration. The aim was to reduce water, lactose and ash contents in the retentate without loss of CMP. UF was performed by using a tubular unit with a feed volume of 9 L. Two PES membranes with MWCO of 9 and 25 kDa were used at pressure of 5 and 3 bar, respectively. Preliminary experiments were performed to obtain sufficient flow rate and hence flux for permeate from the UF unit which was crucial to achieve a high volumetric concentration of retentate. pH of pre-treated whey was changed to values in the range of 3-9 with acid and alkali addition for this purpose. As the pH of pre-treated whey was increased flow rate of permeate was increased. The highest relative mean flux was obtained at pH 9 with the membrane with MWCO of 9 kDa and at a pH of 7.0 with the 25 kDa membrane. A volumetric concentration factor of 5 was possible with both membranes. The flow rate of permeate obtained from 25 kDa membrane was higher than that from 9 kDa membrane, the process took shorter time accordingly. While one diafiltration cycle was possible without significant loss of CMP with 25 kDa membrane, four diafiltration cycles could be performed with 9 kDa membrane. Higher recovery and purity of CMP were obtained with 9 kDa membrane compared to that with 25 kDa membrane. Retentates from both UF/DF runs with two membranes were freeze-dried to obtain CMP concentrates. A CMP concentrate with 9.7% CMP, 2.8% α-lactalbumin, 1.5% β-lactoglobulin, 20.2% protein, 61.8% lactose, 18.1% ash, 0.85% bound sialic acid, 0.29% Phe and 0.34% Tyr in dry matter was obtained from pre-treated whey by using 9 kDa membrane. Although CMP content of the concentrate obtained by using 25 kDa membrane was lower (5.8%), it could be preferred because of lower ash content (7.5%) and shorter process time. In the last part of the study, a rice drink fortified with the CMP concentrate was developed. A base formula containing rice flour (3-8%, w/w) and xanthan gum (0.01-0.05%, w/w) as the structuring components that were heated at different temperatures (80-90oC) for 15 min was developed according to three-factor at three levels CCD by the response surface methodology. All samples were pre-hydrated in water bath 50oC for 20 min, heated, homogenized and cooled. Particle size, zeta potantial, color values, rheological properties (apparent viscosity, flow behaviour index, consistency and thixotropy), sedimentation and phase seperation of the drinks were measured as responses. None of the factors were found to have significant effect on sedimentation. While rice flour and temperature affected all responses significantly, there was no effect of level of xanthan gum on apparent viscosity and particle size. Selected response variables phase separation, particle size, apparent viscosity and zeta potential were targeted to be minimized in optimization of the base-formula and heating temperature. Optimum base formula of rice drink was estimated to contain 3% rice flour and 0.05% xanthan gum that was heated at 80C. The desirability of the values of estimated response from the optimized model with respect to the targeted values of responses was found 84%. Rice drink prepared at the optimum conditions was sweetened with 2.5% (w/w) sucrose to improve its acceptability. Descriptive sensory analysis was performed to compare appearance, flavor and texture of sweetened and unsweetened rice drinks with those of low fat milk. The color, mouthfeel, consistency, particle size, graininess and overall acceptability of rice drinks were found to be significantly different than those of low fat milk. Although the highest scores were given to low fat milk, sweetened rice drink was found to be closer to low fat milk compared to unsweetened rice drink. Isolated CMP concentrate (7%, w/w) was added to sweetened rice drink and then physical properties of the drink was measured. The amount of CMP to be added was calculated by considering the average daily amount of Phe allowed for the diet of children at age of 1-10 years. CMP resulted in reduction of zeta potential, increase in particle size and slight sedimentation in rice drink. It also enhanced apparent viscosity and color values of the drink.