Method of Obtaining Casein from Skim Milk using Oligochitosans

interaction of milk proteins and oligochitosansis based on formation of electrostatic complex between negatively charged casein micelles and positively charged polysaccharide groups. At low concentrations of oligochitosans, partial interaction with milk proteins occurs. Maximum protein coagulation (90 %) is achieved in equivalence zone at the concentration of oligochitosans: 45.3 kDa-0.88×10 -4 M; 25.4 kDa-1.57×10 -4 M; 7.7 kDa – 5.19×10 -4 M; 1.5 kDa-26.67×10 -4 M. In these concentrations, taking into account the molecular weight and degree of deacetylation of oligochitosans, the total content of protonated amino groups is equal 2.4×10 -4 M. When t protein content in skimmed milk is 3.0 % - 3.2%, oligochitosans of any molecular weight and degree of deacetylation can be used for casein coagulation, important that the final concentration of amino groups should be 2.4×10 -4 M. It was determined that the use of the studied samples of oligochitosan allows to obtain coacervate where 1g of oligochitosan binds 6.0 - 6.2g of milk proteins.


Introduction
The combination of harmless nature and biological activity of chitosan creates prerequisites for its wide application in milk processing and casein production [1]. Due to the structure of casein micelle, at pH above 5.0 a large negative charge is concentrated on its surface. Chitosan at pH below 6.5 has a positive charge that enables ionic interaction of chitosan with casein. So adding chitosan to milk we can expect their coagulation and precipitation [2][3][4].
Chitosan is soluble in organic acids that causes difficulties for milk processing [3]. Oligochitosans are low-molecular-weight chitosan derivatives with a molecular weight between 2 and 50 kDa. They are highly soluble in water and do not influence on acidity when added to milk. There is a practical possibility to use oligochitosan to isolate casein from skimmed milk [4,5]. The effectiveness of the interaction of casein with oligochitosans depends on their molecular weight and the number of amino groups. The objective of the research was to obtain casein from skimmed milk using oligochitosans with different molecular weights.

Materials and Methods
The objects of the research were: skimmed cow milk (produced by JSC "Savushkin product", Russia) and oligochitosans with molecular weights of 45.3, 25.4, 7.7 and 1.5 kDa with a degree of deacetylation (SD) of 86%, obtained by the previously described method [6]. The molecular weight of oligochitosans was determined by viscosimetry and calculated using the Mark-Houwink equation; the degree of deacetylation was determined by conductometric titration. To study the process of casein coagulation, water solutions of oligochitosans with different molecular weights were added to skimmilk to the final polysaccharide concentration from 0.02% to Copy@ LR Alieva of casein complexes with chitosan was performed on "TA-4000 Mettler Toledo" (Switzerland). Measurements were performed in the temperature range of 25-600 °C in the air atmosphere at a heating rate of 5°C/min. The activation energy was determined by the Broydeau equation. The protein concentration was measured by biuret method.

Results and Discussions
Milk is a complex polydisperse mixture of casein micelles, fat globules, whey proteins, lactose and minerals. Protein components and fat globules at a pH of 5.0-6.2 are negatively charged and able to interact with oligochitosan. In our research we studied the effect of oligochitosan on milk components with a fat content of 3.2% at a pH of 6.0. The results of the research are presented in the table 1 (below). Analysis of the results shows that adding oligochitosan with a molecular weight of 25.5 kDa to the milk with fat content of 3.2 % results in fractionation into milk fat, casein and whey proteins. Adding oligochitosan to skim milk we can isolate casein fraction.
We investigated the process of skim milk proteins coagulation depending on the concentration of oligochitosans with different molecular weights at pH 6.0. We added water solutions of oligochitosans with molecular weights of 45.3, 25.4, 7.7 and 1.5 kDa in the final concentration from 0.02% to 1% to skimmed milk and observed coagulation. This process took place at 60 °C during 30 minutes.
The protein content in milk, after separation of the coagulate, depended on the concentration and molecular weight of oligochitosan ( Figure 1A). The Figure 1B       The charge of polyioniс complex decreased as the negatively charged macroions of proteins linked to oligochitosan. As a result, electro-neutral complex of oligochitosan with casein and whey proteins was formed. Aggregation of these complexes resulted in their precipitation in the form of coacervate [1,5].
The proposed method for obtaining caseins using water-soluble oligochitosans opens the possibility of increasing the yield of milk protein to 90-92%. In addition the whey obtained in this process has pH close to neutral and can be used for functional food production.