Isoelectric precipitation, casein

Sample Preparation for CE. To dissociate the caseins, all samples were dissolved in a sample buffer containing 8M urea and 10 mM dithiothrei-tol at pH 8, and left for at least Ih at room temperature before filtration (0.22 pm Millex-GVi3, MilUpore) and CE analysis. The isoelectrically precipitated casein from milk and cheese and the purified casein standards were dissolved at lOmg/mL. To most samples, IpL of additional tripeptide Lys-Tyr-Lys (50mg/mL) was added per 50pL of sample as a reference compound. 

Soybean Protein Isolates. Soybean protein isolates, having a protein content of >90 wt%, are the only vegetable proteins that are widely used in imitation dairy products (1). Most isolates are derived from isoelectric precipitation, so that the soybean protein isolates have properties that are similar to those of casein. They are insoluble at thek isoelectric point, have a relatively high proportion of hydrophobic amino acid residues, and are calcium-sensitive. They differ from casein in that they are heat-denaturable and thus heat-labile. The proteins have relatively good nutritional properties and have been increasingly used as a principal source of protein. A main deterrent to use has been the beany flavor associated with the product. Use is expected to increase in part because of lower cost as compared to caseinates. There has been much research to develop improved soybean protein isolates. 

The properties of many dairy products, in fact their very existence, depend on the properties of milk proteins, although the fat, lactose and especially the salts, exert very significant modifying influences. Casein products are almost exclusively milk protein while the production of most cheese varieties is initiated through the specific modification of proteins by proteolytic enzymes or isoelectric precipitation. The high heat treatments to which many milk products are subjected are possible only because of the exceptionally high heat stability of the principal milk proteins, the caseins. 

Initially, it was believed that milk contained only one type of protein but about 100 years ago it was shown that the proteins in milk could be fractionated into two well-defined groups. On acidification to pH 4.6 (the isoelectric pH) at around 30°C, about 80% of the total protein in bovine milk precipitates out of solution this fraction is now called casein. The protein which remains soluble under these conditions is referred to as whey or serum protein or non-casein nitrogen. The pioneering work in this area was done by the German scientist, Hammarsten, and consequently isoelectric (acid) casein is sometimes referred to as casein nach Hammarsten. 

K-Casein is soluble in Ca2+ at all concentrations up to those at which general salting-out occurs. Solubility is independent of temperature and pH (outside the pH range at which isoelectric precipitation occurs). Not only is K-casein soluble in the presence of Ca2+ but it is capable of stabilizing asl-, as2- and /9-caseins against precipitation by Ca2+ (section 4.5.8). 

Although the gelation properties of whey proteins are of great importance in many foods (Mulvihill, 1992) and it is possible to form a weak gel in creams by the formation of a continuous network of fat globules, most important milk gels are those involving casein micelles which can be made to form a gel matrix either by isoelectric precipitation (acid-induced gel) or by the action of a proteolytic enzyme (rennet-induced gel). Both gel types... 

Proteins can be concentrated by isoelectric precipitation, heat precipitation, alcohol precipitation, ultrafiltration, microfiltration, and freeze concentration. Numerous references report differences in function in relation to the method of concentration of the protein. Whey protein produced by acid-heat precipitation (lactalbumin) is essentially insoluble but shows high water binding capacity and for this reason is the whey protein of choice for baking and for formulation of cereal products [89]. The use of ultrafiltration instead of acid precipitation of soy protein provides products with quite different characteristics [108], However, caseinate produced via acid precipitation is nearly as suitable for the production of imitation cheese as is rennet casein produced by the action of chymosin on skim milk [88],... 

There are two principal established methods for the production of casein on an industrial scale isoelectric precipitation and enzymatic (rennet) coagulation. There are a number of comprehensive reviews on the subject (e.g. Muller, 1982 Fox, 1989 Mulvihill, 1992 Fox and Mulvihill, 1992) which should be consulted for references. 

Addition of acetic or mineral acid to skimmed milk to reduce the pH value to 4.6, the isoelectric point, will cause the casein to precipitate. As calcium salts have a buffer action on the pH, somewhat more than the theoretical amount of acid must be used. Lactic acid produced in the process of milk souring by fermentation of the lactoses present by the bacterium Streptococcus lactis will lead to a similar precipitation. 

Kermack and Wright Biochem. J. XVII. 635,1923) have shown that gelatine at a of 4 T exerts but little protective power on a negative gum benzoin sol, acid gelatine precipitates the colloid in small concentrations whilst alkaline gelatine protects it. Similar observations have been made by Zsigmondy on the effect of casein on gold, but its exact isoelectric point was not established. 

Some like casein, salt free globulin and acid albumin are not heavily solvated in solution and are thus readily precipitated at the isoelectric point in a manner similar to the suspensions already considered. Others, e.g. glutin, gelatine and natural albumin, are solvated like silica which at the isoelectric point are not necessarily precipitated being maintained in the sol form by the solvent. On removal of the stabilising water however by the addition of alcohol or neutral salts precipitation will occur and this most readily at the isoelectric point. 

Co-preclpltate is an insoluble milk protein product that is produced by heating skinimllk to high temperatures ( > 90 C) to denature the whey proteins and complex them with the casein micelles. The heated system is subsequently adjusted to isoelectric point conditions of pH 4.5-5 to precipitate the complexed whey protein-casein micelles, centrifuged or filtered to recover the precipitate, washed and dryed. The resulting product, which is virtually insoluble, exhibits only minor functionality in most typical emulsification applications. 

The inorganic colloidal calcium phosphate associated with casein in normal milk dissolves on acidification of milk to pH 4.6 so that if sufficient time is allowed for solution, isoelectric casein is essentially free of calcium phosphate. In the laboratory, best results are obtained by acidifying skim milk to pH 4.6 at 2°C, holding for about 30 min and then warming to 30-35°C. The fine precipitate formed at 2°C allows time for the colloidal calcium phosphate to dissolve (Chapter 5). A moderately dilute acid (1 M) is preferred, since concentrated acid may cause localized coagulation. Acid production by a bacterial culture occurs slowly and allows time for colloidal calcium phosphate to dissolve. The casein is recovered by filtration or centrifugation and washed repeatedly with water to free the casein of lactose and salts. Thorough removal of lactose is essential since even traces of... 

They aggregate and precipitate from solution when the pH is adjusted to the isoelectric point of caseins (c. pH4.6). Precipitation at this pH, which is temperature-dependent (i.e. does not occur at temperatures below 5-8°C and occurs over a wide pH range, perhaps 3.0-5.5, at higher temperatures, e.g. 70°C), occurs owing to the loss of net positive or negative charge as the pH approaches 4.6. 

Commercial casein is usually manufactured from skim milk by precipitating the casein through acidification or rennet coagulation. Casein exists in milk as a calcium caseinate-calcium phosphate complex. When acid is added, the complex is dissociated, and at pH 4.6, the isoelectric point of casein, maximum precipitation occurs. Relatively little commercial casein is produced in the United States, but imports amounted to well over 150 million lb in 1981 (USDA 1981C). Casein is widely used in food products as a protein supplement. Industrial uses include paper coatings, glues, plastics and artificial fibers. Casein is typed according to the process used to precipitate it from milk, such as hydrochloric acid casein, sulfuric acid casein, lactic acid casein, coprecipitated casein, rennet casein, and low-viscosity casein. Differences... 

In the first part of this experiment, you are going to isolate casein from milk which has a pH of about 7. Casein will be separated as an insoluble precipitate by acidification of the milk to its isoelectric point (pH = 4.6). The fat that precipitates along with casein can be removed by dissolving it in alcohol. 

Isolation of Caseins. Isoelectric caseins were obtained by precipitation from whole milk or from 5g of homogenized cheese in 30 mL of water by adding 2M FICl to pH 4.6 followed by centrifugation at 3500 rpm for 15 min. To isolate the casein fractions completely from whey and eliminate the remaining fat, it was washed once with 1M sodium acetate buffer (pH 4.6) and three times with dichloromethane/1 M sodium acetate buffer (pH 4.6) (1 1, v/v). The casein fractions obtained were lyophilized and stored at -20 °C. 

The caseins exist in milk as polydisperse aggregates ranging in size from ca. 40 to 220nm (3), but the size distribution of micelles depends upon the method of measurement. These casein micelles scatter light and are responsible for the whitish, opaque nature of skim milk. The casein micelles are also associated with a colloidal apatite comprised of calcium-phosphate-citrate (CPC) which has a stabilizing influence on the micelle structure. The colloidal CPC is in equilibrium with soluble CPC in the milk serum phase and is solubilized as the pH is reduced. Thus, as the pH is reduced to the isoelectric point of the caseins (4.6), the colloidal CPC solubilizes, and the caseins precipitate (143). This phenomenon should be kept in mind during some of the following discussions. 

White, amorphous powder or granules without odor or taste. Very sparingly sol in water and in nonpolar organic solvents sol in aqueous so]ns of alkalies, levorotatory. The isoelectric zone is around pH 4.7 sol in coned HCI with light violet color. Amphoteric forms salts with both acids and bases. Present in bovine milk as neutral calcium caseinate and in human milk as potassium caseinate. Precipitated from solns satd with metallic salts. Forms a hard, inso] plastic with formaldehyde. 

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