Acid isoelectric precipitation

For laboratory-scale production of casein, HCl is usually used for acidification acetic or lactic acids are used less frequently. Industrially, HCl is also usually used H2SO4. is used occasionally but the resulting whey is not suitable for animal feeding (MgS04 is a laxative). Lactic acid produced in situ by a culture of lactic acid bacteria is also widely used, especially in New Zealand, the principal producer of casein. 

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 

The procedure used for the industrial production of acid (isoelectric) casein is essentially the same as that used on a laboratory scale, except for many technological differences (section 4.15.1).The whey proteins may be recovered from the whey by salting out, dialysis or ultrafiltration. 

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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. 

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. 

Isoelectric precipitation results in loss of whey protein as well as undesirable non-protein components. Gillberg (35) has shown that the cystine composition of the isolate is lower than for the meal extract because non-precipitated whey protein has a relatively larger proportion of the total sulfur amino acids. This observation is reinforced by Mattil (36), who showed that the range for amino acid composition was different for several commercial concentrates as compared to commercial isolates. He found methionine to be lower for isolates than for concentrates. 

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... 

Isoelectric precipitation and acid precipitation are also used to separate antibodies. Isoelectric precipitation (also called euglobulin precipitation) uses the solubility properties of a protein near its isoelectric point.69 When a concentrated protein solution in a low ionic strength buffer is titrated to its isoelectric point, it precipitates very slowly. Table 4 shows a balance of a process for purification of murine monoclonal IgM antibodies.70 The success of precipitation can be followed by the cumulative IgM content in the centrifugate and the sediment. The procedure is very gentle, but very sensitive to environmental conditions. 

The water-extractability of both the 7S and 11S proteins decreases with aging of the soy meal (4). During isoelectric precipitation 15-30% of the globular fraction is denatured and fails to redissolve in neutral buffer (4, 5). As the pH is lowered to 2-3 the 11S component becomes increasingly sensitive to irreversible denaturation. This acid-sensitive fraction not only limits the solubility of such isoelectric protein isolates, and therefore their functional uses, but this fraction may also be responsible for retention of undesirable flavors (5). 

Our studies on 7S globulin isolated under conditions which avoided the acidic denaturation conditions created by isoelectric precipitation showed about 5% helix, 60% g-structure and 35% random coil. It is quite possible that the isolation conditions have determined the secondary order of the isolated protein. 

For preparative purposes, it is perhaps more convenient to start from pancreas acetone powder (pancreatin). Ninty-five per cent pure bovine procarboxypeptidase A has been obtained by ammonium sulfate fractionation of pancreatin extracts and isoelectric precipitations (47). When the proteins precipitated by 0.39 saturated ammonium sulfate are chromatographed on DEAE-cellulose in a concentration gradient of pH 8.0 phosphate buffer, the two last and most acidic peaks contain procarboxypeptidase A in an electrophoretically homogeneous form (48). The molecular weight of the protein determined by light scattering and sedimentation-diffusion is 94-96,000. Its isoelectric point in univalent buffers of 0.2 ionic strength is below 4.5. 

Separation of soy protein by EDBM has specific advantages over the conventional isoelectric precipitation used industrially for the production of soybean protein isolates. This technology does not use any added acids or bases during the process to adjust the pH of the protein solution, and the chemical effluents generated during the process could be reused at different stages in the... 

The precipitation and fractionation of soy protein using CO2 in the pressure range Q.1. 7 MPa was also examined (111,112). Pressurized CO2 allowed better control of the solution pH than the addition of mineral acids and prevented local pH overshoots, which can result in poor quality precipitates. Protein precipitates formed by isoelectric precipitation using CO2 were spherical, whereas those formed using sulfuric acid were irregular (111). The decrease of solution pH to a value close to the isoelectric pH of the proteins was the primary cause of protein precipitation. Volatile acid precipitation is unlike conventional GAS, in which pressures of 5-10 MPa are required to achieve precipitation from an expanded organic solution. 

Hofland GW, de Rijke A, Thiering R, van der Widen LAM, Witkamp G-J. Isoelectric precipitation of soybean protein using carbon dioxide as a volatile acid. J Chromatogr B 2000 743 357-368. 

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],... 

Zhang, J., Li, Y., Li, J., Zhao, Z., Liu, X., Zhang, Y., et al. (2014). Generation of biofunctional biodegradable electrospun nanofibers composed of poly(L-lactic acid) wool isoelectric precipitate. Textile Research Journal, 84(A), 355—367. 

Water-soluble polymers and polyelectrolytes (e.g., polyethylene glycol, polyethylene imine polyacrylic acid) have been used success-hilly in protein precipitations, and there has been some success in affinity precipitations wherein appropriate ligands attached to polymers can couple with the target proteins to enhance their aggregation. Protein precipitation can also be achieved using pH adjustment, since proteins generally exhibit their lowest solubility at their isoelectric point. Temperature variations at constant salt concentration allow for frac tional precipitation of proteins. 

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. 

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