Casein acidification

Milk from cows contains 3.2% protein, about 80% of which is casein. Casein is isolated by a precipitation process from milk, involving heating, rinsing to remove whey, and drying to a powder. The yield is about 3 kg/ 100 kg skim milk. Rennet casein is obtained when the casein is precipitated by chymosin enzyme, also known as rennet, and acid casein is produced when precipitation is accomplished by acidification. Acid casein is usually found in the form of sodium caseinate or calcium caseinate, which are water-soluble salts. Caseinates are made by reacting NaOH or CaOH with a slurry of casein curd or powder and then spray drying (Southward, 2010). 

Dissolution in Phosphate buffer, gel electrophoresis, in gel tryptic digestion, petide extraction (CH3CN/H2O/TFA), cleaning on ZipTip CIS Acidification (pH 4.6 with 5% HAc) to precipitate caseins, centrifugation... 

For cheese sample Ultraturrax homogenization (two cycles) with H2O, centrifugation (for skimming), acidification at pH 4.2. 6 (for casein precipitation), centrifugation, filtration For cheese sample extraction with H2O (sonication) for milk centrifugation, acidification (pH 4.6) to precipitate caseins, centrifugation, filtration... 

The self-assembly of caseins may be readily manipulated by processing methods that affect the integrity of native casein micelles and the character of the casein interactions in aqueous media. Examples of such procedures are (Dickinson, 2006) (i) acidification toward the isoelectric point (p/) (pH 4.6-4.8), leading to a neutralization of the net protein charge (ii) enzyme action, as exploited in the production of cheeses and fermented milks (iii) addition of divalent ions, especially, Ca2+ ions (iv) addition of sucrose or ethanol (v) temperature treatment and (vi) high-pressure treatment. 

We have seen earlier in this chapter how the self-assembly of casein systems is sensitively affected by temperature. Another thermodynamic variable that can affect protein-protein interactions in aqueous media is the hydrostatic pressure. Static high-pressure treatment causes the disintegration of casein micelles due to the dismption of internal hydro-phobic interactions and the dissociation of colloidal calcium phosphate. This phenomenon has been used to modify the gelation ability of casein without acidification as a consequence of exposure of hydrophobic parts of the casein molecules into the aqueous medium from the interior of the native casein micelles (Dickinson, 2006). High-pressure treatment leads to a reduction in the casein concentration required for gelation under neutral conditions, especially in the presence of cosolutes such as sucrose (Abbasi and Dickinson, 2001, 2002, 2004 Keenan et al., 2001). 

Vetier, N., Desobry-Banon, S., Ould Eleya, M.M., Hardy, J. (1997). Effect of temperature and acidification rate on the fractal dimension of acidified casein aggregates. Journal of Dairy Science, 80, 3161-3166. 

Figure 7.23 Influence of low-methoxyl amidated pectin on microstructure of caseinate gels acidified with giucono-8-lactone. The images show the gels (2 wt%) after 6 hours of acidification to pH = 4.0 for different added polysaccharide concentrations (wt%) (a) 0 (b) 0.1 (c) 0.4 (d) 0.8. Scale bar = 10 pm. Reproduced from (Matia-Merino et al., 2004) with permission.

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. 

For laboratory-scale production of casein, HC1 is usually used for acidification acetic or lactic acids are used less frequently. Industrially, HC1 is also usually used H2S04 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... 

Acidification of milk is accompanied by a progressive solubilization of colloidal calcium phosphate and other colloidal salts from casein. Solubilization is complete below about pH 4.9 (Figure 5.11). 

On acidification to pH 4.6, the caseins coagulate, which is the principle used to manufacture of a family of cheeses which represent about 25% of total cheese consumption and are the principal cheeses in some countries (Appendix 10B). Acidification is traditionally and usually achieved by in situ fermentation of lactose by a Lactococcus starter but direct acidification by acid or acidogen (gluconic acid-d-lactone) is also practised. The principal... 

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... 

Originally, the caseins were defined as those phosphoproteins which precipitate from raw skim milk upon acidification to pH 4.6 at 20°C, and the individual families were identified by alkaline urea gel electrophoresis (Whitney et al 1976). With the resolution of their primary structure, it became possible to classify them according to their chemical structure, rather than on the basis of an operational definition. When one does this, it is apparent that not all of the caseins contain phosphorus (Table 3.1) some are also found in the acid whey after removal of the precipitated caseins. 

Because of these chemical and physicochemical changes in the casein micelles upon cooling of milk, the milk becomes more viscous and displays an increased tendency to foam. Also, casein micelles in cold milk commonly exhibit incomplete coagulation upon acidification and treatment with rennet (Harper 1976 Muller 1982A Marshall 1982 Morr 1982). 

Milk proteins are subdivided into random coiled caseins, which can be precipitated by acidification of raw skim milk to pH 4.6 at 20°C, and into more globular whey proteins, which remain in the serum after precipitation of the caseins (42). In Table 8, an overview is given of the molecular structure and basic properties of the major protein fractions present in milk. Some specific properties that might be of importance for their determination in foods and food products are also listed. For the young of mammals, including humans, milk is the first and, for most, the only food ingested for a considerable period of time. With the domestication of animals, it became possible to include milk in the diet of adult humans as well. For much of the world, particularly in the West, milk from cattle (Bos taurus) accounts for nearly all the milk processed for human consumption (43). 

This is formed of the nitrogenous substances (casein, albumin) and fats contained in milk, separated by coagulation (by rennet or by acidification). As a result of special fermentations which occur during the maturation of the cheese, these give rise to soluble albuminoid substances (albumoses, peptones, etc.), amino-adds (phenylaminopropionic add, tyrosine, leucine, etc.), ammoniacal products, fatty adds (lactic, propionic, caproie), etc. Cheese also contains water and mineral salts, including added sodium chloride. 

Bazinet, L., Lamarche, F., Ippersiel, D., and Amiot, J. 1999a. Bipolar membrane electro-acidification to produce bovine milk casein isolate. J. Agric. Food Chem. 47, 5291-5296. 

Historically, ideas of casein micelle structure and stability have evolved in tandem. In the earlier literature, discussions of micellar stability drew on the classical ideas of the stability of hydrophobic colloids. More recently, the hairy micelle model has focused attention more on the hydrophilic nature of the micelle and steric stabilization mechanisms. According to the hairy micelle model, the C-terminal macropeptides of some of the K-casein project from the surface of the micelle to form a hydrophilic and negatively charged diffuse outer layer, which causes the micelles to repel one another on close approach. Aggregation of micelles can only occur when the hairs are removed enzymatically, e.g., by chymosin (EC 3.4.23.4) in the renneting of milk, or when the micelle structure is so disrupted that the hairy layer is destroyed, e.g., by heating or acidification, or when the dispersion medium becomes a poor solvent for the hairs, e.g., by addition of ethanol. 

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. 

In the isolation of casein following the acidification, you removed the precipitate by filtering through a cheese cloth and squeezing the cloth. If you did not squeeze out all the liquids, would your yield of casein be different Explain. 

Acidification to the isolectric pH of casein using lactic acid bacteria or food-grade acids/acidogens, at 20 40 C. and resultant slow quiescent aggregation of the sensitized casein micelles e.g., for cream cheese. [A combination of acidification and rennet-hydrolysis (a smaller quantity of rennet than for rennet-curd cheeses, e.g., 5-100 versus 900-1000 chymosin units per 100 L milk) is normally used for low-fat acid-curd cheeses such as Quark and related varieties (Schulz-Collins and Senge, 2004)]... 

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