Casein alkali-treated

Adverse effects of exposing proteins to alkaline conditions are known. As early as 1913, it was shown that severely alkali-treated casein fed to dogs was eliminated unchanged in the feces, that it was not attacked by putrefactive bacteria and that trypsin or pepsin was unable to hydrolyze it (9). Ten Broeck reported that egg albumin treated with 0.5 N. NaOH for 3 weeks at 37° had no immunological properties (10). The nitrogen digestibility values of 0.2 M and 0.5 M NaOH-treated casein (80°C, 1 hr), as determined in rats, was 71 and 47%, respectively, as... 

The amino acid composition of alkali-treated casein, lactal-bumin, and wheat gluten are given in Tables I-III. The results show that the following amino acids are destroyed to various extents under basic conditions threonine, serine, cystine, lysine, and arginine, and possibly also tyrosine and histidine. The losses of these amino acids is accompanied by the appearance of lysinoalanine and other ninhydrin-positive compounds. 

Tovar (14) performed an experiment designed to evaluate the in vivo effect of D-amino acids in alkali-treated protein without the presence of lysinoalanine. In addition, either lime or caustic soda were used to investigate whether these alkalis had different effects vivo Zein was exposed to O.IN alkali for 4 hours at 85 C. Because lysine is absent from zein, no lysinoalanine formation was observed. Diets were prepared using untreated or alkali-treated zein and were supplemented with casein and free amino acids to meet the nutritional requirements of the... 

In 1969, deGroot and Slump also demonstrated decreases for in vitro digestibility of alkali-treated soy protein Isolate and decreases In absorption of some amino acids by everted Intestinal sacs (14). These workers also observed decreases In net protein utilization for treated soybean meal, treated soy protein Isolate and treated casein. These decreases In net protein utilization were correlated with Increases In LAL formation. In these experiments, therefore. It was possible that protein utilization was hindered by the presence of LAL. 

In 1981, Friedman, Zahnley and Masters measured the in vitro digestibility of alkali-treated casein by trypsin and chymotrypsin as a function of temperature, time and pH of the treatment (17). They also measured LAL formation and the racemlzatlon of aspartate... 

This method revealed significant amounts of dehydroalanine in alkali-treated casein and acetylated casein (Table 10). 

Recently, Bunjapamai et al., (1982) have shown that citracony-lated casein treated with alkali is as highly racemized as treated non-citraconylated casein, although the former contains no LAL. In vitro digestion of the racemized but non-crosslinked casein was Just as restricted as was the digestion of the racemized crosslinked casein. Thus, racemization alone can inhibit in vitro digestibility of alkali-treated proteins. 

Freimuth, U., Krause, W. and Doss, A. (1978). On the alkali treatment of proteins. II. Enzymatic hydrolysis of alkali-treated p-casein and acid casein. Nahrung, 22, 557-568. (German). 

The key to the problem seems then to be the measurement of the enzymatic releasability of protein-bound LAL, as mentioned by Slump (1978). Little on this topic can be found in the literature. Using a closed system (pepsin-pancreatin and pepsin-pronase-prolidase-aminopeptidase), Slump (1978) found an LAL release with treated soybean of 3% (1.3 g LAL/16 g N), and of 2% with treated casein (5.5 g LAL/16 g N content) or 0.5% (1.0 g LAL/16 g N content). Using a different vitro digestion system, Finot et al. (1978) reported a 27% release of LAL from alkali-treated lactalbumine. 

The casein, which is contaminated with calcium phosphate and fats, is filtered off to as small a volume as possible (about 500 cc.) and transferred to a 2-1. beaker. It is then treated with 0.1 M sodium hydroxide, the alkali being added slowly and with stirring through a capillary extending to the bottom of the beaker (Note 4). The addition of alkali is continued until the / ll of the mixture reaches 6.3 (Note 5) 100-150 cc. of the alkali is required. The end-poinl is determined by matching against... 

Manufacture of crystalline lactose from permeate derived by ultrafiltration of lactic casein whey presents special problems because of the low pH, high lactate concentration, and high calcium and phosphate concentrations (Hobman 1984). Research at the New Zealand Dairy Research Institute has led to a pilot-scale process whereby calcium phosphate complexes are partially removed before evaporation by an alkali and heat treatment to precipitate them, followed by centrifugation to clarify the treated permeate. Removal of about 50% of the calcium is sufficient to avoid problems during evaporation. 

Beeby, R. and Kumetat, K. J. 1959. Viscosity changes in concentrated skim milk treated with alkali, urea, and calcium complexing agents. I. The importance of the casein micelle. J. Dairy Res. 26, 248-257. 

For example, the respective values at pH 10.6 are 0.262, 0.494, and 1.04 mole per cent (ratio of about 1 2 4) at pH 11.2 the values are 0.420, 0.780, and 1.32 mole per cent and at pH 12.5 (pH of 1% protein solution in 0.IN NaOH), the respective values are 0.762, 0.780, and 2.62 mole per cent. (Note that the value of casein approaches that of gluten at this pH). The observed differences in lysinoalanine content of the three proteins at different pH values are not surprising since the amino acid composition, sequence, protein conformation, molecular weights of protein chains, initial formation of intra- versus intermolecular crosslinks may all influence the chemical reactivity of a particular protein with alkali. Therefore, it is not surprising to find differences in lysinoalanine content in different proteins treated under similar conditions. These observations could have practical benefits since, for example, the lower lysinoalanine content of casein compared to lactalbumin treated under the same conditions suggests that casein is preferable to lactalbumin in foods requiring alkali-treatment. 

Derived Proteins.—A few primary protein derivatives have been mentioned in connection with the substances described above. Paracasein and fibrin are formed as the result of the action of certain enzymes on casein and fibrinogen, respectively. The metaproteins are obtained by treating proteins with dilute acids or alkalies. Acids form acid-proteins, which are soluble in acids and insoluble in alkalies. The latter form soluble alkali-proteins, which are precipitated by acids. Many soluble proteins are changed into insoluble substances (coagulated) when heated. The derived proteins resemble closely the substances from which they are formed except in solubility. The nature of the change is not understood. It is possible that the conversion of a protein into a primary derivative may be associated with a change in the colloidal condition of the molecule. 

The work of Bunjapamai, Mahoney and Fagerson (21) was the first successful attempt to separate the effects of racemization from crosslinking as measured by vitro digestion. Alkali-treatment of citraconylated (lysine-blocked) or non-blocked casein resulted in racemized only (blocked) or racemlzed and LAL cross-linked (non-blocked) casein. vitro multienzyme digestion of these preparations as well as untreated casein revealed similarly decreased digestibilities for the treated proteins whether crosslinked or not, indicating that the primary cause for reduction of casein digestibility was racemization. 

Hayashi and Kameda have reported 40% to 70% decreases in pepsin-catalyzed hydrolysis of lysozyme, soybean protein, casein and rlbonuclease A due to alkali-treatment under slightly milder conditions than ours (16, 29). Friedman, Zahnley and Masters reported an 80% decrease in digestibility of sodium hydroxide-treated casein measured as hydrolysis by trypsin (17). However, trypsin is specific for lysyl residues and lysine levels decreased to about half control values during the alkali-treatment, with a concomitant Increase in LAL formation. The somewhat lower digestibilities reported by these laboratories compared to our observations may be due to LAL formation in the proteins other than zein. 

Casein is the major component (80 per cent) of milk, with molecular weights between 1 and 20kDa and includes four distinct types a-sl, a-s2, (3, and k. Casein is the predominant phosphoprotein that precipitates at pH 4.6 (20°C) and is characterized by an open, random coil structure. By treating acid-precipitated caseins with alkali solution caseinates are produced. Both caseins and caseinates form transparent films from aqueous solutions without any treatment because of their random coil nature and numerous hydrogen bonds. Caseins have shown to be useful in adhesives, micro encapsulation, food ingredients, and pharmaceuticals [31]. 

---