Casein succinylation

A succinylated casein derivative that has nearly all its amines blocked can be used as a substrate in protease assays (Hatakeyama et al., 1992). As the casein is degraded by a protease, free amines are created from a-chain cleavage and release of a-amino groups. The creation of... 

Soy protein is a low-cost food protein with good nutritional value, but its uses in foods are limited because of inferior functional properties as compared to those of commonly used animal proteins such as casein and albumin (1.2). Therefore, modifications are often required to make soy protein more suitable for food use. Improved functional properties, particularly in the pH range of 3 to 7 where most food systems belong, have been achieved by non-enzymatic methods, including succinylation (3-5), deamidation (6.7), and phosphorylation (8.9). 

Several workers have chemically modified milk proteins ( c-casein, casein) to determine the chemical basis for the stabilization of the casein micelle by c-casein and to elucidate the mechanism of rennin action (35,36). Creamer et al. (37) made several derivatives of sodium caseinate and showed their improved solubility. In preliminary feeding trials in which succinylated casein was the only protein source, supplementation with lysine was required for the normal growth of rats. 

Primary Mode of Reaction and Consequences (9). Bovine a8i-casein (Variant B) was prepared from fresh milk (12) and immediately succinylated to increase water dispersibility (13). This succinylated asi-casein preparation, molecular weight of ca. 25,000 daltons, was used as the hydrophilic protein substrate. The compound L-norleucine 1-13C-dodecyl ester, prepared from K13CN and 1-bromoundecane through four steps (14,15,16), was used as the lipophilic nucleophile. C-13 NMR measurements showed that this sample gave only one signal at a distance of 65.2 ppm from the signal of tetramethylsilane (TMS). 

The enzymatic process was carried out under the following conditions medium, 20% (v/v) acetone in 1M carbonate (pH 9) containing lOmAf 2-mercaptoethanol concentration of succinylated < si-casein in medium, 20% (w/w) concentration of L-norleucine l-13C-dodecyl ester in medium, 0.25M concentration of papain (recrystallized) in medium, 0.02% (w/w) incubation temperature, 37°C and incubation time up to 60 min. For further details refer to the published papers (7,8). 

Another experiment was carried out with the 5-min, 15-min, and 30-min incubation mixtures in order to determine the correlation between their content of the 20,000-dalton product and the emulsifying activity. The result was that the emulsifying activity increased in accordance with the formation of this polypeptide (see Table II). No increase in the emulsifying activity results when succinylated asi-casein is incubated with papain in the absence of L-norleucine n-dodecyl ester. 

All of the data from Table II indicate that, under the unconventional conditions intentionally set in the present study, papain can catalyze the aminolysis of the ES intermediate that probably occurs from succinylated asi-casein (substrate) and papain (enzyme) by L-norleucine n-dodecyl ester (nucleophile), with formation of a surface-active 20,000-dalton product to which this lipophilic nucleophile is attached covalently as illustrated in Figure 1. The observed amphiphilic function of the 20,000-dalton product is probably a result of the formation of a localized hydro-... 

Table I. Properties of a 20,000-Dalton Polypeptide Formed from Succinylated asi-Casein by Treatment with Papain in the Presence of L-Norleucine l-13C-Dodecyl Ester...

We succinylated FPC, SPI, casein, and ovalbumin as well as gelatin, with the result that their whippability, foam stability, and emulsifying activity were improved to a certain extent (see Table V). 

Each of the proteins was dispersed in an alkaline medium (pH 7-9) and then treated with succinic anhydride amounting to 10 times the concentration level (molar basis) of amino groups present in the protein. The degrees of succinylation measured by a usual method (13) were as follows 98.0% for FPC, 95.2% for SPI, 94.0% for casein, 97.7% for ovalbumin, and 98.8% for gelatin. 

Little attention has been given to the biological consequences of such acylation for food use. Creamer et al. (40) reported the result of growth and toxicity trials in rats and mice fed acetyl or succinyl casein and acetyl milk whey protein. These proteins had lower protein efficiency ratios than casein, and added L-lysine hydrochloride did not correct this deficiency. However, a trial with acetyl casein in mice showed no overt toxicity. These workers (40) concluded that none of the milk proteins as modified with anhydrides can be regarded as suitable for inclusion in acidic food products. It is obvious that a thorough biological evaluation is needed to assess whether acylated soy proteins are suitable for food use. 

A surface-active macropeptide (MW 20,000) was produced as well by Toiguchi et al. [149] from succinylated aSl-casein by modification with papain. The reaction was carried out in the presence of L-leucine n-dodecyl ester. This was covalently bound to the peptide during the... 

In order to produce the amphoteric protein-based surfactant, the incorporation of lipophilic amino acid ester was attempted using the one-step method of plastein reaction with papain at pH 9. In a system containing succinylated ttsi-casein as a protein substrate and luecine n-dodecyl ester as a lipophile, the peptide bond between Phe and Tyr of casein was first hydrolyzed, and this is followed by the incorporation of luecine n-dodecyl ester at the same position, forming a new C-terminus [34]. The structure of the macropeptide with respect to the distribution of hydrophilic amino acid residues is shown in Fig. 4 [29,34]. Amphiphilic structure consisting of hydrophilic protein portion and lipophilic luecine n-dodecyl ester was clearly demonstrated. 

For example, succinylated wheat gluten is quite soluble at pH 5 (cf. Fig. 1.40). This effect is related to disaggregation of high molecular weight gluten fractions (cf. Fig. 1.41). In the case of succinylated casein it is obvious that the modification shifts the isoelectric point of the protein (and thereby the solubility minimum) to a lower pH (cf. Fig. 1.42). Succinylation of leaf proteins improves the solubility as well as the flavor and emulsifying properties. 

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