Plasteins

Plasteins ate formed from soy protein hydrolysates with a variety of microbial proteases (149). Preferred conditions for hydrolysis and synthesis ate obtained with an enzyme-to-substrate ratio of 1 100, and a temperature of 37°C for 24—72 h. A substrate concentration of 30 wt %, 80% hydrolyzed, gives an 80% net yield of plastein from the synthesis reaction. However, these results ate based on a 1% protein solution used in the hydrolysis step this would be too low for an economical process (see Microbial transformations). 

Pish protein concentrate and soy protein concentrate have been used to prepare a low phenylalanine, high tyrosine peptide for use with phenylketonuria patients (150). The process includes pepsin hydrolysis at pH 1.5 ptonase hydrolysis at pH 6.5 to Hberate aromatic amino acids gel filtration on Sephadex G-15 to remove aromatic amino acids incubation with papain and ethyl esters of L-tyrosine and L-tryptophan, ie, plastein synthesis and ultrafiltration (qv). The plastein has a bland taste and odor and does not contain free amino acids. Yields of 69.3 and 60.9% from PPG and soy protein concentrate, respectively, have been attained. 

A pepsin hydrolysate of flounder fish protein isolate has been used as the substrate (40% w/v) for plastein synthesis, using either pepsin at pH 5 or alpha chymotrypsin at pH 7, with an enzyme—substrate ratio of 1 100 w/v at 37°C for 24 h (151). The plastein yields for pepsin and alpha chymotrypsin after precipitation with ethanol were 46 and 40.5%, respectively. 

Pish silage prepared by autolysis of rainbow trout viscera waste was investigated as a substrate for the plastein reaction using pepsin (pH 5.0), papain (pH 6—7), and chymotrypsin (pH 8.0) at 37°C for 24 h (152). Precipitation with ethanol was the preferred recovery method. Concentration of the protein hydrolysate by open-pan evaporation at 60°C gave equivalent yields and color of the final plastein to those of the freeze-dried hydrolysate. 

The sulfur amino acid content of soy protein can be enhanced by preparing plasteins from soy protein hydrolysate and sources of methionine or cystine, such as ovalbumin hydrolysate (plastein AB), wool keratin hydrolysate (plastein AC), or L-methionine ethyl ester [3082-77-7] (alkaU saponified plastein) (153). Typical PER values for a 1 2 mixture of plastein AC and soybean, and a 1 3 mixture of alkah-saponified plastein and soybean protein, were 2.86 and 3.38, respectively, as compared with 1.28 for the soy protein hydrolysate and 2.40 for casein. 

Plasteins ate stiU in the experimental stage of development. Eurther work is needed on the scale-up of processing conditions for plastein synthesis which would lead to commercially usefiil products and on the functional utiUty of plasteins as ingredients in foods. 

Currently available proteins are all deficient to greater or lesser extent in one or more of the essential amino acids. The recently advanced plastein reaction (229) has made it possible to use protein itself as substrate and to attach amino acid esters to the protein with high efficiency. By this method, soy bean protein (which is deficient in methionine) has been improved to the extent of having covalently attached L-methionine at 11%. 

H. Determann, J. Heuer, D. Jaworek, Untersuchungen iiber die Plastein-Reaktion VIII. Spezifitat des Pepsins bei der Kondensationsreaktionen , Justus Liebigs Ann. Chem. 

The plastein reaction usually involves two steps hydrolysis of protein and resynthesis of peptide links. Yamashita et al. (20) found similar BV, digestibility, and weight gain for denatured soy meal and soy plastein. This same group (49,50) had described a one-step process by which amino acids may 5 enzymatically incorporated in intact protein to improve protein quality. With soy protein they applied a racemic mixture of D,L-methionine ethyl ester and were able to enzymatically incorporate L-methionine. As Schwimmer (51) has pointed out, one expects the methionine so incorporated to be highly available due to its location at the end of the polypeptide chains. 

The protease-catalyzed synthesis of peptide bonds is known as the plastein reaction ( ). Plastein itself is defined as the product formed by this reaction which is insoluble in trichloroacetic acid solutions 6). The plastein reaction has been most extensively investigated by researchers in Japan (, ... 

Z 2)- These scientists have reviewed various aspects of plastein and the plastein reaction and its importance to protein functionality and nutriture (6, JO). 

The conditions necessary Tor the plastein reaction have been reviewed by Fujimaki et al. ( ), and compared to those necessary for proteolysis by Arai et al. ( ). The substrate for the synthetic reaction must consist of low molecular weight peptides, preferably in the tetramer to hexamer range. These are usually produced from proteins by protease action. A number of proteolytic enzymes and protein substrates have been investigated for producing plastein reaction substrates. The most often used proteases are pepsin JJ), and papain (12,13), but others... 

The properties of plasteins are quite different from those of the starting peptide mixture. In addition to insolubility in... 

Perhaps the most controversial aspect of the plastein reaction has been the molecular weight of resulting products. Early work... 

Hofsten and Lalasidis (15), however, reported that plasteins subjected to gel exclusion chromatography in 50X acetic acid showed no increase in molecular size over that of the reactants. Monti and dost (29) reached the same conclusion based on gel chromatography in DMSO and on analysis of a-amino nitrogen in plasteins. Hofsten and Lalasidis (15) noted that hydrophobic peptides showed unusual elution behavior on sephadex gels in water or dilute buffers, providing a possible explanation for differences in their results compared to those of Arai et al. 

Based on the solubility and chromatographic behavior of plasteins, Hofsten and Lalasidis (15) concluded that gel formation was due to nonpolar interactions of small hydrophobic peptides. 

Figure 5. Scheme for removing impurities from protein substrate by hydrolysis, purification, and resynthesis via the plastein reaction (i)... 

Another major application of the plastein reaction is nutritional improvement of proteins by the incorporation of limiting amino acids (8). A plastein containing approximately 7% methionine was produced from soy protein hydrolyzate and L-methionine ethyl ester in the presence of papain. This material was shown to be utilized as a source of methionine in the rat, producing a PER of 3.38 when incorporated into soy protein diets to give a methionine level of 2.74% of protein. 

Similarly, lysine has been incorporated into gluten hydrolyzate and lysine, threonine and tryptophan have been individually incorporated into zein hydrolyzates. Lysine, methionine, and tryptophan were incorporated simultaneously into hydrolyzates of protein from photosynthetic origin. A very interesting application of this procedure involved the preparation of low-phenylalanine plasteins from a combination of fish protein concentrate and soy protein isolate by a partial hydrolysis with pepsin then pronase to liberate mainly phenylalamine, tyrosine, and tryptophan, which were then removed on sephadex G-15. Desired amounts of tyrosine and tryptophan were added back in the form of ethyl esters and a plastein suitable for feeding to infants afflicted with phenylketonuria was produced. 

Pallavicini et al. (16) utilized a-chymotrypsin immobilized on chitin to catalyze plastein formation from leaf protein hydrolyzates. When analyzed by gel exclusion chromatography, the products were comparable to those produced by soluble enzymes. Modification of Specific Functional Properties... 

Fujimaki (77,78) condensed bitter soybean protein hydrolysates in a Plastein-Reaction (79) and obtained non-bitter protein-like products, unfortunately without determination of molecular weights. 

Enzymatic gelation of partially heat-denatured whey proteins by trypsin, papain, pronase, pepsin, and a preparation of Streptomyces griseus has been studied (Sato et al., 1995). Only peptic hydrolysate did not form a gel. The strength of the gel depended on the enzyme used and increased with increasing DH. Hydrolysis of whey protein concentrate with a glutamic acid specific protease from Bacillus licheniformis at pH 8 and 8% protein concentration has been shown to produce plastein aggregates (Budtz and Nielsen, 1992). The viscosity of the solution increased dramatically during hydrolysis and reached a maximum at 6% DH. Incubation of sodium caseinate with pepsin or papain resulted in a 55-77% reduction in the apparent viscosity (Hooker et al., 1982). 

Preparation of cheeses and soy derivatives Solubilization of protein concentrates Production of protein hydrolysates Gluten modification in bread doughs Chillproofing of beer Plastein formation Tenderization of meats Quality determination of proteins... 

Plastein Formation. Plastein formation is another example of using proteases to modify high-protein food systems to drastically change the properties of that system (II). In the plastein reaction a protease such as papain is used to partially hydrolyze the proteins to about a 10,000-20,000-dalton size at a pH near neutrality. After concentrating the hydrolyzate to 35% (based on protein) and a change in pH, the same protease or a different one is used to catalyze the resynthesis of a few peptide bonds. This may result in a decrease in the solubility of the protein. 

Some potential uses of the plastein reaction are given in Table V. The plastein reaction has been proposed for removing bitter peptides formed through previous hydrolysis of proteins by facilitating the resyn-... 

More details of the plastein reaction and its application to remove pigments such as chlorophyll, or to remove off-flavor components such as the beany taste of soybeans, are shown in Figure 2. The protein of the food system is solubilized and denatured (in order to achieve proteolysis), a protease is added, and the hydrolytic reaction is allowed to proceed. On partial hydrolysis of the protein the pigments and flavor constituents are released from the protein they are removed, the hydrolyzate is concentrated, and resynthesis and/or rearrangement of the amino acid sequence of the polypeptides is catalyzed by the same or a different protease. Resynthesis also can be carried out in the presence of added amino acid esters in order to improve the nutritional/functional properties of the protein. 

The mechanism of the plastein reaction is shown by the equation on page 64. 

Table V. Some Potential Uses of the Plastein Reaction in High-Protein Foods...

Figure 2. A process of enzymatic protein degradation and resynthesis for producing a plastein with improved acceptability and an improved amino acid composition (11)...

Enzyme-catalyzed attachment of amino acids to proteins represents an attractive and interesting way for improving the nutritional value of food proteins. The enzymes that participate in the gastrointestinal digestion of food proteins catalyze exclusively hydrolytic reactions under physiological conditions. However the synthetic activity of proteolytic enzymes was reported first by Danilewski in 1886, and more recently a number of studies have been devoted to plastein formation from con-... 

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