Racemization alkali treatment

The enantiomeric separation of the D- from the L-stereoisomers of amino acids is an area of growing interest. It is generally recognized that heat- and alkali-treatment of proteins can result in the racemization of L-isomers of amino acid residues to the D-analogs. Almost without exception, humans cannot utilize the D-isomers of amino acids, and some are thought to be toxic (although... 

Amino acids in animal and plant proteins appear to occur solely as L-isomers. However, D-amino acids are observed widely in nature as constituents of bacterial cell walls and of several antibiotics ( ) In addition, the heat and alkali treatments used in food processing can produce racemization of amino acids (4-6). 

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. 

Zein, which is the major protein in corn, was chosen because it contains no lysine. This precluded the formation of LAL during alkali treatment. Therefore, any changes in digestibility or uptake could be attributed to racemization effects alone. Additionally, we were interested in comparing the effects of sodium hydroxide treatment with the effects of calcium hydroxide treatment because lime is used in the preparation of corn meal for use in tortillas. If the traditional lime treatment of com meal is unnecessarily harsh, it could have important nutritional consequences because a large segment of the Mexican population obtains much of their dietary protein in the form of tortillas (22). 

In summary, alkali treatment of casein catalyzes racemization of optically active amino acids. Factors that influence racemization include pH, temperature, and time of treatment. Further studies are desirable to assess (a) how these factors operate in structurally different proteins and (b) the presence of D-amino acids in foods and feeds. (See Table 12 for some preliminary findings). 

LAL is, in fact, 15 to 25 times more nephrotoxic than protein-bound LAL (De Groot et al., 1976). Thus, any decrease in the digestibility of the protein due to alkali treatment could then influence the degree of availability of LAL. The release of biologically active LAL from a treated protein is determined by the proteins accessibility to proteolytic enzymes. As demonstrated by Masters and Friedman (1980), alkaline treatment induces amino acid racemization, with each protein and even each protein fraction reacting differently to the same treatment. Since racemization decreases the digestibility of protein, these differences could explain discrepancies among results reported by different authors. 

Br. CHa. CHa. CHa. CH(NHa). CH(CHa). CHa. CHjBr HBr. which on treatment with dilute alkali gives di-heliotridane (II). As the latter contains two asymmetric carbon atoms, two diastereoisomeric racemates might be produced in this reaction but only one was formed. It had density and refractive index in general agreement with those recorded for Z-heliotridane, as were also the melting points of characteristic derivatives. Density Df °0-902, refractive index wf, 1-4638 (<. with Adams and Rogers,3i Df ° 0-935, iijf° 1-4641), picrate, m.p. 234-6° (literature 232-6°), picrolonate, m.p. 162-3°, aurichloride, m.p. 200-1° (Konovalova and Orekhov give for these two constants 152-3° and 199-200° respectively). 

Nutritional and Physiological Effects of Alkali-Treated Proteins. The first effect of the alkaline treatment of food proteins is a reduction in the nutritive value of the protein due to the decrease in (a) the availability of the essential amino acids chemically modified (cystine, lysine, isoleucine) and in (b) the digestibility of the protein because of the presence of cross-links (lysinoalanine, lanthionine, and ornithinoalanine) and of unnatural amino acids (ornithine, alloisoleucine, / -aminoalanine, and D-amino acids). The racemization reaction occurring during alkaline treatments has an effect on the nitrogen digestibility and the use of the amino acids involved. 

For maximizing protein yield and minimizing contaminant nucleic acids, alkali extraction at elevated temperatures is a feasible procedure (63,73). However denaturation of proteins during extraction is a serious problem because it significantly destroys functional properties and limits the food uses of the extracted protein (2, 74). In addition to denaturation, exposure of protein to alkaline treatments may also cause other undesirable effects, i.e. racemization of amino acids, g-elimination and crosslinking or certain amino acids and formation of potentially antinutritive compounds (11,23,75). 

Both of the alkaloids anhalamine (62) from ljophophora mlliamsii and lopkocerine (63) from Ijophocereus schotti were isolated (after the properties of purified mescaline had been noted) in the search for materials of similar behavior. Interestingly, lophocerine, isolated as its methyl ether, after diazometliane treatment of the alkali-soluble fraction of total plant extract, is racemic. It is not known if the alkaloid in the plant is also racemic or if the isolation procedure causes racemization. 

The hydrolysis of the active alkyl phthalates is usually carried out by dissolving them in a 20-25% aqueous solution of sodium hydroxide containing 2.5 moles of alkali and distilling the mixture with steam. Filtration or extraction of the alcohol may be necessary when it is not volatile with steam. No evidence of racemization has been observed with various types of saturated alcohols even on prolonged boiling with concentrated aqueous alkali. Certain alcohols of the allylic type or alcohols otherwise sensitive to alkali or to heating may require more cautious treatment, both in the hydrolysis and in the subsequent final purification. Should the alcohol be foimd to be optically impure at this stage it sometimes may be purified by recrystallization. Either the... 

XLVII), isomeric with hydroxylunacrine (XXXVII), by com jarison of its UV-speetrum with those of model compounds. On treatment with methyl sulfate it forms a quaternary salt which is decomposed by alkali to racemic hydroxylunacridine (XXXVIII) as lunacrinol is ojatically active the reaction must be accompanied by racemization. 

Further support for the structure of methysticin became available when Klohs and co-workers synthesized a racemic mixture of methysticin by the Reformatsky condensation of 3 4 -methylenedioxycinnamaldehyde with methyl 4-bromo-3-methoxycrotonate (Klohs etal., 1959a). The synthetic product exhibited identical IR and UV spectra to those of the naturally occurring methysticin. Further evidence for structural identity was obtained from treatment of both the synthetic and natural materials with alkali to yield identical products corresponding to methystic acid (Klohs etal., 1959a). 

With modification, the aforesaid method can sometimes be applied to the resolution of a neutral compound. Such a resolution is accomplished by first converting the neutral compound into a derivative which can form a salt. Resolution of dl-octanol-2 is an example of the application of this modification. The racemic mixture of the alcohol is converted by treatment with phthalic anhydride into the acid phthalate. In the next step the acid phthalate is reacted with the naturally occurring laevorotatory base, brucine. Fractional crystallisation of the resulting mixture of the brucine salts yields the separated salts. Decomposition of the separated salts with hydrochloric acid removes brucine, and the two resulting acid phthalates are then hydrolysed with alkali to get the d- and 1-forms of octanol-2. 

In addition to the loss of several amino acids by alkaline treatment, and the possibility of toxic compounds being produced, alkali may also cause racemization of amino acids (47), which can also occur with roasting (48). There is need for further nutritive investigations of such racemized products, as well as for fundamental studies on the racemiza-tions of amino acids in different proteins under various conditions (48). 

The first resolution of racemic c/s-2-ACPC was reported only after the isolation of natural cispentacin. Konishi et al. resolved racemic cis-2-benzoyloxycarbonylaminocyclopentanecarboxylic acid by fractional crystallization of the salt formed with (+)-dehydroabietylamine. After repeated crystallizations from acetonitrile, the enantiomers were recovered by treatment with alkali and deprotection by catalytic hydrogenation [2]. 

Racemization of amino acyl residues in food proteins is a reaction that can take place during processing and cooking. This review deals with the occurrence and detection of alkali- and heat-induced racemization in proteins. Differences between calcium hydroxide-and sodium hydroxide-induced racemization and the effects of treatment with these alkalis on protein bioavailability is discussed. 

Recent studies have shown that in addition to the structure of the amino acyl residue, the position of the residue in the peptide (or protein) can have a major effect on racemization (69). Therefore, at the end of an exposure to alkali, and depending on the severity of the treatment, a mixture of the original protein and several D-amino acyl residue-containing proteins is likely to result. The latter are not necessarily Identical, i.e., the D-amlno acyl residues may be located at different positions along the primary structure of the protein, thereby giving rise to a heterogenous mixture of racemized proteins. 

While several laboratories have shown that severe racemiza-tion of proteins can occur during treatment with sodium hydroxide (6,18,22-24,61,62) the effects of other alkalis used in food processing are documented less well. Jenkins, et al. (70) have observed substantial differences in the degree of racemization caused by lime or caustic soda treatment of zein. Lime causes only 50% to 90% of the racemization observed for several amino acyl residues compared to when caustic soda is used. Because a substantial amount of calcium ion remained bound to the protein (approx. 10,000 ppm) compared to l/20th that amount of sodium ion for the caustic soda-treated zein, it is possible that divalent calcium may stabilize the protein making it less susceptible to racemization. Tovar (14) observed increases of 40% to 50% in serine and phenylalanine racemization and a decrease of 30% aspartate racemization for caustic soda-treated fish protein concentrate compared to lime-treated protein (see Table II). These studies indicate that different alkalis have different effects on racemization of proteins specifically, lime may cause less racemization than caustic soda at a similar pH. 

It has been well established that the three major factors involved in racemization are pH, temperature and time of treatment, with Isomerization increasing with increasing pH (above 8), increasing temperature and Increasing time. While exposure to extremes of pH and temperature may be short for most processed materials, it is clear that racemlzatlon of the most labile residues (e.g. serine, aspartate) occurs fairly rapidly. Because alkali-induced racemlzatlon can cause decreased nutritional quality, treated protein materials should be assayed for racemlzatlon. Decreases in nutritional quality due to processing should be a-voided, especially in situations where protein intake may be at marginal levels. Furthermore, the choice of alkali for a particular treatment may affect the level of racemization observed in a protein material. Studies of the effects of different alkalis should be extended to other proteins which are important in the food industry. 

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