Citrus amino acids

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

In the purification of pectinesterase from the fruits of Citrus nat-sudaidai,61 fractional salting-out with ammonium sulfate was followed by chromatography on a column of DEAE-cellulose and by separation of the active fraction on Sephadex G-100. A preparation (purified solution) having a specific activity 460-fold greater than that of the original extract was obtained. Its homogeneity was checked by disc electrophoresis, and its amino acid content was determined and fundamental, kinetic data were obtained. 

Several inferences can be drawn from these data that may suggest the impacts to be expected at the consumer and decomposer levels. Accelerated leaf drop may influence the development of pests—namely, aphids, scale insects, and red citrus mites. Pest populations might be increased if injured leaves had higher concentrations of amino acids or free sugars before abscission (see Chapter 11) or diminished if leaves fell too rapidly. Leaf and fruit drop would provide a larger substrate for populations of decomposer organisms at the soil surface. 

Botanically speaking, citrus is a hesperidium, a berry with a leathery aromatic rind and a fleshy interior divided into sections. As shown by the cross section shown in Fig. 6.1, the exo carp or peel consists of an outer layer called the flavedo which contains oil glands and pigments and a white spongy inner layer called the albedo. The fleshy interior or endocarp of the fruit consists of wedge-shaped sections (segments) filled with multiple fluid-filled sacs or vesicles. These juice sacs constitute the edible portion of a citrus fruit. The cytoplasm contents provide the primary source of the citrus juice. The juice consists primarily of water, sugars, pectins, lipids, terpenes, amino acids, phenolics, carotenoids and minerals. 

The amount of protein in citrus fruit is relatively low (Table II), and the juice and peel have about the same amount (29). Much of the value that is considered as protein is either free amino acids or non-protein constituents which contain nitrogen. 

Among the various free amino acids reported in citrus juices (32), arginine is the only semi-indispensable amino acid that occurs in moderate amounts. The majority of amino acids in citrus are considered to be nonessential according to the classification... 

Table XI. Comparison of Index of Nutrient Quality (INQ) of the Amino Acids of Citrus Products With Those of Various Other Common Foods ...

The protein in citrus is generally associated with the solid portions of the fruit, i.e., the seeds, flavedo, albedo, chroma-tophores, and pulp. Some of these components find their way into the juice along with the available free amino acids during extraction and processing and storage. Studies conducted in our laboratories (42,43,44) and by others (45) have shown that reductions in the pulp content of juice slow the rate of browning. 

Some authors have indicated that the sugar-amino acid reactions of the Maillard type are of minor importance in citrus juices because of the high acidities involved. Studies in our laboratories (42-44) would tend to indicate that, to the contrary, the amino acids and sugars are of more than just minor importance in the darkening of citrus juices. Huffman (42) treated citrus juices with cationic ion-exchange resins to remove amino acids, proteins, and the mineral cations, then restored the cations. 

Other methods of estimating total amino acids exist but have not been widely applied to citrus. Various ninhydrin colorimetric methods are published, but different colors produced by individual amino acids caused quantitation problems. Ting and Deszyck (63J attempted to reduce this problem by using two wavelengths, 400 and 570 nm, for proline and the "ninhydrin blue" amino acids. Differences in molar absorption made the results dependent upon the composition of the standard mixture. 

The development of the commercial amino acid analyzer based on the ion-exchange method led to the rapid accumulation of compositional data on the amino acids in citrus. Many investigators have published amino acid compositional data. A summary from a recent review (76) is presented in Table II. These data make it relatively easy to detect adulterations with amino acids, although the equipment and analyses are expensive for routine testing. 

Table II. Amino Acid Composition (mg/lOOml) of Citrus Juices from...

Other investigators have used maximum and minimum concentration values of specific amino acids as a criteria of authenticity. Brenoe (55) reported means and confidence intervals for all of the amino acids in Greek orange juice. Special emphasis was placed on glycine since it is present in very low levels in citrus juices but is relatively abundant in protein hydrolysates. 

Fuzfai Z and Molnar-Perl I, Gas chromatographic-mass spectrometric fragmentation study of flavonoids as their trimethylsilyl derivatives Analysis of flavonoids, sugars, carboxylic and amino acids in model systems and in citrus fruits. J Chromatogr A 1149 88-101 (2007). 

Although any of several combinations of proteases can be used, ideally, one or more non-specific endopeptidases should be used first to convert the protein into many small peptides. These small peptides can then be degraded to amino acids by aminopeptidases and prolidase (hydrolyzes X-Pro bonds). Sometimes, carboxypeptidases are also used. Although leucine aminopeptidase has been used as the amino-peptidase (see Hill and Schmidt 1962), it may be preferable to use aminopeptidase M (Rohm and Haas, supplied by Henley and Co. of N.Y.), since this enzyme removes most residues at acceptable rates. Leucine aminopeptidase removes hydrophobic residues most rapidly, whereas some other residues are removed very slowly. Most procedures should probably include the use of prolidase (Miles) since many aminopeptidases do not cleave X-Pro bonds at appreciable rates. If it is found that proline is not released quantitatively by these procedures, the use of citrus leaf carboxypeptidase C (Rohm and Haas) can be tried after the initial endopeptidase hydrolysis and before the addition of aminopeptidase M and prolidase. Carboxypeptidase C (also yeast carboxypeptidase Y - see Hayashi et al. 1973) hydrolyzes proline bonds (as well as all others), but if proline is at or adjacent to the NH2 terminus of a peptide, it would probably not be released. In all procedures a control consisting of the enzymes only should be run in parallel with the hydrolyzed sample, and corrections should be made for any amino acids found by analysis of the control. suhic / /< > , mi... 

Amino acid analyses of phosvitin preparations also indicated inhomogeneity. It is striking, however, that an equal number of )3-hydroxyamino acids and of phosphorus atoms were found to be present. The phosphorus is alkali-labile at room temperature. Phosvitin is readily dephosphorylated with the aid of an acid phosphatase from citrus fruits (5). Bone phosphatase, on the other hand, does not liberate phosphorus. Moreover, the base binding capacity of this protein indicates that all of the phosphate groups are present in the form of monoesters with two dissociable hydroxyls. 

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