EFFECT OF VITAMIN B6 ON PHENYLALANINE METABOLISM IN THE BRAIN OF NORMAL AND p-CHLOROPHENYLALANINE-TREATED RATS

Abstract— A phenylketonuria-like state was produced in the preweanling rat, and the metabolism of phenylalanine in the normal and phenylketonuric brain was compared. The effect of B₆ vitamers on the disposition of phenylalanine was also investigated. Phenylalanine was metabolized mainly by transamination and to a lesser extent by decarboxylation in both the normal and phenylketonuric-like brain. Small amounts of amine were detected in all the brains throughout the experimental period. More than 95 percent of the metabolized amino acid appeared as aromatic acids, which steadily accumulated and remained in the brain for the duration of the experiment. No change in the metabolic pattern was produced by pyridoxol. In striking contrast, pyridoxamine prevented the accumulation of acidic metabolites in the brains of all animals tested. We suggest that pyridoxamine phosphate and/or pyridoxamine is actively associated with the removal of excess keto acids and aldehydes from the brain.     

Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.     

Exploring routes to stabilize a cationic pyridoxamine in an artificial transaminase: site-directed mutagenesis versus synthetic cofactors

Two artificial transaminases were assembled by linking a pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between alpha-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The k(cat) values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group.     

The oxidation of Schiff bases of pyridoxamine with α-oxo acids by manganous ions and peroxidase

1. Oxygen was taken up rapidly when pyruvate was added to mixtures of pyridoxamine and Mn(2+) ions after lag periods that were shortened by peroxidase (donor-hydrogen peroxide oxidoreductase, EC 1.11.1.7). 2. The total oxygen uptake was proportional to the pyridoxamine added and was accompanied by the disappearance of pyridoxamine; the pyruvate acted catalytically and hydrogen peroxide was not formed. 3. At pH6 more than half the pyridoxamine that disappeared was accounted for as pyridoxal and ammonia; it is suggested that the primary reaction is the oxidative deamination of the pyridoxamine. 4. Results were similar when alpha-oxobutyrate or glyoxylate were substituted for pyruvate, except that the reactions were slower and the yield of pyridoxal less. 5. The oxidative decarboxylations of alpha-oxoglutarate and phenylpyruvate are catalysed by Mn(2+) ions and these reactions are activated by peroxidase; pyridoxamine increased both the rates and total oxygen uptakes in these reactions, and ammonia was produced. 6. The lag periods in the oxidation of mixtures of pyridoxamine and alpha-oxo acids, catalysed by Mn(2+) ions, were also shortened by traces of colloidal manganese dioxide. 7. It is suggested that the activating effect of peroxidase depends on its catalysis of manganese oxidation.     

Stereochemistry of holoaspartate transaminase after modification of the active site Lys-258.

Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transaminase is a stereospecific process. After carbamylation of the active site Lys-258, the stereospecificity of the reaction in 2H2O is retained. By a correlation method, it is shown that addition occurs from the si side of the complex and the pyridoxamine phosphate produced is deuterated at position pro-S of the pyridoxamine methylene group. These results constitute a demonstration for the stereochemstry of a half-transamination process of the phosphorylated coenzyme under single turnover conditions. They also illustrate that free Lys-258 is not required to maintain stereospecificity and cast doubts on the implication of this residue as a participant in C4 proton addition during catalysis by the native form of this mammalian enzyme.     

Unexpected isomeric equilibrium in pyridoxamine Schiff bases

Pyridoxamine is a vitamin B₆ derivative involved in biological reactions such as transamination, and can also act as inhibitor in protein glycation. In both cases, it has been reported that Schiff base formation between pyridoxamine and carbonyl compounds is the main step. Nevertheless, few studies on the Schiff base formation have been reported to date. In this work, we conduct a comparative study of the reaction of pyridoxamine and 4-picolylamin (a pyridoxamine analog) with various carbonyl compounds including propanal, formaldehyde and pyruvic acid. Based on the results, 4-picolylamin forms a Schiff base as end-product of its reactions with propanal and pyruvic acid, but a carbinolamine with formaldehyde. On the other hand, pyridoxamine forms a Schiff base with the three reagents, but the end-product is in equilibrium with its hemiaminal form, which results from the attack of the phenolate ion of the pyridine ring on the imine carbon. This isomeric equilibrium should be considered in studying reactions involving amine derivatives of vitamin B₆.     

EFFECT OF HYPERPHENYLALANINEMIA ON VITAMIN B6, METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The turnover of the different forms of B₆ vitamers in the brains of normal and hyperphenylalaninemic preweanling rats was compared after administration of a load of [¹⁴C]pyridoxol. Metabolic transformations occurred in the following sequence: oxidation of pyridoxol to pyridoxal, which was in turn phosphorylated to the 5'-phosphate ester. No significant amount of pyridoxamine was formed during the 8-h experimental period. Pyridoxamine 5'-phosphate was derived from pyridoxal 5'-phosphate. The specific radioactivity of pyridoxal phosphate in the hyperphenylalaninemic brain was significantly lower and increased at a slower rate than in control brains. This difference could not be accounted for by either a deficient supply or inhibited activity of the enzyme, pyridoxal kinase. The synthesis of pyridoxamine 5'-phosphate in the experimental animals also lagged behind the controls. Decreased activity of enzymes dependent on pyridoxal phosphate as cofactor would explain the slower turnover of this B₆-coenzyme.     

Inactivation of avian progesterone receptor binding to ATP-Sepharose by pyridoxal 5'-phosphate

The affinity of progesterone receptor from hen oviduct for ATP-Sepharose was diminished by preincubation with pyridoxal 5′-phosphate. This effect was specific for pyridoxal 5′-phosphate since the related compounds, pyridoxal, pyridoxine, pyridoxamine and pyridoxamine 5′-phosphate, were not effectors. The inactivation was easily reversed by the addition of the primary amine, Tris. However, in the presence of the reducing agent NaBH₄, the inhibitory effect of pyridoxal 5′-phosphate was irreversible. The results suggest that pyridoxal 5′-phosphate forms a Schiff base with a critical amino group, presumably at the nucleotide binding site of the progesterone receptor.     

Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase

Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E.

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)     

Amine Transaminase

An enzyme “amine transaminase”, which catalyzed transamination between amines and α-keto acids, was found to occur in certain fermentative bacteria, such as Escherichia coli and Aerobacter aerogenes. Using a partially purified enzyme preparation obtained from cell extract of E. coli, some properties of the enzyme were investigated. α-Ketoglutaric acid appeared to be the most efficient amino acceptor and substitution of α-ketoglutaric acid by other α-keto acid resulted in much lower activity. Putrescine, cadaverine and hexamethylenediamine were found to be active as amino donors, but the other monoamines, diamines and polyamines were inert. Treatment of the enzyme with acid ammonium sulfate resolved the enzyme into apo- and coenzyme. The apoenzyme was well reactivated by pyridoxal phosphate as well as pyridoxamine phosphate. Physiological role of the amine transaminase was suggested in relation to the metabolism of amines in bacterial cells.     

Interaction of aspartate aminotransferase with amino acids

1. The capacity of various amino acids to convert the pyridoxal form of aspartate aminotransferase into the pyridoxamine form has been investigated. 2. Glutamate has the highest converting capacity; aspartate, α-aminopimelate, α-aminoadipate and other amino acids follow. 3. The converting capacity of the various amino acids assayed is connected with their structural features. 4. A possible role of amino acids as secondary substrates of aspartate aminotransferase is suggested.     

Fluorimetric assay of sialic acids.

A fluorimetric assay has been developed for sialic acids in which sialic acids react with pyridoxamine to give fluorescent compounds in the presence of zinc ion and pyridine. This assay method is specific for unbound sialic acids and is a simple and sensitive procedure compared with the thiobarbituric acid assay of sialic acids.     

Fern L-methionine decarboxylase: kinetics and mechanism of decarboxylation and abortive transamination

L-Methionine decarboxylase from Dryopteris filix-mas catalyzes the decarboxylation of L-methionine and a range of straight- and branched-chain L-amino acids to give the corresponding amine products. The deuterium solvent isotope effects for the decarboxylation of (2S)-methionine are D(V/K) = 6.5 and DV = 2.3, for (2S)-valine are D(V/K) = 1.9 and DV = 2.6, and for (2S)-leucine are D(V/K) = 2.5 and DV = 1.0 at pL 5.5. At pL 6.0 and above, where the value of kcat for all of the substrates is low, the solvent isotope effects on Vmax for methionine are 1.1-1.2 whereas the effects on V/K remain unchanged, indicating that the solvent-sensitive transition state occurs before the first irreversible step, carbon dioxide desorption. The enzyme also catalyzes an abortive decarboxylation-transamination reaction in which the coenzyme is converted to pyridoxamine phosphate [Stevenson, D. E., Akhtar, M., & Gani, D. (1990a) Biochemistry (first paper of three in this issue)]. At very high concentration, the product amine can promote transamination of the coenzyme. However, the reaction occurs infrequently and does not influence the partitioning between decarboxylation and substrate-mediated abortive transamination under steady-state turnover conditions. The partition ratio, normal catalytic versus abortive events, can be determined from the amount of substrate consumed by a known amount of enzyme at infinite time, and the rate of inactivation can be determined by measuring the decrease in enzyme activity with respect to time. For methionine, the values of Km as determined from double-reciprocal plots of concentration versus inactivation rate are the same as those calculated from initial catalytic (decarboxylation) rate data, indicating that a single common intermediate partitions between product formation and slow transamination. The partition ratio is sensitive to changes in pH and is also dependent upon the structure of the substrate; methionine causes less frequent inactivation than either valine or leucine. The pH dependence of the partition ratio with methionine as substrate is very similar to that for V/K. Both curves show a sharp increase at approximately pH 6.25, indicating that a catalytic group on the enzyme simultaneously suppresses the abortive reaction and enhances physiological reaction in its unprotonated state. Experiments conducted in deuterium oxide allowed the solvent isotope effects for the partition ratio and the abortive reaction to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stereochemistry of the reactions of glutamate-1-semialdehyde aminomutase with 4,5-diaminovalerate

Conversion of glutamate 1-semialdehyde to the tetrapyrrole precursor, 5-aminolevulinate, takes place in an aminomutase-catalyzed reaction involving transformations at both the non-chiral C5 and the chiral C4 of the intermediate 4,5-diaminovalerate. Presented with racemic diaminovalerate and an excess of succinic semialdehyde, the enzyme catalyzes a transamination in which only the l-enantiomer is consumed. Simultaneously, equimolar 4-aminobutyrate and aminolevulinate are formed. The enzyme is also shown to transaminate aminolevulinate and 4-aminohexenoate to l-diaminovalerate as the exclusive amino product. The interaction of the enzyme with pure d- and l-enantiomers of diaminovalerate prepared by these reactions is described. Transamination of l-diaminovalerate yielded aminolevulinate quantitatively showing that reaction at the C5 amine does not occur significantly. A much slower transamination reaction was catalyzed with d-diaminovalerate as substrate. One product of this reaction, 4-aminobutyrate, was formed in the amount equal to that of the diaminovalerate consumed. Glutamate semialdehyde was deduced to be the other primary product and was also measured in significant amounts when a high concentration of the enzyme in its pyridoxal form was reacted with d-diaminovalerate in a single turnover. Single turnover reactions showed that both enantiomers of diaminovalerate converted the enzyme from its 420-nm absorbing pyridoxaldimine form to the 330-nm absorbing pyridoxamine via rapidly formed intermediates with different absorption spectra. The intermediate formed with l-DAVA (lambdamax = 420 nm) was deduced to be the protonated external aldimine with the 4-amino group. The intermediate formed with d-DAVA (lambdamax = 390 nm) was deduced to be the unprotonated external aldimine with the 5-amino group.     

Pyridoxamine analogues scavenge lipid-derived gamma-ketoaldehydes and protect against H2O2-mediated cytotoxicity

Isoketals and levuglandins are highly reactive gamma-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether pyridoxamine, which has been shown to scavenge alpha-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive gamma-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic gamma-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of pyridoxamine with the 15-E2-isoketal, including a stable lactam adduct. Two lipophilic analogues of pyridoxamine, salicylamine and 5'-O-pentylpyridoxamine, also formed lactam adducts when reacted with 15-E2-isoketal. When we oxidized arachidonic acid in the presence of pyridoxamine or its analogues, pyridoxamine-isoketal adducts were found in significantly greater abundance than the pyridoxamine-N-acyl adducts formed by alpha-ketoaldehyde scavenging. Therefore, pyridoxamine and its analogues appear to preferentially scavenge gamma-ketoaldehydes. Both pyridoxamine and its lipophilic analogues inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic pyridoxamine analogues provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of pyridoxamine and lipophilic pyridoxamine analogues to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions.     

Uptake and metabolism of N-(4'-pyridoxyl)amines by isolated rat liver cells.

Uptake and metabolism of [3H]pyridoxine and 3H-labeled N-(4'-pyridoxyl)amines by isolated rat liver cells were studied at physiological concentration (0.5 microM) of vitamin B6 by using both membrane filtration and centrifugation methods for removal of radiolabeled solutes after incubations with cells. It was found that the characteristics of import of N-(4'-pyridoxyl)amines into liver cells is similar to those of import of natural vitamin B6. Upon entry each 4'(N)-substituted pyridoxamine was converted to its 5'-phosphate and then oxidized to release pyridoxal 5'-phosphate and the original amine. Considerable size of the amine substituent is tolerated for transport and metabolism, but a charged function impedes entry. The amount of released pyridoxal 5'-phosphate (and therefore the amount of released original amine) is controlled partially by the size of the amine affixed to B6 and partially by the enzymatic steps involved. This system illustrates how biologically active amines can be piggybacked onto a vitamin that gains facilitated entry to cells that have the enzymatic means to release the free amine for subsequent effects within the cell.     

Syntheses,properties, and use of fluorescent N-(5′-phospho-4′-pyridoxyl)amines in assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A sensitive and simple fluorometric assay has been developed for detection of pyridoxamine (pyridoxine) 5′-phosphate oxidase. This technique utilizes fluorescent N-(5′-phospho-4′-pyridoxyl)amines as substrates that, upon incubation with the oxidase, release the free fluorescent amine. The substrates were prepared by condensation of pyridoxal 5′-phosphate with fluorescent amines and subsequent hydrogenation of the Schiff bases. Since N-(1-naphthyl)ethylenediamine is 15 times less fluorescent in the intramolecularly quenched substrate than the product amine, the direct increase of fluorescence, as well as selective extraction of more fluorescent product, can be utilized for assay. The apparent K_m value for this substrate is 8 μm, which is slightly less than that of pyridoxamine 5′-phosphate; V is larger than the natural substrate value. The greater sensitivity gained by this fluorimetric method allows detection of the oxidase in smaller quantities than can be determined by the conventional colorimetric assay.     

红枣贮藏期果面微生物对碳源的利用及主成分分析

Biolog方法就是微生物在利用碳源过程中产生的自由电子, 与四唑盐染料发生还原显色反应, 颜色的深浅可以反映微生物对碳源的利用程度。采用Biolog方法, 研究红枣贮藏期果面微生物对FF和ECO微孔板上碳源的利用情况, 进行主成分分析(Principal component analysis, PCA)。羧酸类、吐温类、碳水化合物、酯类、氨基酸类及胺类碳源是红枣贮藏期果面微生物群落在FF和ECO微孔板上利用的主要碳源。随着贮藏时间的延长, 红枣果面微生物对碳源的利用情况差异较大, 用保鲜剂处理过的红枣果面微生物对碳源的利用远远低于未处理的红枣果面微生物, 而且贮藏时间越长, 果面微生物对碳源的利用程度越高。利用ECO微孔板上31种碳源作PCA, 第一主成分特征值的贡献率为78.54%, 第二主成分特征值的贡献率为19.06%。