Chapter 33
Synthesis of L-Ascorbic Acid and Its 2-Phosphate and 2-Sulfate Esters and Its Role in the Browning of Orange Juice Concentrate Eldon C. H. Lee Westreco, Inc., New Milford, CT 06776
The nutritional and health importance, synthesis, stability, and applications of L-ascorbic acid and its derivatives are reviewed. Syntheses and stabilities of the 2-sulfate and 2-phosphate esters of L-ascorbate are described, but only the 2-phosphate ester seems to possess vitamin C activity. L-Ascorbic acid is shown to contribute to the nonenzymatic browning of orange juice concentrate. The oxidative and anaerobic degradation of L-ascorbic acid may be its critical role in this respect.
L-Ascorbic acid is biosynthesized from carbohydrate precursors including glucose and galactose by a variety of plant and animal species. Humans, other primates, and guinea pigs, as well as insects, invertebrates, fishes, and certain bats and birds are not able to synthesize L-ascorbic acid due to the absence of the enzyme L-gulonolactone oxidase (1). The prevention of scurvy has been accepted as thecriteria for estimating the minimal vitamin C requirements (2). Besides the widely accepted roles of L-ascorbic acid in preventing scurvy, facilitating amino acid metabolism, increasing iron absorption, collagen synthesis, and as a biological blocking agent against nitrosamine formation, a recent review on the nutritonal and health aspects of L-ascorbic acid (3) and an October 1991 review in Science by the American Association for the Advancement of Science indicated its important health aspects in increasing immunocompetence, faciliating drug metabolism, and reducing the risk of a wide variety of human disorders including cancer, heart disease, and atherosclerosis. The appropriate intake levels of vitamin C for each of its physiological functions have not yet been fully established. Synthesis of L-Ascorbic Acid Because of its vitamin C potency, useful reducing and antioxidant properties, and low toxicity for pharmaceutical, food, agricultural, and industrial applications (4,5), L-ascorbic acid production has grown continuously. The current commercial
0097-6156/94/0546-0388$06.00/0 © 1994 American Chemical Society
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synthesis of L-ascorbic acid (6,7) and speculation on possible enzymic methods of preparation ( 7) have been recently reviewed. In the Reichstein-Grussner five-step synthesis (8, Figure 1), D-glucose is reduced to sorbitol and then oxidized to L-sorbose by aerobic fermentation. Reaction of L-sorbose with acetone and acid gives 2,3:4,6-m-0-isopropylidene-L-xylo2-hexulofuranose. The free primary alcohol group of the diacetone derivative is oxidized to 2,3:4,6-di-0-isopropylidene-L-xylo-2-hexulosonic acid, which is the converted to L-ascorbic acid by heating with an acid in a non-aqueous medium. The overall yield of L-ascorbic acid from D-glucose is about 50%. A second synthesis is a two-stage fermentative process (9, Figure 2). Here, D-glucose is converted to 2,5-diketo-D-gluconate via a Erwinia sp. and the media is inactivated by adding sodium dodecyl sulfate without isolation. In the second fermentation step the intermediate is reduced to 2-keto-L-gulonic acid via a Corynebacterium sp. The overall conversion of D-glucose to L-ascorbic acid is about 73%. Oxidation of L-Ascorbic Acid L-Ascorbic acid can undergo a two-step oxidation to give dehydroascorbic acid by way of the intermediate monodehydroascorbic acid free radical (10). In aerobic organisms L-ascorbic acid protects biological tissue against activated radicals of oxygen. The autoxidation of L-ascorbate with oxygen in the presence of transition metal ions (77) is important in aerobic systems found in tissues, pharmaceuticals, and foods. In the absence of catalysts, L-ascorbic acid reacts slowly with oxygen (72). The rate of aerobic oxidation of L-ascorbic acid is pH-dependent: the rate of oxidation is more rapid and the degradation is more extensive in an alkaline medium than in an acidic solution (13). A number of enzymes, in particular ascorbic acid oxidase, in foods and biological systems accelerate the oxidation of ascorbic acid (14). These enzymes should be inactivated to prevent oxidative loss of L-ascorbic acid. Synthesis and Stability of the 2-Sulfate and 2-Phosphate Esters of L-Ascorbate Ester and ether derivatives of L-ascorbic acid have been important to determine its chemical structure and biological role, and to modify its solubility and stability (13, 15,16). The reactivity of ascorbic acid toward electrophiles is a function of the ionization and steric environments of the four hydroxyl groups at the C2, C3, C5, and C6 positions. Chemical substitution on the ene-diol hydroxy Is stabilizes the molecule against oxidative decomposition. In aqueous solution, the 3-OH and 2-OH have an ionization constant of pKi=4.17 and pK =11.79, respectively (77). While the 3-OH readily ionizes, the derealization of the electron density among the 0(l)=C(l)-C(2)=C(3)-0(3) ring generating resonance results in low reactivity. Under more basic conditions, the ionization of the 2-OH occurs with the formation of the di-anion, which allows selective substitution of this position with electrophiles in the presence of free hydroxyls at C3, C5, and C6. L-Ascorbate 2-sulfate (18,19) was synthesized in nearly quantitative yield by reacting ascorbate with trimethylamine-sulfur trioxide in alkali (pH 9.5-10.5) at 70°C (Figure 3). The 2-sulfate ester (18,19) was also obtained in 75% yield by 2
390
FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
CH OH -H
HO-
-H
HHO-
CH OH
CH OH
2
HO-
2
2
HO
-OH
100%
HO
Ni, H ,
H
-H
HO
*CHO
l=o
H H OH H
95%
0
H
2
OH
HO
Acetobacter
H * CH OH
* CH OH
2
2
D-Glucose
H
HO
L-Sorbose
Sorbitol
>90%
Acetone H S0 2
4
0°C
ÇH3)2Ç
X
C0 H 2
N
3* | HC 2
V
CH 0 2
Ο I (CH ) C 3
2,3:4,6-di-0-lsopropylideneL-xylo-2-hexulonsonic acid
2
2,3:4,6-di-lsopropylideneL-xylo-2-hexulofuranose
>90%
2-keto-L-Gulonic acid
L-Ascorbic acid
Figure 1. Synthesis of L-ascorbic acid by the Reichstein-Grussner synthesis.
33.
LEE
CH OH H
HO
H
H — O H
ÇH OH
CH OH
2
HO
HO
391
L-Ascorbic Acid and Its 2-Phosphate and 2-Sulfate Esters
2
2
ho Erwiniasp. o
2
HOH-
•H
1) S D S
H
2) C o r y n e b a c t e r i u m s p .
OH
glucose, 0
OH
Q
H
H
H 2
OH
H *CHO
COO" *COO"
D-Glucose
2,5- diketo-D-Gluconate
C0 H 2
2-keto-L-Gulonic acid toluene cone. HCI 110°C * CH OH 2
H-I-OH^
HO
0
OH
L-Ascorbic acid
Figure 2. Synthesis of L-ascorbic acid by the two stage fermentative process.
392
FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
CH OH 2
(CH ) N:SO 3
HO
3
3
OH
L-Ascorbic acid
Trimethylamine-sulfur trioxide
1) alkali (pH 9 . 5 - 1 0 . 5 ) , N , 70°C 2
+
2) c a t i o n - e x c h a n g e resin ( H ) 3) neutralization
CH 0 2
"O
0S0 " 3
L-Ascorbate 2-sulfate
1) D M F , pyridine, 25°C +
2) c a t i o n - e x c h a n g e resin ( H ) 3) neutralization
HO
OH
5,6- O-lsopropylideneL-ascorbic acid
Pyridine-sulf ur trioxide
Figure 3. Synthesis of L-ascorbate 2-sulfate.
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sulfation of 5,6-0-isopropylidene-L-ascorbic acid with pyridine-sulfur trioxide in DMF/pyridine followed by hydrolytic removal of the 5,6-0-isopropylidene blocking group. L-Ascorbate 2-sulfate is approximately 20 times more stable than L-ascorbate towards oxygen in boiling water (18,19). L-Ascorbate 2-sulfate occurs in animals (1), but it has been reported to have vitamin C potency only in finfish (20), not in mammals (21). More recent data (22) contradicts the idea that L-ascorbate 2-sulfate is active in fish. 5,6-O-Isopropylidene-L-ascorbate 2-phosphate was prepared in almost quantitative yield by the action of phosphoryl chloride at 0-5°C on 5,6-0-isopropylidene-L-ascorbate in alkali (pH 12-13) containing a high concentration of pyridine (23,24). After hydrolytical cleavage of the 5,6-acetal group, L-ascorbate 2-phosphate was isolated in 70% yield as its crystalline tricyclohexylammonium salt (Figure 4). L-Ascorbate 2-phosphate has been shown to be an active source of vitamin C in the monkey and guinea pig (25). The 2-phosphate ester, like the 2-sulfate ester, is much more stable in air than L-ascorbic acid (23). The 2-phosphate ester might be used as a stable source of vitamin C for foods and feeds in which the ingredients are free of phosphatase activity. Non-enzymatic Browning in Orange Concentrate Presently, most fruit concentrates are frozen for long term storage in North America. With aseptic packaging, processors could eliminate enormous freezing process and shipping costs. Browning in fruit concentrates on storage at ambient temperatures, however, constitutes a major problem. Wagner (26) reported that the quality of fruit concentrates deteriorates in three or four months. A great deal of research was carried out on the browning of various fruit products during and subsequent to World War II (27). Three hypotheses generally reported were: 1) Maillard or melanoidin condensation (28), 2) ascorbic acid oxidation, and 3) active-aldehyde reaction. Many conflicting results and conclusions, however, have been found. Fruit concentrate browning is mainly due to non-enzymatic browning because the enzyme activity has been thermally inactivated. Orange concentrate, with relatively high levels of ascorbic acid, gives more serious browning than apple, pear, and grape concentrates, which have low levels of ascorbic acid. Therefore, orange concentrate was chosen to investigate ascorbic acid browning. The browning of orange concentrate was studied by using cation and anion exchange fractionation of orange juice components and removal of residual oxygen with glucose oxidase. The study was directed to investigate the three types of reactions: 1) the oxidative browning reaction of ascorbic acid, 2) the anaerobic browning reaction of ascorbic acid at strongly acidic pH, and 3) the Maillard reaction of amino acids and reducing sugars. Effect of Glucose Oxidase on the Oxidative Browning of Ascorbic Acid The use of glucose oxidase for the removal of oxygen from beverages has been suggested (29). The glucose oxidase used (DeeO L-750, Miles Lab.) had an optimum pH range from 4.5 to 6.5 and optimum temperature range from 30 to 65°C. Acid-stable glucose oxidase (e.g. Fermo Biochemical Co., Illinois) might perform more effectively in low-pH orange concentrate.
394
FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
H Ç-O^CH 2
H
0
3
CH
3
HO
OH
5,6- Olsopropylidene-L-ascorbic acid alkali (pH 1 2 - 1 3 ) pyridine 2.5 M 0-5°C POCI 3
H C-0 2
CH
3
H-r-O^CK
HO
OH
5,6- Olsopropylidene-L-ascorbate 2-phosphate
+
1) c a t i o n - e x c h a n g e resin ( H ) 2) neutralization
CH OH 2
H4-OH^°
L-Ascorbate 2-phosphate Figure 4. Synthesis of L-ascorbate 2-phosphate.
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The effect of glucose oxidase on browning is given in Figures 5 and 6. The orange juice syrup with addition of glucose oxidase showed less browning than the untreated control. The aerobic oxidation of ascorbic acid occurs rapidly to dehydroascorbic acid when metal catalysts, particularly copper or iron, are present (77). Dehydroascorbic acid is converted to diketogulonic acid irreversibly, and then to furfural by decarboxylation and dehydration, with the formation of brown pigments by subsequent polymerization (30). Effect of pH on the Anaerobic Browning of Ascorbic Acid The effect of pH on the browning of orange juice syrup in Figure 7 showed that lower pH caused more browning. In strong acid the hydrogen ion catalyzed the decomposition of ascorbic acid by hydrolysis of the lactone ring, and further decarboxylation and dehydration to furfural and acids (31). Cation Exchange Fractionation +
The use of cation ( H form) exchange fractionation removed about 77% of total nitrogen and proteins (Table I), and 99.5% of free amino acids (Table II). Removal of the nitrogenous constituents would eliminate the non-enzymatic browning reactions from the condensation of the nitrogenous constituents, particularly amino acids with sugars (28), dehydroascorbic acid (7), and melanoidin intermediates (27, 28). The cation ( H form) exchange fractionation also removed the metallic cations (86% of total ash in Table I). Removal of the metallic ions, particularly iron and copper, reduces the rate of aerobic oxidation of ascorbic acid (11,12). The effect of cation ( H form) exchange fractionation on the browning of orange juice syrup is shown in Figures 5 and 6, and its effect on the browning of orange juice concentrate is shown in Figure 8. +
+
Cation and Anion Exchange Fractionation The effect of cation and anion exchange fractionation on orange concentrate is given in Table III. The cation ( H form) exchange resin removed the positive-charged proteins (77% of total proteins) and metallic ions whereas the anion (OH- form) exchange resin removed the negative-charged proteins (8% of total proteins), ascorbic acid, acidulants, and inorganic anions. The loss of ascorbic acid during cation and anion exchange fractionation in Table IV shows about 72%. The use of cation and anion exchange fractionation gave the least browning of orange juice concentrate in Figure 8. Apparently, removal of the browning precursors, ascorbic acid, nitrogenous compounds particularly amino acids, and metallic ions, minimized the non-enzymatic browning. +
Conclusion This study has indicated the critical role of ascorbic acid in the non-enzymatic browning of orange juice concentrate and syrup. The contribution of each reaction pathway would depend on the relative amounts of the browning precursors, oxygen, pH, and metallic catalysts. Further study may be suggested to elucidate the reaction pathways and mechanisms. It would be challenge to develop practical solutions for preventing or minimizing the browning problem.
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FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
Figure 6. Effect of storage temperature on the browning of orange juice syrup at pH 3.7 for 45 days.
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Days stored Figure 7. Effect of pH on the browning of orange juice syrup at 38°C.
Figure 8. Effect of ion exchange fractionations on the browning of orange concentrate at pH 3.7 and 37°C.
398
FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
Table I. Cation Exchange Fractionation of Orange Concentration Analysis
Control
Total solids (%) Total nitrogen (%) Total proteins (%) Total ash (%) Brix (20°C) Water activity PH Titratable acidity as citric acid, % a
63.50 0.61 3.50 2.35 64.00 0.83 3.70 4.90
Treatment
Loss (%)
60.60 0.14 0.80 0.33 (3.10 ) 60.30 0.86 2.00 (3.7 ) 4.90 a
a
4.6 77.4 77.1 86.0 5.8 — — 0
After pH adjusted to 3.7 with KOH.
Table II. Effect of Cation Exchange Fractionation on the Free Amino Acids of Orange Concentrate Amino acid Lysine Histidine Arginine Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Total amino acids Free amino acids removed (%)
+
Control (mg/g)
Cation (H ) (mg/g)
0.18 0.07 1.62 PI PI PI 0.10 2.90 0.09 0.48 0.10 0.02 0.03 0.02 0.06 0.17
