Chapter 15 vitamin c

15.2 Chemical Structure, Biopotency, andPhysicochemical Properties 15.2.1 Structure and Potency The term vitamin C is used as the generic descriptor for all compounds exhibiting qualitat

Vitamin C

15.1 Background

Humans, in common with other primates, guinea pigs, and fruit-eating bats, rely on their diet to provide vitamin C in the form of ascorbic acid The functions of ascorbic acid are based primarily on its properties

as a reversible biological antioxidant

Hydroxylase enzymes that require iron or copper ions as cofactors also require the specific presence of ascorbic acid as an electron donor to main-tain these ions in the reduced form In the synthesis of collagen, the initial polypeptide undergoes post-translational modifications to produce the crosslinked triple helix These modifications entail the hydroxylation of proline and lysine residues by enzymes that require ferrous ions Other iron-containing hydroxylases are required for the post-translational modification of osteocalcin (a protein involved in bone mineralization) and protein C (one of the blood-clotting factors) Iron-dependent hydroxylations also feature in the synthesis of carnitine from protein-incorporated lysine Many peptide hormones and hormone-releasing factors are synthesized as precursor molecules that, after a series of copper-dependent modifications, are converted to their active forms A copper-dependent enzyme also catalyzes the hydroxylation of dopamine

to noradrenaline

Ascorbic acid, in collaboration with a-tocopherol and b-carotene, plays

an important role in the defense against cellular damage by oxidants

In this role, ascorbic acid scavenges many types of free radical and also regenerates the reduced form of a-tocopherol

A deficiency of vitamin C results in scurvy, the primary symptoms of which are hemorrhages in the gums, skin, bones and joints, and the failure of wound healing These symptoms are accompanied by listless-ness, malaise, and other behavioral effects

Ascorbic acid is generally regarded as being nontoxic, although exces-sive daily amounts can cause an increased production of oxalic acid in some individuals, leading to an increased risk of kidney stone formation

15.2 Chemical Structure, Biopotency, and

Physicochemical Properties

15.2.1 Structure and Potency

The term vitamin C is used as the generic descriptor for all compounds exhibiting qualitatively the biological activity of ascorbic acid The

acid) is also not found in natural products, apart from its occurrence in certain microorganisms It is, however, a byproduct of biosynthetic vitamin C, produced from glucose by a combined chemical and microbial procedure, and has been detected in beer after addition of commercial vitamin C [1] Erythorbic acid possesses similar reductive properties to

-ascorbic acid in guinea pigs [2,3]

Ascorbic acid is used extensively in food technology as a stabilizer for the processing of beverages, wines, and meat products Ascorbyl palmi-tate is a stable, fat-soluble form of the vitamin which does not occur in

OH HO

OH OH C

CH 2 OH

H

C

CH2OH

OH H

OH OH H

C

CH 2 OH

H HO

OH HO

C

CH 2 OH

H

L -Ascorbic acid D -Ascorbic acid

L -Isoascorbic acid D -Isoascorbic acid

(Erythorbic acid)

Epimers

1 2 3 4 5 6

Enantiomers (mirror images)

Enantiomers (mirror images)

H

FIGURE 15.1

Stereoiosomers of 2-hexenono-1,4-lactone.

nature It exhibits the full antiscorbutic activity of ascorbic acid on a molar basis: that is, 1 g of ascorbyl palmitate is equivalent to the potency of 0.425 g of ascorbic acid Erythorbic acid is cheaper to manufacture on a

sub-stitute erythorbate for ascorbate when, for technological reasons, the anti-oxidant or reducing properties and not the vitamin C activity of the additive is required In the United Kingdom and certain other countries, erythorbate is not permitted as an antioxidant, and it is also prohibited for use with raw and unprocessed meats

acid, forming the ascorbyl radical anion (also known as semidehydroas-corbate) as an intermediate (Figure 15.2) Dehydroascorbic acid possesses full vitamin C activity because it is readily reduced to ascorbic acid in the animal body Dehydroascorbic acid is not an acid in the chemical sense, as

it lacks the dissociable protons that ascorbic acid has at the carbon 2 and 3 positions

15.2.2 Physicochemical Properties

15.2.2.1 Solubility and Other Properties

Ascorbic acid is an almost odorless white or very pale yellow crystalline powder with a pleasant sharp taste and an mp of about 1908C (with decomposition) Pure dry crystalline ascorbic acid and sodium ascorbate are stable on exposure to air and daylight at normal room temperature for long periods of time Commercial vitamin C tablets possess virtually their original potency even after 8-yr storage at 258C [4] Ascorbic acid is readily soluble in water (33 g/100 ml at 258C), less soluble in 95% ethanol (3.3 g/100 ml), absolute ethanol (2 g/100 ml), acetic acid (0.2 g/100 ml), and acetonitrile (0.05 g/100 ml) and insoluble in fat sol-vents [5] A 5% aqueous solution of ascorbic acid has a pH of 2.2 – 2.5, the acidic nature being due to the facile ionization of the hydroxyl

– O

HCOH

H

CH 2 OH

HCOH

H

CH 2 OH

HCOH

H

CH 2 OH

•

(a)

OH

FIGURE 15.2

Oxidation of ascorbate (a) L -Ascorbate (AH2), (b) ascorbyl radical anion (A2†), and (c) dehydroascorbic acid Note the delocalized unpaired electron in the ascorbyl radical anion.

resistant to ionization (pK2¼ 11.79) [6] Sodium ascorbate is freely soluble

in water (62 g/100 ml at 258C; 78 g/100 ml at 758C) and practically insolu-ble in ethanol, diethyl ether, and chloroform; the pH of an aqueous sol-ution is 5.6– 7.0

Ascorbyl palmitate is practically insoluble at 258C in water (0.2 g/100 ml), soluble in ethanol (20 g/100 ml), and slightly soluble in diethyl ether The solubility in vegetable oils at room temperature is very low (30 mg/100 ml) but increases sharply with increasing temperature

The carbonyl enediol group of ascorbic acid confers strong reducing properties to the molecule, as indicated by its ability to reduce Fehling’s

or Tollen’s solution at room temperature The redox potential of the first

15.2.2.2 Stability in Aqueous Solution

Oxidation of ascorbic acid follows first-order kinetics in the pH range 3– 7

in aqueous model systems containing traces of copper Stability is higher

in the pH range 3.0– 4.5 than in the range 5.0 – 7.0 [7] At alkaline pH, ascorbic acid is unstable At pH 1, the ionization of ascorbic acid is sup-pressed, and the fully protonated molecule is relatively slowly attacked

by oxygen Consequently, the rate of oxidation of ascorbic acid accelerates

as the pH is increased from 1.5 to 3.5

Dehydroascorbic acid in solution at neutral or alkaline pH undergoes a nonreversible oxidation to form the biologically inactive, straight-chained compound, 2,3-diketogulonic acid The half-life for this breakdown is

6 min at 378C [8], 2 min at 708C, and less than 1 min at 1008C [9] Dehydroascorbic acid is, however, stable for several days at 48C at pH 2.5 –5.5 [5]

15.3 Vitamin C in Foods

15.3.1 Occurrence

The ascorbic acid and dehydroascorbic acid contents of some vegetables the observed concentrations found in these samples, but they can vary ation, maturity, climate, sunlight, method of harvesting, and storage all can affect the levels of vitamin C

Fresh fruits (especially blackcurrants and citrus fruits) and green veg-etables constitute rich sources of vitamin C Potatoes contain moderate amounts but, because of their high consumption, represent the most

vari-important source of the vitamin in the British diet Liver (containing

10 –40 mg/100 g), kidney, and heart are good sources, but muscle meats and cereal grains do not contain the vitamin Human milk provides enough ascorbic acid to prevent scurvy in breast-fed infants, but preparations of cow’s milk are a poor source owing to oxidative losses incurred during processing Cabbage and other brassica contain a bound form of ascorbic acid known as ascorbigen, which exhibits

15 –20% bioavailability in guinea pigs [12]

TABLE 15.1

Vitamin C Content of Some Vegetables and Fruits

Concentration (mg /100 g) a,b

Vegetables

Broccoli

Cabbage

Cauliflower, fresh, raw 54.0 + 1.0 8.7 + 0.6 63 + 1 Spinach

Microwaved (4 min) 48.3 + 3.7 5.8 + 0.6 54 + 4 Peppers

Potatoes (without skin)

Fruits

Oranges

a Values reported are mean + standard deviation based on three measurements When no values are listed, the concentration was ,1 mg/100 g sample.

b Foods analyzed by HPLC together with robotic extraction procedures.

Source: From Vanderslice, J.T., Higgs, D.J., Hayes, J.M., and Block, G., J Food Comp Anal., 3,

105, 1990 With permission.

15.3.2 Stability

Ascorbic acid is very susceptible to chemical and enzymatic oxidation during the processing, storage, and cooking of food The catalyzed oxi-dative pathway of ascorbic acid degradation is the most important reac-tion pathway for loss of vitamin C in foods In the presence of molecular oxygen and trace amounts of transition metals [particularly copper(II) and iron(III)], a metal –oxygen– ascorbate complex is formed This complex has a resonance form of a diradical that rapidly decomposes

to give the ascorbate radical anion, the original metal ion, and hydrogen peroxide The radical anion then rapidly reacts with oxygen to give dehydroascorbic acid [13]

In the absence of free oxygen, an anaerobic pathway of ascorbic acid degradation leads to the formation of diketogulonic acid [13] The rate

of degradation is maximum at pH 3 –4 and therefore this pathway could be responsible for the anaerobic loss of vitamin C in canned grape-fruit and orange juices, which have a pH of ca 3.5 Degradation of ascorbic acid beyond diketogulonic acid is closely tied to nonenzymatic browning in some food products [13]

The enzyme mainly responsible for enzymatic degradation of ascorbic acid in plant tissues after harvesting is ascorbate oxidase (EC 1.10.3.3), which catalyzes the oxidation of ascorbic acid to dehydroascorbic acid This enzyme exhibits maximum activity at 408C and is almost completely inactivated at 658C [14] Hence rapid heating, such as the blanching

of fruit and vegetables or the pasteurization of fruit juices, prevents

TABLE 15.2

Range of Vitamin C Values in Some Vegetables and Fruits

Sample

Total Vitamin C Content (AA þ DHAA) (mg/100 g)

Oranges

Source: From Vanderslice, J.T and Higgs, D.J., Am J Clin Nutr.,

54, 1323S, 1991 With permission.

the action of this enzyme during post-process storage The ascorbic acid in intact plant tissue is protected from ascorbate oxidase and other enzymes

by cellular compartmentation However, when tissues are disrupted after bruising, wilting, rotting, or during advanced stages of senescence, the enzymes gain access to the vitamin and begin to destroy it

Enzymatic destruction of ascorbic acid in plant tissues begins as soon as

a crop is harvested Maintaining cool conditions during transport and storage can markedly reduce vitamin C loss in some vegetables For example, newly harvested peas retained 70% of their ascorbic acid content after 14 days’ storage at 48C compared with 20% retention at ambient temperature (208C) Corresponding retentions for broccoli were

90 and 30% [15]

Vegetables grown for commercial freezing are processed as soon as possible after harvest Some loss of vitamin C takes place during blanch-ing, but little further loss occurs during deep frozen storage Blanching losses are greatest in green leafy vegetables with large surface areas Thus, steam blanching of broccoli decreased ascorbic acid by about 30%, whereas losses in green beans were only slight [16] In a study of frozen stored green beans [17], no significant oxidation of ascorbic acid occurred during the blanching and freezing steps During frozen storage, there was a progressive conversion of ascorbic acid to

The dehydroascorbic acid was very stable at freezer temperatures, the average loss after 250 days of frozen storage being only 8% Thus vitamin C, in the form of dehydroascorbic acid, is well retained during the frozen storage of green beans

Chemical oxidation of ascorbic acid is lowered during processing by carrying out vacuum deaeration and inert gas treatment where feasible The headspace in containers should be minimized and hermetically sealed systems used Ascorbic acid is very stable in canned or bottled foods after the oxygen in the headspace has been used up, provided the food is not subjected to high-temperature storage or exposed to light In contrast to glass containers, plastic bottles and cardboard cartons are permeable to oxygen, so a lowered vitamin C retention is to be expected Bronze, brass, copper, and iron equipment should be avoided, while sequestering agents such as ethylenediaminetetraacetic acid (EDTA), polyphosphates, and citrates prevent the catalytic action of traces of copper and iron The sulfites and metabisulfites, which are added to

acid in addition to their role as antimicrobial agents The addition of food-grade antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate also protect the vitamin The loss of ascorbic acid from orange juice correlates with the amount

of oxygen initially present in the headspace and that dissolved in the juice,

and with temperature and storage time [18 – 20] In the presence of dissolved oxygen, ascorbic acid decomposition is predominantly aerobic, but when the dissolved oxygen is depleted decomposition con-tinues by an anaerobic pathway that is mainly influenced by temperature [18] The loss of ascorbic acid also correlates with an increase in the browning of the juice [20] Light has little or no effect upon ascorbic acid levels in orange juice [19] The vitamin loss is rapid in the early stage of storage, coincident with the consumption of dissolved oxygen, and then becomes gradual The packaging of orange juice in an exper-imental oxygen-scavenging film reduced ascorbic acid loss in the first 3 days at 258C compared with the loss from juice packaged in a film with

no oxygen scavenger [20] This demonstrated that the oxygen scavenger can remove oxygen from the juice before it has the opportunity to react with the ascorbic acid Furthermore, the residual oxygen-scavenging capacity in the experimental film provided an ongoing barrier to oxygen permeation

The storage of broccoli spears in elevated (20%) carbon dioxide atmosphere suppresses tissue respiration and ethylene production rates, whilst also delaying loss of chlorophyll and ascorbic acid [21] The packaging of broccoli spears in polymeric film modified the atmosphere

by elevating carbon dioxide to 8% and lowering oxygen to 10% While ascorbic acid retention in packaged broccoli dropped about 15% from initial values in the first 48 h, losses were minimal during the following

48 h In contrast, the degradation of ascorbic acid in nonpackaged samples showed a steady decline over time and decreased 31% by

96 h [22]

Ascorbic acid can leach away from fruits and vegetables during proces-sing or cooking This is of little importance with canned, bottled, or stewed fruits where the juice is eaten with the tissue, but may represent

a serious loss with vegetables, where the liquor is drained away before serving If vegetables are steamed or pressure-cooked instead of boiled, the leaching effect is greatly reduced, but a greater loss of ascorbic acid

is to be expected from oxidation Cold water washing or steeping does not normally leach out a significant amount of the vitamin in whole unda-maged fruits and vegetables In jam making, when the fruit is boiled with sugar, ascorbic acid is remarkably stable [23]

Vitamin C in freshly secreted cow’s milk is predominantly in the form

of ascorbic acid, but this is rapidly oxidized by the dissolved oxygen content [24] The photochemical destruction of riboflavin accelerates the oxidation of ascorbic acid in milk through a sensitizing effect [25,26] Losses of vitamin C content during high-temperature-short time (HTST) and ultrahigh temperature (UHT) treatment of milk average 20% [27] Graham and Stevenson [28] studied the effect of ionizing radiation on

source of gamma rays Their HPLC method of analysis permitted the separate measurement of total vitamin C, ascorbic acid, and dehydro-ascorbic acid Fresh strawberries have a relatively short shelf-life, mainly because of fungal spoilage Irradiation at doses of 2 –3 kGy combined with refrigeration has been shown to extend the shelf-life of strawberries Low doses (0.075 – 0.15 kGy) of g-radiation are very effective

in inhibiting the sprouting of potatoes during post-harvest storage Strawberries were assayed immediately after irradiation at doses of 1, 2,

or 3 kGy and then after storage for 5 and 10 days at 68C Irradiation had the effect of increasing the dehydroascorbic acid content in accordance with dose, whilst decreasing the concentrations of total vitamin C and ascorbic acid During the storage of irradiated strawberries, the total vitamin C and ascorbic acid levels increased, whereas dehydroascorbic acid levels decreased Overall, irradiation of the strawberries caused some loss of total vitamin C, which increased during storage (Table 15.3) Raw, boiled, and microwaved potatoes were assayed immediately after irradiation at 0.15 kGy and then after storage for 1, 2, and 5 months at 128C The potatoes were irradiated 1 month after harvesting to allow them to recover from the effects of post-harvest stress Losses of total vitamin C in the raw potatoes immediately after irradiation were about 8%; corresponding losses in the cooked potatoes were about 20% There was no significant difference in the dehydroascorbic acid content between irradiated and nonirradiated samples After 2 and 3 months’ storage, irradiated potatoes contained less vitamin C than nonirradiated potatoes However, after 5 months’ storage, both irradiated and nonirra-diated potatoes had comparable vitamin C levels The vitamin C content

in both irradiated and nonirradiated cooked potatoes showed similar changes on storage to those of raw potatoes Similar findings for potatoes were reported by Shirsat and Thomas [29] The loss of total vitamin C in nonirradiated potatoes after 3 months in storage (158C) was 26–45%, depending on the cultivar Additional losses of 6.5 –13% were

TABLE 15.3

Percent Loss of Vitamin C in Four Varieties of Strawberry Following a 3-kGy Dose of Gamma Radiation

Storage (days) Variety 1 Variety 2 Variety 3 Variety 4 Mean

Source: From Graham, W.D and Stevenson, M.H., J Sci Food Agric., 75, 371, 1997 With permission.

recorded in the irradiated samples stored for 3 months From the fourth month onwards, the vitamin C levels in the irradiated samples began to increase In conclusion, irradiation keeps the potatoes in good marketable condition during proper storage conditions for at least 5 months, with no additional loss of vitamin C after this period

Two studies were carried out to evaluate the possibility of using high hydrostatic pressure (HHP) treatment as an alternative to the blanching

of vegetables Potato cubes retained 90% of their ascorbic acid content after treatment at 400 MPa and 58C for 15 min, but only 35% when the temperature was increased to 508C [30] Green peas retained 82% ascorbic acid after treatment at 900 MPa and 438C for 5 min [31] Other studies have evaluated HHP treatment as a possible alternative to the thermal pasteurization of freshly squeezed fruit juices Ascorbic acid in orange juice and tomato juice was shown to be unstable at a combination of rela-tively high pressure (850 MPa) and temperature (60 –858C) At the same pressure level and lower temperature (508C), no degradation of ascorbic acid occurred within 1 h [32] High-pressure treatment of chilled orange juice (500 and 800 MPa for 5 min) and storage up to 21 days at 48C caused no significant difference in vitamin C [33] Similar treatment of orange juice (800 MPa at 258C for 1 min) greatly extended the shelf-life, with less than 20% loss of ascorbic acid after 3 months’ storage at 48C

or after 2 months at 158C [34] Pressure-processing of guava puree (600 MPa and 258C for 15 min) and storage at 48C for 40 days did not alter initial content of ascorbic acid [35]

Sa´nchez-Morino et al [36] tested three processes that combined high-pressure treatment with heat treatment for their effect on vitamin C retention in orange juice: T0, fresh juice (without treatment); T1,

100 MPa/608C/5 min; T2, 350 MPa/308C/2.5 min; T3, 400 MPa/408C/

1 min Fresh and treated samples were kept refrigerated (48C) and assayed at intervals over 10 days T1 and T3 juices showed a small (,10%) loss of vitamin C just after processing, whereas T2 juices showed

no loss of the vitamin There was no further degradation of vitamin C during the 10-day storage period Therefore, the intermediate pressure/ low-temperature treatment best preserved the vitamin C content

A major factor contributing to the variability in vitamin C content of potatoes is the storage time A sharp decrease in vitamin content was observed during the first 4 months of storage at 78C and 95% relative humidity, followed by either a complete leveling out or a less pronounced decrease [37]

Wang et al [38] reported on the losses of added ascorbic acid during the pilot-scale processing and storage of potato flakes, and during the recon-stitution and holding of the mashed potatoes prior to serving Cumulative losses were: 56% after addition of ascorbic acid to the cooked mashed potatoes followed by drum-drying; 82% storing the flakes 4.3 months