J

Pheophytin

-CO2 CH

-CO2 CH

Zn-pheophytin

Pyropheophytin

-CO2 CH1

Zn-pyropheophytin

FIGURE 9.3 Chemical reactions occurring in heated green vegetables containing Zn2+. (Adapted from LaBorde and von Elbe.11,12)

been used to produce green fruits/vegetables of high quality. In particular, a process called Veri-Green has been patented; this process incorporates metal ions into the coating of cans and the green color of the obtained products has been attributed to the formation of zinc pheophytin and pyropheophytin complexes predominantly of the a derivative. When plant tissue is heated in the presence of Zn2+, chlorophyll reacts with tissue acids to form pheophytin, which may then combine with Zn2+ to form Zn-pheophytin or be decarbomethoxylated to form Zn-pyropheophytin (Figure 9.3; Table 9.1).11 Veri-Green-processed green vegetables have a greener color compared with controls; today, green beans and spinach have been produced by this process and are currently marketed under provision by the FDA that the concentration of Zn2+ in the product be no more than 75 ppm. Unfortunately, the Veri-Green process has not been successful because the amount of Zn2+ required to yield a satisfactory color after processing has resulted in Zn2+ concentrations above the FDA limit of 75 ppm.1,11

Divalent cations decrease the pH of plant tissue by binding with pectic material and displacing hydrogen ions and by hydrolysis of water. Anionic detergents (e.g., sodium dodecyl sulfate, SDS) increase the negative charge of chloroplast membrane surfaces, resulting in the accumulation of H+ ions and an increase in pheophytin formation. Conversely, cationic detergents (e.g., cetyltrimethylammonium bromide, CTAB) decrease the negative surface charge of membrane surfaces, repel H+ ions, and therefore decrease chlorophyll degradation. Moreover, neutral detergents (e.g., Triton X-100) increase chlorophyll retention during heating by displacing negatively charged phospholipids and proteins. In the preparation of pea puree, detergents affect zinc complex formation through their effects on membrane surface charge. Anionic detergents may increase the concentration of Zn2+ at membrane surfaces, facilitating the reaction of chlorophyll derivatives with Zn2+ ions. Moreover, thiocyanate, ben-zoate, oleate, or caprylate anions show similar effects of the anionic detergents (e.g.,

Slight heating produces pheophytin as the major chlorophyll degradation product and color changes from the bright green of chlorophylls to the dull olive green of pheophytin. Mild heat treatment, such as blanching, induces the formation of C-10 decarboxymethoxylated derivatives of pheophytins a and b. As a result, chlorophyll decomposition during canning of vegetables is a two-step process in which pyropheophytin is obtained (Figure 9.2). High-temperature short-time (HTST) processing results in good retention of chlorophylls of fruits and vegetables (Table 9.1).6 Previously, the production of chlorophyllides by enzymatic treatment was suggested as a method for color preservation assuming a higher stability than that of chlorophylls; however, at least with spinach leaves, chlorophyllides have shown lower stability than chlorophylls.618

The control of chlorophyllase activity has also been used as a methodology to preserve green color. Particularly, chlorophyllase inactivation is feasible for the processing of green artichokes (Table 9.1).18 Severe heating treatment induces a decarboxylation reaction to produce pyropheophorbides (Figure 9.2).

Kiwi fruit is processed into various products, primarily canned slices in syrup, frozen pulp and slices, juices, and wines. In canned kiwi fruit, severe changes in the chlorophyll profile are observed as well as in other canned vegetable products where pyropheophytins a and b are the major chlorophyll derivatives. These derivatives have also been observed in plant materials blanched or cooked in a microwave oven. In general, the processing of fruit/vegetables by freezing results in products of high color quality (Table 9.1).7-9

Studies with modified atmospheres have reduced the rate of chlorophyll degradation (Table 9.1); this effect has been associated with inhibition of oxidative enzymes and by reduction in the respiration rate.51017 High-pressure treatment is a good option if processing temperature is not higher than 50°C (Table 9.1).1921

The above-described and other approaches (Table 9.1) have improved the shelf life of various products, but the prolongation is short, not more than a few weeks.

3. Chlorophyll Extraction

The chlorophyll preparations for the food-colorant market are mainly obtained from alfalfa (Medicago sativa), nettles (Urtica dioica), and several pasture grasses. Today, industry is considering obtaining chlorophyll c from brown seaweeds, which is the commercial source of alginates and single-celled phytoplankton; these sources have acquired importance because chlorophyll c has higher stability than chlorophyll a and chlorophyll b. Interestingly, the abundance and self-renewing characteristics of chlorophylls have not been exploited because adequate processing conditions to preserve them have not been established.12

The extraction process for chlorophyll is sketched in Figure 9.4. This must be carried out rapidly and in dim light to prevent degradation reactions such as pho-tobleaching and/or allomerization. Acetone, methanol, ethanol, and chlorinated solvents, among others, are used as extracting vehicles. In particular, the use of aqueous solutions of acetone has been recommended, where the proportion of water must not exceed 10%. Filtration or centrifugation is used to remove solids from the solvent. After solvent elimination, the yield of extraction is around 20% in which chlorophylls, pheophytins, and other degradation products are included.1

Plant tissue

Grinding or blending

Homogenized tissue

Solvent extraction

Filtration or centrifugation r

Chlorophyll extract

FIGURE 9.4 Chlorophyll extraction. (Adapted from Schwartz and Lorenzo.1)

With some plant materials, rapid heating in boiling water followed by an immediate cooling is recommended; this procedure improves the extractability and the stability by the reduction of the oxidation and hydrolytic enzymes. To avoid the effect of acidic conditions, the addition of CaCO3, MgCO3, NaHCO3, Na2CO3, dimethylaniline, or ammonium hydroxide is common.1

The corresponding extract is further processed to obtain water-soluble or oil-soluble preparations. If a dried extract is resuspended in a water-immiscible solvent, an oil-soluble product is obtained; by this procedure metal-free pheophytins or copper pheophytins may be obtained if the process is carried out in acidic conditions and in the presence of copper salts. On the other hand, a dried residue can be saponified, whereby the phytyl group is replaced with sodium or potassium and water-soluble compounds are obtained.1

4. Isolation of Chlorophylls

Extraction may be carried out with acetone23 followed by two precipitation steps using dioxane/water mixtures to obtain partially purified chlorophylls. The mixture is then fractionated by DEAE-Sepharose CL-6B chromatography to separate, first, chlorophylls from carotenoids; in a second stage using the same adsorbent, chlorophyll a is separated from chlorophyll b. The chlorophyll extracts have good purity, and use of cool temperatures is recommended to prevent the formation of C-10 isomers.

5. Chlorophylls as Food Additives

Chlorophylls are approved in the Codex legislation for various applications (Tables 9.2 and 9.3X24 On the other hand, the sodium and potassium salts of the Cu-chloro-phyll complex are approved by the FDA only in the preparation of dentifrices and drugs, but not as food additives.25 26

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