WO2005107479A1

WO2005107479A1 - Oxidation of sulfites with chloroplast

Info

Publication number: WO2005107479A1
Application number: PCT/US2005/015026
Authority: WO
Inventors: George Georgiou; Sung-Chyr Lin
Original assignee: Research Development Foundation
Priority date: 2004-04-29
Filing date: 2005-04-29
Publication date: 2005-11-17

Abstract

The present invention provides a biological method for removing sulfites from food products using chloropast material. In particular embodiments, the present invention provides a biological method of reducing sulfite content in a food product and for preventing or reducing an allergic response in a subject to a sulfite in a food product.

Description

DESCRIPTION

OXIDATION OF SULFITES WITH CHLOROPLAST

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention relates generally to the fields of plant biotechnology, food biotechnology and oenology. In particular, the present invention concerns the reduction or removal of sulfites from a food product such as an alcoholic beverage. More particularly, it concerns the removal of sulfites from wine using chloroplasts.

2. Description of Related Art Sulfites have been used since ancient times for many purposes, including the cleansing of wine receptacles by both Romans and Egyptians. As food additives, they have been used since the 17 century and approved for use in the United States as long ago as the early 1800s. They are currently used for their preservative ability, which includes controlling microbial growth, blanching certain foods, and preventing spoilage of certain perishable foods, beverages and pharmaceuticals. Their antioxidant and anti-microbial properties have gained them an important role in wine making. Sulfites are used to either inhibit or kill bacteria or wild yeast, thus encouraging rapid and clean fermentation of wine grapes. However, sulfites are also a natural and minor by-product of yeast fermentation and thus are produced during the wine fermentation process. While some of the actions of the sulfites are highly desirable (e.g., color stabilization, preservation, and the like), many of the sulfite actions are highly negative. A number of disorders in humans have been proven to be associated with allergic responses to sulfites (Vena et al. 1994). However, allergic responses to sulfites are often difficult to diagnose and discriminate from other allergic responses. For this reason, the number of affected individuals is not known with certainty. The majority of the reactions triggered by sulfites are mild and include dermatologic, respiratory and gastrointestinal symptoms (Vena et al. 1994; Petersen et al. 1992). Other negative attributes include, but are not limited to, allergic reactions which may lead to occasional consequent deaths, inactivation of particular protein-carbohydrate linkages, bronchial constriction and irritation, possible carcinogenicity, and the like. Sulfites, particularly in wines, represents important triggers of asthma and sever bronchospasms (Vally et al. 2000, 2001, 2003). The symptoms are presumed to be mediated by IgE-associated immune responses as well as by genetic defects in mitochondrial enzymes (e.g., rodanase). The symptoms of a sulfite sensitivity reaction may vary from mild to life-threatening. The most common symptoms are mild and involve a skin rash accompanied by redness, hives, itching, flushing, tingling and swelling. Respiratory symptoms may occur that include difficulty breathing, wheezing, and stridor. Gastro-intestinal reactions, which may also occur, involve nausea and stomach cramps. Much less common but more serious signs and symptoms of sulfite sensitivity are low blood pressure, shock, extreme difficulty breathing and loss of consciousness. These symptoms of severe reactions are most apt to occur in the steroid-dependent asthmatic person. The FDA in the United States estimates that one in 100 people is sulfite sensitive to some degree, but for the 10% of the population, specifically asthmatics, up to 5% are at risk of having an adverse reaction to the sulfites. Of those, the ones in whom the most severe reactions have been reported are steroid-dependent and are taking such drugs as prednisone or methylpredmsolone. The number of asthmatic patients that are included in this sulfite sensitive group is estimated to be 500,000 in the United States. Sulfites also pose a greater danger to individuals with a history of allergies, or a deficiency of the liver enzyme sulfite oxidase. Nevertheless, sulfites continue to be used in the wine industry and other food processing industries as an effective color stabilizer and preservative. In recognition of the potential health hazards to the population with sulfite hypersensitivities, the United States FDA regulations require that wines sold in the United States which contain more than 10 ppm equivalents be labeled as "contains sulfites." To circumvent the negative effects of sulfites in foods such as in wine for example, approaches for sulfite removal have been proposed. The use of anion and cation exchangers for the adsorption of sulfites from wines as described in U.S. Patent 5,071,664 has been proposed. The employment of a membrane reactor in conjunction with non-diffusible oxidizing agents for sulfite oxidation has also been provided in the art; see U.S. Patent 5,358,732. However, although both methods claim to be capable of reducing sulfite concentration of wines to below 10 ppm, the nonspecific adsorption and/or nonspecific oxidation of flavor compounds in the wines associated with these two processes can adversely affect the aesthetics of the wines treated. Thus, there is a pressing demand for a process that can specifically oxidize sulfites in sulfite containing food products, such as a wine, without affecting the taste of the wine. SUMMARY OF THE INVENTION

The present invention overcomes the deficiencies in the art for removing sulfites from a food product, such as a wine, without affecting the color or taste of the food product (e.g., a wine). Thus, the present invention provides a method of oxidizing sulfites in a food product comprising (a) obtaining chloroplasts from a chloroplast-containing substance; and (b) contacting the chloroplasts or chloroplast-containing substance with a food product. In particular embodiments of the invention oxidizing sulfites further comprises removing the chloroplasts from the food product. In other embodiments the method of oxidizing sulfites may further comprise an illuminating step to stimulate sulfite oxidation with chloroplasts and/or promote or enhance sulfite removal by chloroplasts. In other embodiments, the present invention provides a pH adjustment step. The pH adjustment step may comprise increasing the pH of a food product within an appropriate range between about, about at least, or about at most, 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4,

3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5,

5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,

7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,

9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9,12.0 or greater. The pH of the wine may be increased to within a range of 7.0 to 9.0. Food products contemplated in the present invention may include, but are not limited to, a beverage such as an alcoholic beverage. An alcoholic beverage may further include a wine such as, but not limited to, a pink or a blush or a rose wine, a red wine or a white wine. Other alcoholic beverages such as beer, distilled liquor, a cocktail mix or a wine cooler are contemplated in the present invention. In some embodiments of the invention, the beverage may be a non-alcoholic beverage such as, but not limited to, lime juice or lemon juice, a dried citrus fruit beverage mix, a fruit juice, or a vegetable juice. The non-alcoholic beverage may contain a sugar syrup or a corn syrup. In yet another embodiment of the present invention, the food product may be a processed food product such as, but not limited to, a baked good. In yet another particular embodiment of the invention, the chloroplasts are obtained from plants or other chloroplast-containing tissues. Chloroplasts may be obtained from wheatgrass or spinach but is not limited to such. Chloroplasts obtained may be contained in a solid support such as, but not limited to, a semi-permeable pouch or capsule or a porous matrix. The porous matrix may be selected from the group consisting of natural polymers, synthetic polymers, inorganic supports, or organic supports. In still yet another embodiment of the present invention, the chloroplast-containing solid supports may be contained in an appropriate reactor. Reactors contemplated by the present invention include, but are not limited to, a packed bed reactor, a fluidized bed reactor, or a membrane reactor. In still yet another embodiment of the present invention, the oxidation of sulfite may be accomplished by passing the food product through the reactor. Sulfites oxidized by the method of the present invention include, but are not limited to, sulfur dioxide or sodium sulfite; bisulfite such as sodium bisulfite or potassium bisulfite; or metabisulfite such as sodium metabisulfite or potassium metabisulfite. In another embodiment, the present invention provides a method of oxidizing sulfites in a food product comprising removing the sulfites from the food product or reducing the sulfites in the food product. In still yet another particular embodiment, the present invention provides a method of reducing or preventing sulfite hypersensitivity in an individual comprising (a) obtaining chloroplasts from a chloroplast-containing substance; (b) contacting the chloroplasts or chloroplast-containing substance with the food product; (c) removing the chloroplasts from the food product; and (d) providing the food product to the individual. In a further embodiment an illumination step may be conducted prior to step (d) to stimulate sulfite oxidation with chloroplasts and/or promote or enhance sulfite removal by chloroplasts. In still yet another embodiment, the present invention comprises oxidizing or reducing sulfites in the food product and/or removing sulfites from the food product. In still a further embodiment, the present invention provides a kit comprising chloroplast and buffer components. The components may be contained in a semi-permeable pouch or bag. In still yet another particular embodiment, the present invention provides a device for oxidizing sulfites in a food product comprising (a) solid supports entrapping chloroplasts or chloroplast-containing substances; and (b) a reactor containing the solid supports. In a further embodiment, the device comprises an illuminating device to stimulate sulfite oxidation with chloroplast and promote or enhance sulfite removal by chloroplasts. In still yet another embodiment, the present invention provides a process of increasing the pH of a wine comprising obtaining chloroplasts from a chloroplast-containing substance and contacting the chloroplasts or chloroplast-containing substance with the wine. The pH of the wine may be increased to between about pH 6.0 to pH 8.0. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIG. 1. Effect of pH on sulfite oxidation with chloroplasts in white wines. White wines with 9.5% alcohol containing 150 ppm sodium sulfite and the required pH buffer systems were incubated with 1 mg/ml wheatgrass chloroplast at 25°C overnight with moderate shaking. The residual sulfite concentration was quantified by HPLC. FIG. 2. Effect of alcohol concentration on sulfite oxidation in white wines.

Chloroplasts at a concentration of 1 mg/ml were added into white wines containing 150 ppm sodium sulfite, 50 mM pH 8.0 phosphate buffer and (B) 5% alcohol, (C) 10% alcohol, or (D) 15% alcohol were incubated at 25°C or three hours with moderate shaking. The residual sulfite concentration was quantified by HPLC. The efficiency of sulfite oxidation in the aqueous solution (A) containing no alcohol was defined as 100%.

FIG. 3. Effect of chloroplast concentration on sulfite oxidation in white wines. White wines with 9.5 % alcohol containing 150 ppm sodium sulfite and 50 mM pH 8.0 phosphate buffer were incubated with chloroplasts at 25°C for one hr with moderate shaking. The residual sulfite concentration was quantified by HPLC.

FIG. 4. Time course of sulfite oxidation with chloroplasts in white wine. Chloroplasts at concentrations of ( ) 1 mg/ml and ( • ) 5 mg/ml were added into white wines containing 9.5% alcohol and 150 ppm sodium sulfite and 50 mM pH 8.0 phosphate buffer and incubated at 25°C with moderate shaking. The residual sulfite concentration was quantified by HPLC.

FIG. 5. Effect of illumination and mixing on sulfite oxidation in white wines. White wines with 9.5%> alcohol containing 150 ppm sodium sulfite and 50 mM pH 8.0 phosphate buffer were incubated with 1 mg/ml chloroplasts at 25°C for three hr (1) with moderate shaking and illumination, (2) with moderate shaking in the dark, (3) in static condition and with illumination, and (4) in static condition and in the dark. The residual sulfite concentration was quantified by HPLC.

FIG. 6. Time courses of sulfite oxidation with chloroplasts in red wines with (o) and without ( • ) illumination. Chloroplasts at concentrations of 1 mg/ml were added into 13.5% alcohol red wines containing 150 ppm sodium sulfite and 50 mM pH 8.0 phosphate buffer and incubated at 25°C with moderate shaking.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A. The Present Invention The present invention provides a biological approach for the removal of sulfites in food products such as alcoholic beverages. The present invention benefits from the knowledge that chloroplasts contain sulfite oxidase and sulfite reductase. Thus, the capability of chloroplast to act as an effective biocatalyst in the oxidation of sulfite to sulfate in wines, thereby removing or reducing the sulfite content, was examined. Chloroplasts were found to be able to carry out the oxidation of sulfites in wines despite the presence of large concentrations of denaturing solvents such as ethanol, and despite the complex composition of wines. The efficiency of sulfite oxidation was promoted by illumination, indicating the participation of light-induced photosynthetic electron transport chain in sulfite oxidation, in addition to sulfite oxidase. The activity of sulfite oxidation of chloroplasts was significantly affected by ethanol concentration. The biological process proposed in the present invention has the advantages over the current art of high specificity and thus of minimal adverse effects on the tastes of the wines treated. Furthermore, unlike other processes that involve the employment of complicated process design, the biological process of the present invention is straightforward and can be easily implemented. The present invention further provides kits such as sulfite oxidation kits containing chloroplasts and buffer components for use by the consumer. For sulfite removal at wine-making or beverage outlet facilities, the present invention contemplates immobilized systems in conjunction with illumination devices that can be developed to enhance efficiency of sulfite removal and reduce the amount of chloroplasts needed. Thus, the present invention provides a simple method that overcomes the deficiencies in the art for removing sulfites from a food product, such as a wine, without affecting the color or taste of the wine. Further, the present invention provides a method of reducing or preventing sulfur hypersensitivity, due to sulfite containing foods, in an individual.

B. Sulfites and Sulfite Oxidation Sulfites are formed naturally in the body during the oxidative degradation of sulfur- containing amino acids, cysteine and methionine, resulting in the accumulation of sulfites which are toxic and must be further oxidized to sulfate to prevent adverse effects. Sulfites are also introduced exogeneously through environmental pollution or from the consumption of processed foods, such as dried fruits. Sulfites as used herein included the salts of sulfurous acids (M₂SO₃), acid-sulfites (MHSO₃, also known as bisulfites), sulfur dioxide (SO₂, also known as sulfurous acid anhydride), metabisulfites (M₂S₂O₅), hydrosulfites (M₂S₂O₄) and the like. Such sulfating agents may further include sodium or potassium sulfite, sodium or potassium bisulfite, and sodium or potassium metabisulfite. Sulfite oxidase (SOi, EC 1.8.3.1) is an essential molybdoprotein that resides in the intermembrane space of the mitochondria and is responsible for the oxidation of sulfite to sulfate, the final reaction in the degradation of sulfur-containing compounds including the amino acids methionine and cysteine (Rajagopalan et al. 1980, 2002; Enemark et al. 2002; Schindelin et al. 2001) and membrane components such as the sulfatides. SO belongs to the family of proteins containing the molybdopterin (MPT) cofactor, which consists of a single molybdenum atom coordinated to apterin derivative through a dithiolene group (Schindelin et al. 2001; Wuebbens et al. 2003). SO₃ ^2"+ H2O + 2(cyt c)₀χ -> O₄ ² + 2 (cyt c)red+ 2H⁺

In the reductive half of the reaction cycle, sulfite binds at the Mo ι center and is oxidized to sulfate generating a transient two-electron reduced MorvFeni species. In the first intramolecular electron transfer (IET), one of the two reducing equivalents generated by sulfite oxidation is transferred from Moiv to the bs-type heme in the N-terminus of SO yielding a Mo_vFen species that can be detected using electron paramagnetic resonance (EPR) spectroscopy (Cohen et al. 1971). In the oxidative half of the reaction cycle, one electron is transferred from the heme Fen to the terminal electron acceptor, cytochrome c_ox. After a second IET from Mo_v to Fem, which produces the MoviFeπ state of the enzyme, the fully oxidized species is regenerated by electron transfer from Feu to a second molecule of cytochrome Co_*. The second IET step (from Moy to Fem) has been extensively studied in reverse after reduction of the heme by one electron using flash photolysis (Feng et al. 2002, 2003; Sullivan et al. 1992, 1993; Pacheco et al. 1999). In plants, during primary sulfate assimilation in the chloroplast, sulfate is reduced via sulfite to organic sulfide, which is essential for cysteine biosynthesis (Leustek et al. 1999). However, there are reports that sulfite can be reoxidized to sulfate e.g., when plants are subjected to SO₂ gas (Heber et al. 1998) or when isolated chloroplasts are fed with radioactively labeled sulfite (Dittrich et al. 1992). Sulfite oxidation in intact chloroplasts is enhanced by light and sensitive to inhibitors of photosynthetic electron transport (Dittrich et al. 1992; Jolivet et al. 1993 and 1995) and thus appear to be partly due to nonenzymatic reactions during electron transport.

C. Sulfite-containing Food Products Sulfites can be found in many foods. Sulfites act as a preservative that controls microbial growth and prevents spoilage of certain perishable foods, beverages and pharmaceuticals. Sulfites are often found in foods and beverages listed as sulfur dioxide, sodium sulfite, sodium bisulfite, potassium bisulfite, sodium metabisulfite and potassium metabisulfite. In addition to their role as preservatives, sulfites are used to bleach food starches, such as corn, potato and sugar beet. Sulfites are also utilized as preventives against rust and scale in boiler water used in making steam that may come into contact with food. Because sulfites inhibit fungal and bacterial growth, they are sprayed on both fresh grapes and those used in wine making. Sulfites are also a normal by-product of wine making. Wines, bottled lemon and lime juices, and sulfur-dried fruits have the highest levels of sulfites. Other foods that contain sulfites include, but are not limited to, alcoholic beverages such as beer, wine coolers, distilled liquors etc.; bakery items such as breads containing dough conditioners, cookies, crackers, pie and pizza crusts, tortillas, waffles; non-alcoholic beverages such as beverages containing sugar or corn syrup, dried citrus fruit beverages, canned bottled, and frozen fruit juices; condiments such as horseradish, relishes, pickles, olives, wine vinegar; dairy such as processed cheese foods; dried foods such as dried herbs and spices, dried fruits, trail mixes; fish and shellfish including fresh shrimp and scallops frozen, canned or dried clams, shrimp, lobster, crab, scallops, dried cod; fruits such as fresh grapes, dried fruits (including raisins and prunes and especially pale fruits that have not discolored), canned, bottled and frozen fruit and juices, maraschino cherries, glazed fruit; gelatins, fillings, frostings including fruit fillings, flavored and unflavored gelatin, pectin, jelling agents, canned frostings and frosting mixes; grain products such as cornstarch, modified food starch, spinach pasta, gravies, hominy, breading, batters, noodle and rice mixes; hard candies; jams and jellies; nuts such as shredded coconut; plant protein product such as soy protein products including tofu, textured vegetable protein, infant formula; snack foods such as filled crackers, dried fruit snacks, tortilla chips, potato chips; sugars such as brown, white, powdered and raw sugars; vegetables such as vegetable juices, canned vegetables (including potatoes), pickled vegetables (including cauliflower, peppers, sauerkraut), "fresh cut" potatoes (as delivered to restaurants), frozen vegetables (including french fries and deli potato salad). Thus, it is contemplated in the present invention that sulfites in a sulfite containing food product, e.g., an alcoholic beverages such as wines, may be reduced or removed by a biocatalyst such as chloroplasts.

D. Sulfites in Wine Making Sulfur has been used in wine making for hundreds of years. Most wineries, conventional and organic, use SO₂ to limit oxidation and bacteria in their wine. When added to water or wine, sulfur dioxide becomes sulfites. Sulfites act as a preservative that controls microbial growth and prevents spoilage of wines. Sulfites added during the various stages of wine making processes lead to high sulfite content in most grape wines of up to 300 ppm. Sulfites are also a natural by-product of yeast fermentation that are produced during the wine fermentation process. Even if no sulfur dioxide is added to wine, fermenting yeasts will produce SO₂ from the naturally occurring inorganic sulfates in all grape juices. Sulfites can be incidentally derived from soil. Thus, it is impossible for any wine to be completely free of sulfur dioxide. SO₂ is usually added to wine before bottling in a gaseous or diluted form. It binds loose oxygen molecules, reducing and delaying oxidation for years. The vast majority of organic winemakers limit the use of sulfites to 90 ppm in red wines, and under 100 ppm in white and sparkling wines; and many organic wines contain less than 40 ppm sulfites. Although technical advances permit the industry to add much less sulfur, most serious winemakers and oenologists concur that to make a consistently stable wine, some sulfites must be added to those naturally present. A handful of winemakers use no added sulfites at all. Although some of these producers have been able to make high quality wines without the use of sulfites, these wines require special handling within the distribution system and do not age as well as wines made with sulfites. The quality of the wine is compromised at best, with extremely short shelf life, and they suffer inordinate product returns compared to the majority of organic wine with added SO₂. The benefits of aging are well known in quality wine; quite the contrary for wines made without sulfites or SO₂ added. Most are designed to be consumed within 18 months of production. They tend to emphasize fresh fruit and are, for the most part, not complex wines. The fact is, wines without sulfites lose fruitiness and are less distinct as they age. Even without adding sulfite and taking great care in avoiding microbial contamination during the process of wine making, a sulfite concentration up to 40 ppm can still be detected in homemade organic wines. Thus, sulfites are intentionally used extensively in the treatment of alcoholic beverages (such as wines). Wines contain about 3 ppm (parts per million) sulfur dioxide produced by yeast metabolism and additionally up to 30 ppm of sulfites added purposefully during wine making. All grape wines (that is, wines made solely from grapes) contain sulfites which are derived from the soil and/or added during picking to prevent spoilage. In the wine making industry, grapes are generally treated with sulfites prior to and after crushing to control undesirable micro-organisms, inhibit browning and serve as an antioxidant. After the fermentation process, sulfites may be added to prevent secondary fermentation. While supplying these benefits during the wine making process, sulfites offer little, if any, benefit after the wine making process is completed and may impart undesirable taste qualities to the wine and prevent those who are allergic to sulfites from enjoying the wine. In addition, storage kegs often are sterilized with burning sulfur candles, and the resulting sulfite can find its way from the keg into the wine or other hard liquors to which it is not added purposefully.

E. Chloroplast and Chloroplast-containing Substances It is desired in the art to reduce or remove sulfites from a food product such as an alcoholic beverage, for example wine. Removal or reduction of sulfites may occur by sulfite oxidation which oxidizes sulfites to sulfates. Chloroplasts, which contain sulfite oxidase and sulfite reductase, are known to be involved in sulfite oxidation. Further, sulfite oxidation in intact chloroplasts may be enhanced by light (Dittrich et al. 1992; Jolivet et al. 1995). Thus, in a particular embodiment of the present invention chloroplast is contacted with wine thereby promoting sulfite oxidation of sulfites to sulfates. The terms 'contact,' 'contacting,' or 'contacted' as used herein encompass the terms 'adding', 'mixing', 'incubating', 'exposing', 'immersing', 'interacting', or 'incorporating' and is understood to have the plain and ordinary meaning to refer to the coming together of the food product, such as a wine, with chloroplasts. Chloroplast for use in the present invention may be obtained from any plant, such as a green plant or from green algae known to one of skill in the art. Chloroplast may be obtained from a herbaceous and/or woody plant but is not limited to such. A chloroplast-containing substance as contemplated by the present invention may be any plant source from which chloroplast, which contains a high level of sulfite oxidase complex, may be obtained or extracted. It is further contemplated in the present invention that any natural product containing high amount of chloroplasts can also be used for sulfite oxidation. Examples of chloroplast-containing substances include, but are not limited to wheatgrass, spinach such as New Zealand spinach or any variety thereof belonging to the spinach family, pea such as a green pea or snow pea or any variety thereof belonging to the pea family, lettuce or any variety thereof belonging to the lettuce family, cabbage, mangold, and tobacco, tomato, chives, leeks, asparagus, broccoli, brussel sprouts, cauliflower, turnips, celery, chard, swiss chard, chicory, collards, dandelions, endive, escarole, garden cress, kale, lettuce, mustard, pak choi or bok choi, parsley, radicchio, watercress, maize, broad bean, barley, wheat, various types of grass, various herbs (cilantro, basil etc.), some strains of Euglena, or Arabidopsis, but is not limited to such. F. Extraction and Purification of Chloroplast Chloroplast for use in the present invention may be obtained by any method known in the art for obtaining, isolating or extracting chloroplast from a source such as plant, e.g., the leaf of a plant or green algae. Generally, "isolated" will refer to an organic molecule or group of similar molecules that have been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Methods of obtaining, isolating or extracting a compound such as chloroplast from a source are well known to those of skill in the art. Isolation or extraction of chloroplasts, such as intact chloroplasts, from green algae or plant tissues, may include mechanical cell wall and membrane breakage, removal of cell debris and unbroken leaf tissue by filtration, collection of total cell chloroplasts by centrifugation, and separation of intact from broken chloroplasts using a layer or gradient as are well known in the art e.g., Percoll® layers or gradients may be used. The present invention provides a simple and rapid method for extracting chloroplast from a chloroplast-containing substance. The chloroplast-containing substance, for example wheatgrass, may be washed in solution such as deionized water, or appropriate buffers to remove undesirable particles and placed in an extracting apparatus such as a juicer, blender, food processor, or homogenizer. The extract is then collected, and may be filtered and the filtrate centrifuged to collect the chloroplasts. The chloroplast pellet may then be rinsed with water or buffers and lyophihzed as described herein. The chloroplast-containing substance may be washed 1, 2, 3 or more times. The chloroplast pellet may be rinsed 1, 2, 3, or more times. Solids supports may also be used for extracting the chloroplast from a sample, such as a plant tissue sample and for maintaining or storing the chloroplast extracted. Such solid supports may include but are not limited to, spin columns, spin filters, vials, test tubes, flasks, bottles, elution columns or devices, filtration columns or devices, syringes and/or other container means. Such supports may further include plastic or glass. The collecting may be in any container such as a plastic, or glass container as is known in the art. More preferably, the collecting may be in a spin column, vial, test tube, flask, or bottle. Filters that may be employed for filtering the extract collected may include, but are not limited to, muslin cloth, cheesecloth, strainers or sieves of various pore sizes, filters or filter pads of various sizes or any other item or membrane known in the art that may be used as a filter or for separating a liquid substance from undesired particles. Another method for extracting chloroplast from a chloroplast-containing substance may involve grinding of plant tissues in a mortar with a pestle in the presence or absence of pinch (small amount) of acid washed sand which facilitates the grinding of the substance into a fine powder or other tissue grinding apparatus such as a Dounce glass tissue grinder into a fine powder. The chloroplast may also be obtained using a mechanical apparatus by subjecting or placing the chloroplast-containing substance in a blender or food processor or juicer to obtain a fine powder of the chloroplast-containing substance. A mechanical mill (Tecator Cyclotec Sample Mill, Model 1093) may also be used. Other methods may be employed that are well known in the art to efficiently and quickly isolate or extract chloroplasts from a chloroplast-containing substance (e.g. plant leaves). In some instances, it may be desired to used differential centrifugation to isolate chloroplasts. Using this method, deveined leaves are grinded, using clean sharp sand, in mortar and pestle to a paste and suspended in 0.5 M sucrose (a blender may be used for >100 mL volumes). The homogenate is then filtered through about two or more layers of clean cheese cloth in a glass funnel into an iced test tube and centrifuge at low speed at 50x g for 10 minutes. The supernatant is decanted into a clean cold centrifuge tube and the sediment discarded. The supernatant is then centrifuged at lOOOx g for 10 minutes to precipitate chloroplasts. The pellet is resuspended in ice-cold 0.5 M sucrose. This method leaves the chloroplasts outer membrane intact. However, the outer membrane may be ruptured by resuspending the chloroplasts in a diluted suspension solution, centrifuging the suspension and collecting the chloroplasts. The chloroplasts may be resuspended in isotonic media such as NaCl (0.35 M is recommended or an undiluted suspension buffer such as sucrose). Mariac et al. (2000) describes a fast and simple method of extracting chloroplast that overcomes the need for differential centrifugation using density gradients. Using this method, the leaves collected or obtained from a plant do not have to be kept in the dark and lyophihzed before extraction. However, lyophilization may also be used. Using this method one can obtain chloroplasts by lysing a cell extract of leaves with a non-ionic detergent, followed by centrifugation to obtain the chloroplast. The product obtained may be further purified to removed or eliminate other proteins in the mixture by treating the mixture with a chloroform and isoamyl alcohol. Intact chloroplast may be isolated or extracted from plant leaves as described by Bourque et al. (1973). Kits available in the art such as a Chloroplast Isolation™ kit are also available from suppliers such as Sigma (St. Louis, MO) for separating intact chloroplasts from ruptured ones. Other methods commonly used in the art for extracting proteins from a plant tissue may also be employed. Such methods involve the homogenization of mortar-grounded material in liquid nitrogen with an extraction buffer (20 mM Tris-HCl, pH 8.0, 5 mM EDTA,

50 M leupeptin, 1 M pepstatin A, 10 M 3, 4-dichloroisocumarine, 1 mM phenylmethylsulfonyl fluoride and 0.05 % SDS). In instances where leaves are used from which to extract chloroplasts, it is preferable to use young, healthy leaves without necrotic areas or lesions, although older leaves which are not senescent may be used. If the midrib of the leave is thick and tough, it may be desirable to remove it prior to extraction of the chloroplast by cutting or excising it from the leaf by any method known to one of ordinary skill in the art. It may be desirable to cut the leaves into smaller pieces to make extraction process easier. However, it is noted that any chloroplast- containing substance may be employed in the present invention such as any part of a plant containing chloroplasts, green algae or any other chloroplast-containing substance known to one of ordinary skill in the art. In some instances, it may be desired to store the leaves prior to the isolation or extraction of chloroplast. In such instances, the leaves may be stored in a container on ice to keep samples cool but to avoid freezing. Any container that is known to one of ordinary skill in the art for storing a substance on ice, such as a plastic container or bag, or glass container etc., may be used. In instances where it is desired, the leaf samples may be quick-freezed using liquid nitrogen. Leaf samples may be stored at -80°C until ready to be lyophihzed. Lyophilization may be performed with the use of a lyophilizer as would be known to one of ordinary skill in the art. Briefly, the fresh or frozen sample containing chloroplast is loaded into a lyophilizer that has a chamber temperature of -60°C and under (pulling) a vacuum. The sample may take about 72 hours to dry. The lyophihzed sample(s) may be stored for days, weeks, months or years. Following extraction and separation of the compounds of the present invention from natural products, purification techniques as are known to those of ordinary skill in the art may be employed. Such techniques may be used to achieve partial or complete purification (or purification to homogeneity). Thus, once isolated or extracted, the chloroplast of the present invention may be purified if desired. A "substantially purified" compound of the present invention will be a composition in which chloroplasts form the major component of the composition, such as constituting about 50%, about 60%, about 70%>, about 80%, about 90%, about 95%) or more of the molecules in the composition. G. Immobilization of chloroplasts Solid supports may be used for the immobilization of chloroplasts extracted. Any method described in the art for the immobilization of enzymes or tissues can be employed for the immobilization of chloroplasts. Examples include matrix-entrapment, membrane- entrapment, encapsulation or adsorption. The matrices, membranes, or capsules used for entrapments may be synthetic or natural, organic or inorganic, and of various pore sizes.

H. Treatment of a Food Product with Chloroplasts It is desired in the art to reduce or eliminate the sulfite concentration in a food product such as a beverage or other foods containing sulfites. In particular embodiments, the present invention contemplates removing, reducing or eliminating sulfites from wine using free chloroplast or immobilized chloroplast as a biocatalyst. As described above, chloroplast may be obtained from a variety of sources using methods described herein and as are known to one of ordinary skill in the art. The chloroplast may be contained or placed in a porous packet, pouch, sachet or bag prior to being contacted with the wine. The chloroplast may be in the form of a pellet, tablet, capsule or powder. Chloroplast may be contacted with the wine contained in a vessel such as an oak barrel, stainless steel tank, bottle (glass, plastic), vat (wood, metal, plastic), concrete vat or tank, bucket, jug or any other vessel of any size that is known in the art, that may be used for wine making in the industry or in the home. At various stages of wine making sulfites may be present or added to the wine. Thus, it is contemplated that the chloroplast of the present invention may be contacted with the wine at various stages of the wine making process as is desired by the wine maker. In the production of white wine, the chloroplast may be contacted with the wine at the stage of juice separation, must treatment, fermentation stage, racking stage or holding stage of production and prior to the bottling stage. In the making of red wine, the chloroplast may be added at the fermentation stage, the oak aging stage or racking stage and prior to the fining and bottling stage. In some instances, it may be more preferred to contact the chloroplast during, at a point before the end of the fermentation stage, or at the end of the fermentation stage of the wine making process but before fining to remove undesired particle such as chloroplast and before the bottling stage. The chloroplast may be contacted with the wine by shaking, mixing, punching or stirring methods that may be employed in wine making. In other instances, immobilized chloroplasts may be contained in a column or a membrane device. The removal of sulfites in wines can be thus accomplished by passing the wines, at various stages as desired by the wine maker or right before consumption at wine outlet facilities or at home, through the device at flow rates that would allow the efficient removal of sulfites. The concentration of the chloroplast added to a food product, such as a wine, may be of about 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml or greater. In a preferred embodiment of the invention, the concentration of the chloroplast may be 2 mg/ml to about 5 mg/ml. The amount of time of chloroplast incubation in a food product, such as a wine, to allow for oxidation of sulfites may be of about 30 minutes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours.

I. Removing Chloroplasts from a Food Product Contemplated in the present invention, are methods of removing chloroplast from a food product after the food product has been contacted with the chloroplast to allow for sulfite oxidation whereby sulfites are removed, reduced, or eliminated in the food product. Methods of removing chloroplasts may involve filtration, centrifugation, sedimentation, or any method known in the art for removing a substance from a food product, such as a wine, prior to the formation of the end product to be packed, bottled, stored or consumed. The chloroplast may also be remove by fining or clarifying methods which may also employ the use of filtration or centrifugation methods known in the art of wine making for removing particles from a wine. Clarification methods are similar for both white and red wines. In the case of wine making, chloroplast that has been added in a porous packet, pouch, sack, sachet, or bag or any such containing apparatus may be removed by removing the apparatus containing the chloroplast sediment from the wine containing vessel. In instances where the chloroplast has been added directly to a food product, such as a wine, the chloroplast may be removed by filtering. The chloroplast of the present invention contacted with the wine may be removed by any filtering method which involves passing the wine through a filter small enough to remove undesirable elements. Various filtering technologies allow great flexibility to wine makers to make stable wines of varying styles. Depth or sheet filtration uses a relatively thick layer of fine material (diatomaceous earth, cellulose powder, perlite, etc.) to trap and remove small particles. Surface or membrane filtration passes wine through a thin film of plastic polymer with uniformly-sized pores that are smaller than the particles. Sterile filtration uses micropore filters which are fine enough to remove such undesired particles as yeast cells used in fermentation. Filters such as mini jet filters, super jet filters, free standing electric bottle filter, filter pads such as Vinebrite™ Filter Pads, Vinebrite™ Filter Pads, micropore filters, muslin cloth or nylon or cheesecloth may be employed. For example, the Buon Vino Super Jet Filter™ can filter 80 gallons of wine per hour, and the Buon Vino Mini Jet Filter™ can filter 5 gal/15 mi. Filters for use in wine making are well known to one of ordinary skill in the art. Chloroplast that has been added in a porous containing apparatus as described above, or directly to a food product, such as a wine, may be removed at the racking stage when the lees are removed from the wine. Racking is usually begun or conducted within one to two weeks after the completion of the fermentation stage. Racking is the oldest technique of clarification that is just one step beyond natural settling. This technique is simply the siphoning off of the relatively clear wine after the lees have settled to the bottom, leaving them behind to discard. The lees are the insoluble matter including dirt and dust, cellulose, dead yeast cells, bacteria, tartrates and pectin. The chloroplast contacted with the wine would also settle at the bottom of the wine solution making it easy to remove in the racking stage. Racking may be done only once or several times before a wine is bottled. Cold stabilization or refrigeration may be considered an adjunct or enhancement to racking. Cold stabilization is accomplished by allowing the wine to warm up to "room temperature" and then chilling it down to about 40° F. This would allow the chloroplast to settle in the wine vessel and the wine drawn off by racking, as described above. The chloroplast of the present invention contacted with the wine may be removed by the fining method used in wine making. Fining is a method of clarifying or chemically stabilizing wine. The procedure begins by stirring into the container of wine a fining agent that is heavier than both water and alcohol and does not dissolve in either. The agent ultimately settles to the bottom of the wine containing vessel (tank or barrel), causing small suspended particles to precipitate out along with the agent. The clarified wine is then separated by siphoning (racking) off the settlings (lees). Physical agents work by absorbing tiny particles and dragging them. Chemical agents work by forming chemical bonds with hydrogen elements in the undesired particles. Fining agents may include egg white, milk, blood, gelatin, carbon, casein (the principal protein constituent of milk and cheese) and isinglass (an extract of sturgeon bladders). Heat stabilization is a fining process that uses bentonite (a clay of hydrated magnesium silicates) to remove protein, which may cloud a wine. Fining agents are well known in the art. In other instances, it may be preferred to centrifuge, or high-speed spin the wine to remove the chloroplast, however, this technique requires careful control to avoid undue oxidation and loss of alcohol during the process. In yet other instances, where the removal of sulfites in wines is accomplished by passing wines through a devise containing immobilized chloroplasts, the aforementioned clarification steps can be omitted without compromising the quality of the wines treated.

J. Kits In further embodiments of the invention, there is provided a kit for the isolation or extraction of chloroplast from a chloroplast-containing substance or sample. Any of the compositions described herein may be comprised in a kit. In a non-limiting example, reagents for digesting and/or extracting chloroplast from a chloroplast-containing substance may be included in a kit. The kits will thus comprise, in suitable container means, any of the reagents or components disclosed herein. It may also include one or more buffers, such as digestion buffer or an extracting buffer, and components for isolating the chloroplast from a chloroplast-containing substance. The components of the kits may be packaged either in aqueous media or in dry form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit (they may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the chloroplast or chloroplast-containing substance, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the chloroplasts or chloroplast-containing substances are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, buffer and/or other diluent. Such kits may also include components that facilitate isolation of the extracted chloroplast. It may also include components that preserve or maintain the chloroplast or that protect against its degradation. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution. A kit will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.

K. Examples The following examples are included to demonstrate the feasibility of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 Experimental Procedures

Preparation of wheatgrass chloroplasts. Wheatgrass was subjected to a manual screw type juicer after rinsing with deionized water. The juice collected was filtered to remove fibers. The supernatant was then centrifuged at 200 x g for 3 minutes to precipitate whole cells. The supernatant was further subjected to centrifugation at 10,000 x g to collect chloroplasts. Upon rinsing the pellets containing chloroplasts were lyophihzed. About 700 ml of juice was obtained from 1 kg of wheatgrass. Upon filtration, centrifugation and lyophilization, about 7 grams of greenish powder was obtained and was used for all subsequent experiments. Sulfite oxidation with wheatgrass chloroplasts. Unless specified otherwise, all reactions were conducted under normal laboratory illumination at 25 °C with moderate shaking. For all experiments aliquots of samples were taken and filtered with 0.2 μl syringe filters for HPLC analysis. HPLC analysis. The concentration of sulfite was determined by an HPLC method previously disclosed in the literature (McFeeters et al. 2003). Five μl of sample was injected to an HPLC system equipped with a 7.8 x 300 mm Rezex Organic Acid column (Phenomenex, CA). A mobile phase of 0.02 N sulfuric acid at a flow rate of 0.8 ml/min was used for isocratic elution. The column effluent was monitored with a UV detector at a wavelength of 210 nm.

EXAMPLE 2 Oxidization of Sulfite by Chloroplast

Results of preliminary experiments with 5%> ethanol solution containing 300 ppm sulfite showed that chloroplasts were capable of oxidizing sulfite, while in the absence of chloroplasts the auto-oxidation of sulfite into sulfate was negligible in the presence of 5 % alcohol.

EXAMPLE 3 Effect of pH on Activity of Chloroplast

Crude chloroplasts, prepared as described above, at a final concentration of 1 mg/ml were added to 9.5%> alcohol white wine (Sutter Home White Zinfandel) spiked with sodium sulfite to a final sulfite concentration of 150 ppm and 50 mM buffer systems at pH values ranging from 4.3 to 9.1. For wine solutions at pH 4.3 to 5.0, citrate buffer was used; for wine solutions at pH 5.5 to 9.0, phosphate buffer was used. The reactions were allowed to proceed overnight. Aliquots of samples were taken and filtered with 0.2 μl syringe filters for HPLC analysis. As shown in FIG. 1, the activity of chloroplasts in sulfite oxidation increased with pH until it reached the optima at around pH 8.5, at which more than 95 % of sulfite was removed after overnight incubation with 1 mg/ml of chloroplasts, leading to a residual sulfite concentration of ca. 7.5 ppm and confirming the feasibility of sulfite removal with chloroplasts. Further increase in pH led to a decline in the efficiency of sulfite removal. EXAMPLE 4 Effect of Alcohol Concentration on Efficiency of Sulfite Removal

The effect of alcohol concentration on sulfite oxidation with a chloroplast concentration of 1 mg/ml was studied by evaluating the efficiency of sulfite oxidation in synthetic wines with alcohol concentrations of 5%, 10%, and 15%>, prepared by mixing 9.5%> alcohol wine containing 300 ppm sulfite and 50 mM pH 8.0 phosphate buffer with equal volume of 0.5%>, 10.5%>, and 20.0% ethanol solutions, also 50 mM pH 8.0 phosphate buffer, respectively. The reactions were allowed to proceed for 3. hr. Aliquots of samples were taken and filtered with 0.2 μl syringe filters for HPLC analysis. As show in FIG. 2, it is evident that the sulfite oxidation activity of chloroplasts decreased with alcohol concentration. As the alcohol content of wines increased from 0% to 5%>, the relative efficiency of sulfite oxidation was reduced by 66.4%>, indicating the denaturing nature of ethanol toward sulfite oxidase and/or the pigment proteins of the photosynthetic electron transport chains. Nevertheless, the extent of inactivation in sulfite oxidation activity of chloroplasts became less significant as the concentration of ethanol was further increased to 10% and 15% and thus validated the process of sulfite oxidation with chloroplast with wines containing alcohol at least up to 15%>.

EXAMPLE 5 Effect of Chloroplast Concentration on Efficiency of Sulfite Removal

The effect of chloroplast concentration on the efficiency of sulfite removal was studied. Wines containing 9.5 %> alcohol with 150 ppm sulfite and 50mM pH 8.0 phosphate buffer were incubated with chloroplasts at concentrations ranging from 1 mg/ ml to 10 mg/ml at room temperature for 1 hr. Aliquots of samples were taken and filtered with 0.2 μl syringe filters for HPLC analysis. As expected the efficiency of sulfite removal increased proportionally with the concentration of chloroplasts, FIG. 3. While only 10 %> of sulfite was removed with a chloroplast concentration of 1 mg/ml, more than 95 %> of sulfite was removed with a chloroplast concentration of 10 mg/ml. EXAMPLE 6 Time Course of Sulfite Oxidation

Time courses of sulfite oxidation with chloroplast concentrations of 1 mg/ml and 5 mg/ml in 9.5% alcohol white wines containing 150 ppm sulfite and 50 mM pH 8.0 phosphate buffer were followed for 6 hours. Aliquots of samples were taken and filtered with 0.2 μl syringe filters for HPLC analysis. As shown in FIG. 4, while less than 60 % of sulfite was oxidized with a chloroplasts concentration of 1 mg/ml after 6 hr incubation, more than 90 %> of sulfite was oxidized with a chloroplast concentration of 5 mg/ml within 3 hr. This result suggests that to be able to effectively remove sulfite within a reasonable time frame, a chloroplast concentration of 2 to 5 mg/ml might be needed.

EXAMPLE 7 Effect of Light and Mixing on Efficiency of Sulfite Oxidation

To investigate the effect of illumination on sulfite oxidation with chloroplasts, sulfite oxidation with a chloroplast concentration of 1 mg/ml in 9.5%> alcohol white wines containing 150 ppm sulfite and 50 mM pH 8.0 phosphate buffer in flasks covered with aluminum foil were performed with and without shaking were performed. The reactions were allowed to proceed for 3 hr. Aliquots of samples were taken and filtered with 0.2 μl syringe filters for HPLC analysis. As shown in FIG. 5, mild agitation that ensured the suspension of chloroplasts particles in wines and probably enhanced the transfer of sulfite increased the efficiency of sulfite oxidation. By comparing the data, it was found that the efficiencies of sulfite removal were reduced by 46.2 % and 34.4 % in light and in dark, respectively. It is also evident that moderate laboratory illumination stimulates sulfite oxidation with chloroplasts. In the absence of illumination, the efficiency of sulfite removal by chloroplast was reduced by about 42 % from 37.3 %> to 21.7 %. Since no measurable enhancements in auto-oxidation of sulfite were observed in the absence of chloroplasts (data not shown), the results may indicate the participation of light-induced photosynthetic electron transport chain of chloroplasts in sulfite oxidation. Since all the experiments in this study were performed under dim light, the light-induced photosynthetic oxidation capability of chloroplasts was not filly exploited and accounted for only ca. 40 %> of the sulfite removed. It is, therefore, reasonable to presume that the efficiency of sulfite removal can easily be further enhanced from 37.3 % to 60.2 % with better illumination. EXAMPLE 8 Sulfite Oxidation with Chloroplasts in Red Wines

The removal of sulfite from red wines (Sutter Home Red Zinfandel) which contains 13.5%) alcohol was studied. As shown in FIG. 6, the effect of illumination on sulfite oxidation was not as significant as that observed in the white wine, used for all other experiments reported in this study, FIG. 5. In red wines, illumination only enhanced sulfite removal from 83%> to 93%>. Illumination-induced sulfite removal accounted for 41.5%) of the total sulfite removal in white wines but only accounted for 10.8% of the total sulfite removal in red wines, indicating that light-induced photosynthetic oxidation participates in sulfite oxidation and does so to a greater extent in white wines. Surprisingly, the efficiency of sulfite removal in red wines was much higher than in white wines. Whereas in the white wine only 15%) of sulfite was removed with a chloroplast concentration of 1 mg/ml after 1.5 hr. incubation, FIG. 3, 93 %> and 100%> sulfite removal from the red wines were achieved with the same amount of chloroplasts within 45 min. and 1.5 hrs., respectively, FIG. 6, indicating the possible presence of positive effectors in red wines for sulfite oxidation with chloroplasts. The results of these examples clearly demonstrate the feasibility of sulfite oxidation/removal from both red wines and white wines with chloroplasts.

All of the compositions and/or methods and/or apparatus disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and/or apparatus and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

US Patent 5,071,664

US Patent 5,358,732

Bourque et al, Biochem. Biophys. Res. Commun., 51(4):9993-999, 1973.

Cohen et al, J. Biol Chem., 246:374-382, 1971.

Enemark et al, in Metal Ions in Biological Systems (Sigel, A., Sigel, H, ed), Marcel Dekker, New York, Vol. 39, pp. 621-654, 2002. Feng et al, Biochemistry, 41 :5816-5821, 2002. Feng et al, J. Biol. Chem., 278:2913-2920, 2003. Guide to Sulfited Foods," Center for Science in the Public Interest. Hebeτ et al, Int. Rev. Cytol, 177:255-286, 1998. Jolivet et al, Phytochem., 34: 1467-1471, 1993. Jolivet et al, Phytochem., 38: 9- 14, 1995. Leustek et al, Plant Physiol, 120:637-644, 1999. Mariac et al, Biotechniques, 28(1): 110-113, 2000 McFeeters, et al, J. Agric. Food Chem., 51 :1513-1517, 2003. Pacheco, et al, J. Biol. Inorg. Chem., 4:390-401, 1999. Petersen, et al, Contact Dermatitis, 27:344-345, 1992. Rajagopalan, in Molybdenum and Molybdenum-containing enzymes, (Coughlan, M. P., ed), Pergamon Press, Oxford, pp. 241-272, 1980 Rajagopalan, et al, in Wiley Encyclopedia of Molecular, Medicine (Creighton, T. E., ed), John Wiley and Sons, New York, pp. 3048-3051, 2002. Schindelin et al, Adv. Protein Chem., 58, 47-94, 2001.

Sulfites: FDA Limits Uses, Broadens Labeling," FDA Consumer, October 1986. Sullivan et al, Biochemistry 32:12465-12470, 1993. Sullivan et al, J. Am. Chem. Soc, 114:9662-9663, 1992. Vally, et al, J. Allergy Clin. Immunol, 105: 462-467, 2000. Vally, et al, Thorax 56: 763-769, 2001. Vally, et al, Addict Biol, 8:3-11, 2003. Vena, et al, Contact Dermatitis 31 :172-175, 1994. Wuebbens et al, J. Biol. Chem., 278, 14523-14532, 2003.

Claims

1. A method of oxidizing sulfites in a food product comprising: (a) obtaining chloroplasts from a chloroplast-containing substance; and (b) contacting said chloroplasts or chloroplast-containing substance with said food product.

2. The method of claim 1, further comprising removing said chloroplasts from said food product.

3. The method of claim 1, further comprising an illuminating step.

4. The method of claim 1, wherein the food product is a beverage.

5. The method of claim 4, wherein the beverage is an alcoholic beverage.

6. The method of claim 5, wherein the alcoholic beverage is a wine.

7. The method of claim 6, further comprising increasing the pH of the wine.

8. The method of claim 7, wherein the pH of the wine is increased to within a range of about 7.0 to about 9.0.

9. The method of claim 5, wherein the alcoholic beverage is a beer, distilled liquor, a cocktail mix or a wine cooler.

10. The method of claim 4, wherein the beverage is a non-alcoholic beverage.

11. The method of claim 10, wherein the non-alcoholic beverage is lime juice or lemon juice, a dried citrus fruit beverage mix, a fruit juice, or a vegetable juice.

12. The method of claim 10, wherein the non-alcoholic beverage contains a sugar syrup or a corn syrup.

13. The method of claim 1, wherein the food product is a processed food product.

14. The method of claim 1, wherein the processed food product is a baked good.

15. The method of claim 1, wherein the chloroplasts are obtained from plants or other chloroplast-containing tissues.

16. The method of claim 15, wherein the chloroplasts are obtained from wheatgrass.

17. The method of claim 15, wherein the chloroplasts are obtained from spinach.

18. The method of claim 1, wherein the chloroplasts are contained in a solid support.

19. The method of claim 18, wherein the solid support is a semi-permeable pouch, capsule or a porous matrix.

20. The method of claim 19, wherein the porous matrix is selected from the group consisting of natural polymers, synthetic polymers, inorganic supports, or organic supports.

21. The method of claim 18, wherein the chloroplast-containing solid supports are contained in a reactor.

22. The method of claim 21, wherein the reactor is a packed bed reactor, a fluidized bed reactor, or a membrane reactor.

23. The method of claim 21, wherein the oxidation of sulfite is accomplished by passing the food product through the reactor.

24. The method of claim 1, wherein the sulfite is sulfur dioxide or sodium sulfite.

25. The method of claim 1, wherein the sulfite is bisulfite or metabisulfite.

26. The method of claim 25, wherein the bisulfite is sodium bisulfite or potassium bisulfite.

27. The method of claim 25, wherein the metabisulfite is sodium metabisulfite or potassium metabisulfite.

28. The me^'tirόd "of claifh' I',"wh'efeιn oxidizing sulfites in a food product comprises removing the sulfites from the food product.

29. The method of claim 1, wherein oxidizing sulfites in a food product comprises reducing sulfites in the food product.

30. A method of reducing or preventing sulfite hypersensitivity in an individual comprising: (a) obtaining chloroplasts from a chloroplast-containing substance; (b) contacting said chloroplasts or chloroplast-containing substance with said food product; (c) removing said chloroplasts from said food product; and, (d) providing said food product to the individual.

31. The method of claim 30, further comprising an illumination step prior to (d).

32. The method of claim 30, comprising oxidizing sulfites in the food product.

33. The method of claim 30, comprising removing sulfites from the food product.

34. The method of claim 30, comprising reducing sulfites in the food product.

35. The method of claim 30, wherein the food product is a beverage.

36. The method of claim 35, wherein the beverage is an alcoholic beverage.

37. The method of claim 36, wherein the alcoholic beverage is a wine, a beer, a distilled liquor, a cocktail mix, or a wine cooler.

38. The method of claim 35, wherein the beverage is a non-alcoholic beverage.

39. The method of claim 38, wherein the non-alcoholic beverage is lime juice or lemon juice, a dried citrus fruit beverage mix, a fruit juice, or a vegetable juice.

40. The method of claim 38, wherein the non-alcoholic beverage contains a sugar syrup or a corn syrup.

41. The^' me^'tfioα-'brclaim ^• U,"wnerein the food product is a processed food product.

42. The method of claim 41 , wherein the processed food product is a baked good.

43. The method of claim 30, wherein the chloroplast is obtained from wheatgrass or spinach.

44. The method of claim 30, wherein the sulfite is sulfur dioxide or sodium sulfite.

45. The method of claim 44, wherein the sulfite is bisulfite or metabisulfite.

46. The method of claim 45, wherein the bisulfite is sodium bisulfite or potassium bisulfite.

47. The method of claim 45, wherein the metabisulfite is sodium metabisulfite or potassium metabisulfite.

48. A kit comprising chloroplast and buffer components.

49. The kit of claim 48, wherein the chloroplasts are contained in a semi -permeable pouch or bag.

50. A device for oxidizing sulfites in a food product comprising: (a) solid supports entrapping chloroplasts or chloroplast-containing substances; and (b) a reactor containing said solid supports.

51. The device of claim 50, further comprising an illuminating device.

52. The device of claim 50, wherein the solid support is a semi-permeable pouch or bag, a capsule or a porous matrix.

53. The device of claim 50, wherein the reactor is a packed bed reactor, a fluidized bed reactor, or a membrane reactor.

54. A process of increasing the pH of a wine comprising: (a) obtaining chloroplasts from a chloroplast-containing substance; and (b) contacting said chloroplasts or chloroplast-containing substance with said wine. The process of claim 54, wherein the pH is increases to between about pH 6.0 to about pH 8.0.