Technical Problem One of the crucial challenges of modern science is the prolongation of life of the population and the retardation of ageing. These endeavors include the important realization that the ageing rate is in a high degree influenced by free radicals. The effect of said radicals may be significantly diminished by the intake of appropriate antioxidants, preferably of natural sources. On one hand, this leads to the tendency to consume antioxidant-rich quality food; on the other hand it leads to the addition of natural supplements to foodstuffs, thereby concurrently substituting synthetic additives (colorants, preservatives, flavoring agents) that often exhibit antioxidant properties as well. Hence there is a need for processes enabling a simple and inexpensive isolation of natural substances having antioxidant properties and other health-beneficial properties, starting from readily available raw materials, possibly various plant residues, and for the discovery of processes for a widespread use of these substances by the population. For improved economy appropriate low-cost, possibly secondary raw materials have to be found, that may be of different sources and exhibit different properties. For each particular raw material the corresponding isolation process has to be developed in conformance with its specific characteristics.

The process has to yield an isolate having desired properties to preserve the natural properties of the isolates. The process and the isolate involved therein influence the applicability of said isolate. In particular, problems arise from the restrictions in the selected solvents and other chemicals, with regard to food regulation.

State of the Art There have been known various types of natural substances having antioxidant and other beneficial effects on human health. Among these substances are of particular interest polyphenols that also include anthocyans. The latter are a large group of natural colorants widespread in the plant kingdom. They are found in flowers, fruits (especially in berries), and in vegetables.

Because of their water-solubility and attractive color anthocyans are currently employed as foodstuff supplements. They exhibit multiplural activity-as antioxidant agents, as antimicrobial agents or as natural pigments. They are potential substitutes for less desirable synthetic preservatives and colorants.

Anthocyans are phenolic plant metabolites belonging to the flavonoid family (Nyman and Kumpulainen, 2001). They are the largest group of water-soluble pigments in the plant kingdom, where they are widespread. In alimentary products for humans they are represented in major quantities in fruits, vegetables, pulse and wine. In nature they are represented in glycoside form, and they are responsible for the red, blue and violet colors of fruits and vegetables (Nyman and Kumpulainen, 2001; Revilla et al, 1998). Bright, intensive colors of anthocyans fulfil a particular purpose in nature. They attract insects that pollinate the plants and disseminate the seeds in the environment.

They also offer mechanisms fulfilling their role in the inhibition of insects'attacks (Wang et al, 1997).

In nature have been discovered 16 anthocyanidins, and six of them are often found in fruits and vegetables. Anthocyanidins differ in the number and the position of-OH and CH3 groups (Goiffon et al, 1999). The most important anthocyanidins are pelargonidin, cyanidin, delphinidin, peonidin, malvidin and petunidin.

Anthocyans are potential antioxidants. The consumation of anthocyan containing food has a beneficial effect on human health. The key factor influencing the antioxidant activity of anthocyans is the pattern substitution in the ring B (difference in-OH and CH3 groups). Different anthocyannidins have different antioxidant values. Consequently it is important to know the structure of antocyanidins in food in beverages, for this reveals their antioxidant value (Nyman and Kumpulainen, 2001).

The investigation of the"French Paradox"stirred up interest for the benefits offered by the consumation of anthocyan-rich food. Antioxidant properties of anthocyans play an important role in the prevention and in part also in the treatment of several chronic diseases, and they diminish the risk of coronar diseases (Degenhardt et al, 2000). The investigation of anthocyans revealed their positive curative effect in the treatment of diabetical diseases, mammary cysts, and their beneficial effect on vision. They inhibit the occurrence of atherosclerosis caused by increased blood cholesterine. The investigation revealed also other possible beneficial physiological effects of anthocyans: protection against radiation, protection against inflammatory processes, and protection against platinum toxicity in chemotherapy, protection of the liver against damage by carbon tetrachloride. They have antimicrobial, antioxidant and numerous other capacities, owing to their various effects on different enzymes and metabolic processes in the body (Da Costa et al, 2000 ; Matsumoto et al, 2001; Wang et al, 1997; Kosir and Kidric, 2002; Bridle and Timberlake, 1997). These outstanding biological properties of anthocyans are supposed to significantly contribute to the beneficial effects of fruit and vegetable consumation.

The feeding of animals with the extracts of crushed red grape residues (pomace) containing predominantly anthocyans, revealed no detrimental effects. Also the consumation of said extracts by humans did not reveal any detrimental effects. Therefore the colorant obtained from grape skins is recognized by the Food and Drug Administration (FDA) as safe and not injurious to health (Da Costa et al, 2000).

Anthocyans have been receiving an increasing attention because of their possible use as natural colorants. The addition of red, blue or pink colored anthocyans is increasingly interesting, especially due to the aspiration for the employment of environment friendly, safe colorants that are not injurious to health (Degenhardt et al, 2000). The major reason is the resistance against the employment of synthetic colorants in alimentary products, owing to the growing awareness of the advantages of healthy alimentation (Malien-Aubert et al, 2001). In Europe the addition of natural anthocyan pigments to nutrients is allowed. These natural colorants are published in EECE163 (Goiffon et al, 1999).

It has been known, that the processing leads to the deterioration of the original color of the products. This may be corrected by the addition of natural pigments.

There is also a need for the coloration of colorless products that as such do not appeal to the consumer (Bridle and Timberlake, 1997).

Because of their attractive color, good solubility in water and their curative effects anthocyans are considered as potential substitutes for synthetic colorants (Morais et al, 2002). As a rule, anthocyans are found in attractively colored, fragrant fruits, such as wine grapes, berry fruits, mango, figs, pomegranates. For this reason they are very suitable as natural food supplements (Francis, 1989).

Because of their antioxidant and antimicrobial effects anthocyans may prolong the shelf life of products to which they are added. Their activity type renders phenolic substances toxic for a very broad spectrum of microorganisms that do not easily become resistant thereto (Nychas, 1995; Nyman and Kompulainen, 2001).

The instability of anthocyans is influenced by oxygen, high temperatures, metal ions and enzymes, to say agents promoting their decomposition and polymerization. Polymeric forms have mostly a brownish hue (Revilla et al, 1998 ; DeMan, 1999; Heredia et al, 1998; Morais et al, 2002).

Grapes are a rich source of polyphenols that are consumed in the form of fresh grapes predominantly, however, as red wine constituents. The anthocyan content in grapes is between 30 and 750 mg/100 g. Like in other plant materials the amount is highly variable dependent upon the plant variety, the season and environmental factors. The major part of anthocyans is found in grape skin.

During maceration they are transferred into the grape'juice and by means of vinification into the vine. During maceration only a portion of the anthocyans is transferred into the grape juice. A considerable portion remains in the pomace that yields a relatively inexpensive source for the production of anthocyans (colorants and medicinal agents) (Tomera, 1999; Tamura and Yamagami, 1994).

Blackcurrants have a three-to five-fold higher C vitamin content than lemons (150 mg or more in 100 g); it is important, as well, that blackcurrants contain relatively large amounts of potassium, calcium, magnesium, phosphorus, iron, copper, manganese, zinc, cobalt, nickel, chromium, selenium, aluminium, and bromine. Blackcurrants contain also about 6% sugars, fruit acids especially citric acid, a lot of vitamin E, a lot of anthocyan colorants and pectine (Cortese, 2000 ; Matsumoto et al, 2001) performed a study in which food was supplemented by the four principal blackcurrants anthocyanins (Del 3G, Cia 3G, Del 3R and Cia 3R). They established the anthocyanins'concentration in the blood and the urine after the intake of the extract. Maximum Cia 3R and Del 3R concentrations were measured two hours after the intake, and then slowly decreased. Maximum Cia 3G and Del 3G concentrations were measured one hour after the intake, and then rapidly decreased. These differences arise probably because of structural differences of the sugar constituents (rutinose is a disaccharide, glucose is a monosaccharide).

Redcurrants contain about 5% sugars; their nutritional excellence is first of all due to their richness with minerals. This is valid for macro elements (especially K, Ca, Mg, P, S), as well as for micro elements (Se, Co, Si and several others).

They contain also a lot of fruit acids acting as body toxin cleaners. The main anthocyanidin in redcurrants is cyanidin with various bound sugars.

Redcurrants are an important source of anthocyanins that are rarely found in nature. Characteristic anthocyanins in redcurrants are Cia 3- (2-glucosyl) rutinoside and Cia 3- (2-xylosyl) rutinoside. Found were also Cia 3soph, Cia 3G and Cia 3R (Goiffon et al, 1999).

The easiest and economically acceptable process for obtaining anthocyans from plant materials is aqueous extraction; the process is, however, complex.

Obtained are large quantities of the aqueous extract (1-2% of dry matter), which has to be concentrated. (Bridle et al, 1997) describe the extraction of pomace with water in the ratio of 1: 1. The extract is separated from admixtures (proteins, high molecular weight phenolic compounds), and concentrated by ultrafiltration on cellulose acetate membranes. This yields an extract containing predominantly anthocyans and sugars (Bridle and Timberlake, 1997). On the market is available the commercially available product enocyanin, a natural colorant obtained by extraction of anthocyans from pomace after the vinification of red grapes. Usually pomace of grape varieties is employed having strong pigmentation (for example Lancellota, Lambrusco, Alicante, and Salamin). The processing is performed in special vessels under addition of SO2 and takes several months. After concentration the liquid is cooled to precipitate potassium tartrate, centrifugated and formulated for the use as a liquid or as a powder after spray drying. In a process known in France as"Sefcal"grape pomace is extracted with 0. 2% SO2 in water continuously at 80 °C. This process requires a minimal solvent volume (1 L solvent for 1 kg pomace).

There are also known anthocyan extracts made of elderberries. For this purpose are used homogenized elderberries, and fermentation at ambient temperature under addition of yeasts without pH adjustment.

The most important characteristics of anthocyan extracts are their color intensity and hue; their stability in foods is, however, often limited. Because of decolorization in contact with S02 and susceptibility to chemical conversions at a pH about 4 their use as foodstuff colorants is often limited. Anthocyans esterified with acids exhibit an improved stability, and are more likely to be used as natural colorants (red cabbage anthocyans) (Degenhardt et al, 2000).

Slovenia is a country where wine is produced in large quantities. Wine production yields relatively large quantities of grape pomace as by-product.

Pomace is mainly discarded, although it contains a fair amount of anthocyans and other flavonoids. There are also numerous plantations of redcurrants and blackcurrants, which are mainly processed into juices. A large amount of anthocyans and other polyphenolic constituents remains in their pomace as well.

Hitherto there have not been known examples for the isolation and use of anthocyan extracts from plant materials of Slovene origin.

Disclosure of technical Problem with Working Examples The invention relates to: o processes for the optimal isolation of phenolic compounds from specific fruit processing residues, such as wine grapes, redcurrants and blackcurrants and the like, wherein the isolates have desired characteristic properties ; the process is simple, inexpensive and does not comprise substances with harmful health effects; * the use of the obtained isolates as supplements to foodstuffs, such as fruit juices, jams and marmalade, candy (bonbons, sweetmeats), dairy desserts and other products, to improve their antioxidant properties or functional sensorial properties (for example color, stability).

As raw materials were used various fruit processing residues, for example crushed redcurrants and blackcurrants residues, and marc obtained from crushed grapes after fermentation, especially from the Modri pinot variety.

We developed a simple process for the isolation of polyphenols by means of extraction during about 1 to 24 hours, at ambient temperature, with polar solvents such as ethanol, methanol or a mixture of either of them with water, or exclusively with water. Preferred are extractive processes employing water or a mixture of ethanol-water because of their better applicability in food processing.

The obtained isolates contain high percentages of key constituents (common polyphenols, anthocyans, sugars), exhibit a potent antioxidant capacity, demonstrated by measuring their free radical scavenging potential (DPPH), and by measuring their (-carotene oxidation inhibition potential. The obtained isolates require no additional treatment (such as separation of admixtures and specific constituents). Optionally, they were concentrated by evaporation in vacuum.

The extracts were added to various fruit juices, such as strawberry, sour cherry, bilberry, and apple juices, and to other alimentary products. Chemical analyses were used for determination of the anthocyans contents, antioxidant values, and the color intensity and hue. Established were the effects of added concentrates on the analyzed parameters, and analyzed sensorial characteristics with specific tests (ranging, pair comparison, score evaluation).

Selected were optimal supplements and their quantities on the base of the staple food properties, the composition and characteristics of the extracts and concentrates, and the acceptability of the supplemented products by the consumer.

The advantages of the present isolation processes are: use of low-cost plant waste materials, . simplicity, additional treatment of the isolates is superfluous, isolates have desired properties and composition, isolates have unimpaired natural properties and do not contain noxious admixtures.

The isolates are suitable as foodstuffs'supplements, and improve the antioxidant and functional properties of such foodstuffs. Also, their sensorial properties remain unaltered or are improved. Because of the intensive red color of the extracts they are"most suitable as supplements to intensively colored foodstuffs, such as strawberry, sour (marasca, morello, mahaleb) cherry, bilberry juices, and other products made of said fruits, and other fruits and vegetables. The color of such foodstuffs is often unstable, which limits their shelf time. The addition of the extracts minimizes the problem in a significant manner.

Isolates-supplemented products possess the following key advantages in comparison with non-supplemented products : improved antioxidant characteristics, o improved functional characteristics, such as stability, health effects and others, o improved sensorial characteristics, such as color, taste, flavor and others.

The invention is illustrated, but not limited by the working Examples.

Working Examples PREPARATION OF EXTRACTS AND CONCENTRATES Concentrate from redcurrant pomace (conc. RR) Water (4 L) was poured onto 2 kg of fresh blackcurrant or redcurrant pomace, shaken on a shaker for 1 hr at room temperature, and filtered by suction filtration. The filter residue was reextracted with half volume of the solvent at identical conditions. The extracts were combined, one half was utilized, and the other half was concentrated by distillation under vacuum to approximately one tenth of its volume, the concentrate was analysed and added to various alimentary products. Analysis results are shown in Table 1.

Concentrate from blackcurrant pomace (conc. BR) Water (4 L) was poured onto 2 kg of fresh blackcurrant pomace, shaken on a shaker for 12 hrs at room temperature, and filtered by suction filtration. The filter residue was reextracted with half volume of the solvent at identical conditions.

The extracts were combined, and concentrated by distillation under vacuum to approximately one tenth of their volume, the concentrate was analysed and added to various juices or alimentary products. Analysis results are shown in Table 1.

Concentrate from grape marc (conc. Gr) An ethanol-water mixture (70: 30 vol./vol., 4 L) was poured onto 2 kg of fresh grape marc (pomace after fermentation), shaken on a shaker for 24 hrs at room temperature, and filtered by suction filtration. The filter residue was reextracted with a half volume of the solvent at identical conditions. The extracts were combined, and concentrated by distillation under vacuum to approximately one tenth of their volume, the concentrate was analysed and added to various juices and alimentary products. Analysis results are shown in Table 1.

Table 1: Total polyphenols'and anthocyans'contents and antioxidant capacity expressed as % AA (the absorbances ratio of the residual 2, 2-diphenyl-l-picril hydrazide in the sample and in the blind sample after 15 minutes at (a = 514 nm-Moure et al, 2001), and KKA (antioxidant capacity coeficient based on absorbance measurements (ß-carotene Moure, et al, 2001).. Total anthocyans Total Material (mg/L) Polyphenols (mg/L) % AA KAA Conc. RC 15. 5 69. 6 4. 3 69 Conc. BC 202. 9 377. 0 38 380 Conc. Gr 74. 8 579. 0 39 444 APPLICATION OF CONCENTRATES IN JUICES Application of concentrates in strawberry juice Strawberry juice (nectar Pago) was supplemented with various portions of concentrated RC, BC, and Gr extracts. Thus, 100 mL juice was supplemented with 0. 5 mL concentrate (in the following text 0. 5%), 1 mL (1%), 2 mL (2%), 3 mL (3%), and 4 mL (4%), and thoroughly mixed.

The samples were subjected to chemical and sensorial analysis-the results are shown in Tables 2,3, 4,6, and 8.

Application of extracts or concentrates in bilberry, apple, and sour cherry juices Employed was the same procedure as for strawberry juice. The quantity and the type of the supplement were selected in dependence on the composition and the sensorial characteristics of the starting juice, as well as the appeal to the consumer. The results are shown in Tables 5 and 7.

Table 2: Anthocyans'contents in strawberry juice with various portions of supplemented BC, RC and Gr concentrates Anthocyans'contents in strawberry juice (mg/L of juice) No suppi. 0. 5% 1% 2% 3% 4% suppl. suppl. suppl suppl. suppl. Conc. BC 98. 9 203. 7 267. 0 411. 4 548. 0 715. 9 Q Conc. RC 98. 9 110. 6 112. 0 123. 7 129. 4 152. 6 25 Conc. Gr 98. 9 116. 4 128. 6 138. 3 139. 4 168. 6 Table 3: Antioxidant activity in strawberry juice with various portions of added concentrates of blackcurrants (BC), redcurrants (RC) and grapes (Gr) Antioxidant activity of strawberry juice (%) No suppl. 0. 5% 1% 2% 3% 4% suppl. suppl. suppl. suppl. suppl. Conc. BC33405431504562586 Conc. RC 33 33. 0 33. 7 36. 2 39. 3 43. 7 rn Conc. Gr 33 38. 2 38. 0 41. 4 43. 9 45. 6 Table 4 : Color intensity of strawberry juice with various supplements of BC, RC and Gr concentrates Color intensity of strawberry juices (total sum of absorbances at 420 and 520 nm) No suppl 0. 5% 1% 2% 3% 4% suppl. suppl. suppl. suppl. suppi. Conc. BC 1. 035 1. 525 1. 676 2. 225 2. 876 3. 444 Conc. RC 1. 035 1. 061 1. 049 1. 018 1. 039 1. 066 (o Conc. Gr 1. 035 1. 081 1. 076 1. 211 1. 250 1. 387 DETERMINATION OF ACCEPTABILITY OF PRODUCTS SUPPLEMENTED WITH ISOLATES OR CONCENTRATES Evaluation of sensorial characteristics Selected alimentary products, supplemented and non-supplemented, were evaluated by a three-membered expert tasting committee, that evaluated the characteristics by means of ponderated scoring, and allocated 0-8 points to the taste, and 1-4 points to each of the following characteristics: color, smell, and homogenousness. (0 points-least-expressed characteristic, 8 or 4 points respectively-optimal-expressed characteristic). The results are shown in Tables 5,6, and 7.

Table 5: Results of apple juice sensorial evaluation Apple juice (No. of points allocated) Possible Non-2% BC 4% RC ! % Gr No. of supplement. points Taste 0-8 6. 8 5. 5 7. 3 6. 7 Color 0-4 3. 7 3. 5 3. 7 3. 3 Smefl 0-4 3. 5 2. 5 3. 3 2. 9 L Homogenousness 0-4 4. 0 3. 7 4. 0 4. 0 Total score (sum) 0-20 18. 0 15. 2 18. 3 16. 9 Table 6: Total No. of points allocated to strawberry juice by the tasting experts Strawberry juice (No. of points allocated) Possible No. of Non-2% BC 4% RC ! % Gr points supplemented A 0-20 18. 0 17. 5 18. 0 16. 0 B 0-20 18. 0 16. 5 18. 5 17. 0 'C020185175180160 I I Table 7: Total No. of points allocated to supplemented and non-supplemented sour cherry juice by the tasting experts Sour cherry juice (No. of points allocated) Possible No. of Non-2% BC 4% RC 1% Gr points supplement. A 0-20 17. 0 18. 0 17. 5 17. 0 1 B 0-20 18. 5 16. 5 19. 0 15. 0 C 0-20 18. 5 17. 5 18. 5 17. 0 Pair comparison The evaluation of the acceptability (appeal to the consumer) was performed by pair comparison. A pair comprised one non-supplemented product and one supplemented product. 15 tasting experts performed the comparison.

The results for strawberry juice are represented in Table 8.

Table 8 : Results of testing the pair strawberry nectar-strawberry nectar supplemented with concentrate (the No. of responses represents the No. of tasting experts, that selected one individual of the pair). PAIR No. 1 PAIR No. 2 PAIR No. 3 Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry juice juice + 4% juice juice + 2% juice juice+1% conc. RC cone. BC conc. Gr No. of responses 8 7 1 14 3 12 Thus, the process for isolating antioxidants from plant waste material, is characterized in that it is performed by extraction with polar solvents, preferably with water or a water-ethanol mixture at a temperature of 10-50 °C, preferably 15-30 °C, and optionally followed by concentration using evaporation under vacuum. The process is performed by means of extraction with polar solvents, preferably with water or a water-ethanol mixture at a temperature of 10-50 °C, preferably 15-30 °C, and optionally followed by concentration using evaporation under vacuum. The plant waste materials used are preferably grapes, redcurrants and blackcurrants.

A further object of this invention is the use of crushed fruit residues, in particular grapes, redcurrant and blackcurrant, for the isolation of substances having antioxidant and beneficial functional and sensorial properties, preferably polyphenols and anthocyans.

The invention comprises isolates and concentrates obtainable by the processes, and containing substances, preferably polyphenols and anthocyans having antioxidant and beneficial functional and sensorial properties, in concentrations enabling the significant improvement of said properties in the foodstuffs supplemented by said isolates and concentrates.

The invention also comprises the use of isolates and concentrates according to the invention for supplementing foodstuffs, preferably fruit and vegetable juices, compotes, jams or marmalade, and other colored foodstuffs subject to color deterioration during processing or storage (high temperatures, oxidation).

Isolates and concentrates are designed as supplements to foodstuffs of vegetable or animal sources, thus improving their antioxidant, and other beneficial properties for humans by the supplement of said isolates and concentrates. Foodstuffs supplemented by the isolates and concentrates are the object and the result of the invention.

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