WO2017222486A2

WO2017222486A2 - Natural food colouring from corn-cockle (berberis) fruit

Info

Publication number: WO2017222486A2
Application number: PCT/TR2017/050276
Authority: WO
Inventors: Olcay KAPLAN INCE; Eren ASAM
Original assignee: Kaplan Ince Olcay; Asam Eren
Priority date: 2016-06-20
Filing date: 2017-06-20
Publication date: 2017-12-28
Also published as: WO2017222486A3

Abstract

Invention is about a natural food coloring from corn cockle fruit. Obtained food coloring maintains its natural color against temperature increasing and its stability alteration is low. Also food dye maintains its natural color against ambient oxygen and by absorbance chancing protecting its stability was determined.

Description

A NATURAL FOOD COLOURING FROM CORN-COCKLE (BERBERIS) FRUIT

TECHNICAL FIELD

Invention is related to obtained natural food colouring from corn-cookie (berberis) fruit. Obtained food colouring; maintains its natural color when temperature increase and its stability change is low. It was also determined from the change in absorbance of the food dye that maintains its natural color against ambient oxygen and maintains its stability.

PRIOR ART

It is known in history firstly the Egyptians colored their food with the coloring matter. About 3500 years ago, the painted sugar called "khand" was presented as a gift to when Alexander the Great come back from India to Europe. In the recent history, firstly, in 1856, the production of synthetic dyestuffs was started with the synthesis of "anilin moru" dye material. In the following years, the World Health Organization (WHO) in 1956 approved a list involve 40 country, containing 1 14 synthetic colorants and 50 natural coloring materials, opening the way for the use of food colorings in the food industry. In the following years, the rapid development of technology in parallel with industrial progress has also accelerated the development of new production techniques and the diversification of consumer taste over time as the use of food colors in the food industry.

The industrial revolution that started in the 18th century brought speed to human life As a result of this, eating habits were affected and ready food consumption became widespread. Despite the widespread use of ready-to-eat foods and the production of high-quality ready-to-eat food, the amount of natural coloring materials to be used in these colors did not supply the increasing demand. Because of the higher coloring power, color range and stability of natural coloring materials compared to natural coloring materials, the use of synthetic coloring in food industry has become widespread. However, synthetic food colorants toxicological and ecological depending on the reasons, the use of natural colorants have increased the share of the market every year.

BRIEF SUMMARY OF THE INVENTION

The invention relates to a new natural food coloring derived from corn-cockle

(berberis) fruit which will be used as a natural food coloring which is useful for health and will not cause toxicological effects. At the beginning of the natural colorings are the anthocyanin based colorings. Although the use of anthocyanins as coloring agents in foods is intended to increase the attractiveness of food, the high preference for anthocyanin-based food coloring because they have positive health impact. In nature, as a source of anthocyanin, some parts of many plant species especially their fruit have been consumed by people. Black raspberries, raspberries, black mulberries are rich fruits in terms of anthocyanins and also, Corncockle (berberis) fruit has rich anthocyanin content. This information is in the light, corn coockle fruit was preferred for this invention as a source of natural food coloring material.

DETAILED DESCRIPTION OF THE INVENTION

Corn cockle (Berberis) fruit grows mainly in Asia, and in many parts of Europe and North America globally, more northerly in temperate regions. In our country, it grows spontaneously in the North Anatolian region without agriculture and has more than 450 species, grows up to 2 m in height, it is called Berberis vulgaris with bushy, flowering and thorny. Some of this Berberis genus is green every season, while some species are thorny in winter. The spines of this plant, which grows from the stem of the stem and has extremely hard spines, come out of the stem of the leaves which form bundles on the branches. Therefore women are called "thorny grapes". Fruit is meat with one or more seeds. In some species, the fruits are found individually, while others are in the form of grape bunches. Unique to Turkey and Iran Is known by the name "Berberis crataegina". It is almost the same length as Berberis vulgaris. But it can easily be distinguished from the foliage and thorns that darken in the autumn. The interior of the body and root is yellow and the fruity gets black when it matures. It is also named with names such as barberry, aged work, amberparis, rabbit bread, thorny grape in different Anatolia regions. Berberis vulgaris and Berberis crataegina have spread over a wide area. Most of the barnyard found in almost every region of Turkey grows spontaneously in Izmir, Ankara, Kirklareli, Kastamonu, Cankin, Sivas, Nigde, Kayseri and Konya regions. Fruits (Berberis crataeginae), tannins, organic acids (malic, tartic, citric acids) and high levels of ascorbic acid (vitamin C) and anthocyanins.

The light, an electromagnetic wave, allows the perceived object to be perceived. When a light beam falls on an object, some of it is reflected while the other is absorbed by the object. These two events occur at the same time. The reflected light waves and the equivalent color are perceived by the eye. Colors can be perceived differently depending on external conditions. The same color can be perceived in different colors under direct incoming or non-direct light. However, the human sense of sight adapts to the source of the light and can make the color be perceived as the same color. The eye reacts to the three basic colors red, green and blue. The eyes perceive other colors, such as red, green, and blue, as other blends of these colors. Colors perceived by the human eye are visible colors at wavelengths between 380 and 770 nm.

Color has an important role in the attractiveness of food. The first impression of the consumer about the quality of food relates to the color. Consumers, food to be consumed items are in usual color expectancy. When food raw materials are processed into the product, there is more or less color loss. Color materials are also important in terms of creating a standard color in food as technologically. Some synthetic or natural coloring are used in order to make the food more attractive and increase the desire to consume, which is a great importance in the selection of food to be consumed by the consumer. But for human health and reliable food more advantages over the past few years due to some advantages and acceptability natural food colorings are preferred. However, due to some advantages and acceptability in terms of human health and reliable food, more natural food colors are preferred in recent years. There are many reasons for adding food colors as a food additive. Some of these are:

• In order to protect the natural color of the food because of the destruction of the natural color of the foods in the production stage (eg fruiting of the fruits) or post- production storage period, • To ensure the standardization of the original color intensity of the new processed grains to be obtained from the different color intensity shades of the food obtained at different times of the natural growing season,

• Giving a more attractive appearance,

· Aromatic drinks, sauces or yoghurt like fruity, consumer; it is applied in the food that the taste and aroma of the product will invoke. Especially when the color of the product is expected to be weaker, it is desired to increase the color density,

• To help protect the taste, aroma and light-sensitive vitamins during the storage of processed foods,

· To ensure the recognizability of the food or to preserve its color-specific authenticity,

• Since color is an important sign of sensory quality of food, external paint is added to help increase the acceptability of food. Some products, such as confectionery, have been added with wine or some herbal extracts to correct the color. In parallel with the development of the industry in time, the food industry has also undergone a rapid change. Food industry has also made use of mineral and metal-based compounds such as red lead (Pb30₄), mercury sulfur (HgS) or copper arsenate (CuAs0₄) for the coloring of foodstuffs. These types of toxic chemicals, which were later identified as harmful over time, have been used in many different foods such as confectionery and brine in the food industry. Studies have shown that these toxic compounds used lead to poisoning or even deaths. Food colorings are collected in 3 groups as synthetic, natural identical and natural coloring.

Synthetic colorants are color materials that are not found in nature due to their chemical form but they are produced by chemical synthesis. Also, food additives can be easily mixed and do not cause sensory defects such as unwanted taste and odor in food. Despite the declining number of non-natural dyes, which are suitable for consumer health in recent years and which are not allowed to be added to food, a wide variety of synthetic food dyes depend on low costs, high efficiency and excellent durability food industry is widely used in all areas of the industry. Compared to natural colorants, they give superiority in terms of factors such as coloring power, color spacing, durability, ease of use and low cost. The artificial colorants have very high water solubility. Many of them are resistant to heat, light, acids, alkalis and also resistant to protective substances. In 1856, Sir William Henry Perkin developed and applied the first synthetic food coloring "muavine" in history. Developed other synthetic coloring materials in the following years will be exactly what the industry anticipates, easy to produce, cheap, easy to mix with food, very good coloring and do not create sensory defects such as unpleasant taste and odor when doing so, synthetic coloring have found wide use all over the world.

The culture of living together with the industrial revolution that started in the 18th century urbanization has begun to gain speed, and as a result, ready-made food consumption has become widespread. Despite the high production of ready-to-eat foods, the production of natural coloring materials to be used in these foods has not received increased demand. In order to meet this need; lead and copper salts, copper sulphate has started to be used as color materials in various chemical substances. Depending on the coloring of these toxic stains and foodstuffs, many deaths have been detected in the UK. Studies on artificial coloring materials have been increased to meet the demand for increased coloring materials. Synthetic colorants are discovered and produced from the mid-19th century these paints replaced the traditional coloring materials traditionally used and synthesized as many artificial color materials as every day. It is a known fact that some synthetic colorants have side effects on health. Naturally identical food dyes are produced from natural sources with various processes. Chlorophyll in copper complex or caramel is included in this group.

Caramel; caramels are viscous liquids or hygroscopic powdery substances in reddish-brown or brown-black colors. Caramels are the oldest and most use among the food colorings. The caramels are divided into four classes (Class I, II, III and IV), taking into account the method used in their production and the reactants used. Although caramel is soluble in water, it does not dissolve in many organic solvents. More than 80% of the caramel produced in America is consumed in food industry, especially in drinks and alcohol colors are preferred. Caramel is used in the coloring of different foods such as dairy products, pre-cooked or dried pasta and pastas; fresh meat, broiler meat, fresh and frozen seafood, crackers, cocoa and chocolate products, baby food, candies and syrups, coffee and tea. During the caramelization process among the low molecular weight components formed in caramels are 4-(5) methylimidazole (4-Mel) III. and IV. class caramels; 2-acetyl-4 (5) -tetrahydroxybutylimidazole (THI) is only present in III class caramels 5-hydroxymethyl furfural (HMF) is found in all caramel classes. While 4- Mel showed neurotoxic effects from these components called caramel markers, some in vitro studies have shown that carcinogenic compounds in human liver inhibit the enzyme cytochrome P450, which plays a role in the oxidation. In another study conducted, 4-Mel was found to induce lung cancer in male and female mice. THI suppresses the immune system and HMF cause damage to the eyes, respiratory system, skin and mucous membranes.

Chlorophyll copper complexes; it is difficult to protect the stability of the chlorophyll pigment and it is difficult to use due to reasons such as water dissolution a colorant. Today, however, a derivative known as "sodium copper chlorophyll" is preferred. This compound is the chlorophyll sodium salt obtained after the substitution of magnesium in the chlorophyll structure with copper. Sodium copper chlorophyll with blue-green color is easily soluble in water and heat treatment to protect the green color of the tissues. The copper in the composition has been reported to have a concentration that is too low to exhibit toxic effects.

The Turkish Food Codex has allowed the use of chlorophylls in very different food groups in the coloring notification used in food. However, the Food and Drug Administration (FDA) does not allow the use of copper chlorophyll complexes as a food additive, while allowing sodium and potassium salts to be used only in dentifrices and medicines. Codex limited the foodstuffs and amounts of using copper chlorophyll complexes. Natural coloring materials are microbial, herbal, animal or mineral origin pigments. These materials, which have limited color range and poor stability, are poorly colored. In addition, their sensitivity to light, temperature, pH and redox agents is high and structural stability is poor.

Up to the middle of the 17th century, coloring materials obtained from natural sources such as saffron, carrot, mulberry, flower, copper and iron ore minerals, animal products and vegetables were used. These coloring materials used in various forms in the past, today's developing technology and coloring properties depending on the increasing necessity have been the subject of research of many natural resources and acquired industrialization. Very little of the natural coloring matter is soluble in water. This limits the color variety of natural coloring materials. For this reason, oil-soluble natural coloring materials can be converted into a structure that can be used in water- based foods by processing with a suitable emulsifying agent.

The prohibition of synthetic food colorants due to toxicological and ecological reasons has increased the share of the use of natural colorants by approximately 4-6% per year.

Although the use of natural food colors has increased due to consumer demands, some parameters have to be taken into account when using natural colors. These can be summarized as follows.

- Legal regulations in countries where a food product to be added with color matter will be produced

- The desired color tone in the food item,

- The pH value of the food which the coloring matter will be exposed to (the pH of the medium affects the color stability and tone of most natural dyes and often distorts the stability)

- The structural form of food, especially if it has an aqueous structure or contains significant amounts of liquid or solid fat (eg the presence of tannins and proteins limits the use of certain colorants such as anthocyanins)

- The process conditions, especially the temperature grade applied during production; and processing times,

- The packaging material of food which added color matter is another important feature. Because ambient oxygen and light are among the other external factors that affect the stability of coloring matter. In particular, the square stoids lose their stability by being affected by ambient oxygen and light,

- Desired physical form for a matter that is more interested in the industry (eg, natural styrenic liquids in terms of price are generally more expensive than dusty ones). Anthocyanins are a coloring matter that is soluble in water and allows many fruits and vegetables to color from red to blue. It has been reported that many fruits such as red and black currants, raspberries, strawberries, apples, sour cherry and black mulberry contain high amounts of anthocyanin. Anthocyanins are one of the subgroups of flavonoids. The basic structure of anthocyanins having glycosides of anthocyanidins is 2-phenylbenzopyrylium (flavium cation).

It is known that more than 500 anthocyanins and 23 anthocyanidins are found in the nature. The differences of anthocyanins depend on the number of hydroxyl groups in the chemical molecule, the degree of methylation of hydroxyl groups, and the type, number and bond forms of molecularly bonded. In addition, factors such as the number and structure of the aliphatic and aromatic acids bound to sugars in the structure cause the difference between the anthocyanins. Anthocyanidins found in plants the most common found are pelargonidin, peonidin, cyanidin, malvidin, petunidin and delfinidin. Among these anthocyanidins cyanide, delphinidin and pelargonidin the most found 10%, 80% of the pigmented leaves, 69% of the fruit and 50% of the flowers. The most common anthocyanins in nature are fruits and vegetables with 50% cyanide, 12% delphinidin, 12% pelargonidin, 12% peonidin, 7% petunidine and 7% malvidin.

Anthocyanins as coloring additives in food industry the biggest problem encountered in the use of is their low stabilization. The color stability of anthocyanin pigments varies depending on pH, temperature, structure and concentration of anthocyanin, and metal ions and phenolic compounds in the environment. Sugars bound to anthocyanidins are in many cases p-cumaric, caffeic, ferulic, sinapic, gallic acid aromatic acids or p-hydroxybenzoic acid or aliphatic acids such as malonic, oxalic, malic or acetic acid. Acylation is highly effective on the stability of anthocyanins. The main principle of color stabilization in plants is that they form complexes called ancophenes with other anthocyanins.

Copigments generally are colorless, but when complexed with anthocyanins they strengthen and stabilize their colors. At the beginning of the copigments are flavonoids. Flavonoids, as well as organic acids, alkaloids, amino acids, nucleotides, polysaccharides, metals and other anthocyanins can be classified as copigment. Anthocyanins show color change according to ambient pH such as indicator. When the pH of the medium is below 2, the anthocyanin dominates the flavium cation and the color tone is red. If the pH is between 4-5, colorless carbinol pseudobase form dominates. If the pH of the medium goes above 5, a quinidal anhydrous form is formed and the ambient color becomes blue. However, these reactions are two-sided reactions, and the colorless form of the anthocyanins can be converted into colored cationic and quinidal forms. Disintegration of anthocyanins mechanism, hydrolysis, oxidation and condensation with other phenolic compounds. The most important parameters for the anthocyanin breakdown are pH and temperature.

Although anthocyanins are the best known natural food dyes, they are difficult to purify and not stable as a chemical structure. They are not widely used for this purpose. Sources of significant anthocyanin include black grape pod, red cabbage, concord grape, black carrot, sweet potato, red radish, potato, purple corn and blueberries. In commercial terms, the sources of anthocyanins are limited to raw materials. Economically, potential sources of anthocyanins include grape bark extracts are important. Red grape skins vary in size from season to season depending on seasonal conditions and environmental conditions. In the United States, only anthocyanins extracted from grapes by the FDA are considered to be food color additives. The commercial preparations are enochyanine and sediment (residue deposited in the wines of the wines) commercial grape extracts are allowed to be used in confectionery products, alcoholic and non-alcoholic beverages. Fruits such as black grapes and byproducts, blueberries, Hibiscus calyces and blackcurrant are known to have high anthocyanin content. However, they are found to exhibit low stability against hydration and pH changes. Desired color characteristics and the sources of the acylated and consumed anthocyanins with high stability are reddish, reddish, red potatoes and black carrots.

In nature, as a source of anthocyanin, it is consumed by structural parts of many plant species, especially fruitful people. The use of anthocyanin as a coloring matter in food anthocyanin-based food colors are preferred due to their positive effects on health. Hibiscus sp. Anthocyanins, hypertension and liver disorders, bilberry (Vaccinium) anthocyanins is used in the treatment of infections, visual defects, diarrhea and various other diseases. In general, in humans and animals, anthocyanins are absorbed in the form of glycosides (unstructured). The glycosides of anthocyanins are not degraded in the digestive system of the mammals and participate directly in the bloodstream. Within the scope of a study, it has been found that less than 1 % of the anthocyanin is absorbed after consumption of black raspberries as a source of anthocyanin at doses of 2.69 ± 0.085 g / day on volunteer subjects. Studies have shown that consumption of food with high phenolic content after 10 minutes, however, the flavonoids reached active levels, but did not accumulate in the plasma. Some flavonoids are excreted in urine 4 hours after consumption. Anthocyanins prevent DNA breakage, inhibit certain enzymes such as estrogenic activity, cyclooxygenase, accelerate cytokine production and strengthen the immune system is specified. Anthocyanins have been found to modulate cognition and motor function, contributing to the prevention of neural diseases that develop memory and age. In addition, anthocyanins can be used for anti-inflammatory, anti- vascular, platelet aggregation-inhibiting, normal vascular permeability-protecting, and are protective against UV radiation. In another study, consumption of anthocyanins extracted from the sources of various anthocyanins has been shown to be more effective in feeding the anthocyanin rich diets as a result of observing the effects of intestinal cells. A NATURAL FOOD COLOURING FROM CORN COCKLE (BERBERIS) FRUIT

The corn cockle fruit is a grape fruit with two small cores covered with a waxy layer, an outer shell with a very thin plate and has high proportion of pulp. Within the scope of the study, fruits were filtered through the thin pore filter cloth and fruit juice was obtained. The lyophilize tubes are filled with 5 ml_ of juice and are pressurized at -87 ° C 1 1 Pa and dried under vacuum (72 hours) in a controlled manner to remove water and pulverized.

In order for the anthocyanins to be stable against oxygen, the powdered fruit juice was purged with nitrogen prior to storage. Then the pulverized food coloring was stored at -18^SC was stored.

For the dry matter analysis of corn cockle, 3 g of samples (3 replicate samples) were weighed and put into porcelain crucibles that were at constant weight and tared and then samples were dried at 105 ^SC until constant weight. After the treatment, the samples were taken in a desiccator and cooled to room temperature dry matter content of the corn cockle samples that are constant weight was calculated using the formula (DM% = [(G2-G) / (G1 -G)] x100). (DM%: Percentage of dry matter, G: Tare of the weighing container, G1 : sample + weighing container, G2: sample after drying + tare container). As a result of the analysis, the dry matter content of the corn cockle fruit was determined as 37.2 ± 0.9 g/100 g dry weight.

2 g sample was weighed in porcelain crucibles that tare was preliminarily determined. 1 ml_ of 95% ethyl alcohol was added to the crucibles and the samples were burned by pre-burning until they became charred. The charring sample was burned in an ash oven at 550 ^SC until no black spots were found. The total amount of ash was expressed as " g/100g ". As a result, the ash amount of the corn cockle fruit was determined to be 1 .01 ± 0.05 g / 100 g ash.

The pH depends on the active hydrogen ion concentration in the solution, and the active hydrogen level in the solution also determines the acidity. The principle of this assay is measuring pH scale of foods' H ions charges (potential) differences.

Due to the nature of the corn cockle fruit, it is a fruit with a high water content, allowing the water to be removed effectively. With the help of a thin pore filter cloth, a mechanical press was applied to remove the juice and homogenous fruit juice was obtained. 20 ml_ was taken from the corn cockle juice and was placed in a 25 ml_ beaker. At 20 ^SC, measurements were made by immersing the pH meter glass electrode was directly into the liquid sample and calculation was done by using the equation pH = -log (H⁺). According to the results the pH value of the corn cockle fruit was determined as 3.25 ± 0.03.

The moisture (water) content of food is not a parameter that can be used alone in microbial growth and chemical reactions. Moisture content, gives information about the total amount of water in the product. Water activity (aw) is the ratio of the vapor pressure of pure water at the same temperature to the vapor pressure of water in the product. In another saying, it is the point that, the sample moisture value balanced the air relative moisture. At this point, no moisture exchange occurs between the product and air. Corn cockle sample was taken till to fill the water activity device reservoir and measurements were done at 25 ^SC. As the result of the analysis, the water activity (aw) value of corn cockle was determined as 0.95 ± 0.01 .

Color measurement of corn cockle juice samples were made by using Hunter (L ^*, a ^*, b ^*) color measurement system with color meter at room temperature under proper light after device reservoir was filled with sample. L ^*, a ^*, b ^* and a ^* / b ^* values were determined.

The brix values of barberry juice were determined by refractometer at 25 ^SC, the results being given as " g / 100 g ". As a result of the analysis made, the brix value of the corn cockle fruit was determined as 27.4 ± 0.2 g/100 g.

Table 1. Some analysis and results of corn cockle fruit

DPPH (2,2-diphenyl-1 -picrylhydrazyl) is a commercially available stabilized, organic nitrogen radical. Transposition of a free electron in the molecule structure allows the formation of a violet color.

When DPPH solution is hydrogen across with a substance (antioxidant), that gives H atom, occurred a reduced Formby loss of dark violet color. Proton transfer reaction by antioxidant (A-H) to the DPPH free radical causes a decrease in absorbance at 517 nm. This process is performed with ultra violet visible spectrophotometer until the absorbance remains constant. The greater the decrease in absorbance, the higher the antioxidant activity. DPPH-H, is the reduced form. A; is the free radical created in the first step. Then this radical goes into other reactions.

The corn cockle fruit was prepared at different concentrations. Take 100 μΙ_ of sample from the different concentrations and add 3 ml_ of methanol and 1 x 10 ⁴ M DPPH (2,2-diphenyl-1 -picrylhydrazyl) solutions. After incubation in the dark for 30 minutes at room temperature, the absorbance values were measured on a spectrophotometer at 517 nm, at which DPPH gave maximum absorbance. DPPH solution and the solvent from which the sample was dissolved, were used as positive control. The results are given as DPPH scavenging activity.

% DPPH scavenging activity = [(ApppH-AExampie) / ADPPH] 100 (ADPPH: Absorbance at 517 nm for DPPH, AExampi_e: Absorbance at 517 nm for example).

Total phenolic content of corn cockle extract was determined according to Singleton and Rossi (1 965) method. For this purpose, gallic acid standards were prepared at different concentrations, 1 , 5, 10, 25, 50, 100 and 300 ppm and pipetting and processing steps for the determination of total phenolic content by Folin reactant were carried out respectively. Absorbance values were measured by using a spectrophotometer at a wavelength of 760 nm. Later absorbance values were plotted against concentrations. The amount of TFC (total phenolic content) of the corn cockle fruit extract according to the drawn graph was calculated taking into account the dilution factor. The results were given as mg GAE / g of fruit juice.

Table 2. Pipetting and processing steps for total phenolic content identification

For sugar analysis 0.5%, 1 %, 5% and 0.75% of mix standards consist of glucose, fructose and sucrose sugars were prepared and injected to device and calibration graphs were obtained. Then, 5 mL of sample was weighed and 45 mL of ultra-pure water was added. The diluted sample was centrifuged at 5000 rpm for 15 minutes then filtered through a 0.45 μιη filter and samples were analyzed by using sample HPLC-RID. The used mobile phase was acetonitrile/water (4:1 ), the used column was 250x4.6 mm amine phase column, column temperature was 30 ° C, and flow rate was 1 mL/min.

5 grams of seedless corn cockle fruit were weighed for phenolic component analysis and 25 ml_ of methanol was added. The mixture was stirred and centrifuged at 5000 rpm in a centrifuge for 15 minutes. Obtained extracts were filtered by 0.45 μιη filters and analyzed by HPLC-DAD.

0.1 g was taken from the powdery food colouring obtained from the corn cockle fruit. Dissolved in 100 ml_ ultra pure water (containing 1 % HCI). To determine the maximum absorption wavelength of anthocyanins in this solution, scanned wavelength in the range of 190-900 nm by using spectrophotometer and maximum absorption wavelength of anthocyanins were determined. Stabilization of the anthocyanin against pH, temperature and ambient oxygen of this solution was then determined by narrowing the wavelength(in the range of 400-600 nm) against to blank (ultra pure water containing 1 % HCI). The results of each analysis were evaluated separately.

In order to determine the stability against pH change, 0.1 g of the powdery food colouring obtained from the corn cockle fruit was taken. Dissolved in 100 ml_ ultra pure water (containing 1 % HCI). The first pH of the solution was measured. 0.1 M NaOH solution was prepared to increase the pH of the solution. pH was adjusted to 3, 5, 7, 9, and 1 1 by adding gradually 0.1 M NaOH on a magnetic stirrer and at each pH the solution was taken by syringe. Measurements were made by using spectrophotometer against blank (ultra-pure water containing 1 % HCI) at a wavelength in the range of 400- 600 nm.

In order to determine the stability against to temperature changes 0.1 g from the food colouring was taken. Dissolved in 100 ml_ ultra pure water (containing 1 % HCI). The first measurement was made at room temperature at the wavelength in the range of 400-600 nm by using spectrophotometer. The temperature is controlled while stirring the solution on the magnetic stirrer heater in the beaker and the solution was taken at 30, 40, 50, 60, 70, 80, 90 and 98 ^SC by syringe. Each sample was allowed to cool down until to reach the room temperature. Absorbance values were measured against to blank (ultra pure water containing 1 % HCI) by using spectrophotometer in the range of 400-600 nm wavelength when each sample temperature reached to room temperature.

In order to determine the stability against to environmental oxygen 0.1 g of powdered food coloring was taken. Dissolved in 100 mL ultra pure water (containing 1 % HCI) and the first measurement was by spectrophotometer in the range of 400-600 nm wavelength. The absorbance values at 1 , 2, 3, 4, 5, 6, 12, 24 and 48 hours were measured against blank (ultra-pure water containing 1 % HCI) 400-600 nm wavelength by spectrophotometer .

The change in DPPH radical scavenging activity of fruit juice obtained from corn cockle juice was calculated using a UV-VIS spectrophotometer to determine the decrease in absorbance and the results were calculated as DPPH radical scavenging activity.

Table 3. Result of DPPH radical scavenging activity analysis of corn cockle juice

Total phenolic content of fruit juice obtained from corn cockle were determined by UV-VIS spectrophotometer. Calibration graph was obtained using the 1 , 5, 10, 25, 50, 100 and 300 ppm gallic acid standards prepared for analysis. The absorbance values of the samples were evaluated using the gallic acid curve. Considering the dilution factor, the total amount of phenolic content calculated as mg GAE (Gallic acid equivalent) /g and given at below table.

0 10 20 30 40 SO 60 TO 80 90 100 11

Co n c entratio n, p p m

Figure 1. Gallic acid calibration graph

Table 4. Total phenolic content

Detected phenolic compounds and concentrations (mg/L) in the analysis were given at below table.

Table 5.The determined phenolic compounds and their concentrations

When we investigate susceptibility of food colouring obtained from corn cockle fruit to pH, temperature, ambient oxygen; the food colouring can not protect its natural color counter to pH change and its color was easily losted. We observed a high change in stability versus pH increase from the absorbance at each pH measurement. We observed that pH 5 and at above values can not be determined any absorbances by UV- VIS spectrophotometer at about 515 nm wavelength. It was determined that food colouring retained its color stability at low pH but it lost its color stability while pH increased. We have determined from the change of absorbance that the color of the food colouring protected its natural color the natural color against the temperature increase and the absorbance alteration showed that its stability chancing was low against the temperature increases. The food colouring protected its natural color against ambient oxygen and is exposed to ambient oxygen alterations in absorbance showed that the change in stability of food colouring was low.

As a result, considering the historical development of food colouring, the use of natural origin food colourings was widespread until the industrial revolution, after the industrial revolution with the increase in technological developments, the use of synthetic food colourings has become widespread.

Parallel to these developments, legal regulations have been introduced for the use of coloring materials. As a result of the researches, the adverse effects of synthetic food colourings on health have been noticed and many synthetic food colourings have been prohibited until the day. Considering the positive effects on health today, the use of natural food colouring has become important. However, the low color stability of natural food colouring limits the area of use.

Natural food dyes are pigments derived from microbial, vegetal, animal or mineral sources. It was found that anthocyanin content of corn cockle (Berberis), the subject of the present invention, was compatibility for food colouring.

Some parameters of the food coloring obtained from fruit and juice were investigated and the results were observed by applying some foods. As a result, the stability of food coloring from corn cockle fruit juice at low pH maintains color stability but at high pH unable to protect its color stabilty, against increases temperature and ambient oxygen maintains stability. It was determined that DPPH radical scavenging activity and TMC (total phenolic content) in the food colouring obtained from the juice of the corn cockle was high. This indicates that the antioxidant capacity is high.

Corn cockle fruit is completely natural since no chemical is used when it is obtained from fruit. In addition, the color intensity of the corn cockle fruit used in the production of food colouring is also very good. Corn cockle grows naturally and the analyses of the results indicate that the antioxidant content of the fruit is high, is another advantage. Antimicrobial properties of this food colouring with high antioxidant properties may also be mentioned. Corn cockle fruit, which is not consumed too much by people, can be used as a food coloring to earn an economy.

Claims

1 . A natural food colouring and it is characterized by the presence of corn cockle powder, obtained by removing water from Corn cockle (Berberis) fruit juice.

2. The corn cockle powder of Claim 1 and it is characterized by comprising 7.90 ± 0.16 mg GAE/g total phenolic content.

3. A method of obtaining natural food colouring, and it's characterized with the steps below;

- Applying mechanical press to corn cockle fruits,

- Then filtered through thin pore filter cloth,

- Thus obtaining fruit juice,

- Filling lyophilization tubes with 5 ml_ of fruit juice and stored at -87 ° C under 1 1 Pa pressure and being dried (72 hours) under vacuum to remove water controlledly,

- Being powdered,

- In order to anthocyanins to be stable against oxygen, passing the fruit juice through nitrogen gas before storage,

- Then being stored at -18^SC as a powdered food coloring.