Field of the Invention The present invention relates to a method for producing natural dyestuffs using elevated sound. In particular, the present invention relates to a natural dyestuff in red color and shades thereof, obtained by ultrasonic cavitation extraction from the plant Berberis crataegina, and an optimized method for obtaining same. Background Art

The characteristics required for artificial dyes, which are used as colorants in the cosmetics, pharmaceutical and textile industries, especially in the food sector, are inexpensiveness and resistance to process conditions. For this reason, artificial ingredients are intensively used in a large part of the colorants industry.

In line with the increasing consumer awareness, researches on artificial dyes have revealed that long-term use of these types of dyes causes allergic reactions and cancer, especially toxic effects. These results have led the academic and industrial community to natural alternatives to said substances.

In addition, certain dyes, such as carmine, have limited commercial appeal due to the fact that the sources and methods of obtaining same are considered strange in some cultures, for example, they are classified as religiously prohibited substances, which is considered to be another triggering factor.

Ultrasound application, i.e., ultrasonic extraction, is an innovative approach that allows high efficiency extraction of intracellular chemicals. There are also studies in which it is used in combination with heat (thermosonication) or pressure treatment (manosonication) in order to increase the extraction efficiency. Elevated sound generates acoustic cavitation that triggers cell fractionation by increasing the molecular contact of a solvent and a sample, thus enabling a more efficient extraction process in a short period of time and with less solvent (Agcam et al., 2017, "Optimization of anthocyanins extraction from black carrot pomace with thermosonication", Food chemistry, 237, 461-470; Rodrigues et al., 2010, "Ultrasound extraction of phenolics and anthocyanins from jabuticaba peel", Industrial Crops and Products, 69, 400-407). The efficiency of the elevated sound treatment is fully dependent on parameters such as time, temperature, intensity and solvent/solute ratio. Therefore, the optimization of such parameters is extremely important for the highest extraction of the desired component (Sharmilla et al., 2019, "Ultrasound aided extraction of yellow pigment from Tecoma castanifolia floral petals: Optimization by response surface method and evaluation of the antioxidant activity", Industrial Crops and Products, 130, 467-477). Although the popularity of the natural dye industry has increased in the last few years, it is a concept that producers and consumers have focused on for the last 10-20 years, and there are many national and international companies producing in this field. The raw materials used by these companies consist of plants with high anthocyanin content. The raw materials that are commonly used are beetroot, berry type fruits, or red fermented rice. The dyes obtained by direct extraction of these raw materials are used in production.

It has been found that all of the studies on the berberis crataegina plant in international databases are related to the health aspect of the plant. It seems that the plant is used, in only one study, to stain the cell walls. Among the publications in the art that can be considered partially relevant to the elements mentioned in the present invention, the following may be cited: Sonmezdag et al., 2018, "Volatile and key odourant compounds of Turkish Berberis crataegina fruit using GC-MS-Olfactometry", Natural product research, 32(7), 777-781; Kaya, et al., 2018, "Production and characterization of chitosan based edible films from Berberis crataegina's fruit extract and seed oil", Innovative food science & emerging technologies, 45, 287-297; Sari, F., 2016, "The Copigmentation Effect of

Different Phenolic Acids on Berberis crataegina Anthocyanins", Journal of Food Processing and Preservation, 40(3), 422-430; and I§ikli and Yilmaz, 2014, "Some physical properties of sun-dried Berberis fruit (Berberis crataegina)" Journal of food science and technology, 51(1), 104-110. A review on the thesis databases revealed the following studies: one characterization study with Berberis crataegina ("Biochemical characterization of Berberis vulgaris L. and Berberis crataegina DC. wild fruits exist naturally in Bayburt city ", Abdulkadir Karabulut), one study on stability ("Determination of stability of Karamuk ( Berberis crataegina ) antocyanins in lemon juice and buffer solutions", Emine idi§), as well as those studies wherein it is used in yoghurt ("Physical and sensory properties of yoghurt produced by addition of concentrated karamuk and kavut", Buket Ate§ Gundogan) and ice cream ("An analysis on the use of anthocyanins of berberis (Berberis crataegina) for the encapsulation and production of ice-cream", Melike Okurkan). None of the thesis studies envisages optimization of the extraction.

Accordingly, none of the publications in the prior art and no combination thereof describes or manifests the whole of the elements that will provide the benefit aimed at the present invention. Objects of the Invention

Principal object of the invention is to provide solutions to the problems mentioned in the prior art.

Another object of the invention is to provide a method for obtaining natural dyestuffs with a high extraction efficiency, low cost and high speed while preserving their chemical structures. Summary of the Invention

The issues as listed in the background section are important factors in the emergence of the present invention, which has the feature of being an alternative to artificial dyes. In view of the above, there is still a need for further improvement of the methods for obtaining natural dyestuffs with a high extraction efficiency, low cost and high speed while preserving their chemical structures. If the health dimension is examined, it is envisaged that in addition to its dye feature, the plant source from which the invention is obtained will also make a contribution to the public health with its phenolic compounds, vitamins and many other bioactive components.

The present invention is a method for extraction of intracellular chemicals, comprising the step of forming cavitation bubbles in an extraction medium by exposing the extraction medium to ultrasound. The temperature of the extraction medium is preferably kept below the temperature at which the chemical substance to be extracted undergoes thermal degradation. The chemical substance to be extracted is preferably anthocyanin. Anthocyanin is preferably extracted from Berberis crataegina. The temperature in the extraction medium can be maintained, for example, at 60°C or lower. For example, the temperature in the extraction medium can be maintained in the range of 50°C to 60°C. Alternatively, the temperature in the extraction medium can be kept below 50°C. For example, the temperature in the extraction medium can be maintained in the range of 35°C to 45°C, for example 40°C. Temperatures around +/- 2°C of the temperatures according to the present invention can be considered equivalent to those specific temperatures. For example, a range of 40°C +/- 2°C can be considered as an equivalent of 40°C. The method may include preparing an extraction medium to contain a slurry, and applying an ultrasound intensity corresponding to an energy consumption of greater than 40W per 50 milliliters of extraction medium, more preferably 120W or higher per 50 milliliters of extraction medium. In the method, the frequency of the ultrasound applied may be, for example, 26 khlz. The extraction medium may, for example, comprise a slurry containing essentially 75% wt. of methanol, 0.1% wt. of formic acid, and the balance distilled water (e.g.: 75% wt. of methanol, 24.9% wt. of distilled water, and 0.1% wt. of formic acid). Brief Description of the Drawings

The drawings, whose brief description is provided below are just given for better understanding of the present invention and as such, are not intended to determine the scope of the claimed subject matter, in the absence of the description, wherein:

Figure 1 is a schematic illustration representing an exemplary embodiment in which the method of the present invention can be applied.

Detailed Description of the Invention Within the scope of the present invention describes a method for obtaining anthocyanin by ultrasonic extraction of a plant, whose Latin name is 'Berberis crataeginb, which the local people call as 'karamuk', which grows naturally in the mountainous regions of the Kayseri city, for example. The plant can be adjusted to varying shades of red, purple and pink with its high anthocyanin content. An adjustment of the optical properties of the plant is achieved through the pH value. The color of the plant is in red tones at strong acidity values (pH<7), whereas it turns to purple where acidity is weaker (pH >7). In order to increase the amount of anthocyanin composition used in obtaining the desired colors, the value(s) are determined at which the most efficient amount of anthocyanin is obtained by applying elevated sound at different values (temperature, time and intensity). In this specification, the term "elevated sound" is used as a synonym for "ultrasound with sufficient intensity to cause cavitation in the liquid extraction medium (20)". In this context, the values for the intensity of the ultrasound (adjustment of a device having a certain power consumption rate at various power consumption rates such as 20%, 60% and 100%), temperature (for example, 20°C, 40°C and 60°C, which allows processing of the raw material and that will not cause the degradation of the extracted material), and extraction time (economically acceptable times such as 10, 15 and 20 minutes) were applied in different ways, and a total of 17 different combinations were extracted, and an extraction method was determined providing the highest yields with the equipment in which the experiments were carried out. In this regard, the amount of anthocyanin is increased with increasing temperature up to a certain value (40±5 °C), intensity and time. As exemplified in the specific experiments below, a combination with the highest efficiency, which is achieved with the equipment at hand, was determined to be 100% intensity, 40°C and 20 minutes.

The cosmetics, pharmaceuticals and textile industries, notably the food industry, frequently use red and its tones, and they lead the sectors where the use of natural dyes would be demanded. In the food industry, the resulting anthocyanin may be directly incorporated into production, especially in confectionery, bakery products and beverages. The resulting anthocyanin can be directly used in the decoration of bakery products (e.g., cakes), which offer optical properties in red tones such as strawberry, raspberry or cherry. In addition, when color change is required, the manufacturer can easily perform modifications for optical properties using an ordinary organic acid additive (for example, ascorbic acid). That is, the method of the present invention and the resulting product have industrial applicability. Therefore, the present invention also provides the use of anthocyanin (for example, the anthocyanin obtained by the method of the invention) as a drug or food colorant, and/or as a nutritional supplement. When an ultrasonic energy is introduced into the liquid extraction medium (20), cavitation bubbles are formed, and with their collapse, high shear stresses and pressure/temperature shocks instantaneously occur around them. These effects not only allow the release of the substance to be extracted by breaking the cell walls, but also accelerate the extraction by mechanically thinning the boundary layers on the extraction interfaces around the collapsed bubbles, and increasing the driving forces for mass transfer.

Thanks to the acceleration effect of the ultrasonic cavitation, the extraction time is reduced. Assuming that thermal degradation is proportional to the time of exposure to temperature, the reduced extraction time can keep the thermal degradation of the substance to be extracted (in this example: anthocyanin) to a minimum, while achieving a high extraction efficiency. EXAMPLES:

The examples herein are provided for easy understanding of the practice of the present invention and how the parameters of the method can be adjusted according to preference, and are not intended to be limiting on the scope of protection.

EXAMPLE-1: DETERMINATION OF THE EXPERIMENTAL PARAMETERS

As mentioned above, the efficiency of the ultrasonic extraction can be increased by adjusting the parameters in the extraction so as to find the most suitable combination. In this regard, 17 exemplary extraction parameters used in our study (temperature (°C), intensity (%) and time (min)) were determined by means of a software called Design Expert. In this study, the term "intensity" refers to a relative value in the range of 0% to 100%, depending on the use of the energy consumption capacity of the device providing the ultrasonic waves. For the specific case in this example, 100% intensity corresponds to a power consumption of 200 W in an ultrasound device (ultrasonic mixer) used as a source of elevated sound (10). The extraction medium (20) used in this example, in which ultrasonic cavitation was achieved, is a mixture prepared in a 50-milliliter chamber, containing 75% wt. of methanol, 0.1% wt. of formic acid, and the balance (24.9% wt.) distilled water. The temperature of the extraction medium (20) was controlled using a temperature measuring and control assembly (30) comprising a water bath (40) in which the extraction medium (20) was immersed and a temperature probe immersed in the extraction medium (20). For temperature monitoring and control, any other set of methods/tools may also be used which are already known to a skilled person being a chemical, mechanical or food engineer. In order to determine the parameters such as power consumption intensity and temperature required to ensure the cavitation that will accelerate extraction in different extraction media, a few trials with the source of elevated sound (10) will be sufficient. Three exemplary values of 20%, 60% and 100% were determined for the intensity, wherein the effects of the ultrasound on extraction were examined. The ultrasonic mixer used in the study as a source of elevated sound (10) is a Hielscher UP200Ht model with a maximum power consumption of 200 W and a frequency of 26 kHz. While determining the intensity values, those intensity values were selected corresponding to the lowest, medium and highest power consumption allowed by the device in operation. It has been determined that at intensities below 20%, the breaking capacity of the device is negligible, and it can move the molecules only by vibration. Hence, at low intensity values such as 20%, it was checked whether there was sufficient cavitation for the extraction, and it is observed that there was no significant cavitation, and an extraction efficiency was obtained, albeit very little. At 60% and 100% intensity values, it has been observed that an increase in efficiency based on the use of ultrasound was effective to the desired extent. That is, the extraction efficiency is increased in parallel with the increase in intensity.

Therefore, with the invention, the extraction efficiency is considerably increased by obtaining ultrasonic cavitation in the extraction medium (20).

In order to examine the effect of temperature, 3 different exemplary values were also used, i.e., 20°C, 40°C and 60°C. In the selection of the temperature values, it has been observed that an increase in temperature at a certain value increases the efficiency of ultrasonic extraction. However, since the components tried to be extracted are anthocyanin derivatives sensitive to temperature, it has been concluded that 60°C, at max., constitutes a good example of extraction temperature, given that exceeding a certain value would damage the components.

Therefore, when determining the ultrasonic extraction parameters of the invention, it is preferable to set the temperature values in such a way that does not cause thermal degradation of the substance to be extracted. More preferably, within this limitation, the temperature can be kept as high as possible. That is, the temperature is preferably just below the thermal degradation temperature of the substance to be extracted (e.g., below 1 to 10°C, preferably below 1 to 5°C).

In exemplary experiments, 10, 15 and 20 minutes were considered appropriate for the extraction times, although not necessarily. For the duration, such values are selected in order to get the highest efficiency from the device in the shortest period of time.

EXAMPLE-2: THE RESULTS AND EVALUATION OF THE EXPERIMENTS PERFORMED WITH THE PARAMETERS IN EXAMPLE-1 In line with the data obtained, the highest efficiency was observed in the example performed at 100% intensity, 40°C and 20 minutes. When the intensity and time is remained constant and the temperature is decreased, the efficiency is decreased. That is, it was observed that the efficiency of the extraction performed at the same intensity and for the same time, at 20°C, was lower. On the other hand, the lowest efficiency was observed in the extraction performed at the lowest intensity, temperature and time (intensity: 20%, temperature 20°C and time 10 min.). The efficiency increases with each extraction where intensity is increased. However, it has been observed that a temperature increases above 40°C reduces the efficiency even in high intensity application. This results from the fact that temperatures above 40°C degrade the anthocyanins in the plant's structure.

That is, in the method that is the subject of the present invention, those temperatures that will not cause thermal degradation of the substance to be extracted may be preferred, especially among them, it is preferable to approach, as much as possible, to the highest temperature that the substance can withstand. Since thermal degradation kinetics is time dependent, even if the temperatures causing thermal degradation are exceeded, the extraction (exposure to temperature) time is considerably reduced by virtue of the accelerating effect of ultrasonic cavitation. Therefore, the thermal degradation at the end of the extraction can be kept at negligible extent, so that the material to be extracted can be obtained with high efficiency (extraction efficiency is high). As a result, as the intensity (%) is increased, the efficiency increases and it is found that the efficiency is directly proportional to the intensity. Increasing the intensity with controlled temperature values will directly increase the efficiency. An increase in temperature increases the efficiency until it reaches 40°C; when it is 10°C above this value, it gradually decreases the efficiency, and at 60°C there is a significant decrease. That is, increasing both the intensity and the temperature decreases the efficiency. 40- 50°C was observed to be the most ideal extraction temperature. As for the intensity, an efficiency can be achieved when an intensity of 20% or more is applied, but it has been observed that the most ideal intensity values specific to the device used in the experiments are between 60% and 100%. In cases where a much more powerful (high power consumption) ultrasound generator is used as a source of elevated sound (10), the appropriate relative intensity value may be comparatively lower, and similar results may be obtained.

At these temperatures, where thermal degradation is considered to be slow or non existent (for anthocyanin, for example, preferably at temperatures not exceeding 60°C, or preferably not exceeding 50°C), the efficiency is increased if the temperature and intensity are kept constant and the time is increased. Hence, the time can be increased as desired at the most ideal temperature and intensity values; however, due to the increased time, it is likely to have problems such as high costs.

According to the method of the invention, increasing the ultrasound intensity reduces the time required to achieve the targeted extraction efficiency. Also in this case, the extraction time can be minimized with high ultrasound intensity (ultrasound device's power consumption) values at said temperatures, so that maximum extraction yields can be obtained with minimum extraction times. With the reduced treatment time, there is no significant degradation in the structures of the extracted cellular chemical components, even when working at temperatures that can cause thermal degradation in long-term exposure. In this way, the properties of said cellular chemical components are preserved, including their nutritional values. With the inventive improvement, the deficiencies of the prior art are eliminated and the aforementioned problems are solved.

In the light of the above, the present invention can be formulated as follows: - The present invention is a method for extraction of intracellular chemicals, comprising the step of forming cavitation bubbles in an extraction medium (20) by exposing the extraction medium (20) to ultrasound.

- The temperature of the extraction medium (20) is preferably kept below the temperature at which the chemical substance to be extracted undergoes thermal degradation.

- The chemical substance to be extracted is preferably anthocyanin.

- Anthocyanin is preferably extracted from Berberis crataegina.

- The temperature in the extraction medium (20) can be maintained, for example, at 60°C or lower. For example, the temperature in the extraction medium (20) can be maintained in the range of 50°C to 60°C.

- Alternatively, the temperature in the extraction medium (20) can be kept below 50°C. For example, the temperature in the extraction medium (20) can be maintained in the range of 35°C to 45°C, for example 40°C. For example, a range of 40°C +/- 2°C can be considered as an equivalent of 40°C. - The method may include the steps of preparing the extraction medium (20) to contain a slurry, and applying an ultrasound intensity corresponding to an energy consumption of greater than 40W per 50 milliliters of extraction medium (20), more preferably 120W or higher per 50 milliliters of extraction medium (20). In the method, the frequency of the ultrasound applied may be, for example, 26 khlz. The extraction medium (20) may contain, for example, a slurry containing substantially

75% of methanol, 0.1% wt. of formic acid, and the balance distilled water. Reference numerals:

10. source of elevated sound 20. extraction medium 30. temperature measuring and control device 40. water bath