METHOD FOR IMPROVING THE EFFICIENCY IN OBTAINING CARMINIC ACID FROM DACTYLOPIUS COCCUS COSTA BY MICROWAVE-ASSISTED EXTRACTION.

Abstract

The present invention relates to a method for obtaining carminic acid from Dactylopius coccus by microwave-assisted extraction. This natural dye comes from the cochineal insect (Dactylopius coccus C.) and has a wide range of applications in the food, pharmaceutical, cosmetic, and craft industries; however, the methods currently used for its extraction are based on conventional methods involving various operations that generate high energy consumption, labor, and waste. Therefore, the present invention provides an alternative method for obtaining carminic acid from Dactylopius coccus that improves efficiency.

Description

METHOD FOR IMPROVING THE EFFICIENCY IN OBTAINING CARMINIC ACID FROM Dactylopius coccus costa BY MICROWAVE-ASSISTED EXTRACTION TECHNICAL FIELD The present invention belongs to the technical field of chemistry. Specifically, it is situated within the area of ​​processes used to obtain organic dyes, more specifically to dyes of natural origin prepared from natural sources, and even more specifically to a method for obtaining carminic acid from Dactylopius coccus by microwave-assisted extraction. BACKGROUND The demand for organic dyes has increased since the 1990s, accelerating at the beginning of this century due to evidence showing that synthetic dyes have caused harm to human health. Cases of allergies have been reported, and many have been found to induce the formation of cancerous tumors. This has led the U.S. Food and Drug Administration (FDA) and the World Health Organization (WHO) to prohibit the use of synthetic dyes in food, cosmetics, and medicine, due to their harmful effects on health. Furthermore, the rising price of petroleum-derived products used to manufacture artificial dyes has contributed to the increased use of natural dyes. One of the most sought-after colorants in the industry is carminic acid, and its demand stems from its current use in the cosmetic, food, pharmaceutical, and textile industries. The use of this product has increased due to the implementation of global regulations prohibiting the use of certain synthetic colorants that are toxic and harmful to human health, primarily those used in the food industry. Carminic acid is extracted from insects such as Dactylopius coccus, better known as cochineal, an insect that lives as a parasite on the leaves of the prickly pear cactus. Carmine is actually the result of an aqueous extraction of the cochineal insect, whose coloring principle is carminic acid. Complexes of carminic acid formed with aluminum or calcium are called lakes, and their main use is in the dairy industry, such as in yogurt and ice cream. Other products that use cochineal and its derivatives include candies, chewing gum, fruits, jellies and jams, soups and sauces, and baked goods. The global food industry uses 75% of production. The cosmetics industry receives 15% of production for products applied to the mouth and eyes, such as eyeshadow, lipstick, and blush. The remaining 10% of production is divided between the pharmaceutical and textile industries.The relevance of cochineal use worldwide is related to the growing demand, estimated at an average annual rate of 6%; and above all to the health of consumers, by replacing synthetic dyes with natural dyes. Several conventional extraction methods for obtaining carminic acid have been reported in the literature. Among the best known is the Japanese method, in which the following compounds are heated in an autoclave at 150 °C and 1 atm of pressure for 10 minutes: 1.5 L of deionized water, 1 g of tartaric acid, 0.2 g of gelatin, and 100 g of ground cochineal (ASTM No. 30 mesh). The liquid is then decanted and the residue filtered. The solution is distilled under reduced pressure to obtain a concentrated extract containing a high percentage of carminic acid. Another known method is that of Forgios, in which 3 L of deionized water, 10 mL of 2N HCl, and 100 g of ground cochineal are boiled for 60 minutes. The solution is then allowed to stand, the liquid is decanted, and the residue is filtered. A re-extraction of the residual cochineal is performed. The solution obtained from this step is concentrated by distillation under reduced pressure. Methanol is added to this extract, and it is subjected to azeotropic distillation again to remove the alcohol. The mixture is then allowed to stand. The resulting extract is filtered and concentrated by distillation under reduced pressure. The final extract is then allowed to stand for the crystallization of carminic acid. The crystals are filtered and allowed to dry. Additionally, the Schunck-Marchlewski method is known, which consists of boiling 1.5 L of deionized water and 100 g of ground cochineal for 30 minutes. The solution is then allowed to stand, and the liquid is decanted and the residue filtered. A second extraction of the residual cochineal is then performed. The resulting liquids are transferred to a kettle to form the metal complex. To perform the complexation of the solution, the carminic acid content is determined in order to calculate the weight of lead acetate required to form lead carminate. The complexation process is carried out by adding neutral lead acetate and boiling for 15 minutes. The resulting solution is then allowed to stand for 24 hours. After this time, the liquid is decanted and the residue is filtered. The product is washed with water. Subsequently, the lead carminate is dispersed in a methane-H₂SO₄ solution and allowed to stand for 24 hours.The liquid is decanted and the residue is filtered. Finally, the alcoholic solution is distilled under reduced pressure to obtain a concentrate, which is then crystallized with H2SO4. A widely used method is the Schutzenberger method, which is reported to yield better results compared to other known methods, and is also the least expensive, since it only uses water as a solvent and boils the solution to extract carminic acid. It consists of boiling 1.5 L of deionized water and 100 g of ground cochineal for 30 minutes without stirring. The solution is then allowed to cool to room temperature and subsequently filtered. The remaining liquid is subjected to two more extractions with the same volume of solvent. However, the drawbacks that are repeated in conventional extraction methods are the use of acids, which, although they help to hydrolyze proteins present in the raw material, can attack the surfaces of stainless steel. On the other hand, there are reports of unconventional methods for carminic acid extraction; Borges et al. (2012) proposed the Supercritical Fluid Extraction (SFE) and Pressurized Liquid Extraction (PLE) techniques. In their study, carminic acid extraction using pressurized liquids was performed with three different solvents (methanol:water, ethanol:water, and ethanol), three temperatures (100, 150°C, and 200°C), a 30-minute extraction time, and pressures up to 10.5 MPa. Extractions were carried out in 11 mL extraction cells, each containing 2.0 g of sample. The SFE method underwent several modifications: an 8 mL extraction cell containing 1 g of sample and 2 g of sea sand was placed in a thermostatically controlled oven, using CO2 as the solvent, pressures between 150 and 300 bar, 40°C, and an extraction time of 240 minutes. However, the operating time is very long. In accordance with the above, there is still a need for new methods for the extraction of this dye, aimed at reducing operating times, costs, and damage to equipment while ensuring high performance. BRIEF DESCRIPTION OF THE INVENTION According to the above, the main objective of the present invention is to provide a method for extracting carminic acid from Dactylopius coccus that allows reducing the extraction times of carminic acid. Therefore, the main object of protection relates to a method for improving the efficiency in obtaining carminic acid from Dactylopius coccus Costa cochineal, characterized in that it comprises the following steps: a) Mix ground granules and deionized water in a ratio of between 1:20, preferably 1:10, 1:15 and 1:20 g / mL; b) Place in a microwave equipment calibrated to a power of no more than 1500 W, preferably 1200 W, for a time no more than 15 min, preferably 5, 10 or 15 minutes at a temperature no greater than 80 °C, preferably selected from between 60, 70 and 80 °C. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Diagram of the post-harvest handling of cochineal from drying. Figures 2A-2B. UV / Visible scan spectrum demonstrating the maximum absorbance peak of carminic acid present at a wavelength of 494 nm. Figure 3. Infrared Spectra of: Conventional Method (CM), Microwave Assisted Extraction (MAE) and Others. DESCRIPTION OF THE INVENTION The present invention relates to a method for obtaining carminic acid from Dactylopius coccus by microwave-assisted extraction. This natural dye comes from the cochineal insect (Dactylopius coccus C.) and has a wide range of applications in the food, pharmaceutical, cosmetic, and craft industries; however, the methods currently used for its extraction are based on conventional methods involving various operations that generate high energy consumption, labor, and waste. Therefore, the present invention proposes microwave-assisted extraction, a green extraction technique that generates electromagnetic radiation in the frequency range of 0.3 to 300 GHz.The energy associated with microwave radiation can heat the surrounding medium; therefore, its use is based on efficient heating and depends on a specific material's ability to absorb microwave energy and convert it into heat. Microwave heating differs from conductive methods because, in the latter, heating is independent of the sample, allowing for the heating of a large number of samples or materials in short periods. For the purposes of the present invention, yield shall be understood as the percentage of carminic acid extracted; and efficiency shall be understood as the time of extraction of carminic acid. The microwave-assisted extraction process of carminic acid comprises the following steps: Sample preparation a) Sacrifice the cochineal insect, b) dry the cochineal sample, c) clean by sieving the sample through a 2 mm sieve; d) degrease by adding hexane to remove excess wax, e) grind to proceed with the extraction. Carminic acid extraction a) Mix ground granules and deionized water in a ratio of between 1:20, preferably 1:10, 1:15 and 1:20 g / mL; b) Place in a microwave equipment calibrated to a power of no more than 1500 W, preferably 1200 W, for a time no more than 15 min, preferably 5, 10 or 15 minutes at a temperature no greater than 80 °C, preferably selected from between 60, 70 and 80 °C. The following presents a preferred embodiment of the present invention and a comparison with other methods. The examples provided are illustrative and not limiting, as a person skilled in the art will understand that there are variations that fall within the scope of protection of the present invention. Sample preparation Sample preparation refers to post-harvest handling which includes the steps of slaughtering, drying, and packing the grain after it has been detached from the cactus. a) Sacrifice the cochineal by some of the following methods well known to those technicians in the matter: Immersion in hot water, application of water vapor, suffocation with application of hexane (100% purity), freezing, suffocation by the use of a plastic bag exposed to the sun for three hours, or by the use of an oven at 38 °C for three hours; Figure 1 (10). b) Dry the dead grain using solar dryers, ovens with spotlights, direct sunlight or in the shade, the use of which is well known to technicians in the field, to avoid changes in the structure, color and final appearance of the product. Figure 1 (20). c) Cleaning (30) using air currents and sieves to remove impurities such as thorns, sand, or other elements. These impurities must be removed to avoid contamination and improve the quality of the grain. Preferably, the grain is passed through a 2 mm mesh. Figure 1 (30). d) Degrease (40) to remove the waxy coating residue (coccerin) that passes through in the previous steps, by solid-liquid extraction using selected organic solvents such as hexane, petroleum ether or ethyl ether. Figure 1 (40). e) Grind (50) the sample using preferably manual, artisanal mills, or mechanical systems specially provided for this purpose until obtaining an optimal granule for efficient extraction. Figure 1 (50). Proximate composition of cochineal 1,400 kg of cochineal was obtained from Santa María Zacatepec, Juan C. Bonilla Municipality in the State of Puebla, Mexico, raised in a greenhouse. 1,400 kg of dried cochineal was sieved to select the highest quality cochineal using a 2 mm sieve. A degreasing process was also carried out to remove excess cochineal. The percentage of carminic acid present in cochineal was determined using the method established by the FCC (Food Chemical Codex, 1981). Exactly 30 mg of the sample were weighed and dissolved in 30 mL of boiling N HCl, then cooled. The solution was quantitatively transferred to a 1 L volumetric flask, diluted to the mark with deionized water, and mixed. The absorbance of the solution was determined in a 1 cm cell at a maximum wavelength of 494 nm using a spectrophotometer, with 0.06 N HCl as a blank. If the absorbance reading was not within the range of 0.20 to 0.25, another solution was prepared and the weight adjusted accordingly. The percentage of carminic acid was calculated according to the following formula: 15*71*100 % of Carminic Acid = —————— 0.262 * W Where A = is the absorbance of the sample in solution (value read at 494 nm). 0.262 = is the absorbance of a carminic acid solution with a concentration of 15 mg / 1000 mL W = is the weight of the sample in mg. Analyzing the chemical composition of cochineal determines its quality. This is important for evaluation because environmental conditions can vary during cochineal development, and changes can occur depending on the fertilization of the prickly pear cactus used. Consequently, the composition of the cochineal can differ, making it important to analyze it to determine if it exhibits average values. The variables typically analyzed in cochineal are moisture, ash, carminic acid content, and protein. Table 1. Proximate composition of the cochineal used in the tests. Composition Percentage Moisture 8.08 Ash 4.73 Lipids 15.53 Carbohydrates 26.84 Proteins 44.69 Carminic acid 20.1 Figure 2 shows the absorption spectrum obtained to determine the percentage of carminic acid and demonstrates its presence at a wavelength of 494 nm (6A). Graph 2A shows similarity to the spectrum in Graph 2B reported by Centeno in 2003; however, since different concentrations were measured, the absorbance is different. Extraction of carminic acid by the Schutzemberqer method Cochineal was mixed with the solvent at a ratio of 1:15 g / mL for extraction and brought to a boil without stirring for 30 minutes. The mixture was decanted and the supernatant filtered using Whatman No. 4 filter paper. The residue remaining on the filter paper, along with the sedimented material, was subjected to two further extractions (Lock, 1997). The resulting extracts were mixed and stored under refrigeration in 50 mL Falcon tubes. The yields were expressed as a percentage, obtaining two yields which are: extract yield and carminic acid yield. The extract yield obtained was 31.9% and the carminic acid yield was 17.8%. Carminic acid extraction and microwave extract A ratio of ground granules and deionized water (1:10, 1:15 and 1:20 g / mL) was placed in vials, which were kept in peek jackets in the carousel of an Anthon Paar, Synthos 3000 microwave equipment with a power of 1200 W. They were kept for 5, 10 and 15 minutes and temperatures of 60, 70 and 80 °C. Advantageously, the microwave extract has the highest yield at 60 °C, 15 min and a solvent ratio of 1:20 g / mL, having a 41% yield. Estimated extract yield by Microwave Assisted Extraction Table 2 shows the maximum increase, where the extract yield is obtained from the average values ​​of the factors. It should be noted that the yield decreases as the temperature increases, as the amount of solvent decreases. The results obtained by optimizing the variables of the factors advantageously yield an optimal performance of 42.1%, this at a temperature of 60 °C, 14.9 min, and 19.9 mL of solvent. Table 2. Estimated extract yield Temperature Time Solvent Prediction for Yield (°C) (min) (mL) (%) 70.0 10.0 15.0 36.5 71.0 10.02 12.9 34.6 72.0 10.3 11. 32.4 Temperature Time Solvent Yield Prediction 73.0 10.6 9.1 29.8 74.0 11.1 7.2 26.9 75.0 11.6 5.4 23.6 Table 3. Extract performance optimization Factor Low High Optimum Temperature 60.0 80.0 60.0 Time 5.0 15.0 15.0 Solvent 10.0 20.0 19.9 By optimizing the variables of the factors, an optimal yield of 23.3% is obtained, achieved at a temperature of 79.7 °C, at a time of 5.04 min and a solvent ratio of 1:20 g / mL. Infrared Spectrum IR spectra were obtained from the solid extract of the cochineal samples studied, obtained by microwave-assisted extraction and the Schutzenberger method, and compared with the spectrum obtained from the commercial carminic acid standard. The infrared spectrum of the 96% pure commercial carminic acid standard allows for the identification of the bonds corresponding to the molecule and comparison with the spectra of the samples obtained by microwave-assisted extraction and the Schutzenberger method; in these, the characteristic peaks of the carminic acid molecule are observed, thus demonstrating that it is not affected by the use of microwave-assisted extraction (Figure 3). Comparison of various extraction methods The Schutzenberger method showed a lower yield than the proposed extraction method, with an extract yield of 31.9%, which is 9.1% less than microwave-assisted extraction. The net yield of carminic acid obtained by the Schutzenberger method was 17.8%, 0.2% less than microwave extraction. The microwave extraction yield was 41% with an efficiency of 27.3 mg / min. The carminic acid yield was 18% with an efficiency of 36.0 mg / min. In contrast, the Schutzenberger method, with an extract yield of 31.9% and an efficiency of 3.5 mg / min, had a carminic acid yield of 17.8% with an efficiency of 2.0 mg / min. Therefore, this process outperforms the conventional method. Table 6 shows a comparison between various extraction techniques, with the proposed invention having better efficiency with respect to the net yield of carminic acid, since it is obtained in only 5 min. Table 6. Comparison of the yields and efficiencies of carminic acid extraction techniques Technique Extract Yield (%) Extract Efficiency (mg / min) Carminic Acid Yield (%) Carminic Acid Efficiency (mg / min) Schutzenberger (MC) 31.9 3.5 17.8 2.0 Pressurized Liquids 42.4 14.1 NR NR Supercritical Fluids (EFS) 39.4 1.6 NR NR Microwave (EAM) 41 27.3 18.0 36.0 NR: Not Reported From the above, it can be seen that the Shutzemberger method yielded a carminic acid yield of 17.8% and an extract yield of 31.9% in relation to the sample weight of 20 cochineal used, compared to the conventional solid-liquid extraction method, which obtained an 18.5% extract yield. The present invention (EAM) yielded an extract of 41%, higher than extraction using the Conventional or Schutzenberger Method and higher than EFS. The carminic acid yield by EAM was 18%, a difference of 0.03% compared to the MC. The amount of solvent used in the EAM was 20 mL per gram of sample, increasing by 5 mL of solvent, since in conventional methods such as the Carre, Japanese, Schutzemberger and German methods, they maintain a ratio of 15 mL per gram of sample, which suggests that with the 1:15 g / mL ratio there is a saturation of carminic acid in the solvent and reduces the extraction yield. This invention presents better efficiency in terms of carminic acid yield, since the time obtained was 5 min, compared to 90 min by the Schutzemberger Method. The proposed microwave extraction method for carminic acid extraction provides superior results compared to non-conventional and MC methods, having higher yield and better efficiency. Although the foregoing description was prepared taking into account the preferred embodiments of the invention, those skilled in the art should be aware that any modification of form and detail will be considered within the spirit and scope of the present invention. The terms in which this specification has been drafted should always be taken in a broad and non-restrictive sense. The materials, form, and description of the elements may be varied provided that this does not alter the essential characteristics of the model.

Claims

1. A method for obtaining natural carminic acid from conditioned Dactylopius coccus Costa cochineal, characterized in that it comprises the following steps: a. Mixing ground Dactylopius coccus Costa cochineal and deionized water in different ratios, such as: 1:10, 1:15 and 1:20 g / mL b. Placing the mixture in a microwave oven at a power of 1200 W, for a time between 5-15 minutes; and a temperature between 60-80 0 C.

2. The method of claim 1, characterized in that the microwave equipment is calibrated to a maximum power of 1500 W.

3. The method of claim 1 for obtaining carminic acid, characterized in that the cochineal is conditioned as follows: a) Killing the cochineal by one of the following methods: immersion in hot water, application of steam, asphyxiation with hexane, freezing, asphyxiation by using a plastic bag exposed to the sun for three hours, or by using an oven at 38 °C for 3 hours. b) Drying the dead cochineal using solar dryers, ovens with spotlights, direct sunlight, or in the shade. c) Cleaning by using an air current and sieves with a 2 mm mesh. d) Defatting by solid-liquid extraction with organic solvents such as hexane, petroleum ether, or ethyl ether; e) Grinding the sample using hand mills, artisanal mills, or mechanical systems.

4. The method of claim 1 for obtaining carminic acid, characterized in that the cochineal used must contain on average 8.08% moisture, 4.73% ash, 15.53% lipids, 26.84% carbohydrates, 44.69% proteins, and 20.1% carminic acid. 5 5. The method of claim 1 for obtaining carminic acid, characterized in that it has a pomegranate extract efficiency of 27.3 mg / min.

6. The method of claim 1 for obtaining carminic acid, characterized in that it has a carminic acid yield of 18%.