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

Abstract

The present invention relates to a method for obtaining carminic acid from Dactylopius coccus by ultrasound-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 and labor consumption and waste. Therefore, the present invention provides an alternative method for obtaining carminic acid from Dactylopius coccus that improves yield.

Description

METHOD FOR IMPROVING THE YIELD IN OBTAINING CARMINIC ACID FROM Dactylopius coccus costa BY ULTRASOUND-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 ultrasound-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 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. One 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 cooled to room temperature and filtered. The residue 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 the extraction of carminic acid; Borges et al. (2012) propose the Extraction techniques by Supercritical Fluid Extraction (SFE) and Pressurized Liquid Extraction (PLE) were used, where carminic acid was extracted using three different solvents (methanol:water, ethanol:water, and ethanol), three temperatures (100, 150°C, and 200°C), for 30 minutes at pressures up to 10.5 MPa. Extractions were performed 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, this method resulted in a very long extraction time. In accordance with the above, there is still a need for new methodologies 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 for improved extraction yield of carminic acid. Therefore, the main object of protection relates to a method for improving the yield 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 an ultrasonication equipment calibrated to a power of no more than 1500 W, preferably 500 W, and a frequency of 20 kHz for a time no greater 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. MC: Conventional Method, UAU: Ultrasound-Assisted Extraction, EAM: Microwave-Assisted Extraction DESCRIPTION OF THE INVENTION The present invention relates to a method for obtaining carminic acid from Dactylopius coccus by ultrasound-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 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 ultrasound-assisted extraction (UAE), which, through the acoustic cavitation effect produced in the solvent by the passage of an ultrasound wave, allows a mechanical effect and greater penetration of the solvent into the tissue, increasing the contact surface area between the solid and liquid phases, resulting in the rapid diffusion of the solute from the solid phase to the solvent. The UAE uses high-frequency sound waves primarily to release the desired compound from the plant material. This accelerates solid and liquid particles under the ultrasonic action, causing the solute to rapidly transition from the solid phase to the solvent, thus facilitating extraction. EAU has a dual effect which allows the mechanical rupture of the cell wall and therefore the release of its contents and the local heating of the liquid, thus increasing the diffusion of the extract. In solid / liquid systems, acoustic acoustic emission (AUE) enhances mass transfer caused by acoustic cavitation induced in a liquid medium. When mechanical waves are transferred through a fluid, the average distance between molecules is modified, causing them to oscillate around their equilibrium position. AUE has two cycles: compression and expansion. In the compression cycle, the intramolecular distance shortens, and in the expansion cycle, the distance lengthens. 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 extraction time of carminic acid. The ultrasound-assisted extraction (UAE) 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 1:10; 1:15 and 1:20 g / mL. b) Place in a jacketed tube of an ultrasonication equipment, preferably Colé Parmer model CP 505 of 500 Watts of power and a frequency of 20 kHz using a 1% microtip; coupled to a cooling system in order to regulate its temperature, for a time of 5, 10 and 15 minutes at temperatures of 60, 70 and 80 °C. The examples presented are illustrative and not limiting, since 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 one of the following methods selected from: immersion in hot water, application of steam, suffocation with application of hexane (100% purity), freezing, suffocation by using a plastic bag exposed to the sun for three hours, or by using an oven at 38 °C for three hours, Figure 1 (10). b) Dry (20) the dead grain preferably by one of the following means: solar dryers, ovens with lights, direct sun or in the shade, to avoid changes in the structure, color and final appearance of the product. Figure 1 (20). c) Clean (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 to remove the waxy coating residues (coccerin) that pass through in the previous steps, by solid-liquid extraction using selected organic solvents of hexane, petroleum ether or ethyl ether Figure 1 (40). e) Grind 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. It was also degreased 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: * 21 * 100 % of Carminic Acid = ~—ttt0.262 * 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. The analysis of the chemical composition of the cochineal determines its quality. It is important for its evaluation because during the development of the cochineal, there can be variable environmental conditions or changes depending on the fertilization of the prickly pear cactus used. Therefore, the composition of the cochineal can vary, making it important to analyze it to determine if it presents 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 (1A). 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 by Ultrasound Assisted Extraction. Ground granules and deionized water were placed in vials in a ratio of between 1:10; 1:15 and 1:20 g / mL. These were placed in a jacketed tube of a Colé Parmer model CP 505 ultrasonication equipment with a power of 500 Watts and a frequency of 20 kHz using a microtip coupled to a cooling system in order to regulate its temperature, for a time of 5, 10 and 15 minutes at temperatures of 60, 70 and 80 °C. Advantageously, the maximum percentage yield of the ultrasonic extract was 49.2% at a temperature of 60 °C in 15 min with a solvent ratio of 1:20 g / mL. Preferably, the yield is obtained from minute 13 at 60 °C to minute 15 at 69 °C, with the same solvent ratio of 1:20 g / mL. Carminic acid yield by Ultrasound-Assisted Extraction The highest yield is preferably obtained at 24% carminic acid at a temperature of 70 °C, for a time of 15 min and a solvent ratio of 20 mL. This can be seen more clearly in the surface contour plots in Figure 3, which were plotted based on a time of 15 min (2 A), a solvent ratio of 1:20 g / mL (2 B) and a temperature of 70 °C (2 C). The optimized variables for achieving optimal carminic acid yield were 67.4 °C, 15 min, and a solvent ratio of 20 mL, resulting in a 24.05% yield. This represents a 0.05% increase over the estimated value. Table 2. Estimated UAE extract yield Temperature Time Solvent Prediction for Yield (mL) (min) (mL) (%) 70.0 10.0 15.0 43.9 71.0 9.8 14.4 43.1 72.0 9.7 14.0 42.5 73.0 9.6 13.6 41.9 74.0 9.5 13.3 41.3 75.0 9.4 13.1 40.6 Table 3. Extract performance optimization Factor Low High Optimum Temperature 60.0 80.0 63.2 Time 5.0 15.0 15.0 Solvent 10.0 20.0 20.0 Estimated extract yield by Microwave Assisted Extraction Table 4 shows the maximum rise, where the extract yield is obtained from the average values ​​of the factors. 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 4. Estimated extract yield Temperature Time Solvent Prediction for Yield (fC) (min) (mL) (%) 70.0 10.0 15.0 36.5 71.0 10.02 12.9 34.6 72.0 10.3 11.3 32.4 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 5. 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 Schutzemberger MC showed a lower yield than the proposed extraction methods, with an extract yield of 31.9%, which is 15.1% less than that obtained by the EAU and 9.1% less than the EAM. The net yield of carminic acid obtained by the MC was 17.8%, which is 8.5% less than the ultrasonic extraction and 0.2% less than the microwave extraction. Similarly, the ultrasonic extraction method yields better extracts than the methods reported by Borges et al. (2012), where they performed ELP and EFS. ELP yielded the highest extract yield at 42.4%, while EFS yielded 39.4%. Thus, EAU continues to show higher yields; EAM was only superior to EFS, with a difference of just 1.4%. Table 6 shows a comparison between various extraction techniques, with the proposed invention having a better extract yield. 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) Schutzemberger 31.9 3.5 17.8 2.0 Pressurized Liquids 42.4 14.1 NR NR Supercritical Fluids 39.4 1.6 NR NR Microwave 41 27.3 18.0 36.0 Ultrasound 47 31.3 26.3 17.5 NR: Not Reported From the above, it can be seen that the Schutzemberger method yielded a carminic acid yield of 17.8% and an extract yield of 31.9% in relation to the weight of the cochineal sample used, compared to the conventional solid-liquid extraction method, which obtained an 18.5% extract yield. The EAU method yielded an extract of 47%, a higher yield compared to the MC Schutzemberger method and the two non-conventional methods: ELP (42.4%) and EFS (39.4%), reported by Borges et al. (2012). Similarly, the carminic acid yield obtained by the EAU method was 26.3%, higher than that obtained by MC. The EAM yielded an extract of 41%, higher than the extraction with the Schutzemberger MC and higher than the EFS; however, it yielded less than the ELP. The carminic acid yield from the EAM was 18%, a difference of 0.03% compared to the MC. Thus, the carminic acid yield obtained by the UAE was the highest at 26.3%, surpassing the 18% obtained by the EAM. Similarly, the UAE had the highest extract yield compared to the ELP, EAM, EFS, and MC. The amount of solvent used in the EAU and 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. The present invention reduces the extraction time to 15 min, compared to the 90 min that elapse in the extraction of MC, making it more efficient for extract yield. The proposed ultrasound and microwave extraction method for carminic acid extraction is a powerful technique as it provides superior results compared to non-conventional methods and MC, 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 with deionized water in different proportions, such as: 1:10, 1:15 and 1:20 g / mL. b) Applying ultrasound waves to the ground cochineal mixture with an ultrasonication device at a power of 500 W and a frequency of 20 kHz for a time range between 5-15 minutes; and a temperature between 60-80 °C.

2. The method of claim 1, characterized in that the ultrasonication equipment is calibrated to a maximum power of 1500 W and uses a microtip coupled to a cooling system.

3. The method of claim 1 for obtaining carminic acid characterized in that the cochineal cochineal is conditioned as follows: a) sacrificing the cochineal cochineal, b) drying the cochineal sample, c) cleaning by sieving the sample on a 2 mm sieve; d) degreasing by adding hexane to remove excess wax, e) grinding to proceed to extraction.

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. The method of claim 1 for obtaining carminic acid characterized in that it has a cochineal extract yield of 47%.

6. The method of claim 1 for obtaining carminic acid characterized in that it has a carminic acid yield of 26.3%.