Methods for obtaining carminic acid

Abstract

A novel, alternative, and low-cost synthetic method for the in vitro production of carminic acid by using hemolymph cells of Dactylopius coccus Costa (cochineal), an insect used in the food, cosmetics, pharmaceutical, and textile dye industries.

Description

[Technical Field]

[0001] The present invention relates to a novel, alternative and low-cost method for the in vitro production of carminic acid (CA) for use in the dye industry, which has wide applications in particular in the food, cosmetic, pharmaceutical and textile industries. [Background technology]

[0002] For many years, the prior art has had a traditional production process involving sowing, growing, and collecting Dactylopius coccus Costa (cochineal) insects within a cycle of approximately 80 to 90 days using greenhouse, shed, and / or open-air systems. This production requires the pairing of plants and insects. Carminic acid extracted from cochineal insects has been known to be used as a red and / or crimson dye in cosmetics (especially lipstick) and in the food industry as food additive E-120 to impart a red color to foods, but it has gradually been replaced by cheaper synthetic dyes. A widely used substitute is cochineal red A, numbered E-124. Carminic acid obtained from indigenous insects has been used in Europe since at least the Iron Age, with evidence found, for example, in Hallstatt culture graves.

[0003] The following processes known in the art all have the same basic steps of preparing the cochineal, drying the material, mechanically separating it, crushing it, and extracting the fats and waxes to extract carminic acid. They differ in the extraction of the fats and waxes. The next step, extraction of carminic acid, is followed by precipitation, sedimentation, filtration, and drying of the carmine.

[0004] Some state-of-the-art solutions for the mass production of carminic acid are disclosed in the following documents:

[0005] Miguel Gonzalez Gonzalez et al., Scientific Note Rev. Fitotec. Mex. Vol. 25 (2): 209-212, 2002, "In vitro Culture of Embryonic Cells of Cochineal (Dactylopius coccus Costa) at Different pH Values," evaluated the determination of the optimum pH at which in vitro cultures of cochineal (Dactylopius coccus Costa) cells recorded maximum acid production. Several pH levels (4.5, 5.0, 5.5, 6.0, and 6.5) and centrifugation (0, 2, 5, and 10 min) fractions were evaluated for macerated cells of cochineal embryos established in Schneider's nutrient medium for culturing insect cell lines. The content of carminic acid present in each sample was determined by high-performance liquid chromatography. The pigment was extracted from the samples using a 2 M HCl solution. The detection limit for the pigment was 1.0 mg L−1. -1 to 120.0 mg L -1 No significant effect of pH was observed on carminic acid content or cell line number, although an intermediate pH (5.5) tended to give better results. However, the paper's conclusions suggest that media with a pH between 4.5 and 6.5 are suitable for in vitro cochineal cell culture, with intermediate pH values ​​tending to favor carmine content and cell line number. Centrifugation times of 2 and 5 minutes were most suitable for establishing D. coccus embryonic cell lines. Carmine production in secondary cultures was low. Therefore, it is suggested that other medium variables, primarily osmolality and components of the nutrient medium, be evaluated.

[0006] US8,919,281 B2 (2013, Means to culture cochineal insects in an artificial medium, Hendrickson, Constance M. Merkle, Denise Lynn) refers to a process for cultivating insects of the genus Dactylopius, which includes heating a mixture containing (a) a cactus additive obtained from a cactus of the genus Opuntia, (b) a polysaccharide, and (c) glucose, combining the heated mixture with a three-dimensional matrix, cooling the combined mixture and matrix to form a hardened medium, and inoculating the hardened medium with a species selected from the group consisting of the genus Dactylopius.

[0007] Some of these patents, such as MX 295682, ARIGEN PHARMACEUTICALS, INC., LYMPHOKINE-ACTIVATED KILLER CELL PROLIFERATION METHOD, Watarai, Shinobu; Nishikawa, Shigeru, refer to a cancer-related cell proliferation / activation process that can be performed at such low cost that it may be applicable to non-human animals. The cell proliferation / activation process of this application includes a step of supplementing a medium with at least 5 to 15 μg / ml of concanavalin A and a growth factor having interleukin-2-like activity during cell culture, and therefore, this process can preferentially proliferate / activate αβ cells. Summary of the Invention [Problem to be solved by the invention]

[0008] However, despite these solutions provided, there remains a need in the art for an effective solution that reduces the production time of carminic acid, allowing for immediate collection, as well as reducing costs in terms of operations and human resources. [Means for solving the problem]

[0009] The present invention solves this problem by providing a novel and unique method for producing carminic acid in vitro, which has the following structure:

[0010] [ka]

[0011] The present invention relates in particular to a method for the in vitro production of carminic acid, based on the isolation, purification and maintenance of a hemocyte cell line obtained from egg-filled adult females of the insect Dactylopius coccus Costa (cochineal), culturing it to give rise to a primary culture (Step I), followed by stimulation of the hemocytes with a mixture of mitogens.

[0012] Advantageously, the production of carminic acid by the novel method of the present invention provides both a reduction in the number of insects used and a reduction in production and collection time, currently estimated at between about 12 and 14 days, as well as reduced operating and human resource costs, large-scale production in a small space, cell management in laboratory conditions without the need for high-input processing, reduced raw material (elimination of host plants), collection in a direct soluble form without the need for drying processes (time and materials), cryopreservation of production cell lines for perpetuation in case of future insect shortages, pest- and natural predator-free production, and the use of fewer insects, about 10 to 15, to produce about 1 kilogram of carminic acid, compared to the approximately 350,000 to 400,000 Dactylopius coccus costa (cochineal) insects required to be sacrificed in the conventional process. Advantageously, production costs are about 50% lower than those using the conventional process, and further, the method provides scalability by replicating the production unit.

[0013] The present invention provides a methodology for isolating, extracting, and maintaining pure CA-producing cell lines to obtain a reproducible and consistent collection of CA production under laboratory conditions and in 1,000 ml vessels, as well as maintaining contaminant-free cell lines in a reproducible and consistent manner that can be repeated before stimulants are administered to the culture. [Brief explanation of the drawings]

[0014] The particular features and advantages of the present invention, as well as other objects, will become apparent from the following description taken in conjunction with the accompanying drawings.

[0015] [Figure 1-2] Figure 1 shows step I of the extraction of hemocytes from the hemolymph of Dactylopius coccus Costa insects. A and B are chromatocytes (separated and enriched in a 70% Percoll gradient) cultured in Schneider's medium for 96 hours. A and B are the same type of cells, but the contrast of their microscopic images is different. In B, the presence of carminic acid granules in the cytoplasm is more evident. C is a young, undifferentiated chromatocyte that begins to produce increased amounts of carminic acid as it matures.

[0016] Figure 2 shows the results of the Percoll gradient (separation phases) and the formation of carbamate (a cellular product) as a dark supernatant on the surface of the last two tubes.

[0017] [Figure 3] FIG. 3 is a phase contrast microscopy study of mitogen-stimulated blood cells after 72 hours of culture in Schneider's medium.

[0018] [Figure 4-5] FIG. 4 shows microscopic observations of bioproduction in terms of cell viability in Schneider's medium cultured for 72 hours after inoculation in an MTT assay on 2 mm plates.

[0019] FIG. 5 shows microscopic observations of granule cells 96 hours after inoculation.

[0020] [Figure 6] FIG. 6 shows a depiction of the production and maintenance process in a flat-bottom vessel according to one embodiment of the present invention.

[0021] [Figure 7] Figure 7 shows the results of inspection of the container 25 days after inoculation.

[0022] [Figure 8] FIG. 8 shows a graph of the results illustrating the absorption spectra (absorbance vs. wavelength in nanometers) for distinguishing CA from live cultures from the table in Example 1 detailed in the Examples section below.

[0023] [Figure 9] FIG. 9 shows a graph of the results illustrating the absorption spectra for identifying purified CA from live cultures from the table in Example 2 detailed in the Examples section below.

[0024] [Figure 10] FIG. 10 is a supplementary figure to Table 2 below, and shows preliminary results for various cell types obtained by Percoll gradients using the method described below (microscopic observation of cells obtained in Percoll gradients).

[0025] [Figure 11-12] FIG. 11 is a photomicrograph of granule cells on day 8 in culture.

[0026] FIG. 12 is a photomicrograph of granule cells at 10 days in culture.

[0027] [Figure 13-14] FIG. 13 shows the effect of anticoagulation in obtaining hemolymph by perfusion.

[0028] FIG. 14 shows the protein integrity obtained during perfusion. DETAILED DESCRIPTION OF THE INVENTION

[0029] In order that the present invention may be better understood, the following definitions are provided.

[0030] The use of the term "approximately" provides some additional range. This term is defined as follows: The additional range provided by this term is approximately ±10%. As a non-limiting example, if "approximately 40 grams" is written, the range is between ±10% of the standard deviation, and similarly for other measurements.

[0031] According to the present invention, the production of carminic acid in vitro involves the extraction, isolation, purification and maintenance of a cell line of hemocytes obtained from egg-filled adult females of the insect Dactylopius coccus Costa (cochineal) in the following general process:

[0032] [Extraction of hemocytes from hemolymph] Hemocytes are obtained by perfusing the hemocoel of egg-filled pregnant strains of Dactylopius coccus with an anticoagulant buffer containing bases, ionic salts, chelating agents, and organic acids until a red pigment solution containing, inter alia, carminic acid (CA)-producing cells is obtained.

[0033] In one embodiment of the invention, the anticoagulant buffer comprises NaOH, NaCl, EDTA and citric acid. The resulting dye solution is precipitated in a sterile container and centrifuged. The supernatant is collected. Meanwhile, a discontinuous Percoll gradient is prepared using phosphate buffered saline. A discontinuous Percoll gradient is pre-run by centrifugation. After the pre-run is complete, the resulting dye solution is added. A centrifugation run is performed to obtain differentiated cells (Table 2). At the end of the run, hemolymph cells are obtained that will be used to generate cultures. The cells are harvested and plated in growth medium for culturing insect cell lines for adaptation.

[0034] [Cell line selection and primary culture acquisition] Under a microscope, cultures showing differentiated cells are identified, scored and selected. Granulocytes and blood cells are selected. The selected cells are plated using growth medium for culturing insect cell lines for adaptation. Finally, the cells are examined and evaluated according to their phenotypic integrity.

[0035] [Stimulation and activation of cell lines] Each culture is stimulated to undergo mitosis by adding a combination of mitogens in phosphate buffered saline. The cultures are incubated until they gradually turn red and are observed for cell division due to the mitogen. It is recommended that cultures that show an increase in population density be selected, grown in containers, and prepared for cryogenic storage.

[0036] [Growth and production in a container] The selected cells are plated using growth medium for culturing insect cell lines for adaptation. The vessels are cultured for approximately 10-14 days (primary line), during which time the cells form a confluent monolayer of cells attached to the vessel and the medium turns red, a positive sign for color production. The vessels are checked daily to assess and confirm the presence of mitoses. At the end of the cycle, the cultures are harvested and the color is squeezed or collected from each culture vessel. This "milking" or "harvest" is carried out in a sterile atmosphere and each harvest is centrifuged in a container to remove cellular debris and collect only the supernatant, which in this case is "the pigment" (carminic acid).

[0037] For purposes of the present invention, the steps of the process described above will be presented in detail.

[0038] [Extraction of hemocytes from hemolymph] Hemolymph is obtained from at least about 20 to about 95 mg of egg-filled pregnant strains of Dactylopius coccus, preferably about 30 to about 85 mg of egg-filled pregnant insects, by perfusing the hemocoel of the strain with an anticoagulant buffer as described in Graham et al., 1986. This anticoagulant buffer contains NaOH in the range of about 0.25 mM to about 0.75 mM (preferably about 0.61 mM), NaCl in the range of about 0.15 M to about 0.50 M (preferably about 0.56 M), EDTA in the range of about 0.10 mM to about 0.25 mM (preferably about 0.16 mM), and citric acid in the range of about 100 mM to about 250 mM (preferably about 100 mM), with a pH in the approximate range of 4.5 to 6.5, as shown in Table 1 below. The resulting solution is a red pigmented solution in which carminic acid (CA)-producing cells and other cell types of the hemolymph (hemocytes) are suspended.

[0039] The resulting dye solution is precipitated in a sterile container, preferably a sterile polypropylene conical tube for the purpose of the present invention, and centrifuged at about 500 to 1,200 revolutions per minute (rpm), preferably about 600 to 1,000 rpm, for about 8 to 17 minutes, preferably about 10 to 15 minutes, at about 2 to 5°C, preferably about 4°C.

[0040] The supernatant is collected in a sterile container, preferably a sterile conical polypropylene tube for the purposes of the present invention, and kept at room temperature.

[0041] Furthermore, as can be seen in Table 2 below and Figure 10 in the drawings section, a discontinuous Percoll gradient (approximately 10-90% range, preferably approximately 20-80% range) is prepared using phosphate buffered saline (1x PBS, pH range of approximately 4.5-5.2, preferably pH approximately 5.5).

[0042] [Table 1]

[0043] The gradient is pre-run by centrifugation at least in the range of about 4,000-5,500 rpm, preferably in the range of about 4,500-5,000 rpm, for about 12 to 22 minutes, preferably about 15 to 20 minutes.

[0044] The resulting dye solution is added in an amount within the range of about 95 to 550 μl (preferably within the range of about 100 to 500 μl) after the pre-run is completed.

[0045] Centrifuging at about 1,000 revolutions per minute (rpm) for about 10 to about 20 minutes, preferably about 15 minutes, at room temperature gives the following results, as can be seen from Table 2 below, reproduced again, and in Figure 10 in the Drawings section:

[0046] [Table 2]

[0047] At the end of the centrifugation run, several phases are obtained, and the interface between Percoll and dye contains purified hemolymph cells, which will be used to generate primary cultures (Step I).

[0048] In a preferred, but non-limiting, process, the cells are plated in about 100 μl to about 500 μl, preferably about 250 μl, of a medium for culturing insect cells, preferably commercially available Schneider's medium, in a 16-well multi-well plate and allowed to adapt for about 20-30 hours, preferably about 24-26 hours, at a temperature in the range of about 20-28° C., preferably 22-26° C.

[0049] [Cell line selection and primary culture acquisition] Approximately 24 hours after culture, the cultures showing better cell integrity and phenotype are inspected under a microscope, evaluated and selected.

[0050] The cell variant "young granulocyte" and the variant "young undifferentiated blood cell" are selected (candidate selection).

[0051] These two variants are selected and re-plated into a plate. In a preferred, but non-limiting, process, for adaptation, the cells are re-plated into a 16-well multiplate using about 100 μl to about 500 μl, preferably about 250 μl, of a medium for culturing insect cell lines, preferably commercially available Schneider's medium, and adapted for about 20 hours at a temperature within the range of about 20-28° C., preferably about 22-26° C.

[0052] Finally, after about 24 hours, the cells are examined and evaluated according to their integrity and maintenance of phenotype.

[0053] [Stimulation and activation of cell lines] To each well containing cells in culture, a combination of mitogens (concanavalin A in the range of about 0.20-0.30 micromolar (preferably about 0.25 micromolar) and phytohemagglutinin in the range of about 0.40-0.60 micromolar (preferably about 0.50 micromolar)) in phosphate buffered saline (1x PBS at a pH between about 4.8-5.2 (preferably about pH 5.0)) is added to promote mitosis in young granulocytes.

[0054] The cultures are cultured at a temperature range of about 20 to 28°C, preferably about 22 to 26°C, for about 20 to 50 hours, preferably about 24 to 48 hours, until they gradually turn red and cell division due to mitogens is observed in the cells.

[0055] Cultures that show an increase in population density are selected and taken to the stage of in vitro growth and production.

[0056] During the container growth stage, it is recommended to prepare for cryogenic storage in a liquid nitrogen tank (within the range of approximately -160 to -180°C, preferably approximately -170°C) for approximately 2 to 3 months.

[0057] [Growth and production in a container] The plated cells are cultured in a container using a medium for growing insect cell lines. For the purposes of this invention, this is approximately 200-300 cm 2 A flat-bottomed vessel having a surface area in the range of about 225 cm is preferred, and more preferably about 225 cm 2 For this purpose, for example, about 200 μl of the plated culture is used to inoculate about 200 to 350 μl, more preferably about 250 to 300 μl, of a medium for culturing insect cell lines, preferably Schneider's medium.

[0058] The vessel is cultured (primary strain) at a temperature ranging from about 20 to 28°C, preferably from about 22 to 26°C, for about 10 to 14 days, preferably from about 12 to 13 days. During this period, the cells attached to the vessel form a confluent monolayer, and the medium turns red, a positive sign of color production.

[0059] The vessels are checked approximately every 24 hours to assess and confirm that the cell line is undergoing mitosis.

[0060] At the end of the cycle, the cultures are harvested and the color is squeezed or collected from each culture vessel.

[0061] This "milking" or "harvest" is carried out in a sanitized environment, and each harvest is centrifuged in a container. For the purposes of the present invention, in order to remove cellular debris and collect only the supernatant, which in this case is the "pigment" (carminic acid), a conical tube with a maximum capacity of about 40-60 ml, more preferably about 50 ml, is preferred, and the centrifugation is carried out at a speed ranging from about 3,500-5,500 rpm, preferably 4,000-5,000 rpm, for about 18-22 minutes, preferably about 20 minutes.

[0062] Importantly, prior art studies using conventional carminic acid production processes have demonstrated that thousands of insects are required to produce approximately 1 kg of carminic acid (CA). Furthermore, high-quality host plants, in this case Opuntia ficus-indica cactus plants, must be constantly replaced or cultivated (costly), and stem prevention and prophylactic phytosanitary control are required, which requires constant cultivation and therefore manpower. Collection requires an 80-90 day growth cycle for the insects to grow, reach adulthood, and be harvested. Furthermore, CA production requires a drying process after collection, during which there is a natural reduction in production, at a ratio of approximately 3:1 (live insects:dried insects), with periods of decline due to the presence of pests and predators. All of the above results in high production costs, and the international price is dominated by countries that produce large amounts of CA, whose prices are set arbitrarily because it is the only form of production and because the biogeographical conditions in these countries allow for large annual production volumes. As a result, large production areas are required to produce attractive quantities of CA.

[0063] In some embodiments of the present invention, growth and carminic acid production in a vessel may be carried out in a vessel such as, but not limited to, a flat-bottom culture bottle, a Roux flask, a benchtop bioreactor, a specialized bioreactor, an industrial bioreactor, a vessel in which a chemical process involving an organism or microorganism or involving biochemically active substances derived from said organism and / or microorganism is carried out, and / or in any other suitable vessel or apparatus capable of carrying out a growth reaction such as those described herein. [Example]

[0064] Example 1. Absorption spectra for identifying CA from living cultures

[0065] [Table 3]

[0066] Filtration was carried out using a PVDF membrane (0.2 mm) heated (55°C) for 30 minutes. Example of calculating CA% using result 3 Formula: %CA=0.5208 / 0.139×0.1g=37.46%

[0067] Example 2. Absorption spectra for identifying CA from purified biological cultures

[0068] [Table 4]

[0069] Example of calculating CA% Formula: %CA=0.5562 / 0.139×0.05g=80.02%

[0070] Although the foregoing embodiments of the present invention have been described in some detail by way of explanation and example for the purpose of improving its understanding, the explanation and examples are not to be construed as limiting the scope of the present invention, but rather as illustrating the spirit and some variations that fall within its scope. As will be apparent to one of ordinary skill in the art, variations or modifications that do not depart from the spirit of the present invention are within its scope. The statements of scientific literature cited herein are expressly incorporated by reference.

Claims

1. 1. A method for producing carminic acid from insects of the genus Dactylopius coccus (cochineal), comprising: extracting hemolymph hemocytes from said insects of the genus Dactylopius coccus of egg-filled peanut strains until a solution containing carminic acid-producing cells is obtained; pre-running a Percoll® gradient; adding the solution containing the carminic acid-producing cells to the pre-run Percoll® gradient to obtain a mixture containing blood cells; purifying the blood cells from the mixture; recovering the purified blood cells; culturing the purified blood cells in a medium for culturing an insect cell line; stimulating and activating the purified blood cells; expanding the carminic acid-producing hemocytes by culturing the cells selected from a culture exhibiting increased population density in a container with a medium for growing insect cell lines; harvesting the culture and recovering the carminic acid produced; A method comprising:

2. extracting blood cells from the hemolymph is performed by perfusing the hemocoagulant cavity with an anticoagulant buffer comprising a base, an ionic salt, a chelating agent, and an organic acid; The Percoll® gradient is prepared using phosphate buffered saline; pre-running the Percoll® gradient by centrifugation; The method of claim 1 , wherein the step of purifying the blood cells from the mixture is performed by centrifugation.

3. The method described in claim 1, comprising the step of selecting young granulocytes and hemocytes and culturing the selected cells using a medium for culturing insect cell lines prior to the step of stimulating and activating the purified hemocytes of Dactylopius coccus.

4. The step of stimulating and activating the purified blood cells of Dactylopius coccus comprises: providing a combination of mitogens in phosphate buffered saline; incubating said Dactylopius coccus blood cells with a combination of added mitogens to produce a culture; and after incubation, selecting the cultures that exhibit an increase in population density.

5. 2. The method of claim 1, wherein the Percoll® gradient is 10-90%.

6. 5. The method according to claim 4, wherein the culturing is carried out at a temperature in the range of 20 to 28°C for 20 to 50 hours.

7. 5. The method of claim 4, wherein the cultures exhibiting increased population density are stored in a cryogenic stage with liquid nitrogen.

8. 8. The method of claim 7, wherein the cryogenic step is carried out at a temperature ranging between -160°C and -180°C for 2 to 3 months.

9. 10. The method of claim 1, wherein the step of extracting hemocytes from hemolymph comprises 20 mg to 95 mg of the insects of the egg-filling strain Dactylopius coccus (cochineal).

10. 3. The method of claim 2, wherein the anticoagulant buffer comprises 0.25 mM to 0.75 mM NaOH, 0.15 M to 0.50 M NaCl, 0.10 mM to 0.25 mM EDTA, 100 mM to 250 mM citric acid, and a pH in the range of 4.5 to 6.

5.

11. 11. The method of claim 10, wherein the anticoagulant buffer comprises 0.61 mM NaOH, 0.56 M NaCl, 0.16 mM EDTA, 100 mM citric acid, and has a pH in the range of 4.5 to 6.

5.

12. 4. The method of claim 3, wherein the culture medium for the insect cell line is Schneider's medium.

13. 5. The method of claim 4, wherein the mitogens are concanavalin A in the range of 0.20 to 0.30 micromolar and phytohemagglutinin in the range of 0.40 to 0.60 micromolar.

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