ANTHOCYANIN PRODUCTION PROCESS FROM ARISTOTELIA CHILENSIS CALLUS CULTURES

Abstract

The invention points to a process of production of anthocyanins from Aristotelia chilensis callus cultures, which is divided into 2 essential steps: the first is the obtaining of biomass in an A. chilensis callus culture, the second step consists of eliciting the production of anthocyanins in the culture, to obtain the desired product. In this way, callus cultivation is presented as a new alternative to provide a nutritional additive rich in anthocyanins appropriate for the food industry, or any other application that requires anthocyanins that does not depend on seasonality or environmental factors.

Claims

1. Process of production of anthocyanins from Aristotelia chilensis callus cultures CHARACTERIZED in that it includes the following steps: (a) growing Aristotela chilensis callus in a vegetable culture medium supplemented with sugars, also containing between 0.1 and 2 mg/L of BAP (6-Benzyl amino purine), between 0.1 to 2 mg/L of NAA (naphthaleneacetic acid); for a period of between 5 and 50 days, with alternating light and darkness; b) eliciting the production of anthocyanins by maintaining the culture of step a) in a growing medium for vegetables, supplemented with sugars, and ethephone between 10 to 500 mg/L; for a period of between 5 and 30 days, with alternating light and darkness.

2. Process according to claim 1 CHARACTERIZED in that in step a) the sugars of the medium are in a total concentration of between 1 to 5% w/v and are chosen between sucrose, glucose and/or fructose.

3. Process according to claim 2 CHARACTERIZED in that in step a) the medium optionally comprises between 20 to 200 mg/L of iron chelate.

4. Process according to claim 2 CHARACTERIZED in that in step a) the crop is maintained in a photoperiod of between 12 to 20 light hours and between 12 to 4 hours of darkness per day, with a light intensity of between 5,000 to 30,000 lux.

5. Process according to claim 1 CHARACTERIZED in that in step b) the sugars of the medium are in a total concentration of between 1 to 20% w/v and are chosen between sucrose, glucose and/or fructose.

6. Process according to claim 5 CHARACTERIZED in that in step b) the medium optionally comprises between 20 to 200 mg/L of chelate of hierro.

7. Process according to claim 5 CHARACTERIZED in that in step b) the crop is maintained in a photoperiod of between 12 to 20 light hours and between 12 to 4 hours of darkness per day, with a light intensity of between 5,000 to 20,000 lux.

8. Process according to claim 1 CHARACTERIZED in that additionally at the end of step b) the anthocyanins are extracted from the crop.

9. Process according to claim 1 CHARACTERIZED in that additionally at the end of step b) the cells of A. chilensis are dried and ground until obtaining a powder rich in anthocyanins.

10. Use of the powder obtained in claim 9 CHARACTERIZED in that it serves as a supplement for food and beverages.

11. Use of the powder obtained in claim 9 CHARACTERIZED in that it serves as a source of anthocyanins in nutraceutical products.

Description

DESCRIPTION OF THE FIGURES

[0009] FIG. 1. Biomass yield graph (mf/m0) of calluses subjected to biomass augmentation treatment, the ratio between the final biomass (mf) and initial biomass (m0) is shown.

[0010] FIG. 2. Graph of anthocyanin concentration expressed in milligrams equivalent of delphinidine per gram of dry weight (mg eq del/pgd) of calluses subjected to an elicitation treatment for the production of anthocyanins.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The invention points to a process of production of anthocyanins from callus cultures of Aristotelia chilensis.

[0012] This process, of anthocyanin production, is divided into 2 essential stages. The first is the obtaining of biomass in the cultivation of calluses of A. chilensis, the second stage consists of the eliciting of the production of anthocyanins in the culture, to obtain the desired product.

[0013] Essentially, the production of anthocyanins from Aristotelia chilensis callus cultures comprises the following steps: [0014] (a) growing calluses of Aristotela chilensis in a vegetable culture medium supplemented with sugars, also containing between 0.1 to 2 mg/L of BAP (6-Benzyl amino purine), between 0.1 to 2 mg/L of NAA (naphthaleneacetic acid) and between 100 to 500 mg/L ascorbic acid; for a period of between 5 and 50 days; with alternating light and dark; [0015] (b) eliciting anthocyanin production by maintaining stage (a) culture in a vegetable growing medium supplemented with sugars and between 10 to 500 mg/L ethephon; for a period of between 5 and 30 days, with alternating light and dark.

[0016] In step a) of the process the sugars in the medium are in a total concentration of between 1 to 5% w/v and are chosen between sucrose, glucose and/or fructose and the medium optionally comprises between 20 to 200 mg/L of iron chelate. And the culture is kept in a photoperiod of between 12 to 20 light hours and between 12 to 4 hours of darkness a day, with a light intensity of between 5,000 to 30,000 lux.

[0017] In step b) of the process the sugars in the medium are in a total concentration of between 1 to 20% w/v and are chosen between sucrose, glucose and/or fructose and the medium optionally comprises between 20 to 200 mg/L of iron chelate; and between 10 to 500 mg/L ethephon; for a period of between 5 and 30 days. And the culture is kept in a photoperiod of between 12 to 20 light hours and between 12 to 4 hours of darkness a day, with a light intensity of between 5,000 to 20,000 lux. Iron chelate can be iron sulfate, hydrated mono, penta or hepta.

[0018] Where, additionally at the end of step b) the anthocyanins are extracted from the calluses in culture. Or alternatively at the end of step b) the A. chilensis calluses are dried, for example, by freeze-drying and ground until a powder rich in anthocyanins is obtained.

[0019] In a preferred embodiment the powder obtained can be used directly as a supplement for food and beverages, and also as a source of anthocyanins for nutraceutical products.

[0020] As indicated, the purpose of step a) of the method of the invention is to obtain biomass in large quantities, the inventors have carried out studies on a laboratory scale, where they have managed to obtain a biomass yield of the calluses of A. chilensis of up to 9.5 times the initial biomass, in 21 days of cultivation.

[0021] The second step of the process of invention consists of the eliciting of the production of anthocyanins in cultures. At this stage, the calluses of A. chilensis in culture are subjected to nutritional stress, so that the metabolic pathway of anthocyanin production is activated and thus achieves the change of coloration of the calluses from green to purple, the characteristic color for the presence of anthocyanins in the plant tissue.

[0022] After step a) of biomass increase, the culture medium is removed and the calluses are kept in vegetable culture medium, supplemented with sugars, and as an elicitor of ethephon. Growing conditions are photoperiod 16 hours light 8 hours darkness, constant agitation of 100 rpm. In these conditions it is possible to obtain completely colored calluses with a high content of anthocyanins.

[0023] Optionally, colored calluses can be processed to obtain an extract of anthocyanins in powder format. To do this, in a preferred embodiment the calluses are subjected to centrifuge for 15 minutes, to remove the excess culture medium. Then they are freeze-dried for 72 hrs. and then they go to grinding in a knife grinder for 10 minutes. At the end of the process, a quality control of the product is carried out, where the anthocyanin content will be measured.

[0024] The product of the invention has the advantage that it is not under the effect of the seasonality of the fruit, since the raw material of the production of anthocyanins is present throughout the year for the case of the invention. It should also be noted that the variability of the fruits, both by genetic variables of the plants or by environmental climatic variables, does not affect the production of anthocyanin in our case, because the production of this is regulated by other factors that make us independent of genetic variability. The process of the invention allows for obtaining a biomass with high content of anthocyanins, which could compete with the biomass of fruit obtained in a season.

[0025] Below are some examples, which show preferred embodiments of the invention. Within the examples there are conditions that give better results than the others tested, however, all options are considered part of the invention. These examples should be considered as illustrative and not limiting the scope of the invention process.

EXAMPLES

[0026] The process of obtaining anthocyanins is divided into the increase of the biomass of calluses through the addition of nutrients and hormones, followed by the eliciting of the production of anthocyanins with the help of elicitors.

Example 1: Increase in Callus Biomass

[0027] For the increase of biomass, calluses of A. chilensis obtained by traditional methods of callogenesis are provided. These calluses were grown in 4 different treatments or media which are specified in Table 1.

[0028] The culture conditions were photoperiod 16 light hours with light intensity of 27,000 lux and 8 hours of darkness, for 28 days.

[0029] FIG. 1 shows the comparison of the results obtained with each treatment used, where after the process it was possible to obtain a yield of 9.5 times increase in initial biomass with treatment N.sup.o 4, this means that for 1 g of inoculated fresh callus 9.5 g of fresh callus is obtained at the end of the process. In the case of treatment N.sup.o 1 and N.sup.o 2 the biomass was tripled or and for the treatment N.sup.o 3 the biomass was quintupled.

TABLE-US-00001 TABLE 1 Description of treatment used for biomass augmentation Treatment N.sup.o1 Treatment N.sup.o2 Treatment N.sup.o3 Treatment N.sup.o4 Medio Murashige & Medio Murashige & Medium Murashige & Modified Gamborg Skoog Skoog Skoog modified B5 medium 2% w/v sucrose 2% w/v sucrose 2% (sucrose, glucose 2% (sucrose, glucose 0.5 mg/L BAP  0.5 mg/L BAP and fructose) and fructose)   1 mg/L NAA   1 mg/L NAA 0.5 mg/L BAP 0.5 mg/L BAP 83.4 mg/L Iron   1 mg/L NAA   1 mg/L NAA chelate (EDTA)

Example 2: Anthocyanin Elicitation

[0030] After the process of increasing cellular biomass, calluses obtained according to treatment 4 described in example 1, were transferred to treatments or means of eliciting the production of anthocyanins, which are specified in Table 2.

TABLE-US-00002 TABLE 2 Description of treatments used for eliciting. Treatment N.sup.o1 Treatment N.sup.o2 Treatment N.sup.o3 Murashige & Murashige & Modified Gamborg B5 Skoog medium Skoog medium medium without nitrogen without nitrogen 2% sucrose 10% sucrose 2% (sucrose, glucose and 100 mg/L ethephon 100 mg/L ethephon fructose) 100 mg/L ethephon

[0031] The growing conditions were photoperiod 16 hours red light with light intensity of 11,000 lux and 8 hours of darkness, for 14 days.

[0032] After 14 days the calluses were harvested and subjected to a process of extraction and quantification of anthocyanins. The calluses were weighed and part of them transferred to Falcon Tubes to quantify and isolate the anthocyanins produced. For this, 5 ml of acidified methanol (0.1% HCl) was added to the Falcon tubes and left stirring at 200 rpm for 1 hour. The mixture was centrifuged at 10,000 g for 15 minutes, the supernatant was recovered and stored at 4° C.

[0033] 3 ml of acidified methanol was added to the remaining pellet and again left stirring for an hour, centrifuged and the supernatant was joined with the previous one. The concentration of anthocyanins in the extracts was quantified through the differential pH method.

[0034] From treatment 1, a callus with reddish colored areas was obtained, the quantification showed that the callus had an average concentration of 2.2 mg eq del/pgd of anthocyanins.

[0035] In the case of treatment 2, a reddish colored callus was obtained and with purple areas, the quantification showed that the callus had an average concentration of 3.8 mg eq del/pgd.

[0036] Finally, for treatment 3 a purple-colored callus was obtained, the quantification showed that the callus had an average concentration of 6.4 mg eq del/pgd of anthocyanins, the results are graphed in FIG. 2.

[0037] The rest of the biomass obtained was freeze-dried for 72 hrs at a temperature of −70° C. and pressure of 0.0007 pascal and a purple dry powder, rich in anthocyanins, was obtained.

[0038] The powder obtained can be mixed with food or beverages providing nutraceutical anthocyanins. Or it can be used as a source of anthocyanins in any composition that requires them.

[0039] The preceding examples demonstrate the effectiveness of the invention process. For the expert in the technique, modifications to these achievements will be evident, which would have similar results to those described, leaving all these modifications included in the scope of the invention, as indicated in the attached claims.