WO2004021798A1

WO2004021798A1 - Method of preparing chitosan microcapsules of astaxanthin and product thus obtained

Info

Publication number: WO2004021798A1
Application number: PCT/MX2002/000085
Authority: WO
Inventors: Inocencio Higuera Ciapara; Leticia Felix Valenzuela; Francisco Martín GOYCOOLEA VALENCIA; Waldo Manuel ARGÜELLES MONAL
Original assignee: Centro De Investigacion En Alimentacion Y Desarrollo A.C.
Priority date: 2002-09-03
Filing date: 2002-09-03
Publication date: 2004-03-18
Also published as: AU2002329108A1

Abstract

The invention relates to a method of preparing microcapsules of astaxanthin in a chitosan matrix crosslinked with glutaraldehyde. The inventive method comprises the following steps: (a) formation of a primary oil-in-water emulsion, of which the dispersed phase is an astaxanthin solution dissolved in dichloromethane and the continuous phase consists of an aqueous chitosan solution with an emulsifier; and (b) formation of an oil-water-oil emulsion, of which the dispersed phase is the aforementioned primary emulsion and the continuous phase consists of mineral oil with an added emulsifier, an aqueous glutaraldehyde solution being added at the end of said step in order to produce the crosslinking reaction of the chitosane; (c) evaporation of the aqueous solvents using heat; (d) recovery of the microcapsules by centrifuging the suspension of same and decantation of the mineral oil; and (e) vacuum drying of the microcapsules obtained.

Description

ASTAXANTINE MICROCAPSU AS PREPARATION METHOD IN QUITOSANE AND PRODUCT OBTAINED

DESCRIPTION TECHNICAL FIELD

The present invention relates to a microencapsulated carotenoid in a chitosan matrix, and a method of preparing the microcapsules. The invention relates to the problem of stabilizing the carotenoid, and converting it into a dry powder, which facilitates its handling and use in food. Specifically, the present invention relates to the process of making astaxanthin microcapsules, with a chitosan matrix crosslinked with glutaraldehyde. BACKGROUND

Carotenoids are a group of pigments widely distributed in nature. They are synthesized by plants, yeasts and some bacteria. There are two groups of carotenoids: carotenes, which are composed only of carbon and hydrogen atoms, and xanthophylls, oxygenated derivatives, to whose group belongs astaxanthin. This xanthophyll is the predominant red pigment in most crustaceans and salmonids. A problem associated with carotenoids is their susceptibility to oxidation by external agents such as oxygen, light and high temperatures. Being highly unsaturated molecules, they are prone to oxidation during food processing and storage. Dried or dehydrated products suffer greater losses of carotenoids, due to the porosity and greater surface area of contact with oxidation promoting agents (Rodríguez-Amaya. Latin American Nutrition Archives. 1999. 49: 385-475). The degradation of these compounds can cause the loss of their nutritional or biological attributes, as well as the formation of colorless products and compounds that contribute with undesirable aromas and flavors. Due to their instability, carotenoids are generally not used in crystalline form but rather in the form of stabilized emulsions, or encapsulated with different matrices.

The encapsulation process consists in developing a polymeric shell or matrix around the carotenoid to protect it from environmental factors. This is one of the aspects in which the utility of the present invention lies. Generally, the simplest encapsulation methods are based on the elaboration of emulsions between the carotenoid (oil phase) and a solution containing the encapsulating material followed by drying. I know it thus obtains a dry product in powder form, in the case of spray drying, or in the form of a solid that can be milled to different particle sizes, as in the case of freeze drying (Desobry et al. Journal of Food Sci. 1997. 62: 1158-1162). For example, Chen and Tang, (J. Agrie. Food Chem. 1998. 46: 2312-2318), made a powdered product of carotenes extracted from carrot pulp. For the above, they emulsified the carotene extract in a gelatin and sucrose substrate, and then spray dried. The disadvantage of the methods that use spray drying is that there are losses of the carotenoid during the process (approximately 11%) and the product obtained does not have very good stability. In addition, the carotenoid can be easily degraded, depending on storage conditions (Desobry et al. Journal of Food Sci. 1997. 62: 1158-1162). Hydrolyzed starch has also been used as an encapsulating matrix of carotenes. For example, a dispersion of β-carotene or lycopene in oil is emulsified in a mixture made from modified starch, sugar and a food grade antioxidant. Subsequently, the emulsion is spray dried to obtain a fine powder for use in food (US5976575). Similarly, powdered or granular products have been produced, containing up to about 2% of astaxanthin extracted from Phaffia rhodozyma yeast, stabilized with gelatin matrix, collagen or gum arabic (Patent EP0628258A1).

Currently, astaxanthin is used in the elaboration of salmon and crustacean aquaculture diets to provide the characteristic orange-red color of these organisms. This is because the cultivated species do not have the natural diet where this pigment is found. In addition, astaxanthin has other biological functions in these organisms, such as: it acts as provitamin A, is associated with the reproduction and development of embryos and with the protection of cells against oxidative effects (Putnam M. Aquaculture and the Environment. European Aquaculture Soc. Special publication. 1991. No. 16. p 245-263).

Although the main use of this pigment is in the aquaculture industry, its use has recently been focused on human consumption, based on the nutritional and physiological properties of this compound. For the above, different preparations have been prepared in the form of powder or emulsions, both astaxanthin free and esterified with fatty acids, obtained from natural sources such as yeasts, algae and crustaceans. For example, JP 10276721 describes the preparation of a food added with astaxanthin and / or esters of astaxanthin for use as a treatment for the prevention of cataracts, or to prevent their progress. Other applications of this pigment, related to human health are: it has been proposed as an anti-inflammatory agent (Patents JP2049091, JP7099924), as an immune system enhancer (Patents WO01 / 24787A1, JP7099924, US6265450), for the prophylactic or therapeutic treatment against infections by H. pylori in the stomach (US6262316), and to treat various symptoms associated with stress (US6265450). Currently there are astaxanthin products (The Astafactor ™, Astacarotene AB ™) made from seaweed flour of the genus Haematococcus. These products are used as a food supplement for humans, for their possible benefits that include: anti-cancer cell effects, tissue protection against photooxidation, anti-inflammatory properties and stimulation of immune system response parameters. There are also studies that propose the use of astaxanthin esters in animals, for example, as a medicine to treat muscle diseases (rhabdomyolysis) in horses (US Pat. No. 6245818), to increase production and decrease the mortality of newborn mammals (pigs, cattle and sheep) (US6054491), as well as to increase egg production and improve the health status of chickens (US5744502).

Two of the main astaxanthin products in the aquaculture market are: Carophyll pink ™ with 5 and 8% astaxanthin respectively. They are made with synthetic astaxanthin, stabilized with antioxidants and encapsulated with a matrix of gelatin and carbohydrates covered with starch. Its main disadvantage is its high cost, as it represents about 15% of the total cost of the diets. In addition, the dissolution of the gelatin at low temperatures is slow, and the storage time of the final product is limited due to the hardening of the gelatin layer.

The present invention describes the use of chitosan as an encapsulating matrix for the stabilization of astaxanthin. Chitosan is a copolymer consisting of 2-N-acetyl-D-glucosamine and 2-D-glucosamine units, linked by β-4-glucosidic bonds. Industrially it is obtained from the deacetylation of chitin. In turn, chitin (N-acetyl-2-amino-2-deoxy-D-glucopyranoside) is a linear polymer of high molecular weight, formed by units of N-acetylglucosamine, which is present in commercial quantities in the exoskeleton of lobsters, crabs and shrimp, which contain between 15-20% chitin. Therefore, crustacean biowastes represent a relatively cheap source of chitosan. Due to the β 1-4 glycosidic bond, the chitosan has a relatively rigid configuration, property that has been the basis for preparing films, gels, fibers and spheres. This biopolymer is currently used in various areas such as biomedical, cosmetic, agricultural, food technology, wastewater treatment, among others. Numerous publications have shown the wide potential of applications of this compound, which include: immobilization of enzymes, as chromatographic support, as adsorbent of metal ions or lipoproteins and in cell cultures.

One of the main applications of chitosan is the development of microcapsules for prolonged release systems of drugs or vaccines. To make such systems, chitosan chains are generally chemically crosslinked. To cite some examples, US6207197 describes the preparation of microcapsules for drug release as a treatment against stomach infection of H. pylori or C. pylori in humans. In this case the microcapsules are made by emulsion and spray drying methods, and include a water insoluble polymer such as ethyl cellulose and an outer layer of chitosan, crosslinked with glutaraldehyde or formaldehyde. The chitosan layer acts as a bioadhesive polymer in the stomach. Also, chitosan has been used in the preparation of semipermeable, water insoluble microcapsules to trap viable cells, ionically cross-linking the amino groups of chitosan, with anionic groups of acids such as polyglutamic or polyaspartic (Patent US4803168). DETAILED DESCRIPTION

The present invention consists of a method for preparing a microencapsulated astaxanthin powder product with a chitosan matrix, so that said pigment is stable towards oxidation by exposure to external agents and to facilitate its handling and use in food. Chitosan microcapsules can be prepared by different methods: emulsion or multiple emulsion with crosslinking, simple or complex coacervation, spray drying and emulsion or multiple emulsion with solvent evaporation. Genta et al. (Chitin Ηandbook. Muzzarelli RAA and Peter M. Eds. European Chitin Soc. 1997. p 391-396) described the latter method for preparing chitosan microcapsules for the release of hydrophilic or hydrophobic drugs in general. Currently, this biopolymer has not been used for microencapsulation of carotenoids. In the present invention, a method similar to that of the previous authors was employed, which consists primarily of the elaboration of a multiple oil / water / oil emulsion to make the astaxanthin microcapsules in chitosan. Subsequently, the aqueous solvents are removed by heat drying under vacuum, and finally the microcapsules are recovered by centrifugation and decantation of the oil phase. This method, although similar, required its adaptations to make it viable for microencapsulation of carotenoids, especially astaxanthin, which are not obvious from the aforementioned documents, but represent the object of the present invention, in which it was proposed to solve the technical problem involved in having a product which will provide stability conditions to this pigment.

The astaxanthin used in the present invention can be of chemical-synthetic origin, or, preferably, come from the extraction of natural sources such as yeasts, algae or crustaceans. In the latter case, the extraction is carried out with organic solvents or with vegetable oils, with already known methodology. In addition, the pigment to be used can be found in three forms: free (without esterification), as in the case of synthetic astaxanthin, esterified with different fatty acids, or in a mixture of the two above, as in the case of algae and crustaceans . In the present invention free astaxanthin, in crystalline form, of synthetic origin (Sigma Chemical, Co.) was used, but the invention is not limited to said pigment source. If the pigment is used as a powder, from any of the aforementioned sources, it is first necessary to solubilize the pigment in an organic solvent such as dichloromethane or chloroform. Any other non-polar solvent, which easily dissolves the pigment, and can evaporate in a temperature range of 50-60 ° C, can also be used to prevent degradation of the pigment at the time of evaporation. The solvent: pigment ratio should be such that the solvent allows it, so that the final product preferably has a high concentration of carotenoid. In addition, it is advantageous to use a raw material that contains high carotenoid content. In the case of synthetic astaxanthin, it contains 98% astaxanthin. The pigment solution thus prepared constitutes the oil phase, to prepare a primary oil / water emulsion. The aqueous phase for preparing the oil / water emulsion consists of a chitosan solution, prepared as follows. First, a non-ionic surfactant, from the group of polysorbates, preferably polysorbate 80, is dissolved in a mixture of acetic acid: methanol. The weight / volume ratio between the surfactant and the solvent mixture is 1.6%. Subsequently, chitosan is added, in a polymer / solvent ratio of 1.6 to 3%. Genta et al. (Chitin Handbook. Muzzarelli RAA and Peter M. Eds. European Chitin Soc. 1997. p 391-396) used a chitosan concentration of 1.6% (w / v) in the same previous aqueous phase, to achieve greater emulsion stability . The lower the concentration of chitosan used, the easier the dissolution of the polymer, and the solution formed is less viscous, which facilitates its handling. In the case of the present invention, the disadvantage of using a low concentration of chitosan is that the polymer shell formed is more fragile, being able to break at the time of forming the emulsion. In addition, the formed chitosan membrane may have a greater diffusion of the pigment to the solvent, which causes losses of the solvent. That is why in the present work a concentration of 3% chitosan is used, to avoid the above problems. Once the two solutions have been prepared, the oil-in-water emulsion is made, in which the dispersed phase is the astaxanthin solution, and the continuous phase is the aqueous chitosan solution. The ratio between the two phases is 1: 2.5 (v / v), but it can be reduced, to increase the concentration of the pigment in the final product, taking care not to compromise the stability of the emulsion.

The multiple oil / water / oil emulsion is prepared by adding the previous emulsion in an oil that can be of vegetable origin such as sunflower, cane or corn, or mineral oil, thermoregulated at 50 ° C, and added a non-ionic emulsifier, as per example sorbitan monolaureate, in a concentration of 1-2% (w / v). The relationship between the two phases may be within the range of 1: 5-1: 9, preferably 1: 9. At the time of preparing the multiple emulsion, the chitosan chain crosslinking agent is added to form the membrane or wall of the microcapsules at the water / oil interface of the emulsion drops (Groboillot et al. Biotechnology and Bioengineering. 1993. 42: 1157-1163). For this purpose, the crosslinking agent that can be used is a dicarboxylic acid such as glutaraldehyde, or a tricarboxylic acid such as citric. Genta et al. (Chitin Handbook. Muzzarelli R. A. A. and Peter M. Eds. European Chitin Soc. 1997. p 391-396) carried out this process in a Nibromixer El equipment, at 50 vibrations / sec / 2h. In our case it is appropriate to use a tissue homogenizer adapted to a laboratory reactor, with a different agitation system, as will be described later in example 1. Subsequently, the aqueous solvents are removed from the suspension of microcapsules obtained in the previous step, by evaporation with heat, under vacuum. Finally, the microcapsules are recovered by centrifugation, decantation of the mineral oil and washing of the microcapsules with petroleum ether to remove oil residues.

An example is presented in order to illustrate the present invention. EXAMPLE 1 Step 1. 48 ml of 2% acetic acid are mixed with 12 ml of methanol. Lg of Polysorbate 80, is perfectly stirred for the complete dissolution of the emulsifier, and finally 1.7 g of chitosan are added. This preparation is left for the necessary time, with mechanical agitation, until all the chitosan has dissolved perfectly. Because as this biopolymer hydrates, the solution becomes increasingly viscous, so additional manual agitation is necessary to complete the dissolution of the chitosan.

Step 2. 200 mg of crystalline astaxanthin powder obtained from Sigma (Sigma Chemical Co.) is dissolved in 20 ml of dichloromethane, in an amber vial, to protect the solution from light. Step 3. An oil-in-water emulsion is prepared. The above solution is dripped slowly over the chitosan solution, as it is stirred at 9500 rpm in an ultraturrax food homogenizer, Janke & Kunkel T25.

Step 4. To 600 ml of mineral oil, 12 g of sorbitan monolaureate are added, stirred, and placed in a 0.8L Janke & Kunkel Ika Labortechnick (Germany) reactor, equipped with a hydraulic jacket and a bath of water circulation, to heat the oil to a temperature of 50 ° C. The oil is stirred at a speed of 100 rpm, provided with the reactor blades, plus 8000 rpm, provided by the ultraturrax homogenizer, which has previously been adapted to the reactor. Once this temperature has been reached, the primary emulsion cited in step 3 is gradually added to the mineral oil. Subsequently, 6 ml of a 50% aqueous solution of glutaraldehyde is slowly added. These conditions are maintained for 5 minutes, stirring of the homogenizer is suspended, and the reaction is allowed to terminate at 50 ° C, stirring at 100 rpm for 1.5 hours. Step 5. The aqueous solvents of the microcapsule suspension are evaporated at 60 ° C, under vacuum, for 6 hours. In this step, the temperature rise from 50 to 60 ° C should be gradual, in a period not less than 30 rnin, so that the stability of the emulsion is not lost (Genta et al. Chitin Handbook. Muzzarelli RAA and Peter M. Eds. European Chitin Soc. 1997. p 391-396).

Step 6. The microcapsule suspension obtained is centrifuged at 1590g for 5 min. The oil is separated by decantation, and the microcapsules are washed 3 times with small portions of petroleum ether to remove oil residues, centrifuging each time, under the conditions already mentioned above, and separating the ether by decantation. Step 7. The microcapsules are dried under vacuum at 45 ° C.

The properties of the microcapsules are as follows: a product is obtained in dust, insoluble in water. The microcapsules have a spherical shape, and size within the range of 5-50 μm, determined by optical microscopy (Cari Zeiss, axiolab model).

Chitosan is part of the natural diet of many aquatic animals, so it is compatible for applications in food for aquaculture. In addition, there are currently numerous chitosan products on the market for human consumption, which are used as dietary fiber, and to trap dietary fat, preventing its absorption by the body. Therefore, together with the nutritional and biological properties of astaxanthin, described above, the present invention proposes the use of chitosan to microencapsulate astaxanthin, to obtain a new product, whose potential for use in human and animal nutrition is very wide, as It has already been described in the state of the art.

Recently, astaxanthin is becoming very important as a food supplement for humans, thanks to its antioxidant properties, considered far superior to those of vitamin E (Miki W. Puré & Appl. Chem. 1991. 63: 141-146). With the present invention a product for human consumption, made with natural astaxanthin and other food grade ingredients, can be obtained. In such a case, chitosan is not hydrolyzed by digestive enzymes, but under conditions of acidic stomach pH, this biopolymer expands its volume without being digested. The previous condition allows astaxanthin not to degrade in the acidic environment of the stomach, which may represent greater bioavailability of the pigment at the intestinal level, once released from the expanded microcapsules.

Having described the method of preparation and the product obtained as above, we claim from our property what is stated in the following claims.

Claims

1. A process for the manufacture of a microencapsulated powder product, containing a carotenoid as an active ingredient, and a chitosan matrix crosslinked with glutaraldehyde, comprising the steps of: 1.1 Preparation of an oil-in-water emulsion by homogenizing the carotenoid dissolved in an organic solvent, in an aqueous solution of chitosan, which contains a non-ionic emulsifier.

1.2 Preparation of an oil / water / oil emulsion, by homogenizing the emulsion obtained in 1.1, in an oil added to an emulsifier. In this step, a chemical agent capable of cross-linking the chitosan polymer chains is subsequently added, stirring the mixture at an appropriate speed, at a temperature and for a period of time to obtain a suspension of microcapsules in oil.

1.3 Evaporation with heat, under reduced pressure, of the aqueous solvents of the microcapsule suspension obtained in the previous step.

1.4 Recovery of the microcapsules by centrifugation and decantation of the mineral oil.

2. Process according to claim 1, characterized in that the concentration of the chitosan solution is in a range of 1.6 to 3%, preferably 3%.

3. Process according to claim 1, wherein the stirring speed to obtain the oil / water / oil emulsion is in two stages.

4. Process according to claim 3, wherein the stirring speed in the first stage is 100 rpm provided by the reactor and 8000 rpm provided by the homogenizer together, during the first 5 minutes.

5. Process according to claim 3, wherein the stirring speed in the second stage is 100 rpm for an additional time of 1 to 1.5h.

6. A microcapsule powder product, which contains a carotenoid as an active ingredient, and a chemically cross-linked matrix or chitosan shell.

7. The product according to claim 6, characterized in that it contains at least 2% by weight of the carotenoid, and a maximum of 8%.

8. The product according to claim 6, characterized in that the carotenoid is non-esterified astaxanthin, or astaxanthin esterified with fatty acids.

9. The product described in claim 6, whose diameter of the microcapsules is in the range of 5-50 μm.

10. Carotenoid microcapsule product obtained from the process of claims 1 to 5.

11. Use of the product of claims 6 to 10 for the manufacture of a food supplement.

12. Use according to claim 11 wherein the nutritional supplement is useful in the treatment of cataracts.

13. Use according to claim 11 wherein the food supplement is useful in the treatment of inflammatory diseases.

14. Use according to claim 11 wherein the food supplement is useful as an immune system enhancer.

15. Use according to claim 11 wherein the food supplement is useful as an antioxidant, to protect the tissues from oxidation.

16. Use according to claim 11 wherein the food supplement is useful as a treatment against H. pylori infections in the stomach.

17. Use according to claim 11 wherein the nutritional supplement is useful in the treatment of muscular diseases in horses.

18. Use according to claim 11 wherein the nutritional supplement is useful for increasing production and reducing mortality of mammals (pigs, cattle and sheep) newborns.

19. Use according to claim 11 wherein the nutritional supplement is useful for increasing egg production and improving the health status of hens.

20. Use according to claim 11, wherein the food supplement is useful as an ingredient in diets, to provide the characteristic orange-red color of salmonids and crustaceans.