Abstract:
The invention relates to a method for the production of a water-dispersible formulation containing carotenoids. The β-carotene was dissolved in n-isobutyl acetate and the hot solvent phase was dispersed in a modified water starch solution. The isobutyl acetate was then evaporated. The solvent traces were removed by water vapor distillation at low pressure. The dispersion as then dried. The β-carotene thus obtained is used as colorant, particularly in the food industry. It has improved characteristics in terms of stability, solubility, color transfer to polar and apolar media and possibility of compressing into tablets with reduced porosity.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of Invention  
         [0002]     The present invention relates to health granulates containing carotenoids and a process for the production thereof. More particularly, the current invention relates to encapsulated carotenoids, especially β-carotene, obtained by utilising various kinds of nutritional starches and formulated in granules excellent in handling properties, compressibility, colour release and appearance, and useful in colouring beverages, formulation of solids and semisolids pharmaceutical preparation and promoting fish growth.  
         [0003]     2. Prior Art  
         [0004]     It is public knowledge in the art that natural β-carotene is utilised in several forms as natural colouring agents for food, cosmetics, fish feed and the like as additives for health food and as a material for regulating vital functions such as a nourishing diet supplement. However, no practical proposal had been made concerning the method of fully health-oriented granulate, i.e., without halogenated solvents or animal proteins.  
         [0005]     Among these carotenoids, β-carotene, one of provitamin A group compounds, is easily oxidizable because it contains a conjugated double bond chains and also a poorly water-soluble compound. These are serious problems in the food industry and especially in the beverage manufacture in which drinks are almost a hundred percent water-based products. Furthermore demanding on health food, mainly from natural sources, is one of the main tasks of Food and Drug Administrations. It has therefore been strongly desired to develop a method by which the carotenoids can be processed without destroying the natural carotene, and the product obtained can be also be preserved in a stable state and in soluble form.  
         [0006]     Solubility problem was mentioned firstly in U.S. Pat. No. 3,998,753 and solved by preparing carotenoids solutions in volatile organic solvents such halogenated hydrocarbons as and emulsifying with a water solution of sodium lauril sulphate. A similar description as found in EP937412 by using a continuous process. These processes, however, involved the use of chloride hydrocarbons, which are well known as toxic products with a limited use in pharmaceutical and food industries, but also environmental risks are derived from the residual solvents.  
         [0007]     A proposal to eliminate these solvents was found in ES2022470, in this carotenoids are added as water dispersable powder, in this process carotenoids were solubilized previously in a food-quality oil. In this formulation the carotenoids is formulated by adding numerous ingredients (stabilisers, emulsifiers, etc.). Thus, this was not a natural product as is demanded by consumers. U.S. Pat. No. 5,364,563 describes a process for producing a powdered carotenoid preparation comprising forming a suspension of a carotenoid in high boiling oil without the presence of an organic solvent. The suspension is superheated with a steam for a maximum period of thirty seconds to form a solution of carotenoid in oil. After that, solution is emulsified in an aqueous solution of a colloid and thereafter spraying and drying the emulsion a powder is formed. However, great losses of carotenoids at the high temperatures used during the treatment with superheated steam and also some isomerizations occur. WO93/04598 proposed the use of natural additives, such as, stabilisers, acidifiers and emulsifiers. U.S. Pat. No. 5,714,658 described the use of a mixture of different solvents including esters of acetic acid, alcohol C1-C4 and oil compatibles with the other solvents. In this process, two problems are involved: firstly, the low solubility of carotenoids in the mixture of solvents, which implies a large amount of oil and solvents required, especially if a high concentration of carotenoids is demanded, and secondly the environmental risks derived from the residual solvents. In EP807431 is described a procedure to obtain a water solution of carotenoids with a protective colloid, at high temperature (180-250° C.) and pressure (96.6-2759 bar) a emulsion is obtained which after drying yield the carotenoid in form of dry powder. However, the equipments and conditions required result in a very expensive process. U.S. Pat. No. 5,780,056 with priority number JP1996000141034 describes microcapsules which are made of a core material comprising natural carotenoid and an edible oil, and a wall which is made of a coating material based on gelatin, the particles have a mean particle size of 0.01-5 μm. The microcapsule has a strength enough to protect natural carotenoid from oxidation and deterioration for a long time and to withstand tableting pressure. The colloid generally used is gelatin originating from warm-blooded animals and such origin is often problematic. For example, preparations based on such gelatin can not be used worldwide for religious reasons.  
         [0008]     In accordance with U.S. Pat. No. 5,478,569 all of these disadvantages can be eliminated when fish gelatin is used in place of gelatin from warm-blooded animals. Also without an expensive manufacturing process of this gelatin, the resulting manufactured preparations do not always have a desired dispersibility in cold water, etc. Furthermore, problems derived by using colloids as gelatine and collagen from animal source are related to the capability of these proteins to be carrier of infections and more particularly of the crazy cow illness.  
         [0009]     In order to obtain greatest benefit from the carotenoids, the particles should either dissolve rapidly or be sufficiently wettable to be easily absorbed. As above-described the first strategy was to manufacture microcapsules as U.S. Pat. No. 5,478,569 and U.S. Pat. No. 5,780,056. However, if a large amount of edible oil is contained for dispersing natural carotenoid, the resulting microcapsules are reduced in strength and the stabilizing effect of gelatin against oxidation as a protective coating is not fully exerted. Natural carotenoid is then vulnerable to oxidation and deterioration. Low microcapsule strength has another problem. When microcapsules are blended with a vehicle and compressed into tablets, the microcapsules can be broken during tableting, allowing carotenoid to leak out. It is thus difficult to blend carotenoid in tablets in a stable manner.  
         [0010]     The second strategy U.S. Pat. No. 3,907,983 could be spray-coated from aqueous solutions with from about 0.5% to about 10% by weight of a hydrophilising material selected from the group consisting of mtehylcellulose, hydroxyethylmethylcellulose and hydroxypropylmethylcellulose. The limitation consists that procedure by which the hydrophilising material is applied to the surface of the individual particles must be carried out in a manner which insures that the particles do not become agglomerated.  
         [0011]     The last strategy deals with granulation process. Thus, U.S. Pat. No. 4,915,966 (priority numbers JP1988000040040754 and JP 1988000040755) granulated dried powder of Dunaliella algae, which is rich in β-carotene, with other materials to make a granulation. The way to process without destroying the natural β-carotene, and preserved in a stable state was by adding cyclodextrin to dried powder of Dunaliella algae in a certain ratio. Granulation was encapsulated in a hard capsule. β-cyclodextrins was used also in WO96/40262 for stabilising β-carotene in a water soluble organic solvent. Additional important improvement, also recently found in EP 8325692A2, was the use of only organic solvent without halogens and in a small extent.  
         [0012]     The present inventors actively investigated possible solutions for the aforementioned problems, especially those related to use of colloids from animal sources. As a result of such investigation, they have completed the present invention by the development of a process in which the natural β-carotene is preserved in a stable state by food-grade starch matrix. Only U.S. Pat. No. 3,790,688 was a previous attempt to formulate β-carotene by using starch, but in this case a precipitate by solvent addition was done instead the emulsion of the process hereby described. The granulation makes the natural β-carotene hydrophilic, dispersible and bioavailable, and therefore acceptable as nutritional supplement in foods. The resulted powder is free flowing as well as amenable to tabletting by direct compression or encapsulated in a hard-capsule are disclosed as well as a method for making such granulation and formulations.  
       SUMMARY OF THE INVENTION  
       [0013]     It is an object of the present to provide water-dispersible carotenoids formulation containing wherein from about 1 to 25 parts by weight of dried powder of carotenoid encapsulated in a matrix of food-quality starch which provides β-carotene included in the encapsulated powder in a stable condition. The formulations containing natural carotenoids having satisfactory physiological activity to the human, body and a high strength enough to prevent the carotenoid from being oxidized or deteriorated during long-term storage. The formulations are suitable to be blended in capsules or tablets or the like in a stable manner, and in such applications as additive agents for foods and pharmaceuticals.  
         [0014]     It is another object of this invention to provide a process for producing the formulation which comprises the following steps: adding β-carotene to mixture of natural or synthetic tocopherols in vegetable oil and dissolving in n-butil acetate or iso-butyl acetate, dissolving a mixture of two different starches in desionized water, preparing a emulsion by admixing both phases and eliminating organic solvent under temperatures between 50-80° and reduced pressure until 15-25% of dry matter is obtained, drying to obtain final product.  
         [0015]     In accordance with the present invention it has been found that both the spray-drying method and, especially fluidised bed method are suitable.  
         [0016]     According to the spray-drying method, the inlet temperature was between 100-190° C., while outlet temperature was 80-150° C.  
         [0017]     According to the fluidised-bed method. The seed material, i.e. typical inert material as sugar particles, as well as dust or fine powder obtained from previous granulation processes or from spray-dried process are placed in a fluidized-bed granulator. Particles are maintained in motion by means of air, the input and output temperature of which is adjusted according between 30-90° C. A solution of the liquid formulation (15-25% of dry matter) is sprayed into the drier at a rate, which ensures that the particles to be coated do not become too damp and conglomerate. After completion of the spraying, the particles can be coated.  
         [0018]     The invention is also concerning with the spray-coating from about 0.5% to about 10% by weight aqueous contained sugar solutions or from of a hydrophilising material selected, i.e. methylcellulose, hydroxyethyl-methylcellulose and hydroxypropylmethylcellulose. Particles can be dried directly by a further input of suitably pre-warmed air.  
         [0019]     It is a further object of the invention to provide a hard capsule containing granulate which comprises the following, step: blending dried powder of granulate with fillers and/or lubricants and encapsulating in the light-impermeable hard capsules, thereby keeping the β-carotene included in the encapsulated form in a stable condition.  
         [0020]     The invention is also concerned with pharmaceutical preparations in a powdered form or in the form of tablets containing carotenoid granulation in combination with conventional pharmaceutically acceptable tabletting adjuvants and/or excipients. Suitable pharmaceutically acceptable tabletting adjuvants and excipients are micro-crystalline cellulose, dextrose, lactose, sucrose, mannitol, glucose, sorbitol; lubricants such as calcium stearate, stearic acid or magnesium stearate as well as mixtures thereof. Talc, cornstarch etc. can be mixed with the lubricants. Flavoring agents and coloring agents can also be used. The process for producing tablets containing β-carotene comprises the following steps: blending and/or granulating the watter-dispersible formulation with adjuvants and/or excipients and tabletting in an eccentric or rotary tablet press at applied pressure between 30 and 200 MPa. The β-carotene remained in the starch matrix after compression in a stable condition and release β-carotene in a very short time.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0021]     The products of the present invention are water-dispersible solid formulations of β-carotene. The term water dispersible means that the formulations of the present invention shows UV-visible spectrum characteristic of β-carotene after dissolution and filtration. Because of the interaction of light with very small particles crystals appear the more yellow the smaller they become. β-carotene crystals of about 3-5 μm size are tomato red. Color changes for orange if particle size is about 1 μm and the dispersion appear yellow if the particle diameter becomes as small as about 0.6 μm. This effect is caused by shifts in the absorption spectrum of light.  
         [0000]     β-Carotene  
         [0022]     β-carotene is a naturally occurring precursor of Vitamin A and is often used as an important colour for the beverage industry. β-carotene is typically derived by extraction from fermentation sources or synthesised using known chemical process. However, β-carotene is easily degraded when subjected to air, light or temperatures. Thus, β-carotene is stabilised and available in this form from, for example, BASF Corporation or Hoffman LaRoche. In the present invention, it is preferred to use β-carotene crystals.  
         [0023]     The β-carotene is dissolved in an organic solvent. The β-carotene have been suspended in Iso-Butyl Acetate (IBA). The suspension has been milled in a ball mill (Netzch minizetal) at 1500 rpm for about 10-15 minutes. The viscous paste has been transferred into warm and continuously stirred IBA at 60° C. The IBA suspension has been rapidly heated to 105° C. The temperature was hold for 5 min. Afterwards the solution of β-carotene is formed and cool down to 85-90° C.  
         [0024]     The composition of the present invention where from about 1 to 25 part of β-carotene by weight of dried powder, the remainder being OSA-starches.  
         [0000]     Starches  
         [0025]     The starches applied are Purity Gum 2000, modified food starch from National Starch &amp; Chemical, this permits easy emulsification of the organic phase in water. After solvent removal the dispersion meets the color shade of β-carotene. This modified food starch does not give enough redispersability of dried powder. Thus, another starch is also added.  
         [0026]     The second variety of food grade starch is Hi-Cap 100 (National Starch &amp; Chemical), this provides a good dispersability of the final product.  
         [0000]     Additional Ingredients  
         [0027]     Because β-carotene is sensible to oxidation, antioxidants may be dissolved in the solvent containing the β-carotene to enhance the stability against deterioration. Any antioxidant approved for food may be used in the present invention, including but not limited to a-tocopherol from natural or synthetic source. The level of antioxidant would be sufficient for β-carotene protection. This should be from 0.1 to 0.3 times the amount of β-carotene. Ascorbyl palmitate can be also added to the formulation due to the synergistic antioxidative effect of the combination of both antioxidants.  
         [0000]     Method of Preparation  
         [0028]     After dissolving β-carotene in Iso Butyl Acetate the next step is the emulsification. In this, the solution of β-carotene with antioxidants in the mentioned solvent at 85-90° C. is poured into the starch solution in water. The dark brown solution has a temperature below 50° C. and has immediately afterwards homogeneized by a rotor-stator system (IKA MF45) for 10 min. The emulsion became dark orange within the first minute.  
         [0029]     The solvent evaporation is at 40-45° C. and reduced pressure (approx. 65 mbar) within 30 minutes. Solvent residues is removed with the water evaporated during several hours.  
         [0030]     For obtaining the solid formulation the preferred method is the fluidized-bed. In this, the seed material, i.e. typical inert material as sugar particles, as well as dust or fine powder obtained from previous granulation processes or from spray-dried process are placed in a fluidized-bed granulator. Particles are maintained in motion by means of air, the input and output temperature of which is adjusted according between 30-90° C. A solution of the liquid formulation (15-25% of dry matter) is sprayed into the drier at a rate, which ensures that the particles to be coated do not become too damp and conglomerate.  
       EXAMPLE 1  
       [0031]     20 g of crystalline β-carotene have been suspended together with 3 g of D-tocopherol extract (Nutrilo) in 800 mL n-butyl acetate. The suspension has been refluxed for 15 min at 130° C. A mixture of 90 g Hi-Cap 100 and 40 g Purity Gum 2000 (National Starch) has been dissolved in 650 g of cation exchange water. The hot organic phase was emulsified in one step in the aqueous phase using an Ultraturrax (IKA). The dispersion has been diluted with 2 L of water. Together with 800 mL of n-butyl acetate these two liter of water has been evaporated from 5 mL flask of a rotatory Evaporator (Buchi). The flask has been heated to about 80° C. and the pressure gradient was as rapid as possible from 1024 mbar down to 60 mbar. Three times 1 L of water has been added and again evaporated to remove all the traces of solvent. 776 g of dispersion (19.1% of dry mass) with a β-carotene content of 2.4% in solution (12.7% β-carotene referred to dry mass) is obtained. The dispersion is granulated and dried in fluidized bed (UniGlatt) of sugar crystals. A free-flowing powder water-wettable and very fast dispersible was obtained.  
                                                       TABLE 1                           Ingredients of β-carotene formulation.                Dry mass (%)   Typical quantity   Ingredients                            58.8%   90   g   Hi-Cap 100           26.1%   40   g   Purity Gum 2000           13.1%   20   g   Crystalline β-carotene           2.0%   3   g   Em 70 (nutrilo)               650   g   Cation exchanged water               800   mL   n-butyl acetate                      
 
       EXAMPLE 2  
       [0032]     396 g of crystalline β-carotene have been suspended together with 3.96 g of synthetic a-tocopherol (Roche) in 800 mL isobutyl acetate. The suspension has been milled in a Netzsch Mini-Zeta (Glass beads, 0.8-1.2 mm) at 1500 rpm for about 10-15 min. The viscous paste has completely transferred into warm and continuously stirred Iso-Butyl Acetate (60° C.). The total amount of Iso-Butyl Acetate was 15840 g. The IBA suspension has been rapidly heated to 105° C. The temperature was hold for 5 min. Afterwards the solution of β-carotene is formed and cool down to 85-90° C. within the next 10 min. A mixture of 1287 g Hi-Cap 100 and 1287 g Puritv Gum 2000 (National Starch) has been dissolved in 12870 g of cation exchange water. The hot organic phase was emulsified in one step in the aqueous phase using a rotor-stator system (IKA MF45). The dispersion has been diluted with water and Iso-butyl acetate has been rapidly evaporated within 30 min. at 40-45° C. at 65 mbar. Solvent residues have been removed with the water evaporated during several hours. The dispersion is granulated and dried in continuous fluidized bed granulator AGT 150 (Glatt) without seed material. A free-flowing powder water-wettable and very fast dispersible was obtained.  
                                                       TABLE 2                           Ingredients of β-carotene formulation.                Dry mass (%)   Typical quantity   Ingredients                            42.76%   1287   g   Hi-Cap 100           42.76%   1287   g   Purity Gum 2000           13.16%   396   g   Crystalline β-carotene           1.31%   39.6   g   Synthetic α-tocopherol                       (Roche)               12870   g   Cation exchanged water               15840   Ml   iso-butyl acetate