Abstract:
The present application discloses microbially-stable solutions of water-solubilized lipophilic vitamins, such as Vitamin E. These solutions contain from about 0.5% to about 30% of a salt selected from potassium chloride, magnesium chloride, calcium chloride, potassium phosphate, calcium phosphate, potassium sulfate, calcium phosphate, magnesium phosphate, magnesium sulfate, potassium iodide, and mixtures of these salts. The method of providing microbially-stable solutions of such water-solubilized lipophilic vitamins, as well as food and beverage products containing those microbially-stabilized water-solubilized lipophilic vitamins, are also disclosed.

Description:
This application relates to and claims priority from U.S. Provisional Application Ser. No. 61/230,148, filed Jul. 31, 2009, incorporated herein by reference. 
    
    
     TECHNICAL FIELD OF THE INVENTION 
     The present invention relates to microbially-stable vitamin solutions, particularly Vitamin E solutions that can be used in food and beverage products. 
     BACKGROUND OF THE INVENTION 
     When formulating food or beverage compositions, it is frequently useful to add vitamins to round out the nutritional value of the product. Vitamins are either water-soluble (hydrophilic) or fat-soluble (lipophilic). It is a particular challenge to formulate fat-soluble vitamins (such as vitamins A, D, E or K) into aqueous-based products, such as health beverages. 
     One known approach for formulating lipophilic vitamins into aqueous solutions is to add hydrophilic side chains onto the lipophilic base molecule. By doing that, the hydrophilic-lipophilic balance (HLB) of the entire molecule can be adjusted toward water-solubility, making the molecule water-soluble. An example of such a water-soluble variant of Vitamin E (Tocopherol) is Vitamin E succinate polyethylene glycol (PEG) 1000 (also referred to as Vitamin E TPGS) which has the following formula: 
     
       
                 
         
             
             
         
      
     
     Additional water-soluble variants of Vitamin E can be made by substituting other dicarboxylic acids (such as carbonic acid, oxalic acid, malonic acid, glutaric acid, adipic acid, phthalic acid, glycolic acid, lactic acid, mendelic acid, citric acid and mixtures thereof) for the succinic acid. In addition, the polyethylene glycol component can be adjusted such that it contains from about 200 to about 2000 (preferably about 1000) subunits. Also, polyether materials other than PEG, such as polypropylene glycol containing from about 200 to about 2000 (preferably about 1000) subunits can be used. Further, the PEG and PPG compounds can be provided as monolaurate, monomyristate, monopalmitate or monostearate derivatives. Specific commercially-available water-soluble Vitamin E materials include Vitamin E succinate polyethylene glycol (PEG) 1000. 
     While these materials can be placed into aqueous solution, they do potentially raise microbial contamination issues. Specifically, upon storage, they can provide an ideal medium for the growth of yeast, bacteria and mold. That contamination issue must be addressed before the materials can be used in food and beverage products. One way to address the issue is to pack the materials aseptically. The additional processing required to do that makes such packaging very expensive. Another way to address the issue is to place broad spectrum preservatives in the solution. While those may be effective, it is preferable not to include preservatives in one component of a finished product, since they might not be optimal for use in the finished product or there might be a desire to formulate the finished product without preservatives. A third way to address this issue is to maintain the pH of the Vitamin E solution very low (i.e., between about 2 and about 4) This, however, causes acid hydrolysis of the Vitamin E, resulting in vitamin loss. 
     SUMMARY OF THE INVENTION 
     The present invention provides for aqueous solutions which comprise from about 0.5% to about 30% (by weight) of a water-solubilized derivative of a fat-soluble vitamin selected from Vitamins A, D, E and K (particularly Vitamin E), and from about 0.5% to about 30% (by weight) of a salt selected from KCl, MgCl 2 , CaCl 2 , K 3 PO 4 , Ca 3 (PO 4 ) 2 , K 2 SO 4 , CaSO 4 , Mg 3 (PO 4 ) 2 , MgSO 4 , KI, and mixtures thereof (particularly KCl or KI). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides a simple, effective and inexpensive way to address this microbial stability issue. This is accomplished by placing an anti-microbially effective amount of potassium chloride (KCl) into the aqueous solution containing the solubilized Vitamin E material. Other salts may be used in place of KCl, as long as they are relatively neutral in pH (pH of from about 6 to about 8, preferably about 7) in solution since, if they are outside this range, they might hydrolyze the ether or ester bonds in the hydrophilic side chain, thereby rendering the vitamin insoluble or ineffective. Examples of such salts that may be used in place of KCl include magnesium chloride (MgCl 2 ), calcium chloride (CaCl 2 ), potassium phosphate (K 3 PO 4 ), calcium phosphate (Ca 3 (PO 4 ) 2 ), potassium sulfate (K 2 SO 4 ), calcium sulfate (CaSO 4 ), magnesium phosphate (Mg 3 (PO 4 ) 2 ), magnesium sulfate (MgSO 4 ), potassium iodide (KI), or combinations of these materials. In addition to microbially stabilizing the soluble Vitamin E component, the salt can serve as a source of minerals (e.g., potassium, magnesium, calcium) if the material is used in a food or beverage product. While sodium salts can be used herein from an efficacy point of view, such salts are generally avoided because of the health issues (e.g., hypertension) associated with sodium in the diet. 
     Generally, the aqueous solutions of the present invention will include a concentration of from about 0.5% to about 30% (by weight) of the solubilized Vitamin E component. One embodiment of the solution contains about 20% by weight of the solubilized Vitamin E component. Although inclusion of the solubilized Vitamin E is generally limited by its critical micelle concentration (CMC), the presence of the salt may salt out the micelles formed, even above the CMC, making the solution less viscous and allowing for the use of higher Vitamin E concentrations in the solution. The salt is generally used in the solution at a level of from about 0.5% to about 30% (by weight); in one embodiment, the salt is used at from about 5% to about 20% (by weight), and in another embodiment, the salt is used at from about 10% to about 15% (by weight). 
     Water-solubilized derivatives of fat-soluble vitamins are known in the art. They can be formulated by adding hydrophilic side chains onto the lipophilic base molecule. By doing that, the hydrophilic-lipophilic balance (HLB) of the entire molecule can be adjusted toward water-solubility, making the molecule water-soluble. An example of such a water-soluble variant of Vitamin E (Tocopherol) is Vitamin E succinate polyethylene glycol (PEG) 1000, which has the following formula: 
     
       
                 
         
             
             
         
      
     
     This material is also known as Vitamin E TPGS. Additional water-soluble variants of Vitamin E can be made by substituting other dicarboxylic acids (such as carbonic acid, oxalic acid, malonic acid, glutaric acid, adipic acid, phthalic acid, glycolic acid, lactic acid, mendelic acid, and mixtures thereof) for the succinic acid. In addition, the polyethylene glycol component can be adjusted such that it contains from about 200 to about 2000 (preferably about 1000) subunits. Also, polyether materials other than PEG, such as polypropylene glycol (PPG) containing from about 200 to about 2000 (preferably about 1000) subunits can be used. Further, the PEG and PPG compounds can be provided as monolaurate, monomyristate, monopalmitate or monostearate derivatives. Specific commercially-available water-soluble Vitamin E materials include Vitamin E succinate polyethylene glycol (PEG)1000. A similar approach can be taken to prepare water-soluble versions of other lipophilic vitamins, including Vitamins A, D and K. 
     Other materials frequently and conventionally found in vitamin and nutritional supplement solutions that can support microbial action can be included, at their art-established levels, in the solutions of the present invention. 
     One embodiment of the present invention is an aqueous solution which contains about 20% (by weight) Vitamin E succinate-PEG1000, and about 15% (by weight) potassium chloride. The product is clear, has good viscosity and inhibits the growth of mold, yeast and bacteria for at least 24 months. Each gram of this solution delivers to the user 2.5-3.0 RDI of d-alpha-tocopherol and 0.022 RDI of potassium. 
     This technology may be used with fat-soluble vitamins other than Vitamin E, specifically with Vitamins A, D and K. 
     The vitamin solutions defined herein may be added, in nutritionally effective levels, to food and beverage products to provide supplementation of those products. Conventionally known food and beverage products may be used. 
     The following non-limiting examples are illustrative of the present invention. 
     Example 1 
     Control 
     A solution is prepared by utilizing 800 grams of de-ionized water to which 200 grams of chipped Vitamin E TPGS is added. The mixture is warmed to a temperature of 37° C. and mixed until all of the Vitamin E has dissolved and a uniform solution is produced. Samples of this mixture are then assessed for microbial susceptibility by subjecting the solution to a 3 week micro challenge utilizing  Candida albicans, Aspergillus brasiliensis, Escherichia coli  and  Staphylococcus aureus . Results show that organisms can proliferate in the unpreserved Vitamin E solution. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 CHALLENGE STUDY RESULTS 
               
               
                 TEMPERATURE = AMBIENT (20-25° C.) 
               
             
          
           
               
                   
                   
                 INITIAL COUNT 
                   
               
               
                   
                 INOCULUM 
                 CFU/gm 
               
               
                 INOCULATING 
                 LEVEL (CFU/gm 
                 (prior to inoculation) 
                 TIME INTERVAL 
               
             
          
           
               
                 ORGANISM(S) 
                 of sample) 
                 APC 
                 Yeast 
                 Mold 
                 Week O 
                 Week 1 
                 Week 2 
                 Week 3 
               
               
                   
               
               
                 
                   Candida albicans 
                 
                 3.4 × 10 5   
                 660 
                 30 
                 60 
                 8.2 × 10 5   
                 8.6 × 10 5   
                 1.2 × 10 6   
                 9.6 × 10 5   
               
               
                 ATCC 10231 
               
               
                 
                   Aspergillus 
                 
                 1.0 × 10 5   
                   
                   
                   
                 2.8 × 10 5   
                 3.4 × 10 5   
                 2.6 × 10 5   
                 2.3 × 10 5   
               
               
                 
                   brasiliensis 
                 
               
               
                 ATCC 16404 
               
               
                 
                   Escherichia coli 
                 
                 4.3 × 10 6   
                   
                   
                   
                 3.1 × 10 6   
                 1.6 × 10 7   
                 8.0 × 10 6   
                 5.9 × 10 6   
               
               
                 ATCC 9896 
               
               
                 
                   Staphylococcus 
                 
                 6.5 × 10 6   
                   
                   
                   
                 1.3 × 10 7   
                 1.2 × 10 6   
                 2.7 × 10 5   
                 1.7 × 10 6   
               
               
                 
                   aureus 
                 
               
               
                 ATCC 6538 
               
             
          
           
               
                 APPEARANCE 
                   
                   
                   
                 Normal 
                 
                   Aspergillus 
                 
                 
                   Aspergillus 
                 
                 
                   Aspergillus 
                 
               
               
                   
                   
                   
                   
                   
                 
                   brasiliensis 
                 
                 
                   brasiliensis 
                 
                 
                   brasiliensis 
                 
               
               
                   
                   
                   
                   
                   
                 visible 
                 visible 
                 visible 
               
               
                   
                   
                   
                   
                   
                 mold growth 
                 mold growth 
                 mold growth 
               
               
                   
                   
                   
                   
                   
                   
                   
                   S. aureus : turbid 
               
               
                   
               
             
          
         
       
     
     Example 2 
     In a second test, a solution is prepared by utilizing 700 grams of de-ionized water to which 100 grams of potassium chloride is added and solubilized. The mixture is warmed to a temperature of 37° C. To this, 200 grams of chipped Vitamin E TPGS is added. The mixture is mixed until all of the Vitamin E has dissolved and a uniform solution is produced. Samples of this mixture are then assessed for microbial susceptibility by subjecting the solution to a 12 week micro challenge utilizing  Candida albicans, Aspergillus brasiliensis, Escherichia coli  and  Staphylococcus aureus . Results show control of the tested organisms, thereby demonstrating hostility. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 CHALLENGE STUDY RESULTS 
               
               
                 TEMPERATURE = AMBIENT (20-25° C.) 
               
             
          
           
               
                   
                   
                 INITIAL COUNT 
                   
               
               
                   
                 INOCULUM 
                 CFU/gm 
               
               
                 INOCULATING 
                 LEVEL (CFU/gm 
                 (prior to inoculation) 
                 TIME INTERVAL 
               
             
          
           
               
                 ORGANISM(S) 
                 of sample) 
                 APC 
                 Yeast 
                 Mold 
                 Week O 
                 Week 1 
                 Week 2 
                 Week 3 
                 Week 4 
                 Week 8 
                 Week 12 
               
               
                   
               
               
                 
                   Candida albicans 
                 
                 2.2 × 10 5   
                 &lt;10 
                 &lt;10 
                 &lt;10 
                 2.7 × 10 5   
                 7.0 × 10 4   
                 1.2 × 10 5   
                 9.4 × 10 4   
                 5.8 × 10 4   
                 6.0 × 10 5   
                 5.1 × 10 4   
               
               
                 ATCC 10231 
               
               
                 
                   Aspergillus 
                 
                 3.0 × 10 5   
                   
                   
                   
                 4.4 × 10 5   
                 4.0 × 10 5   
                 8.6 × 10 5   
                 7.4 × 10 5   
                 6.8 × 10 5   
                 4.6 × 10 4   
                 7.4 × 10 4   
               
               
                 
                   brasiliensis 
                 
               
               
                 ATCC 16404 
               
               
                 
                   Escherichia coli 
                 
                 1.1 × 10 7   
                   
                   
                   
                 1.0 × 10 7   
                 2.2 × 10 4   
                 2.8 × 10 3   
                 &lt;10 
                 &lt;10 
                 &lt;10 
                 &lt;10 
               
               
                 ATCC 8739 
               
               
                 
                   Staphylococcus 
                 
                 2.0 × 10 7   
                   
                   
                   
                 1.3 × 10 7   
                 1.7 × 10 6   
                 4.9 × 10 3   
                  20 
                 &lt;10 
                 &lt;10 
                 &lt;10 
               
               
                 
                   aureus 
                 
               
               
                 ATCC 6538 
               
             
          
           
               
                 APPEARANCE 
                   
                   
                   
                 Normal 
                 Normal 
                 Normal 
                 Normal 
                 Normal 
                 Normal 
                 
                   A. brasiliensis 
                 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 growth settled 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 to bottom of 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 tube. All 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 others normal. 
               
               
                   
               
             
          
         
       
     
     Example 3 
     In a third test, a solution is prepared by utilizing 650 grams of de-ionized water to which 150 grams of potassium chloride is added and solubilized. The mixture is warmed to a temperature of 40° C. To this, 200 grams of chipped Vitamin E TPGS is added. The mixture is mixed until all of the Vitamin E has dissolved and a uniform solution is produced. Samples of this mixture are then assessed for microbial susceptibility by subjecting the solution to a 12 week micro challenge utilizing  Candida albicans, Aspergillus brasiliensis, Escherichia coli  and  Staphylococcus aureus . Results show good control of the tested organisms, thereby demonstrating hostility. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 CHALLENGE STUDY RESULTS 
               
               
                 TEMPERATURE = AMBIENT (20-25° C.) 
               
             
          
           
               
                   
                   
                 INITIAL COUNT 
                   
               
               
                   
                 INOCULUM 
                 CFU/gm 
               
               
                 INOCULATING 
                 LEVEL (CFU/gm 
                 (prior to inoculation) 
                 TIME INTERVAL 
               
             
          
           
               
                 ORGANISM(S) 
                 of sample) 
                 APC 
                 Yeast 
                 Mold 
                 Week O 
                 Week 1 
                 Week 2 
                 Week 3 
                 Week 4 
                 Week 8 
                 Week 12 
               
               
                   
               
               
                 
                   Candida albicans 
                 
                 1.0 × 10 6   
                 20 
                 &lt;10 
                 &lt;10 
                 6.0 × 10 5   
                 8.6 × 10 4   
                 1.6 × 10 3   
                 1.0 × 10 2   
                 1.0 × 10 2   
                 &lt;10 
                 &lt;10 
               
               
                 ATCC 10231 
               
               
                 
                   Aspergillus 
                 
                 6.6 × 10 4   
                   
                   
                   
                 1.9 × 10 5   
                 1.8 × 10 5   
                 2.5 × 10 5   
                 3.1 × 10 5   
                 3.0 × 10 5   
                 2.6 × 10 5   
                 5.0 × 10 4   
               
               
                 
                   brasiliensis 
                 
               
               
                 ATCC 16404 
               
               
                 
                   Escherichia coli 
                 
                 8.0 × 10 6   
                   
                   
                   
                 8.0 × 10 6   
                 2.0 × 10 1   
                 &lt;10 
                 &lt;10 
                 &lt;10 
                 &lt;10 
                 &lt;10 
               
               
                 ATCC 8739 
               
               
                 
                   Staphylococcus 
                 
                 3.2 × 10 6   
                   
                   
                   
                 4.2 × 10 6   
                 1.2 × 10 5   
                  20 
                 &lt;10 
                 &lt;10 
                 &lt;10 
                 &lt;10 
               
               
                 
                   aureus 
                 
               
               
                 ATCC 6538 
               
             
          
           
               
                 APPEARANCE 
                   
                   
                   
                 Normal 
                 Normal 
                 Normal 
                 Normal 
                 Normal 
                 Normal 
                 Normal 
               
               
                   
               
             
          
         
       
     
     Example 4 
     A solution of vitamin E TPGS is prepared in advance and stored by combining 650.0 grams of deionized water, 150.0 grams of potassium chloride and 200.0 grams of vitamin E TPGS. The mixture is heated to 35° C., mixed until uniform, cooled to ambient and stored for several weeks before use. 
     A beverage product is prepared by combining and mixing the following ingredients: 
     
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Ingredients 
                 Wt % 
               
               
                   
                   
               
             
             
               
                   
                 Citric acid 
                 0.130 
               
               
                   
                 Preservative 
                 0.096 
               
               
                   
                 Sucralose 
                 0.036 
               
               
                   
                 Fiber 
                 0.0570 
               
               
                   
                 Potassium citrate 
                 0.024 
               
               
                   
                 Potassium chloride 
                 0.104 
               
               
                   
                 Flavor 
                 0.080 
               
               
                   
                 Potassium ascorbate 
                 0.020 
               
               
                   
                 Calcium D-pantothenate 
                 0.002 
               
               
                   
                 Vitamin E solution (stabilized) - defined above 
                 0.100 
               
               
                   
                 Deionized water 
                 QS 
               
               
                   
                   
               
             
          
         
       
     
     Example 5 
     A solution of vitamin E TPGS is prepared and stored by combining 650.0 grams of deionized water, 149.5 grams of potassium chloride, 0.5 grams of potassium iodine and 200.0 grams of vitamin E TPGS. The mixture is heated to 30° C., mixed until uniform, cooled to ambient and stored for use. 
     A beverage product is prepared by combining and mixing the following ingredients: 
     
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Ingredients 
                 Wt % 
               
               
                   
                   
               
             
             
               
                   
                 Citric acid 
                 0.120 
               
               
                   
                 Preservative 
                 0.080 
               
               
                   
                 High Fructose Corn Syrup 
                 0.110 
               
               
                   
                 Natural Color 
                 0.008 
               
               
                   
                 Potassium citrate 
                 0.024 
               
               
                   
                 Potassium chloride 
                 0.104 
               
               
                   
                 Flavor 
                 0.080 
               
               
                   
                 Potassium ascorbate 
                 0.020 
               
               
                   
                 Calcium D-pantothenate 
                 0.002 
               
               
                   
                 Vitamin E solution (stabilized) - defined above 
                 0.110 
               
               
                   
                 Deionized water 
                 QS