Abstract:
Disclosed is an invention which relates to synthesizing immobilized and crosslinked proteases derived from plants for use as skin care agents. The resulting stabilized protease will minimally penetrate the skin because of its immobilized nature. It will retain activity because of its crosslinked nature and, in certain embodiments, due to its stabilization via physical additives. The present invention relates in particular to a linked papain product used in topical skin applications.

Description:
PRIORITY 
     This application claims priority to U.S. Provisional Application No. 61/296,052, filed Jan. 19, 2010. 
    
    
     SUMMARY 
     Disclosed is an invention which relates to synthesizing immobilized and crosslinked proteases derived from plants for use as skin care agents. The resulting stabilized protease will minimally penetrate the skin because of its immobilized nature. It will retain activity because of its crosslinked nature and, in certain embodiments, due to its stabilization via physical additives. The present invention relates in particular to a linked papain product used in topical skin applications. 
     BACKGROUND 
     The activity of proteases is important in epidermal homeostasis. Thus, proteases have various potential benefits when applied to the skin, but are subject to certain limitations. Papain is a powerful protease derived from papaya and certain other plants. However, it loses activity rapidly in a solution state. This is because papain, similar to all proteases, digests itself as well as undergoes denaturation. In addition, other difficulties with conventional papain products may be encountered when these papain products are used as topical skin care agents in connection with skin penetration and skin irritation. It is highly desirable to develop protease products, and more particularly, a papain product, for use in skin care which do not have such limitations. 
     To overcome the above-mentioned difficulties in the conventional art, exemplary embodiments of the present invention provide a modified stable crosslinked protease product via the techniques set forth described herein, and particularly a modified stable crosslinked protease product. 
     For example, in some embodiments of the present invention, in order to obtain a stable crosslinked papain product, papain is immobilized on a polymer like, for example, a carbomer or carbopol in a primary cross-linking reaction and then a secondary crosslinking reaction is subsequently performed by adding a homobifunctional crosslinking reagent of low molecular weight which is amine reactive such as, for example, Dimethyl adipimidate (DMA), Bis(Sulfosuccinimidyl) suberate (BS3), Dimethyl Suberimidate (DMS), Dimethyl pimelimidate (DMP) and Disuccinimidyl suberate (DSS). 
     In other exemplary embodiments of the present invention, the primary crosslinking reaction and the secondary crosslinking reaction are performed to obtain a stable crosslinked papain product and then the stable crosslinked papain is further stabilized using physical stabilizers such as, for example, sugars or sugar polymers. For example, sodium alginate may be used as a physical stabilizer in accordance with exemplary embodiments of the present invention. 
     The above crosslinked stabilized papain product may still even be further stabilized by including the above stabilized papain product in certain preservative systems or an oil in water formulation. 
     The benefits of the immobilized, crosslinked and stabilized protease (e.g. papain) complex according to exemplary embodiments of the present invention include but are not limited to:
         1) minimal skin penetration,   2) retention of proteasic activity in solution or dry form   3) minimal skin irritation, and   4) ease of formulation       

     In still other embodiments of the present invention, cosmetic, personal care, and pharmaceutical compositions comprising stabilized proteases are described. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       The following figures are presented for the purposes of illustration only, and are not intended to be limiting. 
         FIG. 1  illustrates crosslinking reactions for forming a stable crosslinked protease in accordance with the present invention. 
         FIG. 2  illustrates a stable crosslinked papain product in accordance with an exemplary embodiment of the present invention which has undergone the crosslinking reactions illustrated in  FIG. 1 . 
         FIG. 3  illustrates the percentage of day 0 activity after storing samples of a stabilized papain in accordance with an exemplary embodiment of the present invention at various temperatures for up to 12 weeks. 
         FIG. 4  illustrates the percentage of day 0 activity after storing the no DMA samples at various temperatures for up to 12 weeks. 
         FIG. 5  illustrates a Sigma papain standard curve 
         FIG. 6(   a ) illustrates the percentage of day 0 activity retained after storing stabilized papain samples at various temperatures for up to 12 weeks in accordance with an exemplary embodiment of the present invention. 
         FIG. 6(   b ) illustrates the percentage of day 0 activity retained after storing the no DMA samples at various temperatures for up to 12 weeks. 
         FIG. 7  is a chart which illustrates that no free active papin was found in a stable crosslinked papain product in accordance with an exemplary embodiment of the present invention. 
         FIG. 8  is a chart which illustrates the percentage of activity after 1 week at different temperatures of samples which contain 0.2% of BS3 and control samples which do not contain any BS3. 
         FIG. 9  is a chart which illustrates the percentage of retained activity after 4 weeks of samples of stabilized papain with and without 0.1% alginate. 
         FIG. 10  is a sigma papain standard curve. 
         FIG. 11  is a chart which illustrates the effect of sugar or sugar polymers on the stability of free papain after 10 days at 25° C. and 40° C. 
         FIG. 12  is a chart which illustrates the percentage of activity retained after 2 weeks of 0.4% papain. 
         FIG. 13(   a ) illustrates a stable crosslinked papain product which has been crosslinked by DMA and has been further reacted with sodium alginate. 
         FIG. 13(   b ) is a chart which illustrates the percentage of activity retained after storing 1% DMA samples at various temperatures for up to 12 weeks. 
         FIG. 13  ( c ) a chart which illustrates the percentage of day 0 activity after storing no DMA samples at various temperatures for up to 12 weeks. 
         FIG. 14  is a graph which illustrates the effect of DMA on activity retention of linked papain. 
         FIG. 15  is a chart which illustrates the activity in linked papain samples after 12 weeks at different temperatures. 
         FIG. 16  is a Sigma papain standard curve. 
         FIG. 17  is a chart which illustrates activity of linked papain (with DMA) retained in formulation. 
         FIG. 18  is a chart which illustrates activity of linked papain (without DMA) retained in formulation. 
         FIG. 19  is a graph illustrating the activity of linked papain retained inside of formulations 
         FIG. 20  is a standard curve for sigma papain in placebo formulation. 
         FIG. 21  is a chart which illustrates the activity of linked papain retained in an oil and water formulation. 
     
    
    
     DETAILED DESCRIPTION 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of which this invention belongs. Although methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
     Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. As will be apparent to one of skill in the art, specific features and embodiments described herein can be combined with any other feature or embodiment. 
     The epidermis is comprised of several strata or layers. The outermost layer, the Stratum corneum, is made up of dead cells which have migrated upward over the course of several days from the strata below. These dead cells are normally shed from the surface of the skin via a process called epidermal desquamation which stimulates growth of newer cells at a deeper level. Younger skin is more efficient at this process than aged or damaged skin. As a result, aged skin appears dull, thick and less toned. This may be exacerbated by environmental factors, such as exposure to sunlight; hormonal influences, such as androgens, estrogens, and epidermal growth factor; and vitamin deficiencies, such as deficiencies in vitamins A and D. Protease activity is a key factor in the desquamation process. Thus, the application of proteases to the skin for cosmetic effects, such as skin smoothing and anti-aging, is desirable. However, use of proteases in cosmetic and other applications is known to have three major limitations: instability, potential for allergenicity, and skin penetration. 
     The invention is based, at least in part, on the discovery that proteases can be stabilized through certain crosslinking reactions to form a protease carbomer copolymer, also referred to herein as a “stabilized” protease. Such stabilized proteases comprise a protease crosslinked to a carbomer, wherein the primary amines of the protease are crosslinked to the carboxyl groups of the carbomer and wherein the amines of the protease are further crosslinked by an amine reactive crosslinking agent. In one embodiment of the invention, the stabilized protease further comprises a physical stabilizer, for example a sugar or sugar polymer. 
     A method of forming such stabilized protease product is also disclosed wherein a primary crosslinking reaction is performed to crosslink the primary amines of the protease to the carboxyl groups of a carbomer and a secondary crosslinking reaction is performed via an amine reactive crosslinking reagent. 
     Proteases 
     Proteases are enzymes which catalyse the breakdown of proteins. Proteases, being proteins themselves, have a tendency to undergo autodegradation and are unstable in nature. This proteinaceous nature also renders them allergenic. Additionally, because of their ability to degrade proteins, they may penetrate to the deeper layers of the epidermis and cause damage to the underlying strata. Suitable proteases for use in the present invention include papain, ficin, bromelain, and actinidain. 
     Ficin is a nonspecific sulfhydryl protease isolated from the latex of the fig, the fruit of the  Ficus  tree. Ficin is most commonly obtained from  Ficus carica  and  Ficus glabatra . However, ficin may also be isolated from the fruits of other  Ficus  species, such as  Ficus elastica  and  Ficus insipida . Bromelain is a sulfhydryl protease isolated from the stem and/or fruit of the pineapple ( Ananas comosus ). Actinidain, also known as actinidin is a cysteine protease obtained from the kiwi fruit ( Actinidia deliciosa ). Papain is a cysteine protease obtained from papaya ( Carcia papaya ) and mountain papaya ( Vasconcellea cundinamarcensis ), most often from the latex of the green, unripe fruit. The proteases are present in a concentration between about 0.1% and 5% by weight. 
     Carbomers 
     The protease is first immobilized on a carbomer. Carbomers are homopolymers of acrylic acid having a high molecular weight which is crosslinked with any of several polyalcohol allyl ethers (e.g., allyl ether pentaerythritol, allyl ether of sucrose, or allyl ether of propylene). Examples of suitable carbomers are Carbomer 910, Carbomer 934, Carbomer 934p, Carbomer 940, and Carbomer 941, wherein the numberical suffix indicates the average molecular weight of the polymer chains. As used herein, an “immobilized” protease or “linked” protease refers to a protease that has been reacted with the carbomer via the primary crosslinking reaction. Suitable crosslinking reagents for this primary crosslinking reaction are reagents capable of coupling carboxyl groups to primary amines. One example of a suitable crosslinking reagent is the water soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. EDC is a zero-length cross-linking agent used to couple carboxyl groups to primary amines. EDC reacts with a carboxylic acid groups to give O-acylisourea groups, which form cross-links after reaction with free amine groups. EDC is a primary cross-linker in this embodiment which gets converted to a disubstituted urea during the course of the reaction. The NHS is an acid ester and a catalyst which increases the rate of the cross-linking and remains unchanged during the cross-linking reaction. 
     Chemical Crosslinkers 
     Once immobilized on the carbomer, the immobilized protease is reacted with an amine reactive crosslinking reagent in a secondary crosslinking reaction to form a protease carbomer copolymer, also referred to herein as a “stabilized protease”. The crosslinking reagent is preferably a low molecular weight crosslinker such that the crosslinker will completely react or nearly completely react in the secondary crosslinking reaction. Examples of suitable crosslinkers that can be used include imidoester crosslinkers such as dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3 dithiobis. The amine reactive crosslinkers may be provided in various concentrations ranging from about 0.05% to about 5% by weight, preferably between about 1% and 5%. In certain embodiments, DMA is utilized. 
     Physical Stabilizers 
     After chemical crosslinking, the immobilized and crosslinked protease is optionally further stabilized with a physical stabilizer, for example sugar or sugar polymers. Examples of suitable sugars or sugar polymers are sodium alginate, trehalose, mannitol, glycerol, and Xanthan. In one embodiment, sodium alginate is utilized. Such physical stabilizers may be included in concentrations between about 0.1% and 5% by weight. 
     An illustration of crosslinking reaction for forming one embodiment of the stabilized proteases of the present invention is are shown in  FIG. 1 . An illustration of a stabilized papain product is shown in  FIG. 2 . 
     Applications/Formulations 
     Stabilized proteases of the present invention have miminal skin pentetration, retain proteasic activity in solution or dry form, have minimal skin irritation, and are relatively easy to provide in a formulation. Accordingly, exemplary embodiments of the present invention provide a more stable and much safer protease products in comparison to the protease products of the conventional art, particularly stabilized papain products embodiments. 
     Stabilized proteases are suitable for use in cosmetic and personal care formulations, for example, exfoliating preparations, anti-wrinkle and/or anti-aging preparations, bath additives, hair care preparations, liquid and sold soaps, cleansing solutions, moist cleansing cloths, oils or powders, anti-acne preparations. Stabilized proteases are particularly suitable for treating dry, aged or damaged skin by applying one or more of the stabilized proteases of the present invention to dry, aged or damaged skin in need of treatment. 
     Cosmetic and/or personal care formulations may be in the form, for example, of water-in-oil or oil-in-water emulsion, an alcoholic or alcohol containing formulation, a vesicular dispersion of an ionic or non-ionic amphiphilic lipid, or a gel. Exemplary cosmetic and/or personal care formulations comprise between about 0.5% and 5% stabilized protease, by weight, preferably between about 1% and 3%. 
     The stabilized proteases of the present invention are also suitable for pharmaceutical applications, for example, debridement applications. Debridement is the removal of dead or damaged tissue from wounds, for example ulcerative wounds, or burns in order to assist healing. In one embodiment of the invention, one or more of the stabilized proteases of the present invention are applied to skin wounds or burns in need of debridement. Exemplary formulations and products comprising stabilized proteases for treatment of wounds or burns include bandages/dressings, patches, wash solutions, ointments or gels, or synthetic tissues. In certain embodiments, debridement compositions, for example, bandages, dressings or patches, may optionally include antimicrobials. For debridement applications, the amount of stabilized protease included in the pharmaceutical compositions will be an amount which effectively debrides necrotic tissue and liquefies pus in wounds, and which effects removal in a reasonable time (for example, over a seven day period, of substantially all of such materials. 
     In addition to skin care applications, the stabilized proteases of the present invention may also be suitable in other applications known in the art wherein a stable form of a protease would be desired. One exemplary application is oral care compositions. 
     Topical compositions comprising stabilized proteases may further comprise a variety of other ingredients that are conventionally used in cosmetic, personal care, or pharmaceutical formulations provided they do not unacceptably alter the benefits of the invention. Nonlimiting examples of optional conventional ingredient classes include fragrances, pigments, colorings/colorants, essential oils, astringents, anti-aging agents, anti-acne agents, anti-caking agents, antifoaming agents, antimicrobial agents, antioxidants, binders, pH adjusters, skin bleaching and lightening agents, skin conditioning agents, sunscreens, preservatives, anti-inflammatory agents, moisturizers, thickeners, and vitamins. 
     For certain embodiments of cosmetic, personal care, and/or pharmaceutical applications, it is desirable to further include a preservative system with the stabilized protease. Enzymes are known to denature under harsh conditions like higher temperatures and the presence of incompatible chemicals like strong emulsifiers. Two example preservative systems suitable for use with stabilized proteins, and particularly suitable for stabilized papain, are:
         (a) Phenoxyethanol+Benzoic acid   (b) Diocide (Blend of Phenoxythanol+Capryl glycol+Hexylene Glycol)       

     EXAMPLES 
     The invention is further described in the following example, which does not limit the scope of the invention described in the claims. 
     Example 1 
     A stable papain product which retains its proteasic activity and is safer than conventional papain products which contain free papain was produced by performing a primary crosslinking reaction in which 1% papain was crosslinked to Carbopol using carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC) together with N-hydroxysulfosuccinimide (NHS). Next, a secondary cross-linking reaction was then performed in which the immobilized papain is reacted with dimethyl adipimidate (DMA) to further crosslink the crosslinked papain to thereby form a stable cross-linked papain product. DMA was provided in concentration of 1% and the papain was provided in a concentration of 1% by weight. 
     Three (3) experimental lab batches of the above stabilized papain product were conducted to determine the effect of 1% of DMA has on the enzyme activity/stability of the primary crosslinked papain over a storage period over 12 weeks. The reaction conditions for performing the experiments are set forth in Table 1. 
     
       
         
               
             
               
               
               
               
             
               
             
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Assay protocol using Invitrogen E6639 EnzChek ® 
               
               
                 kit for enzyme activity 
               
               
                   
               
             
             
               
                 Papain standard curve 
               
             
          
           
               
                 STD # 
                 Stock solution(U/mL) 
                 Stock (1 U/mL) 
                 Assay buffer 
               
               
                   
               
               
                 1 
                 1 
                 1000 
                 0 
               
               
                 2 
                 0.5 
                 500 
                 500 
               
               
                 3 
                 0.25 
                 250 
                 750 
               
               
                 4 
                 0.1 
                 100 
                 900 
               
               
                 5 
                 0.05 
                 50 
                 950 
               
               
                 6 
                 0.01 
                 10 
                 990 
               
               
                 7 
                 0.005 
                 5 
                 995 
               
               
                 8 
                 0 
                 0 
                 1000 
               
               
                   
               
             
          
           
               
                 Prepare 1 Unit/mL stock solution of papain 
               
               
                 10 mg Sigma papain 
               
               
                 25 ml dl 1× digestion 
               
               
                 buffer 
               
               
                 Material &amp; buffer requirement: 
               
               
                 0.1M Sodium Bicarbonate, pH 8.3 
               
               
                 1× Digestion buffer: PBS pH6 + 50 ul of TX-100 
               
               
                 1 Mg/ml stock of BODIPY TRX casein = 
               
               
                 1 vial of substrate and dissolve in 0.2 ml of Bicarbonate buffer 
               
               
                 10 ug/mL working solution of BODIPY casein: 
               
               
                 Add 0.2 mL of stock substrate in 19.8 ml of Digestion buffer 
               
               
                 in 50 ml falcon 
               
             
          
           
               
                 Assay: 
                  20 ul of STD/sample 
               
               
                   
                  80 ul of 1X digestion biffer 
               
               
                   
                 100 ul of 10 ug/mL working solution of BODIPY 
               
               
                   
                 casein 
               
               
                 Samples: 
                 50 ul of linked papain mixed with 150 ul of 1× 
               
               
                   
                 digestion buffer containing 0.1% Triton X-100 
               
               
                 Procedure: 
                 Incubate the plate at room temperature for one hour with 
               
               
                   
                 moderate agitation. Protect the plate from light. Read 
               
               
                   
                 the flurescence in a fluorescence microplate reader. Using 
               
               
                   
                 standard fluorescein filters excitation 530/25 nm, emission 
               
               
                   
                 620/40 nm. 
               
               
                   
               
             
          
         
       
     
     The results of the three separate lab batches for determining the retained activity of immobilized papain with 1% DMA and without DMA at various temperatures over a 12 week period are set forth below in Tables 2(a)-5(b). Table 2(a) depicts the percentage of retained activity of linked immobilized papain in the experimental samples which also contain 1% DMA. Table 2(b) depicts the percentage of retained activity of immobilized papain in the control samples which do not include DMA. The experimental samples and the control samples were essentially the same except that the control samples did not contain any DMA. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2(a) 
               
             
             
               
                   
               
               
                 Immobilized Papain crosslinked with 1% DMA 
               
             
          
           
               
                 Time 
                 4° C. 
                 25° C. 
                 40° C. 
                 50° C. 
               
               
                   
               
             
          
           
               
                   
                 100 
                 100 
                 100 
                 100 
               
               
                 2 week 
                 103.927 
                 106.806 
                 92.670 
                 75.654 
               
               
                 4 week 
                 101.832 
                 98.168 
                 69.372 
                 26.70 
               
               
                 6 week 
                 97.906 
                 95.812 
                 55.236 
                 13.089 
               
               
                 0 week 
                 96.073 
                 92.670 
                 36.911 
                 2.356 
               
               
                 12 week  
                 95.812 
                 84.296 
                 12.923 
                 2.068 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2(b) 
               
             
             
               
                   
               
               
                 Immobilized Papain (without DMA) - Control 
               
             
          
           
               
                 Time 
                 4 C. 
                 25 C. 
                 40 C. 
                 50 C. 
               
               
                   
               
             
          
           
               
                 0 week 
                 100 
                 100 
                 100 
                 100 
               
               
                 2 week 
                 96.419 
                 68.286 
                 53.708 
                 25.064 
               
               
                 4 week 
                 91.049 
                 57.545 
                 47.826 
                 8.440 
               
               
                 6 week 
                 90.537 
                 45.013 
                 26.087 
                 2.558 
               
               
                 8 week 
                 85.166 
                 27.877 
                 12.788 
                 0.256 
               
               
                 12 week  
                 78.772 
                 21.043 
                 6.115 
                 0.305 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3(a) 
               
             
             
               
                   
               
               
                 Immobilized Papain Crosslinked with 1% DMA 
               
             
          
           
               
                 Time 
                 4 C. 
                 25 C. 
                 40 C. 
                 50 C. 
               
               
                   
               
             
          
           
               
                 0 week 
                 100 
                 100 
                 100 
                 100 
               
               
                 2 week 
                 107.320 
                 110.000 
                 87.530 
                 65.430 
               
               
                 4 week 
                 95.620 
                 92.650 
                 65.430 
                 29.76 
               
               
                 6 week 
                 91.420 
                 87.210 
                 56.540 
                 10.430 
               
               
                 8 week 
                 87.240 
                 84.320 
                 30.120 
                 1.340 
               
               
                 12 week  
                 85.350 
                 80.460 
                 10.870 
                 1.000 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3(b) 
               
             
             
               
                   
               
               
                 Immobilized Papain (without DMA) - Control 
               
             
          
           
               
                 Time 
                 4 C. 
                 25 C. 
                 40 C. 
                 50 C. 
               
               
                   
               
             
          
           
               
                 0 week 
                 100 
                 100 
                 100 
                 100 
               
               
                 2 week 
                 92.340 
                 63.450 
                 62.870 
                 20.870 
               
               
                 4 week 
                 88.010 
                 53.450 
                 44.620 
                 5.670 
               
               
                 6 week 
                 84.320 
                 44.670 
                 28.980 
                 1.650 
               
               
                 8 week 
                 81.760 
                 22.970 
                 10.960 
                 0.430 
               
               
                 12 week  
                 72.350 
                 17.650 
                 4.300 
                 0.010 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4(a) 
               
             
             
               
                   
               
               
                 Immobilized Papain crosslinked with 1% DMA 
               
             
          
           
               
                 Time 
                 4 C. 
                 25 C. 
                 40 C. 
                 50 C. 
               
               
                   
               
             
          
           
               
                 0 week 
                 100 
                 100 
                 100 
                 100 
               
               
                 2 week 
                 99.000 
                 99.980 
                 92.670 
                 68.520 
               
               
                 4 week 
                 97.620 
                 96.210 
                 62.650 
                 22.01 
               
               
                 6 week 
                 96.420 
                 92.510 
                 52.980 
                 10.650 
               
               
                 8 week 
                 90.610 
                 82.100 
                 31.000 
                 2.420 
               
               
                 12 week  
                 88.010 
                 80.110 
                 10.650 
                 0.890 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4(b) 
               
             
             
               
                   
               
               
                 Immobilized Papain (without DMA) - Control 
               
             
          
           
               
                 Time 
                 4 C. 
                 25 C. 
                 40 C. 
                 50 C. 
               
               
                   
               
             
          
           
               
                 0 week 
                 100 
                 100 
                 100 
                 100 
               
               
                 2 week 
                 98.870 
                 71.820 
                 50.980 
                 27.870 
               
               
                 4 week 
                 86.340 
                 52.340 
                 41.620 
                 5.750 
               
               
                 6 week 
                 80.730 
                 40.210 
                 22.320 
                 1.230 
               
               
                 8 week 
                 81.530 
                 20.410 
                 11.876 
                 0.910 
               
               
                 12 week  
                 75.610 
                 15.620 
                 3.110 
                 0.030 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5(a) 
               
             
             
               
                   
               
               
                 Mean value for % of retained activity of immobilized papain crosslined 
               
               
                 with 1% DMA of Batch 1 (Table 3), Batch 2 (Table 4) and Batch 3 
               
               
                 (Table 5) 
               
             
          
           
               
                 Time 
                 4 C. 
                 25 C. 
                 40 C. 
                 50 C. 
               
               
                   
               
             
          
           
               
                 0 week 
                 100 
                 100 
                 100 
                 100 
               
               
                 2 week 
                 103.4 
                 105.6 
                 91.0 
                 69.9 
               
               
                 4 week 
                 98.4 
                 95.7 
                 65.8 
                 26.2 
               
               
                 6 week 
                 95.2 
                 91.8 
                 54.9 
                 11.4 
               
               
                 8 week 
                 91.3 
                 86.4 
                 32.7 
                 2.0 
               
               
                 12 week  
                 89.7 
                 81.6 
                 11.5 
                 1.3 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5(b) 
               
             
             
               
                   
               
               
                 Mean value for % of retained activity of immobilized papain (without 
               
               
                 DMA) of Batch 1 (Table 3), Batch 2 (Table 4) and Batch 3 (Table 5) 
               
             
          
           
               
                 Time 
                 4 C. 
                 25 C. 
                 40 C. 
                 50 C. 
               
               
                   
               
             
          
           
               
                 0 week 
                 100 
                 100 
                 100 
                 100 
               
               
                 2 week 
                 95.88 
                 67.85 
                 55.85 
                 55.85 
               
               
                 4 week 
                 88.47 
                 54.44 
                 44.69 
                 44.69 
               
               
                 6 week 
                 85.20 
                 43.30 
                 25.80 
                 25.80 
               
               
                 8 week 
                 82.82 
                 23.75 
                 11.87 
                 11.87 
               
               
                 12 week  
                 75.58 
                 18.10 
                 4.51 
                 4.51 
               
               
                   
               
             
          
         
       
     
     As can be seen from the results of tables 2(a)-5(b), the activity of immobilized papain which was cross-linked with 1% DMA in the experimental samples was significantly greater over a variety of temperatures than the activity of immobilized papain in the control samples which was not reacted with DMA. Accordingly, papain which has undergone a primary crosslinking reaction to cross-link papain to a carbomer and then undergone a secondary cross-linking reaction with 1% DMA is significantly more stable than control samples of papain which has undergone the primary cross-linking reaction but has not undergone a secondary cross-linking reaction with DMA. Furthermore,  FIGS. 3 and 4  illustrate the results of tables 2(a)-5(b) that the activity of immobilized papain which has been crosslinked with 1% DMA is significantly greater over a variety of temperatures than the activity of immobilized papain in the control samples which was not reacted with DMA. 
     As set forth below in Tables 6 and 7, papain activity in the experimental samples and the control samples were determined based upon fluorescence change per unit sample of the immobilized papain. A Sigma papain activity standard curve as shown in  FIG. 5  was developed for assistance in quantitating the activity of immobilized papain crosslinked with 1% DMA in accordance with the present example. As noted above, an EnzChek® protease assay kit (E-6639) was used to obtain the standard curve and the fluorescence change per unit sample of the experimental of the present invention. 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Fluorescent Values for Immobilized Papain 
               
               
                 with 1% DMA and without DMA (Control) 
               
               
                 Linked Papain samples with or without 
               
               
                 1% DMA (12 week samples) 
               
               
                   
               
             
             
               
                 4 C. 
               
             
          
           
               
                 1 
                 LP-1% DMA 
                 1:8 
                 5674 
                 5432 
                 5876 
                 5660.7 
                 4415 
                 0.458 
               
               
                 2 
                 LP-Control 
                 1:8 
                 4598 
                 5165 
                 5120 
                 4961.0 
                 3715 
                 0.385 
               
             
          
           
               
                 25 C. 
               
             
          
           
               
                 1 
                 LP-1% DMA 
                 1:8 
                 5432 
                 4987 
                 4976 
                 5131.7 
                 3886 
                 0.403 
               
               
                 2 
                 LP-Control 
                 1:8 
                 2204 
                 2252 
                 2359 
                 2271.7 
                 1026 
                 0.103 
               
             
          
           
               
                 40 C. 
               
             
          
           
               
                 1 
                 LP-1% DMA 
                 1:8 
                 1903 
                 1885 
                 1849 
                 1879.0 
                 633 
                 0.062 
               
               
                 2 
                 LP-Control 
                 1:8 
                 1580 
                 1571 
                 1575 
                 1575.3 
                 329 
                 0.030 
               
             
          
           
               
                 50 C. 
               
             
          
           
               
                 1 
                 LP-1% DMA 
                 1:8 
                 1334 
                 1345 
                 1474 
                 1384.3 
                 138 
                 0.010 
               
               
                 2 
                 LP-Control 
                 1:8 
                 1274 
                 1315 
                 1324 
                 1304.3 
                 58 
                 0.001 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Activity of Immobilized Papain 1% with 1% DMA and immobilized 
               
               
                 papain without DMA (Control) measured in units/ml. 
               
             
          
           
               
                   
                 Activity 
                   
                   
               
               
                   
                 units/ml 
                 Units/ml 
               
               
                   
               
             
          
           
               
                 0 week 
                 LP-1% DMA 
                 3.82 
                 3.82 
                 3.82 
                 3.82 
               
               
                 data Apr. 16, 2008 
                 LP-Control 
                 4.91 
                 4.91 
                 4.91 
                 4.91 
               
               
                   
               
               
                   
                   
                 4 C. 
                 25 C. 
                 40 C. 
                 50 C. 
               
               
                   
               
               
                 2 week 
                 LP-1% DMA 
                 3.97 
                 4.08 
                 3.54 
                 2.89 
               
               
                 data Apr. 30, 2008 
                 LP-Control 
                 3.77 
                 2.67 
                 2.1 
                 0.98 
               
               
                 4 week 
                 LP-1% DMA 
                 3.89 
                 3.75 
                 2.65 
                 1.02 
               
               
                 data May 14, 2008 
                 LP-Control 
                 3.56 
                 2.25 
                 1.87 
                 0.33 
               
               
                 6 week 
                 LP-1% DMA 
                 3.74 
                 3.66 
                 2.11 
                 0.5 
               
               
                 data May 28, 2008 
                 LP-Control 
                 3.54 
                 1.76 
                 1.02 
                 0.1 
               
               
                 8 week 
                 LP-1% DMA 
                 3.67 
                 3.54 
                 1.41 
                 0.09 
               
               
                 data Jun. 11, 2008 
                 LP-Control 
                 3.33 
                 1.09 
                 0.5 
                 0.01 
               
               
                 12 week  
                 LP-1% DMA 
                 3.66 
                 3.22 
                 0.49 
                 0.08 
               
               
                 data Jul. 02, 2008 
                 LP-Control 
                 3.08 
                 0.82 
                 0.24 
                 0.01 
               
               
                   
               
             
          
         
       
     
     In other experiments, crosslinked 1% papain was stored with 1% DMA and crosslinked 1% papain was stored without DMA at the temperatures of 4° C., 25° C. and 45° C. over a 12 week period to determine the stability of these papain samples at these temperatures. The results of this experiment was obtained by a papain activity assay using Invitrogen EnzChek® assay kit, N=3. 
     As shown in  FIGS. 6(   a )- 6 ( b ), the crosslinking of immobilized papain with DMA significantly improved the stability of the product at 25° C. and 45° C. in comparison to the control sample in which linked papain was not stored with DMA.  FIG. 6(   a ) illustrates the percentage of activity retained after storing samples for up to 12 weeks containing linked papain crosslinked by DMA.  FIG. 6(   b ) illustrates control samples which are stored for up to 12 weeks containing linked papain with no DMA It is further noted that as illustrated in  FIG. 6(   a ), it was determined that the linked papain product crosslinked by DMA retains over about 80% activity at 4° C. and 25° C. for 12 weeks and about 50% at 45° C. 
     In still other experiments, as shown in  FIG. 7 , a determination of free active papain in stabilized papain was conducted. The results of the experiment was that it was determined that there was no free active papain found in the papain that had been crosslinked by EDC/NHS in a primary cross-linking reaction and then further crosslinked by 1% DMA in a secondary crosslinking reaction in accordance with an exemplary embodiment of the present invention. On the other hand, there was significant activity of free papain in the papain of the control samples which was non-crosslinked. 
     Example 2 
     In example 2, instead of DMA being used as the secondary cross-linker, 0.2% of the crosslinker BS3 was used to crosslink the immobilized papain product in a secondary cross-linking reaction. The protocol, reactants, reaction conditions and results for determining the percentage of activity of the experimental samples of immobilized papain stored with 0.2% BS3 and control samples of immobilized papain stored without BS3 are illustrated in tables 8-10 and discussed below. 
     
       
         
               
             
               
               
               
               
               
             
               
             
               
               
             
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 Assay protocol using Invitrogen E6639 EnzChek kit for enzyme activity 
               
               
                   
               
             
             
               
                 Papain standard curve 
               
             
          
           
               
                   
                   
                 Stock Solution 
                 Stock 
                   
               
               
                   
                 STD # 
                 (U/ml) 
                 (1 U/ml) 
                 Assay Buffer 
               
               
                   
               
               
                   
                 1 
                 1 
                 1000 
                 0 
               
               
                   
                 2 
                 0.5 
                 500 
                 500 
               
               
                   
                 3 
                 0.25 
                 250 
                 750 
               
               
                   
                 4 
                 0.1 
                 100 
                 900 
               
               
                   
                 5 
                 0.05 
                 50 
                 950 
               
               
                   
                 6 
                 0.01 
                 10 
                 990 
               
               
                   
                 7 
                 0.005 
                 5 
                 995 
               
               
                   
                 8 
                 0 
                 0 
                 1000 
               
               
                   
               
             
          
           
               
                 Prepare 1 Unit/ml stock solution of papain 
               
               
                 10 mg Sigma papain 
               
               
                 25 ml dl 1X digestion buffer 
               
               
                 Material &amp; buffer requirement: 
               
               
                 0.1M Sodium Bicarbonate, pH 8.3 
               
               
                 1x Digestion buffer: PBS pH 6 + 50 ul of TX-100 
               
               
                 1 Mg/ml stock of BODIPY TRX casein = 1 vial of substrate and dissolve 
               
               
                 in 0.2 ml of Bicarbonate buffer 
               
               
                 10 ug/ml working solution of BODIPY casein: 
               
               
                 Add 0.2 mL of stock substrate in 19.8 ml of Digestion buffer in 50 ml 
               
               
                 falcon 
               
             
          
           
               
                 Assay: 
                 20 ul of STD/sample 
               
               
                   
                 80 ul of 1X digestion buffer 
               
               
                   
                 100 ul of 10 ug/mL working solution of BODIPY casein 
               
             
          
           
               
                 Samples: 50 ul of linked papain mixed with 150 ul of 1X digestion buffer 
               
               
                 containing 0.1% 
               
               
                 Triton X-100 
               
               
                 Procedure: 
               
               
                 Incubate the plate at room temperature for one hour with moderate 
               
               
                 agitation. Protect the plate from light. Read the fluorescence in a 
               
               
                 fluorescence microplate reader. Using standard fluorescence filters 
               
               
                 excitation 530/25 nm, emission 620/40 nm. 
               
               
                   
               
             
          
           
               
                   
                 Sample ID 
                   
                   
                   
               
               
                   
                 Linked Papain 
                   
                   
                 1X 
               
               
                 Tube # 
                 Samples 
                 Dilution Filter 
                 Sample 
                 digestion buffer 
               
               
                   
               
               
                 1 
                 LP-BS3 4 C. 
                 1:4 
                 50 
                 150 
               
               
                 2 
                 LP-BS3 25 C. 
                 1:4 
                 50 
                 150 
               
               
                 3 
                 LP-BS3 40 C. 
                 1:4 
                 50 
                 150 
               
               
                 4 
                 LP-BS3 50 C. 
                 1:4 
                 50 
                 150 
               
               
                 5 
                 LP-no BS3 4 C. 
                 1:4 
                 50 
                 150 
               
               
                 6 
                 LP-no BS3 25 C. 
                 1:4 
                 50 
                 150 
               
               
                 7 
                 LP-no BS3 40 C. 
                 1:4 
                 50 
                 150 
               
               
                 8 
                 LP-no BS3 50 C. 
                 1:4 
                 50 
                 150 
               
               
                   
                   
                 1:4 
                 50 
                 150 
               
               
                   
               
               
                 Linked papain samples were made with or without 0.2% BS3 
               
             
          
         
       
     
     
       
         
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 Tube 
                   
                 Dilution 
                   
                   
                   
                   
                 average- 
                   
                   
               
               
                 # 
                 Sample ID 
                 Factor 
                 Read 1 
                 Read 2 
                 Read 3 
                 Average 
                 negative 
                 units/ml 
                 units/ml 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 LP - BS3 4 C. 
                 1:4 
                 10954 
                 10883 
                 12916 
                 11584.3 
                 10231 
                 1.034 
                 4.14 
               
               
                 2 
                 LP - BS3 25 C. 
                 1:4 
                 12196 
                 12865 
                 12959 
                 12673.3 
                 11320 
                 1.156 
                 4.62 
               
               
                 3 
                 LP - BS3 40 C. 
                 1:4 
                 13135 
                 13636 
                 14267 
                 13679.3 
                 12326 
                 1.268 
                 5.07 
               
               
                 4 
                 LP - BS3 50 C. 
                 1:4 
                 11009 
                 11025 
                 11031 
                 11021.7 
                 9668 
                 0.971 
                 3.89 
               
               
                 5 
                 LP - no BS3 4 C. 
                 1:4 
                 8174 
                 19586 
                 12 
                 9257.3 
                 7904 
                 0.775 
                 3.10 
               
               
                 6 
                 LP - no BS3 25 C. 
                 1:4 
                 5345 
                 12494 
                 13 
                 5950.7 
                 4597 
                 0.406 
                 1.62 
               
               
                 7 
                 LP - no BS3 40 C. 
                 1:4 
                 3191 
                 7654 
                 12 
                 3619.0 
                 2265 
                 0.146 
                 0.58 
               
               
                 8 
                 LP - no BS3 50 C. 
                 1:4 
                 2383 
                 5583 
                 14 
                 2660.0 
                 1306 
                 0.039 
                 0.16 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                   
                 Activity retained after 1 
                   
                   
               
               
                   
                 week - % of 4 C. activity 
                 No BS3 
                 0.2% BS3 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                  4 C. 
                 99.957 
                 99.912 
               
               
                   
                 25 C. 
                 52.389 
                 111.643 
               
               
                   
                 40 C. 
                 18.847 
                 122.479 
               
               
                   
                 50 C. 
                 5.052 
                 93.851 
               
               
                   
               
             
          
         
       
     
     As can be seen from the results of table 10 and  FIG. 8 , the activity of immobilized 1% papain which has been further crosslinked with 0.2% BS3 was significantly greater over a variety of temperatures than the activity of immobilized papain in the control samples which was not reacted with BS3. 
     Example 3 
     In this example, 1% stabilized papain and 0.1% sodium alginate were combined. The protocol, reactants, reaction conditions and results for determining the % of activity of experimental samples of stabilized papain stored with 0.1% sodium alginate and control samples of stabilized papain product stored without sodium alginate are discussed below. 
     
       
         
               
             
               
               
             
               
             
           
               
                 TABLE 11 
               
               
                   
               
             
             
               
                 Papain standard curve 
               
               
                   
               
               
                 Prepare 5 Unit/mL stock solution of papain 
               
               
                 10 mg Sigma papain 
               
               
                 5 ml dl 1X digestion buffer 
               
               
                 Material &amp; buffer requirement: 
               
               
                 1X assay buffer: 50 mM borate pH 8.5 
               
               
                 Substrate: 2 mg/ml succinylated casein in 1X assay buffer, dissolve 
               
               
                 1 vial (10 mg) in 5 ml assay buffer TNBSA working soln: 100 ul of 
               
               
                 TNBSA stock to 14.9 ml of assay buffer 
               
             
          
           
               
                 Assay: 
                 50 ul of STD/sample 
               
               
                   
                 100 ul of substrate (2 mg/ml), incubate the plate for 
               
               
                   
                 20 min at RT 
               
               
                   
                 50 ul of TNBSA solution, incubate the plate for 20 mins 
               
               
                   
                 at room temp 
               
               
                   
                 Read absorbance at 450 nm 
               
             
          
           
               
                 Samples tested (4 week linked papain samples - prepared on 
               
               
                 101607 papain concentration 1%) 
               
               
                 No treatment 4 C. 
               
               
                 0.1% Alginate 4 C. 
               
               
                 No treatment 25 C. 
               
               
                 0.1% Alginate 25 C. 
               
               
                 No treatment 50 C. 
               
               
                 0.1% Alginate 50 C. 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                   
                 value 
                   
                   
                   
                 ave-neg 
               
               
                 standard papain 
                 1 
                 value 2 
                 average 
                 negative 
                 absorbance 450 nm 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 3 
                 1.636 
                 1.677 
                 1.6565 
                 0.676 
                 0.9805 
               
               
                 2 
                 1.161 
                 1.16 
                 1.1605 
                 0.427 
                 0.7335 
               
               
                 1 
                 0.554 
                 0.55 
                 0.552 
                 0.232 
                 0.32 
               
               
                 0.5 
                 0.245 
                 0.245 
                 0.245 
                 0.12 
                 0.125 
               
               
                 0.25 
                 0.123 
                 0.127 
                 0.125 
                 0.071 
                 0.054 
               
               
                 0.1 
                 0.073 
                 0.075 
                 0.074 
                 0.049 
                 0.025 
               
               
                 0.05 
                 0.06 
                 0.06 
                 0.06 
                 0.042 
                 0.018 
               
               
                 0 
                 0.054 
                 0.061 
                 0.0575 
                 0.031 
                 0.0265 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 13 
               
               
                   
               
               
                 samples tested (4 week linked papain samples - prepared 
               
               
                 on Oct. 16, 2007 papain concentration 1%)) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 value 1 
                 value 2 
                 average 
                 negative 
                 ave-neg 
                 units/ml 
               
               
                   
               
               
                 No treatment 4 C. 
                 1.455 
                 1.15 
                 1.3025 
                 0.7145 
                 0.588 
                 1.754939546 
               
               
                 0.1% Alginate 4 C. 
                 1.328 
                 1.329 
                 1.3285 
                 0.6123 
                 0.7162 
                 2.132999115 
               
               
                 No treatment 25 C. 
                 0.758 
                 0.629 
                 0.6935 
                 0.5605 
                 0.133 
                 0.413152462 
               
               
                 0.1% Alginate 25 C. 
                 0.936 
                 0.9 
                 0.918 
                 0.563 
                 0.355 
                 1.0678266 
               
               
                 No treatment 50 C. 
                 0.691 
                 0.778 
                 0.7345 
                 0.734 
                 0.0005 
                 0.022412268 
               
               
                 0.1% Alginate 50 C. 
                 0.791 
                 0.746 
                 0.7685 
                 0.6776 
                 0.0909 
                 0.289000295 
               
               
                   
               
             
          
           
               
                 week 1 
               
             
          
           
               
                   
                 units/ml 
                 % of week o activity 
               
             
          
           
               
                   
                 week 0 
                 week 1- 
                 week 1- 
                 week 1 - 
                 week 1- 
                 week 1- 
                 week 1 - 
               
               
                   
                 units/ml 
                 4 C. 
                 25 C. 
                 50 C. 
                 4 C. 
                 25 C. 
                 50 C. 
               
               
                   
               
               
                 No treatment 
                 2.690 
                 2.292 
                 0.777 
                 0.143 
                 85.22 
                 28.9 
                 5.31 
               
               
                 alginate 
                 2.795 
                 2.581 
                 1.546 
                 0.986 
                 92.36 
                 55.32 
                 35.26 
               
             
          
           
               
                 week 2 
               
             
          
           
               
                   
                 units/ml 
                 % of week o activity 
               
             
          
           
               
                   
                 week 0 
                 week 2- 
                 week 2- 
                 week 2 - 
                 week 2- 
                 week 2- 
                 week 2 - 
               
               
                   
                 units/ml 
                 4 C. 
                 25 C. 
                 50 C. 
                 4 C. 
                 25 C. 
                 50 C. 
               
               
                   
               
               
                 No treatment 
                 2.690 
                 2.113 
                 0.673 
                 0.032 
                 78.56 
                 25 
                 1.2 
               
               
                 alginate 
                 2.795 
                 2.550 
                 1.258 
                 0.894 
                 91.23 
                 45 
                 32 
               
             
          
           
               
                 week 3 
               
             
          
           
               
                   
                 units/ml 
                 % of week o activity 
               
             
          
           
               
                   
                 week 0 
                 week 3- 
                 week 3- 
                 week 3 - 
                 week 3- 
                 week 3- 
                 week 3 - 
               
               
                   
                 units/ml 
                 4 C. 
                 25 C. 
                 50 C. 
                 4 C. 
                 25 C. 
                 50 C. 
               
               
                   
               
               
                 No treatment 
                 2.6902 
                 1.89 
                 0.60 
                 0.03 
                 70.24 
                 22.32 
                 1.03 
               
               
                 alginate 
                 2.7950 
                 2.24 
                 1.13 
                 0.59 
                 80.23 
                 40.32 
                 21.01 
               
             
          
           
               
                 week 4 
               
             
          
           
               
                   
                 units/ml 
                 % of week o activity 
               
             
          
           
               
                   
                 week 0 
                   
                 week 4- 
                 week 4 - 
                 week 4- 
                 week 4- 
                 week 4 - 
               
               
                   
                 units/ml 
                   
                 25 C. 
                 50 C. 
                 4 C. 
                 25 C. 
                 50 C. 
               
               
                   
               
               
                 No treatment 
                 2.690 
                 1.7549 
                 0.4132 
                 0.0224 
                 65.2394 
                 15.3588 
                 0.8332 
               
               
                 alginate 
                 2.795 
                 2.1330 
                 1.0678 
                 0.2890 
                 76.3148 
                 38.2049 
                 10.3399 
               
               
                   
               
             
          
           
               
                   
                 % of week 0 activity 
               
             
          
           
               
                   
                   
                 week 4- 
                 week 4- 
                 week 4 - 
               
               
                   
                   
                 4 C. 
                 25 C. 
                 50 C. 
               
               
                   
               
               
                   
                 No treatment 
                 65.239 
                 15.359 
                 0.833 
               
               
                   
                 0.1% Alginate 
                 76.315 
                 38.205 
                 10.340 
               
               
                   
               
             
          
         
       
     
     As can be seen from the results of table 13, the activity of stabilized papain which was stored with 0.1% sodium alginate is significantly greater over a variety of temperatures over 4 weeks than the activity of stabilized papain in the control samples which was not stored with sodium alginate. Accordingly, linked papain reacted with 0.1% sodium alginate is significantly more stable than the control samples of papain mentioned above which do not include sodium alginate. Furthermore,  FIG. 9  illustrates the results of table 13 that the activity of stabilized papain with sodium alginate was significantly greater over a variety of temperatures over 4 weeks than the activity of crosslinked papain in the control samples which did not include sodium alginate. 
     Furthermore, as shown in  FIG. 10 , a Sigma papain activity standard curve using absorbance vs. units/ml was developed for assisting in quantitating the activity of linked 1% papain together with 0.1% sodium alginate. 
     In other experiments, as shown in  FIG. 11 , the effect of sugar and sugar polymers on the stability of free papain after days at 25° C. and 40° C. were conducted. The sugar or sugar polymers included were 0.1% sodium alginate, 5% trehalose, 5% Mannitol, 5% Glycerol, 0.1 xanthan gum, 0.1% HA, 5% sucrose, 5% sorbitol and a no sugar control. From the results it is noted that 0.1% sodium alginate was found to have a stabilizing effect on free papain. This stabilizing effect of 0.1% sodium alginate was also observed on the stabilized papain of the present invention as well. 
     In still other experiments, as shown in  FIG. 12 , the percentage of the activity retained after 2 weeks of stabilized papain made with 0.4% papain was determined. In some samples of these experiments, the linked papain was treated with sodium alginate, in some samples the linked papain was treated with anhydride, in some samples the linked papain was treated with anhydride and sodium alginate and in some samples the linked papain was not treated at all with any of the above. As can be gleaned from  FIG. 12 , the samples which were treated with both sodium alginate and anhydride had the greatest % activity retained after each of the two weeks and the samples which were not treated at all had the lowest % activity retained after each of the two weeks. 
     Example 4 
     In example 4, immobilized papain was reacted with 1% DMA for secondary crosslinking and with 0.1% sodium alginate for further stability and included as part of a preservative system which comprises 1.2% phenoxyethanol+0.2% benzoic acid. The protocol, reactants, reaction conditions and results for determining the % of activity of experimental samples of stabilized papain in accordance with one embodiment of the present invention and control samples of a linked papain product (without DMA or sodium alginate) shown in Table 14 and are discussed below. 
     
       
         
               
             
               
               
               
               
             
               
             
               
               
             
               
               
               
             
               
               
             
               
             
           
               
                 TABLE 14 
               
               
                   
               
               
                 Assay protocol using Invitrogen E6639 EnzChek kit for enzyme activity 
               
               
                   
               
             
             
               
                 Papain standard curve 
               
             
          
           
               
                 STD # 
                 Stock solution (U/mL) 
                 Stock (1 U/mL) 
                 Assay buffer 
               
               
                   
               
               
                 1 
                 1 
                 1000 
                 0 
               
               
                 2 
                 0.5 
                 500 
                 500 
               
               
                 3 
                 0.25 
                 250 
                 750 
               
               
                 4 
                 0.1 
                 100 
                 900 
               
               
                 5 
                 0.05 
                 50 
                 950 
               
               
                 6 
                 0.01 
                 10 
                 990 
               
               
                 7 
                 0.005 
                 5 
                 995 
               
               
                 8 
                 0 
                 0 
                 1000 
               
               
                   
               
             
          
           
               
                 Prepare 1 Unit/mL stock solution of papain 
               
               
                 10 mg Sigma papain 
               
               
                 25 ml dl 1X digestion buffer 
               
               
                 Material &amp; buffer requirement: 
               
               
                 0.1M Sodium Bicarbonate, pH 8.3 
               
               
                 1x Digestion buffer: PBS pH 6 + 50 ul of TX-100 
               
               
                 1 Mg/ml stock of BODIPY TRX casein = 1 vial of substrate and dissolve 
               
               
                 in 0.2 ml of Bicarbonate buffer 
               
               
                 10 ug/mL working solution of BODIPY casein: 
               
               
                 Add 0.2 mL of stock substrate in 19.8 ml of Digestion buffer in 
               
               
                 50 ml falcon 
               
             
          
           
               
                 Assay: 
                 20 ul of STD/sample 
               
               
                   
                 80 ul of 1X digestion biffer 
               
               
                   
                 100 ul of 10 ug/mL working solution of BODIPY casein 
               
             
          
           
               
                 Samples tested contain 
                 1% DMA samples 
                 control 
               
               
                   
                 1% papain 600 (ESP) 
                 1% papain 600 (ESP) 
               
               
                   
                 1% DMA 
                 no DMA 
               
               
                   
                 0.1% Alginate 
                 0.1% Alginate 
               
               
                   
                 1.2% phenoxyethanol 
                 1.2% phenoxyethanol 
               
               
                   
                 0.2% Benzoic acid 
                 0.2% Benzoic acid 
               
             
          
           
               
                 Samples: 
                 25 ul of linked papain mixed with 150 ul of 1X 
               
               
                   
                 digestion buffer containing 0.1% Triton X-100 
               
             
          
           
               
                 Procedure: 
               
               
                 Incubate the plate at room temperature for one hour with moderate 
               
               
                 agitation. Protect the plate from light. Read the flurescence in a 
               
               
                 fluorescence microplate reader. Using standard fluorescein filters 
               
               
                 excitation 590/25 nm, emission 620/40 nm. 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
             
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 15 
               
               
                   
               
             
             
               
                 Linked papain samples with varying conc of other crosslinker (12 week samples) 
               
               
                 4 C. 
               
             
          
           
               
                 1 
                 LP - 1% DMA 
                 1:8 
                 25 
                 175 
                   
                   
                   
                   
                   
               
               
                 2 
                 LP-control 
                 1:8 
                 25 
                 175 
                   
                   
                   
                   
                   
               
             
          
           
               
                 25 C. 
               
             
          
           
               
                 1 
                 LP - 1% DMA 
                 1:8 
                 25 
                 175 
                   
                   
                   
                   
                   
               
               
                 2 
                 LP-control 
                 1:8 
                 25 
                 175 
                   
                   
                   
                   
                   
               
             
          
           
               
                 45 C. 
               
             
          
           
               
                 1 
                 LP - 1% DMA 
                 1:8 
                 25 
                 175 
                   
                   
                   
                   
                   
               
               
                 2 
                 LP-control 
                 1:8 
                 25 
                 175 
                   
                   
                   
                   
                   
               
             
          
           
               
                 Linked papain samples with or without 1% DMA (12 week samples) 
               
               
                 4 C. 
               
             
          
           
               
                 1 
                 LP - 1% DMA 
                 1:8 
                 5321 
                 5467 
                 5562 
                 5450.0 
                 4223 
                 0.580 
                 4.64 
               
               
                 2 
                 LP-control 
                 1:8 
                 4578 
                 4879 
                 5098 
                 4851.7 
                 3625 
                 0.485 
                 3.88 
               
             
          
           
               
                 25 C. 
               
             
          
           
               
                 1 
                 LP - 1% DMA 
                 1:8 
                 5098 
                 5291 
                 5120 
                 5169.7 
                 3943 
                 0.535 
                 4.28 
               
               
                 2 
                 LP-control 
                 1:8 
                 2789 
                 2908 
                 2871 
                 2856.0 
                 1629 
                 0.169 
                 1.35 
               
             
          
           
               
                 45 C. 
               
             
          
           
               
                 1 
                 LP - 1% DMA 
                 1:8 
                 2987 
                 2871 
                 3091 
                 2983.0 
                 1756 
                 0.189 
                 1.51 
               
               
                 2 
                 LP-control 
                 1:8 
                 1765 
                 1775 
                 1879 
                 1806.3 
                  579 
                 0.002 
                 0.02 
               
               
                   
               
             
          
           
               
                   
                 12 week data 
                 4 C. 
                 25 C. 
                 45 C. 
               
               
                   
               
               
                   
                 LP - 1% DMA 
                 4.638 
                 4.283 
                 1.511 
               
               
                   
                 LP-control 
                 3.880 
                 1.350 
                 0.019 
               
               
                   
               
             
          
           
               
                   
                 4 C. 
                 25 C. 
                 45 C. 
               
             
          
           
               
                   
                 units/ml 
               
               
                   
               
             
          
           
               
                 activity units/ml 
               
             
          
           
               
                   
                 0 week 
                 LP - 1% DMA 
                 4.87 
                 4.87 
                 4.87 
               
               
                   
                 data 
                 LP-control 
                 4.61 
                 4.61 
                 4.61 
               
               
                   
                 2 week 
                 LP - 1% DMA 
                 5.01 
                 5.25 
                 4.31 
               
               
                   
                 data 
                 LP-control 
                 4.81 
                 2.67 
                 2.1 
               
               
                   
                 4 week 
                 LP - 1% DMA 
                 5.12 
                 4.98 
                 3.11 
               
               
                   
                 data 
                 LP-control 
                 4.67 
                 2.01 
                 1.87 
               
               
                   
                 6 week 
                 LP - 1% DMA 
                 4.98 
                 4.61 
                 2.31 
               
               
                   
                 data 
                 LP-control 
                 4.18 
                 1.76 
                 1.02 
               
               
                   
                 8 week 
                 LP - 1% DMA 
                 4.81 
                 4.44 
                 1.98 
               
               
                   
                 data 
                 LP-control 
                 4.01 
                 1.35 
                 0.5 
               
               
                   
                 12 week  
                 LP - 1% DMA 
                 4.64 
                 4.28 
                 1.51 
               
               
                   
                 data 
                 LP-control 
                 3.88 
                 1.35 
                 0.02 
               
             
          
           
               
                 % activity retained after storing 
               
             
          
           
               
                   
                 2 week 
                 LP - 1% DMA 
                 102.87 
                 107.80 
                 88.50 
               
               
                   
                 data 
                 LP-control 
                 104.34 
                 57.92 
                 45.55 
               
               
                   
                 4 week 
                 LP - 1% DMA 
                 105.13 
                 102.26 
                 63.86 
               
               
                   
                 data 
                 LP-control 
                 101.30 
                 43.60 
                 40.56 
               
               
                   
                 6 week 
                 LP - 1% DMA 
                 102.26 
                 94.66 
                 47.43 
               
               
                   
                 data 
                 LP-control 
                 90.67 
                 38.18 
                 22.13 
               
               
                   
                 8 week 
                 LP - 1% DMA 
                 98.77 
                 91.17 
                 40.66 
               
               
                   
                 data 
                 LP-control 
                 86.98 
                 29.28 
                 10.85 
               
               
                   
                 12 week  
                 LP- 1% DMA 
                 95.24 
                 87.94 
                 31.02 
               
               
                   
                 data 
                 LP-control 
                 84.16 
                 29.28 
                 0.43 
               
               
                   
               
             
          
         
       
     
     As noted above, the experimental samples in accordance with an exemplary embodiment of the present invention includes 1% DMA, 1% papain 600 (ESP), 0.1% sodium alginate, 1.2 phenoxyethanol+0.2% benzoic acid. The control samples included all the same components as the experimental formulation sample, except the control samples did not include DMA. The results for effect of DMA on the retention activity of linked papain over a 12 week period at various temperatures are set forth below. Moreover, the chemistry of the DMA, linked papain and the sodium alginate are illustrated in  FIG. 13(   a ) 
     As can be gleaned from the above data and  FIGS. 13  ( b ),  13 ( c ),  14  and  15 , the crosslinking of linked papain with 1% DMA significantly improved the stability of the linked papain product over a variety of temperatures over a 12 week period in comparison to the control samples which did not contain any DMA. 
     Furthermore, as set forth above in table 15, papain activity in the experimental sample and the control were determined based upon fluorescence change per unit sample of the linked papain. A Sigma papain standard curve as shown in  FIG. 16  was developed for assistance in quantitating the activity of immobilized 1% papain which was further crosslinked with 1% DMA in accordance with the present embodiment. As noted above, an EnzChek® protease assay kit (E-6639) was used to obtain the standard curve and the fluorescence change per unit sample of the experimental samples of immobilized papain with DMA in accordance with the present exemplary embodiment and the control samples without DMA. 
     Example 5 
     In another exemplary embodiment, immobilized papain was reacted with 5% DMA for further crosslinking and with 0.1% sodium alginate and included as part of the preservative system which comprises 1.2% phenoxyethanol+0.2% benzoic acid. The protocol and results for this embodiment are set forth below. The placebo formulation contains all of the components of the experimental formulation, except for DMA. 
     
       
         
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 16 
               
               
                   
               
             
             
               
                 11 week 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Formulation 
                 Dilution 
                   
                   
                   
                   
                 average- 
                   
                   
               
               
                 samples 
                 Factor 
                 Read 1 
                 Read 2 
                 Read 3 
                 Average 
                 negative 
                 units/ml 
                 units/ml 
               
               
                   
               
             
          
           
               
                 4 C. 
               
             
          
           
               
                 963030/1 
                 1:4 
                 1245 
                 1342 
                 1290 
                 1292.3 
                 151 
                 0.055 
                 0.22 
               
               
                 963030/3 
                 1:4 
                 1677 
                 1549 
                 1682 
                 1636.0 
                 495 
                 0.244 
                 0.97 
               
             
          
           
               
                 25 C. 
               
             
          
           
               
                 963030/1 
                 1:4 
                 1245 
                 1232 
                 1310 
                 1262.3 
                 121 
                 0.039 
                 0.15 
               
               
                 963030/3 
                 1:4 
                 1581 
                 1621 
                 1590 
                 1597.3 
                 456 
                 0.222 
                 0.89 
               
             
          
           
               
                 45 C. 
               
             
          
           
               
                 963030/1 
                 1:4 
                 1245 
                 1154 
                 1167 
                 1188.7 
                 48 
                 −0.002 
                 −0.01 
               
               
                 963030/3 
                 1:4 
                 1576 
                 1582 
                 1501 
                 1553.0 
                 412 
                 0.198 
                 0.79 
               
               
                   
               
             
          
           
               
                   
                 11 week 
                   
                   
                   
               
               
                   
                 formulation 
                   
                   
                   
               
               
                   
                 samples 
                 4 C. 
                 25 C. 
                 45 C. 
               
               
                   
               
               
                   
                 963030/1 
                 0.22 
                 0.15 
                 −0.01 
               
               
                   
                 963030/3 
                 0.97 
                 0.89 
                 0.79 
               
               
                   
               
             
          
           
               
                   
                   
                 formula 
                   
                   
                   
               
               
                   
                   
                 number 
                 4 C. 
                 25 C. 
                 45 C. 
               
               
                   
               
               
                 0 week samples 
                 placebo 
                 963030/1 
                 0.98 
                 0.98 
                 0.98 
               
               
                   
                 5% DMA 
                 963030/3 
                 1.09 
                 1.09 
                 1.09 
               
               
                   
                 sample 
                   
                   
                   
                   
               
               
                 2 week samples 
                 placebo 
                 963030/1 
                 0.85 
                 0.82 
                 0.41 
               
               
                   
                 5% DMA 
                 963030/3 
                 1.12 
                 1.21 
                 1.05 
               
               
                   
                 sample 
                   
                   
                   
                   
               
               
                 4 week samples 
                 placebo 
                 963030/1 
                 0.61 
                 0.71 
                 0.31 
               
               
                   
                 5% DMA 
                 963030/3 
                 1.02 
                 1.02 
                 0.92 
               
               
                   
                 sample 
                   
                   
                   
                   
               
               
                 8 week samples 
                 placebo 
                 963030/1 
                 0.44 
                 0.31 
                 0.15 
               
               
                   
                 5% DMA 
                 963030/3 
                 1.00 
                 0.95 
                 0.85 
               
               
                   
                 sample 
                   
                   
                   
                   
               
               
                 11 week samples  
                 placebo 
                 963030/1 
                 0.22 
                 0.15 
                 −0.01 
               
               
                   
                 5% DMA 
                 963030/3 
                 0.97 
                 0.89 
                 0.79 
               
               
                   
                 sample 
                   
                   
                   
                   
               
             
          
           
               
                 % 0f week 0 activity 
               
             
          
           
               
                 2 week samples 
                 placebo 
                 963030/1 
                 86.73 
                 83.67 
                 41.84 
               
               
                   
                 5% DMA 
                 963030/3 
                 102.75 
                 111.01 
                 96.33 
               
               
                   
                 sample 
                   
                   
                   
                   
               
               
                 4 week samples 
                 placebo 
                 963030/1 
                 62.24 
                 72.45 
                 31.63 
               
               
                   
                 5% DMA 
                 963030/3 
                 93.58 
                 93.58 
                 84.40 
               
               
                   
                 sample 
                   
                   
                   
                   
               
               
                 8 week samples 
                 placebo 
                 963030/1 
                 44.90 
                 31.63 
                 15.31 
               
               
                   
                 5% DMA 
                 963030/3 
                 91.74 
                 87.16 
                 77.98 
               
               
                   
                 sample 
                   
                   
                   
                   
               
               
                 11 week samples  
                 placebo 
                 963030/1 
                 22.47 
                 15.75 
                 −0.74 
               
               
                   
                 5% DMA 
                 963030/3 
                 89.38 
                 81.60 
                 72.67 
               
               
                   
                 sample 
               
               
                   
               
             
          
         
       
     
     As can be gleaned from the above data table and  FIGS. 17-19 , crosslinking of immobilized papain with 5% DMA significantly improved the stability of the immobilized papain product over a variety of temperatures over a 12 week period in comparison to the control samples which did not contain any DMA. 
     Furthermore, as set forth in the above data tables, papain activity in the experimental sample and the control were determined based upon fluorescence change per unit sample of the immobilized papain. A standard curve for Sigma papain the in placebo formulation as shown in  FIG. 20  was developed for assistance in quantitating the activity of immobilized 1% papain cross-linked with 5% DMA in accordance with the present embodiment. As noted above, an EnzChek® protease assay kit (E-6639) was used to obtain the standard curve and the fluorescence change per unit sample of the experimental sample of linked papain with DMA in accordance with the present exemplary embodiment and the control sample without DMA. 
     Examples 6-8 
     The following represent nonlimiting examples of formulations that can be prepared using one or more stabilized proteases. A wide variety of similar formulations are known in the art into which one or more stabilized proteases can readily be incorporated at various concentrations. 
     Example 6 
     An exemplary elastic cream gel has, for example, the composition shown in the table below: 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 PHASE 
                 % 
                 SUPPLIER 
                 RAW MATERIAL 
                 INCI NOMENCLATURE 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 A 
                 63.300 
                 Local 
                 Deionized Water 
                 Water 
               
               
                 A 
                 0.050 
                 Local 
                 Disodium EDTA 
                 Disodium EDTA 
               
               
                 A 
                 1.100 
                 Clariant 
                 Aristoflex AVC/USA 
                 Ammonium Acryloyldimethyltaurate/VP Copolymer 
               
               
                 A 
                 0.150 
                 Clariant 
                 Aristoflex HMB 
                 Ammonium Acryloyldimethyltaurate/Beheneth-25 
               
               
                   
                   
                   
                   
                 Methacrylate Crosspolymer 
               
               
                 A 
                 3.000 
                 Local 
                 Butylene Glycol 
                 Butylene Glycol 
               
               
                 A 
                 2.000 
                 Local 
                 Glycerin 
                 Glycerin 
               
               
                 B 
                 15.000 
                 Momentive 
                 Velvesil DM 
                 Dimethicone and Cetearyl Dimethicone Crosspolymer 
               
               
                 B 
                 3.000 
                 Dow Corning 
                 Dow Corning 1413 
                 Dimethicone 
               
               
                 C 
                 1.200 
                 Seppic 
                 Simulgel NS 
                 Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate 
               
               
                   
                   
                   
                   
                 Copolymer and Squalane and Polysorbate 60 
               
               
                 D 
                 3.000 
                 BASF 
                 Stabilized papain of the 
                   
               
               
                   
                   
                   
                 present invention 
                   
               
               
                 D 
                 1.000 
                 BASF 
                 Germazide PSB 
                 Phenoxyethanol, Chlorphenesin, Benzoic Acid, Butylene 
               
               
                   
                   
                   
                   
                 Glycol, Sorbic Acid 
               
               
                 E 
                 2.000 
                 Alzo 
                 CUPL PIC 
                 PPG-2 Isoceteth-20 Acetate 
               
               
                 E 
                 2.000 
                 Firmenich, Inc. 
                 Hedione 
                 Methyldihydrojasmonate 
               
               
                 F 
                 0.100 
                 BASF 
                 Cloisonne Red 424C 
                 Mica and Titanium Dioxide and Carmine 
               
               
                 F 
                 0.100 
                 BASF 
                 Timica Silver Sparkle 
                 Mica and Titanium Dioxide 
               
               
                   
                   
                   
                 5500 
                   
               
               
                 F 
                 3.000 
                 Local 
                 Water 
                 Water 
               
               
                   
               
             
          
         
       
     
     In a main vessel, combine Phase A. Homo-mix and sweep-mix until powder is hydrated and batch is uniform. When Phase A is uniform, add phase B to main vessel while homo-mixing. Mix until uniform. Add phase C to main vessel while homo-mixing. Mix until uniform. Add phase D to main vessel while homo-mixing. Mix until uniform. Warm phase E ingredients to 150 to 40° C. until melted. Premix Phase E and add to main vessel while homo-mixing. Premix Phase F. Mix until all pearls are suspended and there are no clumps. Continue mixing during addition. Add Phase F to main vessel while homo-mixing. Turn off homo-mixer after addition and sweep-mix until uniform. 
     Example 7 
     An exemplary lotion has, for example, the composition shown in the table below 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 PHASE 
                 % 
                 SUPPLIER 
                 RAW MATERIAL 
                 INCI NOMENCLATURE 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 A 
                 85.50 
                 Local 
                 Deionized Water 
                 Water 
               
               
                 A 
                 0.050 
                 Local 
                 Disodium EDTA 
                 Disodium EDTA 
               
               
                 A 
                 3.0 
                 Local 
                 1,3 Butylene Glycol 
                 Butylene Glycol 
               
               
                 A 
                 1.0 
                 Clariant 
                 Aristoflex AVC/USA 
                 Ammonium Acryloyldimethyltaurate/VP Copolymer 
               
               
                 B 
                 8.0 
                 Inolex 
                 Lexol GT 865 
                 Caprylic/Capric Triglyceride 
               
               
                 B 
                 0.25 
                 Arlacel 165V 
                 Croda 
                 Glyceryl Stearate and PEG-100 Stearate 
               
               
                 C 
                 3.0 
                   
                 Stabilized papain of the 
                   
               
               
                   
                   
                   
                 present invention 
                   
               
               
                 D 
                 1.2 
                 BASF 
                 Germazide PSB 
                 Phenoxyethanol and Chlorphenesin and Benzoic Acid 
               
               
                   
                   
                   
                   
                 and Butylene Glycol and Sorbic Acid 
               
               
                 F 
                 3.000 
                 Local 
                 Water 
                 Water 
               
               
                   
               
             
          
         
       
     
     Mix phase A until homogenous while heating to 65-70° C. In separate vessel, premix phase B. Heat to 70-75° C. or until homogenous. When both phases A and B are at desired temperature, add phase B to phase A and homomix until uniform. When Phase AB is uniform, begin cooling add Phase C at 35-30° C. and mix under prop mixer until uniform. Add pre-mixed Phase D to Phase ABC under homomixer. Mix until uniform. 
     Example 8 
     In this embodiment, the stability of linked papain at various temperatures for up to 12 weeks in an O/W formulation. The results were obtained by a papain activity assay using an Invitrogen EnzChek assay kit after suspending the formulation in 0.1% Triton X-100, n=3. 
     As shown in  FIG. 21 , the linked papain product is further stabilized by an oil in water formulation. About 75% activity is retained after 12 weeks at 45 C. Formulation was found stable at all temperatures tested. These results indicate that when crosslinked papain products are included in an oil and water formulation that this product is even further stabilized. 
     Having described the exemplary embodiments of the present invention, it is further noted that it is readily apparent to those of reasonable skill in the art that various modifications may be made without departing from the spirit and scope of the invention which is defined by the metes and bounds of the appended claims.