Abstract:
Single-solution adhesive resin formulations are made of predominantly dimethacrylates or diacrylates containing predominantly) two carboxyl groups and a functionalized methacrylate or acrylate diluent monomer. A novel light-activated initiator can be used for photopolymerization of such adhesives.

Description:
This application is a divisional of U.S. Ser. No. 09/019,604, filed Feb. 6, 1998, pending. 
    
    
     This invention was made under National Institute of dental Research grant R37 DE05129-19 and P50 DE09322-07 from the National Institutes of Health. The government therefore retains certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Most bonding systems that contain carboxylated monomers for priming and hydrophilic monomers for bonding require a number of consecutive procedural steps after conditioning with an acid which are aimed at optimizing the adhesion to dentin. These include (1) mixing of a primer monomer with a co-monomer/co-initiator, usually a secondary or tertiary aromatic amine, e.g. mono- or bivalent salts of bis-(N-tolyl glycine glycidyl methacrylate) and (2) priming of the prepared dentin surface with this mixture(Bowen, 1985; Bowen, 1986; Bowen, 1987; Venz and Dickens, 1991; Venz and Dickens, 1993; Bowen, 1993; Bowen, 1994; Ibsen et al., 1994; Suh et al., 1994). The third step comprises application of an unfilled bonding resin, which will then be light-cured. The bonding resin consists mainly of 2,2-bis[p(2′-hydroxy-3′-methacryloxypropoxy)phenylene]-propane (bis-GMA) and 2-hydroxyethyl methacrylate (2-HEMA). 
     A different approach was taken by Waknine (1992) who disclosed a three-step adhesive system that contains a nonacidic carbonate monomer instead of the carboxylated resin and a different type of amine accelerator. The bond strengths reported by Waknine are on the order of about 10 MPa. 
     Recently, abstracts describing bonding to dentin with one-component (better described as one-solution) adhesive resin systems have been reported (Jia et al., 1996; Kelsey et al., 1996; Latta et al., 1996; Qian et al., 1996; Dickens et al., 1997). More recently, a patent (Qian et al., August 1997, U.S. Pat. No. 5,658,963) has been issued describing one-step formulations. These adhesive resin formulations comprise in a single solution the components that were applied in the multistep system in several sequential steps as described above. The components are: the bonding resin consisting of a viscous monomer, bis-GMA, and the diluent monomer 2-HEMA, as well as light-sensitive polymerization catalysts, an amine accelerator, and acetone and/or ethanol as the solvent(s). 
     Various U.S. patents disclose use of other one-solution systems. Bunker and Fields, (1985); Aasen, (1988); Bunker, (1994) describe polymerizable phosphate esters. However, these bonding systems yielded a relatively low bond strength to dentin of about 9 MPa Also, these bonding systems contain a multitude of polymerizable components in their systems, among which are phosphate esters. These esters can hydrolyze, thus releasing free acid, which can, in turn, lead to hydrolysis of methacrylate esters resulting in the release of methacrylic acid and loss of the polymerizable functionality on the resin Thus, phosphate esters are less stable than the carboxylic acid esters described in this invention. Bunker et al (1985), reported mixing the adhesive with a solution of sodium benzene sulfinate as a coinitiator, which adds another step to these so-called one-step formulations. 
     James (1987) describes adhesive compositions for tooth enamel, but does not teach adhesion to dentin. 
     Blackwell and Huang (1987, 1989) and Huang and Blackwell (1990) describe phosphate esters of mono- or dipentaerythritol. However, use of the experimental formulations reported by Blackwell and Huang (1987, 1989) resulted in relatively low bond strengths to dentin (about 8.4 MPa). Higher bond strengths were reported by Huang and Blackwell (1990) but only if an unfilled bonding resin was applied as a second step after priming Also, the storage stability of these phosphate-containing resins was less than desirable, possibly due to the above described hydrolysis of the ester group. 
     There is a need in the art for new methods and compositions for adhesives for use in restoring teeth in which the bond strength is good, the steps and reagents are kept to a minimum, and the time required for the restoration is short. 
     SUMMARY OF THE INVENTION 
     It is an object of the invention to provide an adhesive resin formulation. 
     It is another object of the invention to provide a method of bonding a composite resin to a prepared surface. 
     It is still another object of the invention to provide a method of producing a formulation for bonding a composite resin to a prepared surface. 
     It is yet another object of the invention to provide kits for bonding a composite resin to a prepared surface. 
     These and other objects of the invention are provided by one or more embodiments described below. In one embodiment a single-solution adhesive resin formulation for bonding a composite resin to enamel, dentin or nonprecious metal is provided. The formulation can also be used for bonding a ceramic or a nonprecious metal to dentin. The formulation comprises: not more than two polymerizable monomers and a photoinitiator, wherein a first of the two monomers is an acidic monomer and the second of the two monomers is a diluent monomer. 
     In another embodiment of the invention a single-solution adhesive resin formulation for bonding a composite resin to enamel, dentin or nonprecious metal is provided. The formulation can also be used for bonding a ceramic or a nonprecious metal to dentin. The formulation comprises: not more than two polymerizable monomers, a solvent, and a photoinitiator, wherein a first of the two monomers is pyromellitic glycerol dimethyacrylate (PMGDM) monomer, the second of the two monomers is 2-HEA monomer, and the photoinitiator is 2, 4, 6-trimethylbenzoyldiphenylphosphine oxide. 
     In another embodiment of the invention a method of bonding a composite resin to enamel, dentin or nonprecious metal is provided. The method can also be used for bonding a ceramic or a nonprecious metal to dentin. The method comprises: applying an adhesive resin formulation onto a prepared surface selected from the group consisting of enamel, dentin or nonprecious metal; air drying the adhesive resin formulation; repeating the steps of applying and air drying between 0 and 2 times; photopolymerizing the adhesive resin formulation; contacting the formulation with a composite resin; and photopolymerizing the composite resin to form a bond having a shear bond strength of at least 20 MPa. 
     According to another aspect of the invention a method of producing a formulation for bonding a composite resin to enamel, dentin, or nonprecious metal, or a ceramic or a non-precious metal to dentin, is provided. The formulation can also be used for bonding a ceramic or a nonprecious metal to dentin. The method comprises the step of: combining not more than two monomers and a photoinitiator, wherein a first of the two monomers is an acidic monomer and the second of the two monomers is a diluent monomer. 
     In yet another embodiment of the invention a kit for bonding a composite resin to enamel, dentin or metal is provided. The kit can also be used for bonding a ceramic or a nonprecious metal to dentin. The method comprises: 
     a formulation which comprises not more than two monomers and a photoinitiator, wherein a first of the two monomers is a carboxylic acid monomer and the second of the two monomers is a diluent monomer, and a composite resin. 
     The present invention thus provides the art with a single-solution adhesive resin formulation for bonding to enamel, dentin, or nonprecious metal, or for bonding a ceramic or a nonprecious metal to dentin. This formulation simplifies and economizes the bonding procedure by allowing application of the bonding system to conditioned tooth surfaces or to nonprecious metal surfaces in a single step, in contrast to the multistep procedure employed in conventional systems. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     It is a discovery of the present inventor that a successful adhesive resin formulation can be made by simply combining a single acidic primer monomer with a single functionalized diluent monomer in a solvent. The mixture preferably also contains a photoinitiator (camphorquinone) and a tertiary amine accelerator (such as 4-N,N-(dimethylamino)benzoate (4-EDMAB), or, as an alternative, a photoinitiator that does not require an amnine accelerator as does camphorquinone. The solution can be painted onto the dentin once, air dried and then optionally applied two more times, gently dried and photopolymerized. In addition to the time saved by the dental practitioner by requiring only one solution to prepare the tooth surface for bonding, this solution comprises only two resin components and the solvent, as opposed to three resin components and solvent employed in conventional multistep systems. 
     The alternative photoinitiator employed here does not require a tertiary amine accelerator. Tertiary amine accelerators may contribute to unwanted radical formation by the acidic monomers during storage. They may also cause premature curing of the formulation, which results in decreased shear bond strengths of up to 30% after storage of the bonding solution at elevated temperatures (37° C.) for several months (U.S. Pat. No. 5,658,963, at Example 27). Thus elimination of tertiary amines greatly improves the shelf life of the single-solution adhesive resin system. 
     The present invention is based on only two polymerizable monomers, as opposed to three monomers described in U.S. Pat. No. 5,658,963. Acidic monomers which can be used include carboxylic acid monomers, pyromellitic dianhydride-HEMA adduct PMDM), pyromellitic glycerol dimethyacrylate (PMGDM), biphenyl dianhydride-HEMA adduct (BPDM), 3,3′4,4′-tetracarboxylic dianhydride BPGDM and 5-(2,5dioxotetrahydro-3-furanyl)-3-cyclohexene-1,2-dicarboxylic anhydride glycerol dimethacylate adduct (B4400-GDM). Diluent monomers include 2-hydroxyeylmethyacrylate, glycerol dimethacrylate (GDM), ethyl-α-hydroxymethyl acrylate EHMA), and tetrahydro furfurylmethacrylate (THFM). Selection of suitable monomers is well within the skill of the art. The adhesive resin systems of the present invention yield bond strengths between 20 and 40 MPa. These compare favorably with prior art one-solution formulations (One-step; Bisco Inc.), which were reported to have dentin shear bond strengths between 22 MPa and 30 MPa, and bond strengths to enamel or nonprecious metal of 27 MPa to 28 MPa (U.S. Pat. No. 5,658,963, at Examples 1-24). 
     Photoinitiators for polymerization of the adhesive resin can be any known in the art, such as camphorquinone and a tertiary amine. The photoinitiator preferably employed in this invention is 2,4,6-trimethylbenzoyldiphenylphosphine oxide, also known as photoinitiator 8728 (Ward Blenkinsop, Cheshire, UK). It is an acyl phosphine oxide. Reactions of various phosphines with olefinic double bonds have been reported by Morgan et al. as early as 1978. Use of acylphosphine oxides as photointiators have been reported in various German patents (Nos. 2,830,927 and 2,909,994) and in U.S. Pat. No. 4,710,523. An acylphosphine compound for use in polyenepolythiol dental impression compositions has been described in U.S. Pat. No. 5,100,929. However, monoacylphosphine oxides do not cure composite resins to a great depth; they cause a rather shallow curing. This is mainly due to their maximum absorbance at about 380 nm, which is a considerably shorter wavelength than that at which camphorquinone has its maximum absorbance. The absorbance of camphorquinone at 470 nm causes camphorquinonelteruary amine-activated composite resins to cure after 20 s irradiation to a depth of about 5.2 mm compared to 2.9 mm for the monoacylphosphine oxide-activated composite resin. However, use of the monoacylphosphine oxides as initiators for adhesive resin systems as described in this invention does not require a significant curing depth, since adhesive resins are usually applied in thin layers up to a thickness of 200 μm. Monoacylphosphine oxides have not been used previously in adhesive resin systems. Bis-acylphosphine oxides have been described in European Patent No. 184 095. Since they absorb at a slightly higher wavelength than the monoacylphosphine oxides, their depth of cure is somewhat greater and they can be used in conventional dental composite resins. 
     Conditioning of dentin or enamel prior to the application of the single-solution adhesive resin system of the present invention can be achieved by any method known in the art. For example, etching with phosphoric acid can be used, as is conventional. Alternatively, a polymerrible conditioner can be used as described by Dickens (1996). Nonprecious metals which can be used as substrates for the adhesive resin system include nickel-chromium alloys (e.g.,Rexillium®, and chromium cobalt alloys). 
     The adhesive resin PMGDM can be prepared from 1 part pyromellitic dianhydride (PMDA) and two parts glycerol dimethacrylate (GDM). The preferred ratio of PMDA to GDM is 1:2.05 giving a slight excess of GDM to avoid the remaining of unreacted toxic dianhydride. 
    
    
     The following examples are provided for exemplification purposes only and are not intended to limit the scope of the invention which has been described in broad terms above. 
     METHODS USED IN THE EXAMPLES 
     Sample Preparation 
     Dentin samples were prepared and tested by following the procedure outlined by Venz and Dickens (1993). Extracted, noncarious human molars that had been stored in 0.2% sodium azide at 5° C. were cut with a slowly rotating diamond blade (Isomet, Buehler Ltd., Lake Bluff Ill.) under running water until a smooth dentin surface was exposed. The human teeth with exposed coronal dentin, bovine enamel slabs or cast Rexliun® (Jeneric/Pentron, Inc., Wallingford, Conn.) stubs for testing adhesion to metal were mounted in cylindrical polycarbonate holders with self-curing acrylic. The dentin and enamel specimens were stored in distllled water at room temperature until they were used for bonding. Prior to bonding all specimens were ground under water irrigation on 320 grit SiC paper to generate standardized surface conditions. The metal specimens were then sandblasted with 50 μm Al 2 O 3  powder and rinsed twice with 95% ethanol. 
     Bonding Protocol 
     The conditioners used to prepare the tooth surfaces prior to the application of the adhesive resin system were either 37% 1  phosphoric acid gel (Myron International, Kansas City, Kans.), the polymerizable conditioner PYROMELLITIC BIS-[P(2′-HYDROXY-3′-METHACRYLOXY-PROPOXY) PHENYLENE]PROPANE, which is a monomer that has been synthesized from a bifunctional dimethacrylate resin and a carboxylic acid dianhydride, or PYROMELLITIC BIS-[P(2′-HYDROXY-3′-METHACRYLOXY-PROPOXY)PHENYLENE]PROPANE combined with vinyl phosphonic acid. The phosphoric acid gel was rinsed off after 20 seconds. The polymerizable conditioners were agitated on the tooth surfaces for 1 minute and then rinsed. After removing excess water, all surfaces were kept under moist tissue paper. Two drops of the single-solution adhesive were dispensed into a well. A small brush saturated with the solution was pressed with circular movements against the tooth surface, thus allowing the solvent to evaporate during the agitation. A short blast of air was used to speed up the evaporation of the solvent. After that two more applications with the applicator brush were applied, followed by careful drying to remove residual solvent without blowing the resin off the surface. The control (multistep) treatment comprised the following steps: The priming resin PMGDM, which is the addition product of pyromellitic dianhydride and glycerol dimethacrylate (Venz and Dickens, 1991 and 1993), was activated by combining 40 μL (two drops) of 20% PMGDM in acetone and 20 μL (one drop) of 5% of the polymerization catalyst, magnesium bis(N-p-tolylglycine glycidylmethacrylate) [Mg(NTG-GMA) 2 ] in acetone in a dappen dish. Five coatings of the mixture were painted onto the tooth surface, allowing the acetone to evaporate between applications. After five coats the primed surface was carefully dried to remove residual solvent. After application of the activated PMGDM, photopolymerizable unfilled resin was dabbed on the primed surfaces, thinned with an air stream and light-cured for 20 s with a commercial dental curing light (the MAX, Caulk/Dentsply, Milford, Del.). A TEFLON®-coated metal iris with an internal diameter of 4 mm was placed directly on the treated surface. The cavity in the iris was filled with composite (APK Caulk/Dentsply, Miford, Del.), which was then irradiated for 1 min with the curing light. The assembly was left undisturbed for an additional 4 min and then stored in distilled water for 24 h at room temperature prior to testing in shear mode at a cross-head speed of 0.5 mm per min. 
     Example 1 
     Shear bond strengths to dentin mediated by the single-solution formulation were evaluated after phosphoric acid conditioning and compared to the multi-step control. 
     
       
         
               
             
               
               
               
               
             
           
               
                   
               
               
                 Dentin Shear Bond Strengths (SBS) in MPa with the 
               
               
                 Single-Solution Adhesive Resin System 
               
             
          
           
               
                   
                   
                 Sec- 
                   
               
               
                   
                   
                 ond 
               
               
                   
                   
                 (sep- 
                   
               
               
                   
                 Resin composition 
                 arate) 
                 SBS in MPa 
               
               
                 Adhesive system 
                 in acetone (first step) 
                 step 
                 mean (n = 5)  sd* 
               
               
                   
               
               
                 Single-solution A 
                 33% PMGDM #   
                 none 
                 25 ± 5 
               
               
                   
                 10% 2-HEMA 
               
               
                 Single-solution B 
                 38% PMGDM #   
                 none 
                 26 ± 4 
               
               
                   
                 5% 2-HEMA 
               
               
                 Single-solution C 
                 33% PMGDM #   
                 none 
                 22 ± 4 
               
               
                   
                 10% THFM a   
               
               
                 Single solution D 
                 38% PMGDM #   
                 none 
                 20 ± 2 
               
               
                   
                 5% THFM 
               
               
                 Multistep 
                 20% PMGDM #   
                 Un- 
                 25 ± 7 
               
               
                 (Control) 
                 5% Mg(NTG- 
                 filled 
               
               
                   
                 GMA) 2   
                 Bond- 
               
               
                   
                   
                 ing 
               
               
                   
                   
                 resin 
               
               
                   
               
               
                 dentin was conditioned with 37% phosphoric acid gel  
               
               
                   # ali single-solution resins were activated with 0.2% camphorquinone and 0.8% 4-EDMAB;  
               
               
                   ## the multistep primer was activated with 0.2% camphorquinone and 2% benzoyl peroxide  
               
               
                 *standard deviation  
               
               
                   a tetrahydrofurfuryl methacrylate  
               
             
          
         
       
     
     Example 2 
     The experiments with singe-solution adhesives A to D from Example 1 were repeated. 
     
       
         
               
             
               
               
               
               
             
           
               
                   
               
               
                 Dentin Shear Bond Strengths (SBS) in MPa with the 
               
               
                 Single-Solution Adhesive Resin System 
               
             
          
           
               
                   
                   
                 Sec- 
                   
               
               
                   
                   
                 ond 
               
               
                   
                   
                 (sep- 
               
               
                   
                 Resin composition 
                 arate) 
                 SBS in MPa 
               
               
                 Adhesive system 
                 in acetone (first step) 
                 step 
                 mean (n = 7)  sd* 
               
               
                   
               
               
                 Single-solution A 
                 33% PMGDM #   
                 none 
                 35 ± 5 
               
               
                   
                 10% 2-HEMA 
               
               
                 Single-solution B 
                 38% PMGDM #   
                 none 
                 27 ± 7 
               
               
                   
                 5% 2-HEMA 
               
               
                 Single-solution C 
                 33% PMGDM #   
                 none 
                 25 ± 6 
               
               
                   
                 10% THFM a   
               
               
                 Single solution D 
                 38% PMGDM #   
                 none 
                 27 ± 6 
               
               
                   
                 5% THFM 
               
               
                   
               
               
                 dentin was conditioned with 37% phosphoric acid gel  
               
               
                   # all single-solution resins were activated with 0.2% camphorquinone and 0.8% 4-EDMAB;  
               
               
                 *standard deviation  
               
               
                   a tetrahydrofurfuryl methacrylate  
               
             
          
         
       
     
     Example 3 
     The experiments with single-solution adhesive A (33% PMGDM and 10% 2-HEMA) were repeated using either phosphoric acid gel or the polymerizable conditioner PMBGA for conditioning. The single-solution adhesive A was activated either with camphorquinone and 4-EDMAB or Lucerin 8728. 
     
       
         
               
             
               
               
               
               
             
           
               
                   
               
               
                 Dentin Shear Bond Strengths (SBS) in MPa with 
               
               
                 Single-Solution Adhesive Resin System A 
               
               
                 (33% PMGDM and 10% 2-HEMA) varying the 
               
               
                 Photoinitiator and the Conditioner 
               
             
          
           
               
                 System 
                   
                   
                 SBS in MPa 
               
               
                 Code 
                 Photoinitiator 
                 Conditioner 
                 mean (n = 5)  sd* 
               
               
                   
               
               
                 A/CQ-ED 
                 0.2% camphorquinone 
                 PMBGA 
                 36 ± 6 
               
               
                   
                 0.8% 4-EDMAB 
               
               
                 A/LR 
                 1% Lucerin 8728 
                 PMBGA 
                 36 ± 5 
               
               
                 A/CQ-ED 
                 0.2% camphorquinone 
                 37% phos- 
                 36 ± 4 
               
               
                   
                   
                 phoric acid 
               
               
                   
                   
                 gel 
               
               
                   
                 0.8% 4-EDMAB 
                 acid 
               
               
                 A/LR 
                 1% Lucerin 8728 
                 37% phos- 
                 39 ± 4 
               
               
                   
                   
                 phoric acid 
               
               
                   
                   
                 gel 
               
               
                   
               
               
                 *standard deviation  
               
             
          
         
       
     
     Example 4 
     Single-Solution Adhesive Resin System A (33% PMGDM and 10% 2-HEMA) activated with Lucerin 8728 was tested on the nonprecious metal Rexillium® and on bovine enamel, which was conditioned with the polymerizable conditioner PMBGA+vinyl phosphonic acid as described in the U.S. patent application Ser. No. 08/785,174 (Dickens, 1997). 
     
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 Shear Bond Strengths (SBS) to Enamel and Metal with Single- 
               
               
                 Solution Adhesive Resin System A/LR (33% PMGDM and 10% 
               
               
                 2-HEMA, activated with 1% Lucerin 8728) 
               
             
          
           
               
                   
                   
                 SBS in MPa 
               
               
                   
                 Substrate 
                 mean (n = 5)  sd* 
               
               
                   
                   
               
               
                   
                 Rexillium ® 
                 27 ± 5 
               
               
                   
                 Bovine Enamel 
                 35 ± 9 
               
               
                   
                   
               
               
                   
                 *standard deviation  
               
             
          
         
       
     
     Example 5 
     Single-Solution Adhesive Resin System A (33% PMGDM and 10% 2-HEMA) activated with Lucerin 8728, Lucerin TPO or Darocur 4265, a two-component photoinitiator was tested on dentin. 
     
       
         
               
             
               
               
             
               
               
               
             
           
               
                   
               
               
                 Shear Bond Strengths (SBS) to Dentin 
               
             
          
           
               
                   
                 SBS in MPa 
               
               
                   
                 mean (n = 5)  sd* 
               
               
                   
                   
               
             
          
           
               
                   
                 System A/Lucerin 8728 
                 37 ± 9  
               
               
                   
                 System A/Darocur 4265 
                 47 ± 12 
               
               
                   
                 System A/Lucerin TPO 
                 37 ± 12 
               
               
                   
                   
               
               
                   
                 *standard deviation  
               
             
          
         
       
     
     Example 6 
     Shear bond strengths to phosphoric acid-conditioned dentin mediated by the Single-Solution Adhesive System A, which was activated with Lucerin TPO. In this example the adhesive was applied in either 1, 2, or 3 applications with the applicator brush; irradiation times were 10 s or 20 s, respectively. 
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Dentin Shear Bond Strengths (SBS) in MPa with the Single-Solution 
               
               
                 Adhesive System A, activated with Lucerin TPO. 
               
             
          
           
               
                   
                 SBS in MPa (mean** ± SD*) 
                   
               
             
          
           
               
                 No. of applications 
                 10 seconds 
                 20 seconds 
               
               
                   
               
               
                 1 
                 30 ± 13 
                 28 ± 8 
               
               
                 2 
                 37 ± 2  
                  34 ± 10 
               
               
                 3 
                 36 ± 14 
                 31 ± 4 
               
               
                   
               
               
                 dentin was conditioned with 37% phosphoric acid gel  
               
               
                 **n = 4 for each group; *standard deviation  
               
             
          
         
       
     
     Example 7 
     Shear bond strengths to phosphoric acid-conditioned dentin mediated by nonaged and aged (4 days at 45.8° C.) Single-Solution Adhesive System A, which was activated with Lucerin TPO or camphorquinone and tertiary amine. In this example, the 10 s. adhesive was applied in 2 applications with the applicator brush; irradiation times were 10s. 
     
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Dentin Shear Bond Strengths (SBS) in MPa with the Single-Solution 
               
               
                 Adhesive System A, activated with Lucerin TPO. 
               
             
          
           
               
                   
                 SBS in MPa 
               
             
          
           
               
                   
                 (mean** ± SD*) 
                   
               
             
          
           
               
                   
                 No. of applications 
                 nonaged 
                 aged 
               
               
                   
                   
               
             
          
           
               
                   
                 System A/Lucerin/TPO #   
                 37 ± 12 
                 39 ± 10 
               
               
                   
                 System A2/Lucerin/TPO ##   
                 46 ± 11 
                 36 ± 7  
               
               
                   
                 System A2/Lucerin/CQ-4-ED ##   
                 31 ± 8  
                 32 ± 15 
               
               
                   
                   
               
               
                   
                 #aged for 4 days at 46° C.  
               
               
                   
                 ##aged for 7 days at 42° C.  
               
               
                   
                 **n = 5 for each group; *standard deviation