Patent Publication Number: US-5153229-A

Title: Process for producing reference bactericidal endpoint (RBE) limonene

Description:
TECHNICAL FIELD 
     The object of this invention is to show an economical process of producing oxidized limonene which has a potent, rapidly acting antimicrobial activity that persists when stored, and which is superior in antimicrobial activity to that of auto-oxidized limonene. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     During the study of fresh limonene as a hand cleaner, the applicants found that fresh limonene is an excellent solvent, but it is not bactericidal. Conversely, limonene which has undergone auto-oxidation, unexpectedly, was found to have a minimal, inconsistent antibacterial activity which varied in potency from batch to batch. Therefore, it was decided to purposely oxidize limonene to try to enhance and stabilize the antimicrobial properties of the limonene. 
     The applicants found that by bubbling air (or oxygen) into limonene to purposely oxidize (or oxygenate limonene which is the same) the oil, that it became bactericidal at progressively decreasing concentrations until it reached a point beyond which further oxidation caused a decrease in its bactericidal activity. As the purposeful oxidation of limonene progressed, it was noted that the physical properties of the limonene changed as its antimicrobial activity increased. Limonene, which was purposely oxidized was found to be effective in killing bacteria, yeasts, and fungi and its antimicrobial activity persisted unchanged when the purposely oxidized limonene was stored at 2°-4° C. in the dark, for periods exceeding one year. 
     The ability to produce limonene with a stable antimicrobial activity was unexpected and is not appreciated nor delineated in the prior art. In fact it is recognized in the prior art that the addition of air (or oxygen) to terpenes produces volatile mixtures of oxides and peroxides that are unstable. Limonene which undergoes auto-oxidation has an inconsistent antibacterial effect that is not rapidly bactericidal. It evaporates to a thick, non-usable gel before it becomes an effective antimicrobial when auto-oxidation is allowed to occur over a large surface area of the oil. On the other hand, limonene that is purposely oxidized becomes an excellent, stable bactericide and fungicide that is bactericidal and fungicidal in bactericidal and fungicidal concentrations and its activity can be demonstrated with even short interval application. 
     Thus, the object of this invention is to economically produce oxidized limonene: (1) with a potent, rapidly acting antimicrobial activity, (2) that will retain its antimicrobial activity when stored, and (3) that is superior in its antimicrobial activity to the antimicrobial activity of auto-oxidized limonene. 
     Limonene is a terpene with the following formula: ##STR1## 
     Limonene is an oil with a terpenic aroma. It is soluble in alcohol and miscible in oil, but it is insoluble in glycerine. Limonene is usually derived from either a pine or citrus origin. The l-isomer is derived from pine and has a characteristic pine aroma while the d-isomer is derived from citrus and has a characteristic citrus aroma. The l-isomer is also made synthetically, but the synthetic l-limonene has no characteristic aroma. 
     Commercial limonene is usually obtained from the peel of citrus fruit. Pressed liquor is obtained from the peel of any citrus fruit, but most commonly from oranges and grapefruit, after which it is passed through a flash evaporation at 240° F. at atmospheric conditions. Limonene is collected from the condensation of the flash vaporization. Limonene is used commercially as a flavoring agent, is used in large quantities to produce carvone which has a spearmint flavor, is used as a solvent, can be used as a base for soap or perfume, is used in the manufacture of rubber, and is useful as a penetrating oil. 
     Limonene undergoes auto-oxidation when it is exposed to air or oxygen. After exposure to air at room temperature, limonene becomes rancid with a foul smelling odor. The temperature has to be above about 40° F. for auto-oxidation to occur while temperatures above 90° F. increase the rate of evaporation. Because auto-oxidation limits the commercial value of limonene by causing it to become rancid with a foul smelling odor, limonene is usually stored in 55 gallon drums that have their interior painted with phenolic resin base enamel especially formulated for terpenes. The drums are completely filled and capped in order to limit exposure to air (and oxygen) that causes auto-oxidation. Auto-oxidized limonene develops antibacterial activity during auto-oxidation but does not become a rapidly acting bactericide. Auto-oxidation does not produce the potent, rapidly bactericidal limonene described in this patent. 
     2. Description of the prior art 
     The oxidation of limonene produces several chemical compounds that have been enumerated by several different investigators. For instance Bain (in U.S. Pat. No. 2,863,882 and 3,014.047) showed a method of producing and recovering terpenic oxidation products in which he delineated the oxidation products of limonene as: limonene-1,2-oxide, limonene-8,9-oxide, 1-menthene-9-ol, α-2,8-p-menthadiene-1-ol, β-2,8-p-menthadiene-1-ol, dihydrocarvone, β-cymenol, carvone, cis-carveol, and trans-carveol. He never studied the antimicrobial activity of oxidized limonene nor oxidized limonene to the point that it became stable with a potent antimicrobial activity. In fact, because limonene is chemically unstable during its oxidation, Bain thought that in order to obtain the maximum number of oxidation products from limonene, that he should add alkali to limonene when the peroxide value was between 1000 and 2000 and should cease adding air or oxygen to limonene. He thought the peroxide value was the sole indicator of the formation of limonene oxidation products (oxides and peroxides) and that these products could be obtained only when the oxidation of limonene was stopped as the peroxide value rose to its maximum. He never realized that the maximal activity achievable through the oxidation of limonene is obtained before maximum peroxide formation occurs. 
     Blumann listed the compounds formed by the auto-oxidation of limonene in Chemical Abstracts, Volume 63, 1965, n page 1819, and found minute amounts of cis and trans-carveol, trans-p-menth-8-ene-1,2-diol, limonene 1,2-epoxide, limonene 8,9-epoxide, cis and trans-p-mentha-2,8-dien-1-ol, and perillyl alcohol, but he never tried to purposely oxidize limonene and never tried to develop limonene with a stable, rapidly bactericidal activity. 
     Bardyshev studied the chemical compounds in photo-initiated, auto-oxidized, l-limonene and found tiny amounts of the following compounds: carvone, carveol, trans-p-menth-8-ene-1,2,-diol, hydroperoxide, p-menthane, p-cymene, terpinolene, p-mentha-2,4(8)-diene, cyclohexene, cuminic aldehyde, dihydrocarvone, piperitone, p-mentha-2,8-dien-1-ol, pulegone, carvomenthone, PhCOMe, 4-MeC 6  H 4  COMe, cyclohexanone, perillyl alcohol, and p-CH 2  :CMeC 6  H 4  CH 2  OH as was outlined in Chemical Abstracts, Volume 80, 1974, page 359. Like Blumann, Bardyshev never purposely oxidized limonene and never studied the antimicrobial activity of fresh nor auto-oxidized limonene. 
     Zuckerman studied the effect of d-limonene on bacteria and found that fresh limonene had no effect on bacteria. He found that when limonene became auto-oxidized, it developed minimal, inconsistent, weakly inhibitory (not bactericidal) properties. He noted that, at best, auto-oxidized limonene was only weakly inhibitory in that it only &#34;shifts the time of the lag phase of bacterial growth without any influence on the logarithmic phase of bacterial growth.&#34; He noted that auto-oxidized limonene is unstable, and loses its inhibitory effect on keeping as was discussed in Nature 168: 517 (1951). He also recognized that the addition of alkali, as advocated by Bain in his method of oxidizing limonene, destroys the bacteriostatic properties of auto-oxidized limonene. He never recognized that limonene can be purposely oxidized to a point that it becomes a potent, stable antimicrobial which retains its antimicrobial activity when stored. In short, he could not make oxidized limonene with a stable, rapidly bactericidal activity. 
     In U.S. Pat. No. 3,595,975 Gauvreau showed a means of producing disinfecting compositions by combining cetyl pyridinium with terpenes to form antiseptics, but he never learned that limonene which had been purposely oxidized, becomes a potent, stable bactericide and fungicide. The active ingredient in his disinfecting compositions was cetyl pyridinium chloride (and not the terpenes). 
     DISCLOSURE OF THE INVENTION 
     This invention relates to an economical and safe process of producing oxidized limonene that has a potent, stable antimicrobial activity which is retained when it is stored. Its antimicrobial activity is superior to, and distinct from, the antimicrobial activity of auto-oxidized limonene. In one preferred use the oxidized limonene of this invention is a microbicide. Wherever used herein, the term &#34;microbe&#34; as used alone or in derivatives such as microbicide and antimicrobial, means a microscopic organism, such as bacteria, fungi, yeast, viruses, and rickettsia. 
     During the purposeful oxidation of limonene it was found that oxidized limonene became rapidly bactericidal as oxidation progressed. When the bactericidal activity of oxidized limonene: (a) became greater than that which could be accomplished by the auto-oxidation of limonene and, (b) could be demonstrated at a concentration of 0.06 or less against Staphylococcus aureus ATCC 25923 when incubated for sixty minutes, the oxidized limonene was defined as reference bactericidal endpoint limonene (i.e. RBE limonene). RBE limonene is a potent, stable, rapidly bactericidal compound. 
     The production of oxidized limonene that is bactericidal at a concentration of 0.06 or less against Staphylococcus aureus ATCC 25923 when incubated for sixty minutes can be produced by continuously bubbling air (method one) or oxygen (method two) through limonene at room temperature for three to eight weeks. The applicants prefer to bubble air into limonene because oxygen is more expensive to use and oxygen has a great potential to explode, making it much more hazardous to handle. After limonene is oxidized and becomes a potent, stable bactericide, regardless of whether it is produced by the addition of air or oxygen, it retains its antimicrobial activity when stored, maintains its pleasant terpenic aroma (as opposed to the rancid odor of auto-oxidized limonene), and changes physical characteristics with an increased viscosity and an increased specific gravity. While the majority of limonene specimens which are purposely oxidized develop a potent, rapidly bactericidal activity, occasional limonene specimens do not become bactericidal with oxidation regardless of the length of, or the method of, oxidation. Thus, every batch of limonene which is purposely oxidized (to produce RBE limonene) must be individually tested for bactericidal activity. In short, the applicant&#39;s preferred method of producing oxidized limonene with a potent, stable bactericidal activity is to bubble air into limonene, because bubbling air through limonene is less expensive than using oxygen, and is less hazardous to use than oxygen. 
     Oxidized limonene is slightly viscous and readily adheres to glass, metal, wood, cloth, rope, book covers, cement, canvas, ceramic tile, plant surfaces, skin, mucous membranes and teeth leaving a film of oil. In practice, any surface on which it is desirable to kill or prevent bacterial or fungal growth, is contacted directly with bactericidal or fungicidal concentrations of limonene which has been oxidized until it has developed a minimum bactericidal concentration of at least 0.06 or less for Staphylococcus aureus ATCC 25923 when incubated for sixty minutes. It can be applied by swabbing, wiping, painting, washing, brushing, spraying, or any other direct application technique. Alternatively, it can be incorporated in creams, ointments, tinctures, gels, suppositories, paints, sprays, aerosols, toothpastes, solutions, emulsions, surgical soaps, mouthwashes, or antiseptics and can be applied anywhere it is desirable to kill or prevent the growth of bacteria, fungi, or yeasts. 
    
    
     BEST MODE FOR CARRYING OUT THE INVENTION 
     The following examples are illustrative of the best mode for carrying out the invention. They are, obviously, not to be construed as limitative of the invention since various other embodiments can readily be evolved in view of the teachings provided herein. 
     EXAMPLE 1 
     In Example 1, the several methods of producing purposely oxidized limonene that is bactericidal are demonstrated. In the first example, the process of bubbling air into limonene to produce oxidized limonene that is bactericidal at a concentration of 0.06 or less for Staphylococcus aureus ATCC 25923 when incubated for 60 minutes is shown in Table A below. The method of producing auto-oxidized limonene is shown in Table B. 
     The process of producing purposely oxidized limonene and auto-oxidized limonene was as follows: Four gallons of fresh d-limonene (Florida Chemical Company, Inc., Lake Alfred, Florida) were divided into one gallon portions in small-mouthed metal container. Two portions were treated at room temperature (˜25° C.) while two were treated at 50° C. (in a water bath). For each temperature, one portion was left open to room air (auto-oxidized) while the second portion was actively oxidized by continuously bubbling air through it with a Second Nature Challenger I Pump aerator (purposely oxidized). Aliquots were removed from each of the four portions at 0, 2, 4, 6, 8, 10, 12, 14, and 16 weeks. For each aliquot, the viscosity, specific gravity, peroxide value and the antibacterial activity against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were determined as outlined in Tables A, B, D, and E below. 
     For those portions treated at room temperature (Tables A and B), there was a clear difference in the type and potency of the antibacterial activity produced through the purposeful oxidation and auto-oxidation of d-limonene. The purposely oxidized limonene (Table A) showed maximum bactericidal activity by 6 to 8 weeks of treatment. It was rapidly bactericidal (10 minutes exposure) at dilutions up to 1:6.25 for S. aureus ATCC 25923 and 1:800 for E. coli ATCC 25922. It was bactericidal at dilutions of 1:1600 for both organisms with more prolonged exposure time (24 hours). 
     
                                           TABLE A                                 
__________________________________________________________________________
Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity 
of d-Limonene.sup.1                                                       
During The Purposeful Oxidation of the Oil at Room Temperature.           
Purposeful                                                                
          Bactericidal Concentration.sup.3 For                            
Oxidation.sup.2 at                                                        
          S. aureus ATCC 25923 After                                      
                         E. coli ATCC 25922 After                         
Room Temperature                                                          
          Incubation for Incubation for Peroxide                          
                                             Specific                     
                                                  Kinematic               
in Weeks  10 Min.                                                         
               60 Min.                                                    
                    24 Hr.                                                
                         10 Min.                                          
                              60 Min.                                     
                                   24 Hr.                                 
                                        Value.sup.4                       
                                             Gravity.sup.5                
                                                  Viscosity.sup.6         
__________________________________________________________________________
 0(fresh) &gt;1.sup.7                                                        
               &gt;1   0.2  1    1    0.10  50.2                             
                                             0.8439                       
                                                  1.035                   
 2         1   0.5  0.0025                                                
                         0.24 0.02 0.0012                                 
                                        344.3                             
                                             0.8535                       
                                                  1.176                   
 4         1   0.10 0.0006                                                
                         0.06 0.0012                                      
                                   0.0006                                 
                                        327.2                             
                                             0.9118                       
                                                  2.847                   
 6         0.16                                                           
               0.06 0.0012                                                
                         0.0025                                           
                              0.0012                                      
                                   0.0006                                 
                                        353.5                             
                                             0.9679                       
                                                  10.910                  
 8         0.24                                                           
               0.06 0.0006                                                
                         0.0012                                           
                              0.0006                                      
                                   0.0003                                 
                                        334.2                             
                                             1.0122                       
                                                  51.85                   
10         1   0.10 0.0012                                                
                         0.10 0.0025                                      
                                   0.0012                                 
                                        405.8                             
                                             1.0420                       
                                                  199.7                   
12         0.4 0.10 0.0025                                                
                         0.005                                            
                              0.0025                                      
                                   0.0006                                 
                                        389.8                             
                                             1.0633                       
                                                  500.4                   
14         0.4 0.10 0.0025                                                
                         0.06 0.010                                       
                                   0.0012                                 
                                        848.3                             
                                             1.0665                       
                                                  1,268.00                
16         0.4 0.16 0.0025                                                
                         0.10 0.005                                       
                                   0.0006                                 
                                        929.9                             
                                             1.0784                       
                                                  2,562.00                
__________________________________________________________________________
 .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida                  
 .sup.2 Air was bubbled continuously into 1 gallon of oil in a smallmouthe
 metal container with an aerator (Second Nature Challenger I Pump).       
 .sup.3 Bactericidal concentration shown as the highest dilution of       
 limonene (i.e. lowest concentration) killing at least 99.99% of the      
 original inoculum.                                                       
 .sup.4 Milliequivalents peroxide/kg sample                               
 .sup.5 at 25° C.                                                  
 .sup.6 at 25° C. cSt                                              
 .sup.7 Undiluted oil was not bactericidal for 99.99% of the original     
 inoculum.                                                                
 
    
     In contrast, limonene that had been allowed to auto-oxidize at room temperature (Table B) showed maximum bactericidal activity at 14 to 16 weeks of treatment, but never reached the potency of the purposely oxidized oil. Rapid bactericidal activity (at 10 minutes) against S. aureus ATCC 25923 was never seen with any dilution of the auto-oxidized oil and only weakly diluted oil (1:2.5-1:10) was rapidly bactericidal for E. coli ATCC 25922. Even with prolonged incubation (24 hours), the auto-oxidized oil&#39;s bactericidal potency was 17 times less than that of the purposely oxidized oil against S. aureus ATCC 25923 and four times less against E. coli ATCC 25922. Increasing the treatment time of the oil beyond 16 weeks resulted in an overall decrease in potency. 
     
                                           TABLE B                                 
__________________________________________________________________________
Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity 
of d-Limonene.sup.1                                                       
During Auto-Oxidation of the Oil at Room Temperature.                     
          Bactericidal Concentration.sup.3 For                            
Auto-Oxidation.sup.2 at                                                   
          S. aureus ATCC 25923 After                                      
                        E. coli ATCC 25922 After                          
Room Temperature                                                          
          Incubation for                                                  
                        Incubation for Peroxide                           
                                            Specific                      
                                                 Kinematic                
in Weeks  10 Min.                                                         
               60 Min.                                                    
                    24 Hr.                                                
                        10 Min.                                           
                             60 Min.                                      
                                  24 Hr.                                  
                                       Value.sup.4                        
                                            Gravity.sup.5                 
                                                 Viscosity.sup.6          
__________________________________________________________________________
 0(fresh) .sup. &gt;1.sup.7                                                  
               &gt;1   0.2 1    1    0.10  50.2                              
                                            0.8439                        
                                                 1.035                    
 2        &gt;1   &gt;1   0.01                                                  
                        1    0.24 0.005                                   
                                       163.5                              
                                            0.8440                        
                                                 1.066                    
 4        &gt;1   &gt;1   0.01                                                  
                        0.5  0.16 0.0025                                  
                                       199.5                              
                                            0.8477                        
                                                 1.082                    
 6        &gt;1   &gt;1   0.01                                                  
                        1    0.06 0.0025                                  
                                       229.5                              
                                            0.8486                        
                                                 1.090                    
 8         1   1    0.01                                                  
                        0.5  0.06 0.0012                                  
                                       288.2                              
                                            0.8510                        
                                                 1.124                    
10        &gt;1   1    0.005                                                 
                        1    0.06 0.0012                                  
                                       336.9                              
                                            0.8548                        
                                                 1.167                    
12         1   0.5  0.01                                                  
                        0.10 0.01 0.0025                                  
                                       325.7                              
                                            0.8570                        
                                                 1.198                    
14         1   0.4  0.01                                                  
                        0.40 0.0025                                       
                                  0.0012                                  
                                       366.1                              
                                            0.8587                        
                                                 1.247                    
16         1   0.4  0.01                                                  
                        0.10 0.01 0.0025                                  
                                       393.8                              
                                            0.8626                        
                                                 1.299                    
21        &gt;1   1    0.005                                                 
                        0.16 0.005                                        
                                  0.0025                                  
__________________________________________________________________________
 .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida                  
 .sup.2 1 gallon of oil in smallmouthed metal container left open to room 
 air.                                                                     
 .sup.3 Bactericidal concentration shown as the highest dilution of       
 limonene (i.e. lowest concentration) killing at least 99.99% of the      
 original inoculum.                                                       
 .sup.4 Milliequivalents peroxide/kg sample                               
 .sup.5 at 25° C.                                                  
 .sup.6 at 25° C. cSt                                              
 .sup.7 Undiluted oil was not bactericidal for 99.99% of the original     
 inoculum.                                                                
 
    
     For the purposely oxidized limonene (Table A), neither the highest peroxide value nor kinematic viscosity corresponded to the greatest antibacterial potency. This suggested that the antibacterial activity was not due solely to peroxides formed during oxidation nor was it due to mere concentration of antibacterial substances resultant from the evaporation that occurred during treatment. For the auto-oxidized limonene, antibacterial activity followed the peroxide value and kinematic viscosity up to 16 weeks. This suggested that the antibacterial may have been due, in large part, to peroxides and concentration of antibacterial substances during treatment. A comparison of the antibacterial effects of the two oils when each had a similar peroxide value clearly reveals the distinct nature of the activity of the purposely oxidized limonene (Table C). The auto-oxidized oil was not bactericidal at any dilution for either test organism after short exposure (10 minutes) while the purposely oxidized oil was bactericidal at a dilution of 1:4.2 for S. aureus ATCC 25923 and 1:800 for E. coli ATCC 25922. Even with prolonged exposure (24 hours), the bactericidal potency of the auto-oxidized oil was four to eight times less than that of the purposely oxidized oil. 
     
                                           TABLE C                                 
__________________________________________________________________________
Comparison of the Antibacterial Effect of Purposely Oxidized              
d-Limonene and Auto-Oxidized d-Limonene..sup.1                            
                      Bactericidal Concentration For                      
                      S. aureus ATCC 25923                                
                                    E. coli ATCC 25922 After              
                      After Incubation For                                
                                    Incubation For                        
d-Limonene Preparation                                                    
              Peroxide Value                                              
                      10 Min.                                             
                           60 Min.                                        
                                24 Hr.                                    
                                    10 Min.                               
                                         60 Min.                          
                                              24 Hr.                      
__________________________________________________________________________
Purposely Oxidized, 8 Weeks                                               
              334.2   0.24 0.06 0.0006                                    
                                    0.0012                                
                                         0.0006                           
                                              0.0003                      
Auto-Oxidized, 10 Weeks                                                   
              336.9   &gt;1   1    0.005                                     
                                    1    0.06 0.0012                      
__________________________________________________________________________
 .sup.1 Compiled from Tables A and B                                      
 
    
     Increasing the -temperature of oxidation from room temperature (˜25° C.) to 50° C. did not uniformly decrease the time necessary to produce potent, antibacterial activity in the oils (Tables D and E). For the purposely oxidized limonene, maximum bacterial activity was reached after only 3 weeks oxidation (Table D). This was in contrast to the 6 to 8 weeks required for purposeful oxidation at room temperature (Table A). However, the rapid bacterial potency (10 minutes exposure) was 6 to 83 times less for oil purposely oxidized at 50° C. in comparison to that purposely oxidized at room temperature. Thus, the two processes appeared to be generating oils with unique antibacterial effects. The oil purposely oxidized at 50° C. became so viscous at 6 weeks, it could not be tested further for antibacterial activity since it would not mix with the broth diluent in the assay. 
     
                                           TABLE D                                 
__________________________________________________________________________
Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity 
of d-Limonene.sup.1                                                       
During The Purposeful Oxidation of the Oil at 50° C..              
           Bactericidal Concentration.sup.3 For                           
           S. aureus ATCC 25923 After                                     
                          E. coli ATCC 25922 After                        
Purposeful Oxidation.sup.2                                                
           Incubation for Incubation for Peroxide                         
                                              Specific                    
                                                   Kinematic              
at 50° C. in Weeks                                                 
           10 Min.                                                        
                60 Min.                                                   
                     24 Hr.                                               
                          10 Min.                                         
                               60 Min.                                    
                                    24 Hr.                                
                                         Value.sup.4                      
                                              Gravity.sup.5               
                                                   Viscosity.sup.6        
__________________________________________________________________________
 0(fresh)  &gt;1.sup.7                                                       
                &gt;1  0.2   1    1    0.1   50.2                            
                                              0.8439                      
                                                   1.035                  
 2          1   0.16                                                      
                    0.0012                                                
                          0.1  0.0006                                     
                                    0.0006                                
                                         286.1                            
                                              0.9549                      
                                                   8.203                  
 4          1   0.5 0.0012                                                
                          0.1  0.0025                                     
                                    0.0006                                
                                         273.2                            
                                              1.0300                      
                                                   141.9                  
 6.sup.8    1   0.4 0.01  0.01 0.005                                      
                                    0.0025                                
                                         307.9                            
                                              1.0670                      
                                                   1293                   
 8                                       226.2                            
                                              1.0784                      
                                                   3149                   
10                                       241.9                            
                                              1.0863                      
                                                   10,526                 
__________________________________________________________________________
 .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida                  
 .sup.2 Air was bubbled continuously into 1 gallon of oil in a smallmouthe
 metal container with an aerator (Second Nature Challenger I Pump).       
 .sup.3 Bactericidal concentration shown as the highest dilution of       
 limonene (i.e. lowest concentration) killing at least 99.99% of the      
 original inoculum.                                                       
 .sup.4 Milliequivalents peroxide/kg sample                               
 .sup.5 at 25° C.                                                  
 .sup.6 at 25° C. cSt                                              
 .sup.7 Undiluted oil was not bactericidal for 99.99% of the original     
 inoculum.                                                                
 .sup.8 Oil too viscous to permit testing for antibacterial activity after
 6 weeks and for other parameters after 10 weeks.                         
 
    
     Auto-oxidation of limonene at 50° C. produced an oil with even more distinct antibacterial activity (Table E). Fourteen weeks of treatment were required to maximize its rapid bacterial effects (10 minutes exposure) while two to four weeks of treatment were required to maximize its longer interval (24 hours) killing. This was similar to the time interval required to maximize the rapid and longer interval bactericidal effects of limonene auto-oxidized at room temperature (Table B). However, for the limonene auto-oxidized at 50° C., the bactericidal activity determined after long interval exposure (24 hours) decreased after 4 weeks treatment when the test organism was S. aureus ATCC 25923 but not when it was E. coli ATCC 25922. These results indicated that auto-oxidation of limonene at 50° C. could not produce the same bacterial activity as purposeful oxidation of limonene at room temperature. 
     
                                           TABLE E                                 
__________________________________________________________________________
Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity 
of d-Limonene.sup.1                                                       
During Auto-Oxidation of the Oil at 50° C..                        
          Bactericidal Concentration.sup.3 For                            
          S. aureus ATCC 25923 After                                      
                         E. coli ATCC 25922 After                         
Auto-Oxidation.sup.2                                                      
          Incubation for Incubation for Peroxide                          
                                             Specific                     
                                                  Kinematic               
at 50° C. in Weeks                                                 
          10 Min.                                                         
               60 Min.                                                    
                    24 Hr.                                                
                         10 Min.                                          
                              60 Min.                                     
                                   24 Hr.                                 
                                        Value.sup.4                       
                                             Gravity.sup.5                
                                                  Viscosity.sup.6         
__________________________________________________________________________
 0(fresh) .sup. &gt;1.sup.7                                                  
               &gt;1   0.2  1    1    0.1   50.2                             
                                             0.8439                       
                                                  1.035                   
 2        &gt;1   1    0.0025                                                
                         0.5  0.02 0.0012                                 
                                        308.8                             
                                             0.8743                       
                                                  1.559                   
 4        &gt;1   1    0.0025                                                
                         0.4  0.02 0.0012                                 
                                        268.0                             
                                             0.8906                       
                                                  2.010                   
 6        &gt;1   1    0.02 0.16 0.0025                                      
                                   0.0025                                 
                                        217.5                             
                                             0.9005                       
                                                  2.406                   
 8         1   1    0.01 0.2  0.0025                                      
                                   0.0012                                 
                                        189.6                             
                                             0.9056                       
                                                  2.700                   
10         1   1    0.01 0.1  0.005                                       
                                   0.0012                                 
                                        182.5                             
                                             0.9135                       
                                                  3.168                   
12        &gt;1   1    0.02 0.1  0.0025                                      
                                   0.0025                                 
                                        192.6                             
                                             0.9201                       
                                                  3.748                   
14         1   0.5  0.02 0.01 0.0025                                      
                                   0.0025                                 
                                        211.0                             
                                             0.9282                       
                                                  4.544                   
16         1   0.5  0.06 0.1  0.0025                                      
                                   0.0025                                 
                                        219.6                             
                                             0.9363                       
                                                  5.516                   
21        &gt;1   1    0.02 0.06 0.005                                       
                                   0.005                                  
__________________________________________________________________________
 .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida                  
 .sup.2 1 gallon of oil in smallmouthed metal container left open to room 
 air.                                                                     
 .sup.3 Bactericidal concentration shown as the highest dilution of       
 limonene (i.e. lowest concentration) killing at least 99.99% of the      
 original inoculum.                                                       
 .sup.4 Milliequivalents peroxide/kg sample                               
 .sup.5 at 25° C.                                                  
 .sup.6 at 25° C. cSt                                              
 .sup.7 Undiluted oil was not bactericidal for 99.99% of the original     
 inoculum.                                                                
 
    
     A summary of the maximal bactericidal activity obtained following treatment of limonene by the four processes described in the previous paragraphs is given in Table F. This activity has been separated into rapid bactericidal effects (10 minutes exposure) and longer-interval bactericidal effects (24 hours exposure) to emphasize the unique nature of the activity produced by the purposeful oxidation of limonene at room temperature. From this summary it is clear that the rapid bacterial activity can be produced only with purposeful oxidation of limonene at room temperature. The substances in the oil responsible for the rapid bactericidal activity appear to be heat labile (since they were not generated at 50° C.) and are clearly not peroxides. Longer interval bactericidal effects can be produced in limonene by a variety of treatments, and these too appear unrelated to peroxide content of the oils. However, only purposeful oxidation of limonene at room temperature results in an oil with both rapid and longer interval bactericidal activity at low concentrations. 
     
                                           TABLE F                                 
__________________________________________________________________________
Maximal Bactericidal Activity and Peroxide Value of Various Limonene      
Preparations..sup.1                                                       
                      Rapid.sup.2 Bactericidal                            
                                      Long-Interval.sup.3 Bactericidal    
                      Activity Against                                    
                                      Activity Against                    
                      S. aureus                                           
                              E. coli S. aureus                           
                                              E. coli                     
Limonene Preparation  ATCC 25923                                          
                              ATCC 25922                                  
                                      ATCC 25923                          
                                              ATCC 25922                  
__________________________________________________________________________
A.                                                                        
  Maximal Bactericidal Activity.sup.4                                     
  Purposely Oxidized, Room Temperature                                    
                      0.16                                                
                           (6).sup.6                                      
                                0.0025                                    
                                    (6)                                   
                                      0.0012                              
                                          (6) 0.0006                      
                                                  (6)                     
  Purposely Oxidized, 50° C.                                       
                      1    (2)                                            
                              0.1   (2)                                   
                                      0.0012                              
                                          (2) 0.0006                      
                                                  (2)                     
  Auto-Oxidized, Room Temperature                                         
                      1   (16)                                            
                              0.1  (16)                                   
                                      0.01                                
                                          (2) 0.0025                      
                                                  (4)                     
  Auto-Oxidized, 50° C.                                            
                      1   (14)                                            
                               0.01                                       
                                   (14)                                   
                                      0.0025                              
                                          (2) 0.0012                      
                                                  (2)                     
B.                                                                        
  Peroxide Value.sup.5 of Oils with Maximal                               
  Bactericidal Activity                                                   
  Purposely Oxidized, Room Temperature                                    
                      353.5   353.5   353.5   353.5                       
  Purposely Oxidized, 50° C.                                       
                      286.1   286.1   286.1   286.1                       
  Auto-Oxidized, Room Temperature                                         
                      393.8   393.8   163.5   199.5                       
  Auto-Oxidized, 50°  C.                                           
                      211.0   211.0   308.8   308.8                       
__________________________________________________________________________
 .sup.1 Data from Tables A, B, D, and E                                   
 .sup.2 Measured after 10 min. exposure of the test organism to the oil   
 .sup.3 Measured after 24 hr. exposure of the test organism to the oil    
 .sup.4 Shown as highest dilution of oil killing 99.99% of bacterial      
 inoculum                                                                 
 .sup.5 Millequivalents peroxide/kg sample                                
 .sup.6 Number in parentheses indicates weeks of treatment of the oil.    
 
    
     The appearances of the oils following six weeks of the four treatment regimens are quite different. For purposely oxidized limonene, the oil treated at 50° C. was darker yellow and more viscous than that treated at room temperature. For auto-oxidized, the oil treated at room temperature was essentially unchanged in its appearance from untreated (fresh) limonene. Limonene auto-oxidized at 50° C. was similar in appearance to that purposely oxidized at room temperature. 
     From the data presented in Tables A, B, D, and E, a single point was selected as a reference point to indicate that any future oxidized limonene was &#34;fully oxidized&#34;. The reference bactericidal endpoint (RBE) selected was a bactericidal activity at a dilution of at least 1:16.7 (0.06) when tested against S. aureus ATCC 25923 for 60 minutes exposure. This end point was selected because it (a) was found to be highly reproducible and required a greater dilution of oil than any 10 minute exposure endpoint, and (b) was an indicator for the presence of a rapid bactericidal effect against S. aureus and E. coli, and (c) was not obtained with any of the treatments of d-limonene except purposeful oxidation at room temperature. Thus, hereafter, all oxidized limonene achieving this reference bacterial endpoint is referred to as RBE limonene. 
     The standard assay used to test the bactericidal activity against Staphylococcus aureus ATCC 25923 or Escherichia coli ATCC 25922 in Tables A, B, C, D, E, F, H, I, &amp; J was as follows: various volumes of the desired limonene were added to Mueller-Hinton broth, mixed vigorously (Vortex Genie Mixer, Scientific Products, Evanston, Ill.), inoculated with 10 6  colony forming units (CFU)/ml of Staphylococcus aureus ATCC 25923 or Escherichia coli ATCC 25922, remixed and incubated at 37° C. in air. In varying the volume of the desired limonene to broth, concentrations of 1 (undiluted) to 0.01 (1:100 dilution) could be tested in a final volume of one to five ml (Table G). Higher dilutions of limonene (up to 1:51,200) were tested by preparing serial two-fold dilutions from the 0.01 dilution (i.e. equal volumes of limonene-containing broth and broth were mixed serially). At various time intervals, each tube was mixed vigorously and a 0.01 aliquot removed and subcultured on 5% sheep blood agar plates to determine the approximate number of viable CFU/ml in each tube. Results of these assays were expressed as the highest dilution of limonene with no detectable viable colonies on subculture i.e. the bactericidal concentration. This represented the lowest concentration killing 99.99 percent of the original inoculum. 
     
                       TABLE G                                                     
______________________________________                                    
Dilutions of limonene used in fixed volume assays                         
Dilutions Tested                                                          
              Limonene Concentration                                      
______________________________________                                    
Undiluted     1                                                           
1:2           0.5                                                         
1:2.5         0.4                                                         
1:3.3         0.3                                                         
1:4.2         0.24                                                        
1:5           0.2                                                         
 1:6.25       0.16                                                        
1:10          0.1                                                         
 1:6.25       0.06                                                        
1:50          0.02                                                        
 1:100        0.01                                                        
______________________________________                                    
 
    
     To ascertain if a more potent bactericide could be produced through auto-oxidation of d-limonene when the process occurred over a large surface area of the oil, the following experiment was performed: Five hundred milliliters of fresh d-limonene (Intercit Inc., Safety Harbor, Florida) was equally divided into 250 ml portions. One portion was placed into a 500 ml Erlenmeyer flask while the second portion was placed in a 500 ml beaker. The beaker was left open, exposed to air at room temperature (auto-oxidized), while the sample in the flask was purposely oxidized at room temperature by continuously bubbling air through it with a Second Nature Challenger I Pump aerator (purposely oxidized limonene). An aliquot of 5 ml was removed from each (beaker or flask) at one, two, and three weeks. The viscosity, specific gravity, peroxide value, and the antibacterial activity against S. aureus ATCC 5923 of each aliquot was determined as previously outlined. In this experiment, auto-oxidation could take place over a larger surface area of the oil than in the prior experiment (performed in the small-mouthed container). Purposeful oxidation was still performed in a small-mouthed container in this experiment to prevent any ancillary auto-oxidation. 
     As shown in Table H, auto-oxidation of d-limonene at room temperature over a large surface area of the oil, did not produce a highly bactericidal oil. The characteristics of the oil found at three weeks were most similar to those of limonene auto-oxidized over a small surface area after six weeks (Table B). However, oxidation over a large surface area could not be continued for prolonged periods because evaporation of the oil led to a product too viscous to examine for bactericidal activity. Thus, auto-oxidation of limonene over a large surface area did not product a rapidly bactericidal oil and evaporation precluded long-term oxidation under these conditions. 
     
                                           TABLE H                                 
__________________________________________________________________________
Changes in the physical parameters, peroxide value, and bactericidal      
activity of d-limonene.sup.1 during auto-oxidation of the                 
oil over a large surface area.                                            
Auto-oxidation                                                            
         Bactericidal Concentration.sup.2                                 
at Room Temper-                                                           
         After Incubation for                                             
                        Peroxide.sup.3                                    
                             Specific.sup.4                               
ature in Weeks                                                            
         10 Min                                                           
              60 Min                                                      
                   24 Hrs                                                 
                        Value                                             
                             Gravity                                      
                                  Viscosity.sup.5                         
__________________________________________________________________________
0 (Fresh)                                                                 
         .sup. &gt;1.sup.6                                                   
              &gt;1   &gt;1    68.7                                             
                             0.8432                                       
                                  1.024                                   
1        &gt;1   &gt;1   &gt;1   131.2                                             
                             0.8438                                       
                                  1.036                                   
2        &gt;1   &gt;1   &gt;1   170.5                                             
                             0.8434                                       
                                  1.052                                   
3        &gt;1   &gt;1   0.01 231.3                                             
                             0.8454                                       
                                  1.071                                   
__________________________________________________________________________
 .sup.1 250 ml of fresh limonene (Intercit Inc., Safety Harbor, Fla. lot  
 509801) in an open beaker at room temperature                            
 .sup.2 Bactericidal concentration shown as the highest dilution (i.e.    
 lowest concentration) killing at least 99.99% of the original inoculum of
 S. aureus ATCC 25923                                                     
 .sup.3 Millequiv. peroxide/kg sample                                     
 .sup.4 at 25° C.                                                  
 .sup.5 cSt at 25° C.                                              
 .sup.6 Undiluted oil was not bactericidal                                
 
    
     The bactericidal activity of purposely oxidized limonene in this experiment is shown in Table I. As was seen before, a rapidly bactericidal oil was produced by this process. The source of the oil was completely different than that shown in Table A, and the reference endpoint (i.e. bactericidal at a concentration of 0.06 or less for S. aureus ATCC 25923) was reached sooner than the previous experiment. These results suggested that different lots of limonene may require different treatment times for purposeful oxidation to reach the defined reference endpoint. 
     
                                           TABLE I                                 
__________________________________________________________________________
Changes in the physical parameters, peroxide value, and bactericidal      
activity of d-limonene.sup.1 during purposeful oxidation of the oil.      
Purposeful oxida-                                                         
           Bactericidal Concentration.sup.3                               
tion at Room Temper-                                                      
           After Incubation for                                           
                           Peroxide.sup.4                                 
                                Specific.sup.5                            
ature in Weeks.sup.2                                                      
           10 Min                                                         
                60 Min                                                    
                     24 Hrs                                               
                           Value                                          
                                Gravity                                   
                                     Viscosity.sup.6                      
__________________________________________________________________________
0 (Fresh)  &gt;1.sup.7                                                       
                &gt;1   &gt;1     68.7                                          
                                0.8432                                    
                                     1.024                                
1           0.1 0.1  0.1   131.2                                          
                                0.8438                                    
                                     1.036                                
2           0.1 0.1  0.0025                                               
                           170.5                                          
                                0.8434                                    
                                     1.052                                
3           0.1 0.1  0.00125                                              
                           231.3                                          
                                0.8454                                    
                                     1.071                                
__________________________________________________________________________
 .sup.1 Intercit Inc., Safety Harbor, Florida, lot 509801                 
 .sup.2 Air bubbled continuously into 250 ml of limonene in a 500 ml      
 Erlenmeyer flask with an aerator (Second Nature Challenger I Pump) at roo
 temperature.                                                             
 .sup.3 Bactericidal concentration shown as the highest dilution (i.e.    
 lowest concentration) killing at least 99.99% of the original inoculum of
 S. aureus ATCC 25923.                                                    
 .sup.4 millequiv. peroxide/kg sample                                     
 .sup.5 at 25° C.                                                  
 .sup.6 cSt at 25° C.                                              
 .sup.7 Undiluted oil was not bactericidal                                
 
    
     The ability of purposeful oxidation of limonene to produce a rapidly bactericidal oil was determined using limonene from various sources. As shown in Table J, not all samples of limonene became bactericidal with purposeful oxidation at room temperature. Two samples from Aldrich did not acquire bactericidal activity even after purposeful oxidation for five weeks while one sample obtained from this same source became rapidly bactericidal in as short as three weeks. Also, as shown in Table J, both d- and l- isomers of limonene can be purposely oxidized to become a rapid bactericide. These data emphasize the need to test every batch of purposely oxidized limonene after three to eight weeks treatment to ensure that the reference bactericidal endpoint has been reached. 
     
                       TABLE J                                                     
______________________________________                                    
Antibacterial Activity Of Various Limonene Samples As                     
Determined Against Staphylococcus aureus ATCC                             
25933 During Purposeful Oxidation at Room Temperature.                    
           Duration                                                       
           of Pur-                                                        
                  Bactericidal Concentration                              
           poseful                                                        
                  After Incubation For                                    
Sample  Source   Oxidation                                                
                          10 Mins                                         
                                 60 Mins                                  
                                        24 Hours                          
______________________________________                                    
d-limonene                                                                
        Aldrich.sup.1                                                     
                 5 weeks  &gt;1.sup.5                                        
                                 &gt;1     &gt;1                                
l-limonene                                                                
        Aldrich.sup.1                                                     
                 5 weeks  &gt;1.sup.                                         
                                 &gt;1     &gt;1                                
l-limonene                                                                
        SCM.sup.1                                                         
                 3 weeks  &gt;1.sup.                                         
                                 &gt;1     &gt;1                                
d-limonene                                                                
        Intercit.sup.3                                                    
                 3 weeks   0.1   0.005  0.0003                            
l-limonene                                                                
        SCM.sup.1                                                         
                 3 weeks   0.1   0.005  0.0006                            
d-limonene                                                                
        Aldrich.sup.1                                                     
                 3 weeks   0.005 0.0024 0.0003                            
d-limonene                                                                
        FCC.sup.1                                                         
                 6 weeks   0.16  0.06   0.0012                            
______________________________________                                    
 .sup.1 Aldrich, Milwaukee, Wisconsin                                     
 .sup.2 SCM Glidden Organics, Jacksonville, Florida                       
 .sup.3 Intercit, Inc., Safety Harbor, Florida                            
 .sup.4 Florida Chemical Company, Inc., Lake Alfred, Florida              
 .sup.5 Undiluted oil was not bactericidal                                
 
    
     The second method of producing RBE limonene is by continuously bubbling oxygen into limonene as outlined in Table K below. This process, like bubbling air through limonene produces a rapidly bactericidal oil and requires 6 to 7 weeks treatment for the reference endpoint to be achieved. 
     
                       TABLE K                                                     
______________________________________                                    
Bactericidal Activity of d-limonene.sup.1 During Oxygenation As           
Determined Against Staphylococcus aureus ATCC 25923                       
Days d-Limonene                                                           
             Bactericidal Concentration.sup.2                             
Oxidized.sup.3 at                                                         
             Following Incubation For:                                    
25° C.                                                             
             15 Min      60 Min  24 Hours                                 
______________________________________                                    
 0 days      &gt;1.sup.4    &gt;1      &gt;1                                       
 1 day       &gt;1          &gt;1      0.01                                     
 2 days      &gt;1          &gt;1      0.01                                     
 3 days      &gt;1          &gt;1      0.01                                     
 4 days      &gt;1          &gt;1      0.01                                     
 7 days      &gt;1          &gt;1      0.005                                    
 9 days      &gt;1          &gt;1      0.005                                    
15 days       0.5        0.5     0.005                                    
17 days       0.5        0.5     0.001                                    
24 days      &gt;1          0.16    0.005                                    
36 days       0.5        0.10    0.0006                                   
45 days       0.16       0.06    0.001                                    
49 days       0.16       0.06    0.003                                    
53 days       0.3        0.06    0.003                                    
59 days       0.10       0.02    0.003                                    
66 days       0.16       0.01    0.003                                    
73 days       0.01       0.01    0.003                                    
______________________________________                                    
 .sup.1 Fresh dlimonene (Intercit Inc., Safety Harbor, Florida lot 509801)
 .sup.2 Results expressed as the highest dilution of limonene killing at  
 least 99.99% of the original inoculum.                                   
 .sup.3 Oxygen bubbled continuously into dlimonene at a rate of 3 liter pe
 min at 25° C..                                                    
 .sup.4 Undiluted oil was not bactericidal.                               
 
    
     EXAMPLE 2 
     In Example 2 the bactericidal and fungicidal activity of RBE d-limonene and RBE l-limonene was determined against bacteria, yeasts, and fungi as outlined in Tables L &amp; M below. 
     
                                           TABLE L                                 
__________________________________________________________________________
BACTERICIDAL AND FUNGICIDAL ACTIVITY OF RBE d-LIMONENE                    
ORGANISM                                                                  
__________________________________________________________________________
                    BACTERICIDAL CONCENTRATION                            
                    AFTER INCUBATION FOR                                  
                    10 Min                                                
                         60 Min                                           
                              24 Hours                                    
__________________________________________________________________________
A.                                                                        
  BACTERIA                                                                
  Staphylococcus aureus,                                                  
                    0.3  0.06 0.0012                                      
  ATCC 25923                                                              
  Staphylococcus aureus 3*                                                
                    1.00 0.10 0.001                                       
  Staphylococcus aureus 27**                                              
                    1.00 0.10 0.001                                       
  Staphylococcus aureus 37*                                               
                    0.30 0.06 0.0025                                      
  Staphylococcus aureus 39**                                              
                    0.30 0.06 0.0015                                      
  Staphylococcus aureus 52T                                               
                    0.30 0.06 0.0012                                      
  Staphylococcus aureus 53T                                               
                    0.30 0.06 0.0025                                      
  Staphylococcus aureus 54T                                               
                    0.30 0.06 0.0025                                      
  Staphylococcus epidermidis 10                                           
                    1.00 0.10 0.001                                       
10.                                                                       
  Sarcinia lutea    1.00 0.01 0.001                                       
  Streptococcus mitis 15 (alpha)                                          
                    0.01 0.01 0.001                                       
  Streptococcus pyogenes 1 (group A)                                      
                    0.01 0.01 0.01                                        
  Streptococcus salivarius (gamma)                                        
                    0.01 0.01 0.01                                        
  Streptococcus mutans                                                    
                    0.16 0.06 0.0012                                      
  Streptococcus faecalis 15                                               
                    1.00 0.10 0.01                                        
  Lactobacillus casei M7                                                  
                    0.01 0.01 0.01                                        
  Listeria monocytogenes M9                                               
                    0.01 0.01 0.01                                        
  Bacillus subtilis ATCC 6633                                             
                    0.01  0.001                                           
                              0.001                                       
  Escherichia coli 7                                                      
                    0.01 0.01 0.01                                        
20.                                                                       
  Klebsiella SP12   0.01 0.01 0.001                                       
  Salmonella 14 (para B)                                                  
                    0.01 0.01 0.01                                        
  Shigella sonnei 8 0.01  0.001                                           
                              0.001                                       
  Proteus mirabilis 2                                                     
                    0.01 0.01 0.01                                        
  Proteus vulgaris 127                                                    
                    0.10 0.01 0.01                                        
  Proteus rettgeri 106                                                    
                    0.10 0.01 0.10                                        
  Proteus morganii 99                                                     
                    0.01 0.01 0.01                                        
  Providencia stuartii 97                                                 
                    0.01 0.01 0.01                                        
  Enterobacter cloacae 20                                                 
                    0.01 0.01 0.10                                        
  Citrobacter SP8   0.01 0.01 0.10                                        
30.                                                                       
  Pseudomonas aeruginosa 115                                              
                    0.10 0.10 0.01                                        
  Pseudomonas mendocina 129                                               
                    1.0  0.10 0.01                                        
  Neisseria gonorrhea 5                                                   
                     0.001                                                
                          0.001                                           
                              0.001                                       
  Neisseria gonorrhea (Pen R)*                                            
                     0.001                                                
                          0.001                                           
                              0.001                                       
  Hemophilus influenzae (Amp R)**                                         
                    0.01  0.001                                           
                              0.001                                       
  Hemophilus influenzae (Amp S)***                                        
                     0.0001                                               
                          0.001                                           
                              0.001                                       
   Neisseria meningitidis                                                 
                     0.001                                                
                          0.001                                           
                              0.001                                       
  Xanthomonas campestris PV                                               
                     0.0012                                               
                          0.0003                                          
                              0.0003                                      
  vesicatoria 1                                                           
  Xanthomonas campestris PV                                               
                     0.0012                                               
                          0.0003                                          
                              0.0003                                      
  vesicatoria 2                                                           
  Xanthomonas (SP) *+ isolated                                            
                     0.0025                                               
                          0.0003                                          
                              0.0003                                      
  from a Nebraska farmer                                                  
40.                                                                       
  Xanthomonas campestris PV citri                                         
                     0.005                                                
                          0.0025                                          
                              0.0006                                      
  Mycobacterium tuberculosis                                              
                     0.001                                                
                          0.001                                           
                              0.0001                                      
  var hominis H37 RV                                                      
  Mycobacterium kansasii Tam 30                                           
                    0.1  0.01 0.0001                                      
  Mycobacterium scrofulaceum                                              
                    0.1  0.01 0.0001                                      
  Mycobacterium intracellulare                                            
                    0.1  0.10 0.0001                                      
  Mycobacterium fortuitum ATCC                                            
                    1.0  0.10 0.0001                                      
  6841                                                                    
B.                                                                        
  YEASTS                                                                  
  Candida albicans  0.01  0.001                                           
                              0.001                                       
  Sacchromyces SP   0.01 0.01 0.001                                       
                    FUNGICIDAL CONCENTRATION                              
                    AFTER INCUBATION FOR                                  
                    10 Min                                                
                        60 Min                                            
                             24 Hours                                     
                                  7-45 Days                               
__________________________________________________________________________
C.                                                                        
  FUNGI                                                                   
  Epidermophyton floccosum        0.001                                   
  Microsporum audouini            0.001                                   
  Microsporum canis 0.01                                                  
                         0.0025                                           
                             0.0006                                       
  Trichophyton mentagrophytes     0.001                                   
  Aureoblasidium pullulans                                                
                    0.01                                                  
                        0.005                                             
                             0.0012                                       
  OM 279C                                                                 
  Cladosporium cladosporiodes                                             
                    0.01                                                  
                        0.005                                             
                             0.0012                                       
  OM 489                                                                  
  Philopohora lignicola                                                   
                    0.10                                                  
                        0.01 0.0025                                       
  OM 5922                                                                 
__________________________________________________________________________
 *Penicillin resistant                                                    
 **Ampicillin resistant                                                   
 ***Ampicillin sensitive                                                  
 *+Species unidentified                                                   
 
    
     
                                           TABLE M                                 
__________________________________________________________________________
BACTERICIDAL AND FUNGICIDAL ACTIVITY                                      
OF RBE l-LIMONENE                                                         
ORGANISM                                                                  
__________________________________________________________________________
                    BACTERICIDAL                                          
                    CONCENTRATION                                         
                    AFTER                                                 
                    INCUBATION FOR                                        
                    10 Min                                                
                         60 Min                                           
                               24 Hours                                   
__________________________________________________________________________
A.                                                                        
  BACTERIA                                                                
  Staphylococcus aureus ATCC 25923                                        
                    0.5  0.06  0.0025                                     
  Staphylococcus aureus 37 (Pen R)*                                       
                    0.5  0.40  0.005                                      
  Staphylococcus aureus 39 (Meth R)**                                     
                    0.3  0.06  0.0025                                     
  Staphylococcus aureus 52T                                               
                    0.5  0.10  0.01                                       
  Staphylococcus aureus 53T                                               
                    0.5  0.10  0.0025                                     
  Staphylococcus aureus 54T                                               
                    0.5  0.06  0.01                                       
  Staphylococcus epidermidis 10                                           
                    0.1  0.1   0.0025                                     
  Streptococcus mutans M42                                                
                    0.3  0.16  0.005                                      
  Streptococcus faecalis 15                                               
                    0.1  0.01  0.0025                                     
10.                                                                       
  Bacillus subtilis ATCC 6633                                             
                    0.1  0.1   0.1                                        
  Escherichia coli 7                                                      
                     0.005                                                
                         0.0025                                           
                               0.0012                                     
  Salmonella 14 (Para B)                                                  
                      0.0025                                              
                         0.0025                                           
                               0.0006                                     
  Shigella sonnei 8  0.01                                                 
                         0.0025                                           
                               0.0012                                     
  Pseudomonas aeruginosa 115                                              
                    0.1  0.1   0.005                                      
  Neisseria meningitidis 40                                               
                      0.0012                                              
                         0.0006                                           
                                0.00004                                   
  Xanthomonas campestris                                                  
                      0.0025                                              
                         0.0012                                           
                               0.0012                                     
  vesicatoria 1                                                           
  Xanthomonas campestris                                                  
                      0.0025                                              
                         0.0012                                           
                               0.0003                                     
  vesicatoria 2                                                           
  Xanthomonas SP isolated                                                 
                      0.0006                                              
                         0.0006                                           
                               0.0003                                     
  from a Nebraska farmer*+                                                
  Xanthomonas campestris PV citri                                         
                     0.005                                                
                         0.0025                                           
                               0.0006                                     
20.                                                                       
  Mycobacterium tuberculosis                                              
                      0.0005                                              
                          0.000125                                        
                                0.000125                                  
  var hominis H37 RV                                                      
  Mycobacterium fortuitum ATCC                                            
                    0.1  0.1   0.0006                                     
  6841                                                                    
B.                                                                        
  YEASTS                                                                  
  Candida albicans    0.0012                                              
                         0.0012                                           
                               0.0006                                     
                    FUNGICIDAL                                            
                    CONCENTRATION                                         
                    AFTER                                                 
                    INCUBATION FOR                                        
                    10 Min                                                
                         60 Min                                           
                               24 Hours                                   
__________________________________________________________________________
C.                                                                        
  FUNGI                                                                   
  Microsporum canis 0.1  0.0025                                           
                               0.0006                                     
  Aureoblasidium pullulans OM 279C                                        
                     0.01                                                 
                         0.005 0.0012                                     
  Cladosporium cladosporiodes OM 489                                      
                    0.1  0.005 0.0012                                     
  Philopohora lignicola OM 5922                                           
                     0.10                                                 
                         0.01  0.005                                      
__________________________________________________________________________
 *Penicillin resistant                                                    
 **Methicillin resistant                                                  
 *+Unidentified species                                                   
 
    
     The standard assay used to test the bactericidal activity of the d and l isomers of limonene which had been oxidized until they reached the reference bactericidal endpoint was as follows: various dilutions of the desired limonene were separately prepared in an appropriate broth medium for the test strain. An inoculum of 10 6  colony-forming units (CFU)/ml was used. Each test was incubated at the proper temperature for each organism and subcultured (0.01 ml) at 10 minutes, 60 minutes and 24 hours onto agar media free of limonene. 
     Results were expressed as the bactericidal concentration, i.e., the lowest concentration of RBE limonene (ml limonene/total ml of test) killing at least 99.99% of the bacterial inoculum. 
     The activity of RBE limonene was evaluated against Mycobacteria using undiluted oil, and oil diluted up to 1:8000 in Proskauer Beck liquid medium. Each test was inoculated with 10 6  CFU/ml of Mycobacteria and incubated at 37 degrees C. in 7% CO 2  in air. At various time intervals, each test was shaken vigorously and a 0.01 ml aliquot removed. This was subcultured onto Dubos oleic acid-albumin agar plates to determine the number of viable Mvcobacteria remaining. Each test was sampled in this fashion after incubation for 10 minutes, 60 minutes, 24 hours, 1, 2, 3, and 4 weeks. The bacterial concentration was defined as the lowest concentration of oil killing at least 99.99% of the original inoculum. 
     The standard assay used to test certain fungi (dermatophytes) was as follows: RBE d-limonene and RBE 1-limonene were tested in air on sabouraud dextrose agar slants supplemented with 0.05% yeast extract. Each of the two compounds was individually added to different samples of molten agar in dilutions of 1:10, 1:15, 1:100, 1:1000 and then mixed vigorously after which the agar was allowed to solidify. The inoculum was prepared as follows: 10 ml of saline (0.85%) and four drops of tween 80 were added to sabouraud dextrose agar slants containing the fungus grown for 14 days at room temperature. After vigorous shaking, the culture was filtered through sterile gauze and adjusted to the density of a MacFarland Standard No. 1 with 0.85% saline. The limonene containing slants were inoculated with 0.1 ml of this adjusted suspension, incubated at room temperature and checked for growth daily for 43 days. After 7 days, confluent growth of each fungus was observed on limonene-free control slants. The fungicidal concentration of RBE limonene was defined as the lowest concentration preventing growth on the slants for 43 days. 
     The test conditions used to assay the antimicrobial activity of RBE limonene are listed in Table N which follows. 
     
                                           TABLE N                                 
__________________________________________________________________________
                        SUBCULTURE                                        
                                  INCUBATION                              
ORGANISM      BROTH MEDIUM                                                
                        AGAR MEDIUM                                       
                                  CONDITIONS                              
__________________________________________________________________________
  Staphylococcus,                                                         
              Mueller-Hinton                                              
                        5% sheep blood                                    
                                  air @ 37° C.                     
  Sarcina, Bacillus,                                                      
  Enterobacteriacea                                                       
  and Pseudomonas                                                         
  Streptococcus, Lac-                                                     
              Todd-Hewitt                                                 
                        5% sheep blood                                    
                                  10% CO.sub.2 in                         
  tobacillus, Listeria            air @ 37° C.                     
  Neisseria meningi-                                                      
              Mueller-Hinton                                              
                        5% sheep blood                                    
                                  10% CO.sub.2 in                         
  tidis                           air @ 37° C.                     
  Neisseria gonorrhea                                                     
              Eugon     chocolate 10% CO.sub.2 in                         
                                  air @ 37° C.                     
  Hemophilus influenza                                                    
              Levinthal chocolate 10% CO.sub.2 in                         
                                  air @ 37° C.                     
  Yeast       Sabouraud 5% sheep blood                                    
                                  air @ 37° C.                     
              dextrose                                                    
  Xanthomonas campes-                                                     
              Nutrient  blood agar                                        
                                  air @ 25° C.                     
  tris vesicatoria &amp;                                                      
  citri                                                                   
  Xanthomonas species                                                     
              Mueller-Hinton                                              
                        blood agar                                        
                                  air @ 37° C.                     
  isolated from Nebraska                                                  
  farmer (species not                                                     
  identified)                                                             
  Mycobacteria                                                            
              Proskauer Beck                                              
                        Dubos-oleic                                       
                                  7% CO.sub.2 @                           
                        acid-albumin                                      
                        agar                                              
10.                                                                       
  Fungi       Sabourauds dex-     37° C.                           
              trose with 0.05%                                            
              yeast extract                                               
__________________________________________________________________________
 
    
     In example 3, formulations, containing RBE limonene are listed. 
     RBE d-limonene and RBE l-limonene are prepared in the following formulations including liquids, gels, soaps, paints, pastes, creams, ointments, suppositories, tampons, aerosols, and emulsion. When bacteria, fungi or yeasts are treated with formulations that contain RBE d-limonene or RBE l-limonene, the formulations kill or prevent the growth of bacteria, yeasts, and fungi. 
     
         __________________________________________________________________________
CHEMICAL                    % OF TOTAL                                    
                                    RANGE  ACTION                         
__________________________________________________________________________
A.                                                                        
  LIQUIDS                                                                 
1.  SOLUTIONS OR SPRAYS                                                   
    a.                                                                    
      RBE d-Limonene        6.0%    0.1-50%                               
                                           bactericide                    
      Corn Oil              94.0%     50-99.9%                            
                                           diluent                        
                            100.0%                                        
    b.                                                                    
      RBE l-Limonene        1.0%    0.1-50%                               
                                           fungicide                      
      Ethyl Alcohol         99.0%     50-99.9%                            
                                           diluent                        
                            100.0%                                        
2.  MOUTHWASH                                                             
a.    RBE d-limonene        50.0%   0.1-50%                               
                                           antiyeast                      
      Flavor                2.0%    1-5%   flavor                         
      Ethyl Alcohol         48.0%     45-98.9%                            
                                           diluent                        
                            100.0%                                        
B.                                                                        
  DENTIFRICE                                                              
1.  LIQUID                                                                
Liquid soap concentrate     5.0%     2-10% surfactant                     
Saccharin                   0.2%    0.1-1.0%                              
                                           flavor                         
Clove Oil                   1.0%    0.5-3.0%                              
                                           flavor                         
Cinnamon Oil                0.5%    0.5-3.0%                              
                                           flavor                         
Peppermint Oil              0.5%    0.5-3.0%                              
                                           flavor                         
Ethyl Alcohol               42.6%   29.5-95.3%                            
                                           diluent                        
Color                       0.2%    0.1-0.5%                              
                                           color                          
RBE l-limonene              50.0%    1-50% fungicide                      
                            100.0%                                        
2.  GEL                                                                   
Sodium monofluorophosphate  0.8%    0.5-1.5%                              
                                           antiplaque                     
RBE d-limonene              50.0%    1-50% antibacterial                  
Hydrated silica xerogel     10.0%    8-15% abrasive                       
Hydrated thickening silica  8.5%     5-10% binder                         
Sorbitol 70% solution       18.8%     5-73.3%                             
                                           humectant                      
Polyethylene glycol 32      5.0%    3-7%   bodying agent                  
Sodium lauryl sulfate       1.5%    1-2%   surfactant                     
Carboxymethyl cellulose     1.0%    0.5-2% binder                         
gum                                                                       
S D alcohol                 1.0%    0.5-2% stabilizer                     
Flavor                      3.0%    2-4%   flavor                         
Saccharin                   0.2%    0.1-0.5%                              
                                           flavor                         
F D &amp; C Green #3            0.1%    0.1-0.5%                              
                                           color                          
F D &amp; C Green #10           0.1%    0.1-0.5%                              
                                           color                          
                            100.0%                                        
3.  PASTE                                                                 
Sodium monofluorophosphate  0.8%    0.5-1.5%                              
                                           antiplaque                     
RBE l-limonene              50.0%    1-50% fungicide                      
Dicalcium phosphate         22.0%   20.4-30%                              
                                           abrasive                       
dihydrate                                                                 
Water                       16.0%   11.1-69.5%                            
                                           diluent                        
Glycerine                   5.1%     4.5-12.5%                            
                                           bodying agent                  
Flavor                      2.0%    2-3%   flavor                         
Sodium lauryl sulfate       1.5%    1-2%   surfactant                     
Carboxymethyl cellulose gum 1.4%    0.5-2.0%                              
                                           binder                         
Tetrasodium pyrophosphate   1.0%    0.5-2.0%                              
                                           binder                         
Sodium saccharin            0.2%    0.1-0.5%                              
                                           flavor                         
                            100.0%                                        
C.                                                                        
  OINTMENTS &amp; SUPPOSITORIES WITH AND WITHOUT HYDROCORTISONE               
1.  OINTMENT WITH HYDROCORTISONE                                          
RBE d-limonene              1.0%     0.1-15.0%                            
                                           bactericide                    
Polyethylene glycol         59.0%   48.5-59.7%                            
                                           bodying agent                  
3350                                       &amp; emulsifier                   
Polyethylene glycol         39.0%   31.5-39.7%                            
                                           bodying agent                  
400                                        &amp; emulsifier                   
Hydrocortisone              1.0%    0.5-5.0%                              
                                           anti-                          
                            100.0%         inflammatory                   
2.  OINTMENT WITHOUT HYDROCORTISONE                                       
RBE l-limonene              1.0%     0.1-15.0%                            
                                           fungicide                      
Polyethylene glycol         59.5%    51.0-59.95%                          
                                           bodying agent                  
3350                                       &amp; emulsifier                   
Polyethylene glycol         39.5%    34.0-39.95%                          
                                           bodying agent                  
400                                        &amp; emulsifier                   
                            100.0%                                        
3.  SUPPOSITORY WITHOUT HYDROCORTISONE                                    
RBE l-limonene              6.0%    0.1-15%                               
                                           bactericide                    
Polyethylene glycol         56.5%    51.0-59.95%                          
                                           bodying agent                  
1000                                       &amp; emulsifier                   
Polyethylene glycol         37.5%    34.0-39.95%                          
                                           bodying agent                  
3350                                       &amp; emulsifier                   
                            100.0%                                        
4.  SUPPOSITORY WITH HYDROCORTISONE                                       
RBE d-limonene              1.0%    0.1-15%                               
                                           antiyeast                      
Polyethylene glycol         74.0%   60.0-75.2%                            
                                           bodying agent                  
1000                                       &amp; emulsifier                   
Polyethylene glycol         24.0%   20.0-24.2%                            
                                           bodying agent                  
3350                                       &amp; emulsifier                   
Hydrocortisone              1.0%    0.5-5.0%                              
                                           anti-                          
                            100.0%         inflammatory                   
D.                                                                        
  CREAMS WITHOUT HYDROCORTISONE                                           
1.  RBE l-limonene          6.0%     0.1-15.0%                            
                                           bactericide                    
Cetyl alcohol               15.0%   12.0-18.0%                            
                                           thickener                      
Arlacel 165**               5.0%    3.5-7.5%                              
                                           emulsifier                     
Sorbitol 70% solution       5.0%    3.5-8.0%                              
                                           humectant                      
Water                       69.0%   51.5-80.9%                            
                                           diluent                        
                            100.0%                                        
2.  RBE l-limonene          1.0%     0.1-15.0%                            
                                           fungicide                      
    Spermaceti wax          12.5%   10.0-15.0%                            
                                           thickener                      
    Sorbitan monostearate*  10.0%    7.5-12.5%                            
                                           emulsifier                     
    Sorbitol monostearate*  6.0%    4.0-8.0%                              
                                           emulsifier                     
    Polyethylene 20                                                       
    Water                   70.5%   49.5-78.4%                            
                                           diluent                        
                            100.0%                                        
E.                                                                        
  CREAMS WITH HYDROCORTISONE                                              
1.  RBE d-limonene          1.0%     0.1-15.0%                            
                                           fungicide                      
    Cetyl alcohol           15.0%   12.0-18.0%                            
                                           thickener                      
    Arlacel 165**           5.0%    3.5-7.5%                              
                                           emulsifier                     
    Sorbitol 70% solution   5.0%    3.5-8.0%                              
                                           humectant                      
    Hydrocortisone          1.0%    0.5-5.0%                              
                                           anti-                          
                                           inflammatory                   
    Water                   73.0%   46.5-80.4%                            
                                           diluent                        
                            100.0%                                        
F.                                                                        
  TAMPONS                                                                 
1.  RBE d-limonene 2 cc 2 Gm                                              
                             8%      1-15% bactericide                    
    Tampon 23 Gm            92%     85-99% reservoir for                  
                            100%           fungicide                      
G.                                                                        
  AEROSOLS WITHOUT HYDROCORTISONE                                         
1.  RBE l-limonene          6.0%    0.5-50%                               
                                           bactericide                    
    Ethyl alcohol           94.0%    50-99.5%                             
                                           diluent                        
                            100.0%                                        
    Pressurized nitrogen                                                  
    propellant at 100-125                                                 
    psig                                                                  
2.  RBE d-limonene          10.0%    0.5-50.0%                            
                                           fungicide                      
    Soybean Oil             90.0%   50.0-99.5%                            
                                           diluent                        
                            100.0%                                        
    Pressurized nitrogen                                                  
    propellant at 100-125                                                 
    psig                                                                  
H.                                                                        
  AEROSOL WITH HYDROCORTISONE                                             
1.  RBE l-limonene          6.0%    0.5-50%                               
                                           bactericide                    
    Soybean oil             93.0%     45-99.0%                            
                                           diluent                        
    Hydrocortisone          1.0%    0.5- 5.0%                             
                                           anti-                          
                                           inflammatory                   
                            100.0%                                        
    Pressurized nitrogen                                                  
    propellant at 100-125                                                 
    psig                                                                  
I.                                                                        
  OIL IN WATER EMULSION                                                   
1.  RBE d-limonene          0.1%    0.1-50%                               
                                           fungicide                      
2.  Corn oil                10.0%   10-15% oil                            
    Arlacel 40**            2.0%    1-3%   emulsifier                     
    Tween 40                3.0%    2-4%   emulsifier                     
3.  Water                   84.9%     28-86.9%                            
                                           diluent                        
                            100.0%                                        
Heat 2 to 70° C.. Heat 3 to 72° C.. Add 3 to 2 with         
continuous agitation. When 3 and 2 cool to                                
40° C., add 1 with continuous agitation until room tempertature is 
reached.                                                                  
J.                                                                        
  OIL IN WATER EMULSION WITH SOAP (FUNGICIDAL SOAP)                       
1.  RBE d-limonene          10.0%   0.1-25%                               
                                           bactericide                    
2.  Corn oil                21.0%   20.0-40.0%                            
                                           oil                            
    Arlacel 40**            2.0%    1.0-3.0%                              
                                           emulsifier                     
    Tween 40                3.0%    2.0-4.0%                              
                                           emulsifier                     
    Liquid soap concentrate 3.5%    2.5-5.0%                              
                                           surfactant                     
3.  Water                   60.5%     23-74.4%                            
                                           diluent                        
                            100.0%                                        
Heat 2 to 70° C.. Heat 3 to 72° C.. Add 3 to 2 with         
continuous agitation. When 3 and 2 cool to                                
40° C., add 1 with continuous agitation until room tempertature is 
reached.                                                                  
K.                                                                        
  WATER IN OIL EMULSION                                                   
1.  RBE l-limonene          1.0%    0.1-25%                               
                                           fungicide                      
2.  Arlacel 186**           3.0%    2.0-4.0%                              
                                           emulsifier                     
    Soybean oil             15.0%   10.0-25.0%                            
                                           oil                            
    Ceresin wax             0.5%    0.3-0.6%                              
                                           thickener                      
    Beeswax                 0.5%    0.3-0.6%                              
                                           thickener                      
    Tween 40                0.5%    0.3-0.6%                              
                                           emulsifier                     
3.  Water                   79.5%   44.2-87.0%                            
                                           diluent                        
                            100.0%                                        
Heat 2 to 70° C.. Heat 3 to 72° C.. Add 3 to 2 with         
continuous agitation. When 3 and 2 cool to                                
40° C., add 1 with continuous agitation until room tempertature is 
reached.                                                                  
L.                                                                        
  PAINT                                                                   
1.  ENAMEL                                                                
    RBE l-limonene          1.00%    1-10% fungicide                      
    Titanium dioxide        14.91%  12-16% pigment                        
    Calcium carbonate       29.83%  25-35% pigment                        
    Silicate                4.81%   3-6%   pigment                        
    Soya alkyd resin        25.72%  22-28% pigment                        
                                           (binder)                       
    Mineral spirits         23.73%   5-37% solvent                        
                            100.00%        (thinner)                      
2.  LATEX                                                                 
    RBE l-limonene          1.00%    1-10% fungicide                      
    Titanium dioxide        10.76%   8-12% pigment                        
    Silicate                12.91%  10-16% pigment                        
    Calcium carbonate       20.91%  15-25% pigment                        
    Vinyl acrylic resin     12.22%  10-16% vehicle                        
    solids                                 (binder)                       
    Glycol                  8.30%    6-10% solvent                        
                                           (thinner)                      
    Water                   34.00%  12-50% solvent                        
                            100.00%        (thinner)                      
__________________________________________________________________________
 *Croda, Inc., 51 Madison Ave., New York, New York 10010                  
 **Glycerol monstearate and polyoxyethylene stearate ICI of America       
 (Formerly Atlas Chemcial Industries), Wilmington, Delaware 19899         
 
    
     While only certain preferred embodiments of this invention have been shown and described by way of illustration, many modifications will occur to those skilled in the art and it is, therefore, desired that it be understood that it is intended herein, to cover all such modifications that fall within the true spirit and scope of this invention.