Isolation of Pseudomonas alcaligenes for bio-decaffeination of caffeine containing solutions

Abstract

The present invention relates to a simple, safe, and efficient process for the complete bio-decaffeination of caffeine-containing solutions using fungus Pseudomonas alcaligenes CFR 1708, a method of isolating pseudomonas alcaligenes CFR 1708 useful for the bio-decaffeination of caffeine-containing solutions, a Pseudomonas alcaligenes strain of accession number CFR 1708, and a decaffeinated solution obtained by aforementioned process.

1 - 9 . (canceled)  
     
     
         10 . A method of isolating  Pseudomonas alcaligenes  CFR 1708 useful for the bio-decaffeination of caffeine-containing solutions, said method comprising steps of: 
 a. mixing soil sample comprising microbes obtained from coffee processing units with caffeine liquid medium to obtain a culture,    b. incubating the culture for about 70-74 hr at about 28-30° C. in a shaker at 140-160 rpm,    c. making serial dilutions of the above grown culture,    d. inoculating diluted cultures into caffeine agar medium petri plates,    e. incubating the inoculated plates at about 35-39° C. for about 1-3 days, and    f. isolating the well-separated colonies of the caffeine degrading bacteria growing on the incubated plates to obtain pure cultures.    
     
     
         11 . A method as claimed in  claim 10 , wherein the caffeine liquid medium comprises about 40-60 g/L of disodium hydrogen orthophosphate septahydrate, about 10-15 g/L of potassium dihydrogen orthophosphate, about 1-5 g/L of sodium chloride, about 2-7 g/L ammonium chloride, and about 0.1-0.5 g/L Caffeine.  
     
     
         12 . A method as claimed in  claim 10 , wherein the serial dilutions are ranging between 10 −1  to 10 −9 .  
     
     
         13 . A  Pseudomonas alcaligenes  strain of accession number CFR 1708.  
     
     
         14 . A strain as claimed in  claim 13 , wherein the characteristics of the strain are as given below with sign + shows positive for growth, and sign − shows negative for growth: 
 Cultural and biochemical tests for the identification of the isolate:                                                Biochemical Test   Result                   Catalase   +         Oxidase   −         Nitrate reduction   −         Indole Production   −         Methyl red test   −         Voges Proskauer test   −         Starch hydrolysis   −         Citrate utilization   +         Oxidation   +         Fermentation   +         Motility   +         Malonate Utilization   +         Arginine dihydrolase   +         Gelatin hydrolysis   −         Growth at 41° C.   +         Growth at 4° C.   −         Acid slant   −         Acid butt   −         Alkaline slant   +         Alkaline butt   +         H2S production   + (Weak)         Gas production   −         Urease   +         Dextrose utilization   −         Mannitol utilization   −         Polyhydroxy alkanoate (PHB)   −         accumulation                                                                         
     
     
         15 . A decaffeinated solution obtained by a process comprising: 
 g. culturing strain  Pseudomonas alcaligenes  CFR 1708 in caffeine agar plates,    h. transferring actively growing culture into nutrient broth containing 0.1-0.5 g/L caffeine,    i. incubating culture of step (b) at about 28-30° C. for about 20-24 hrs under agitation of between about 50-200 rpm to obtain pre-inoculum,    j. transferring the pre-inoculum to nutrient broth comprising about 0.5-2 g/L caffeine,    k. incubating the pre-inoculum at about 28-30° C. for about 20-24 hours under agitation between about 50-200 rpm to accumulate biomass,    l. harvesting the biomass by centrifugation for about 10-20 min at about 10,000-16,000 g at temperature ranging between about 0-4° C. to obtain pellet,    m. inducing the pellet for caffeine degradation by aseptically transferring the pellet into sterilized caffeine liquid medium of pH about 5-9,    n. incubating the induced pellet at about 25-35° C. under agitation between about 50-200 rotations per minute for time duration of about 36-60 hours, and decaffeinating caffeine-containing solution by incubating with the pellet of step (h).    
     
     
         16 . The solution of  claim 15  wherein concentration of caffeine in nutrient broth is ranging between 0.2-0.4 g/L.  
     
     
         17 . The solution of  claim 15  wherein the sterilized caffeine liquid medium comprises about 40-60 g/L of disodium hydrogen orthophosphate septahydrate, about 10-15 g/L of potassium dihydrogen orthophosphate, about 1-5 g/L of sodium chloride, about 2-7 g/L ammonium chloride, and about 0.1-0.5 g/L Caffeine.  
     
     
         18 . The solution of  claim 15  wherein the induction time is ranging between about 40-48 hr.  
     
     
         19 . The solution of  claim 15  wherein the concentration of caffeine in caffeine-containing solution is ranging between about 0.01-5 g/L of caffeine.  
     
     
         20 . The solution of  claim 15  wherein the caffeine degradation is complete in about 1-14 hours.  
     
     
         21 . The solution of  claim 15  wherein incubating at temperature ranging between 28-30° C. occurs in step (i).  
     
     
         22 . The solution of  claim 15  wherein the nutrient broth comprises about 0.5-4 g/L of yeast extract, about 3-8 g/L of peptone, about 3-8 g/L of sodium chloride, and about 0.08-0.7 g/L of caffeine.  
     
     
         23 . The solution of  claim 15  wherein the sterilized caffeine liquid medium has a pH of about 9.