Rapid method of detection and enumeration of sulfide-producing bacteria in food products

Abstract

A rapid method for detecting spoilage of a food sample, particularly a fish sample, by detecting and enumerating sulfide-producing bacteria (SPB). A growth medium containing iron and sulfur is combined with the food sample forming an incubation mixture which is incubated for a period of time. In one embodiment, a plurality of fluorescence measurements are taken during an incubation period of about 3 hours to 17 hours at 30° C. SPB are determined to be present in the sample if the fluorescence measurement initially increases and then decreases to form a fluorescence maximum (peak). The time to detection of the fluorescence peak can be used with a correlation schedule to enumerate the SPB in the food sample. In another embodiment, a visual test can also be used to identify color changes in the incubation mixture to provide a semi-quantitative enumeration of SPB effective after about 3 hours to 17 hours of incubation.

1 . A method of detecting or enumerating sulfide-producing bacteria in a fish sample, comprising: 
 preparing the fish sample as a liquified fish sample;    forming an incubation mixture by combining the liquified fish sample with a growth medium having a color and comprising iron and sulfur, and wherein an iron precipitate is formed in the incubation mixture when sulfide-producing bacteria act upon the iron and sulfur in the growth medium;    incubating the incubation mixture at an incubation temperature; and    determining a time to color change at which time the color of the incubation mixture changes from yellow to black due to an accumulation of the iron precipitate in the incubation mixture.    
   
   
       2 . The method of  claim 1  comprising the additional step of estimating the number of sulfide-producing bacteria in the fish sample by comparing the time to color change to a correlation schedule.  
   
   
       3 . The method of  claim 1  wherein in the step of incubating the incubation mixture, the incubation temperature is from about 28° C.±0.5° C. to about 35° C.±0.5° C.  
   
   
       4 . The method of  claim 1  wherein in the step of incubating the incubation mixture, the predetermined incubation period is one of 3±0.5 hours, 4±0.5 hours, 5±0.5 hours, 6±0.5 hours, 7±0.5 hours, 8±0.5 hours, 9±0.5 hours, 10±0.5 hours, 11±0.5 hours, 12±0.5 hours, 13±0.5 hours and 14±0.5 hours.  
   
   
       5 . The method of  claim 1  wherein in the step of forming an incubation mixture, the iron is provided as ferric citrate or ferrous sulfate.  
   
   
       6 . The method of  claim 1  wherein in the step of forming an incubation mixture, the sulfur is provided as cysteine and thiosulfate.  
   
   
       7 . The method of  claim 1  wherein in the step of estimating the number of sulfide-producing bacteria, the correlation schedule comprises:  
     
       
         
               
               
             
                   
               
                   
               
                 Detection Time (hr) 
                 Number of SPB (CFU/g) 
               
                   
               
                 3-6 
                 5 × 10 6   
               
                  7-10 
                 5 × 10 5 -5 × 106 
               
                 11-12 
                 10 3 -5 × 10 5   
               
                 >12 
                 <10 3   
               
                   
               
                   
               
           
              
              
              
              
             
             
              
              
              
              
              
              
             
          
         
       
     
   
   
       8 . A method of detecting and/or enumerating sulfide-producing bacteria in a food sample, comprising: 
 providing an intact food sample;    providing a growth medium comprising iron and sulfur and disposing the food sample into the growth medium forming an incubation mixture, and wherein an iron precipitate is formed in the incubation mixture when sulfide-producing bacteria from the food sample act upon the iron and sulfur in the growth medium;    incubating the incubation mixture at an incubation temperature; and    determining a time to color change at which time the color of the incubation mixture changes to black due to an accumulation of the iron precipitate in the incubation mixture.    
   
   
       9 . The method of  claim 8  wherein in the step of providing the food sample, the food sample is selected from the group comprising beef, lamb, pork, poultry, fish, shellfish, molluscs, marine animals, crustaceans, or any product derived therefrom.  
   
   
       10 . The method of  claim 8  comprising the additional step of estimating the number of sulfide-producing bacteria on the food sample by comparing the time to color change to a correlation schedule.  
   
   
       11 . The method of  claim 8  wherein in the step of incubating the incubation mixture, the incubation temperature is from about 28° C.±0.5° C. to about 35° C.±0.5° C.  
   
   
       12 . The method of  claim 8  wherein in the step of incubating the incubation mixture, the predetermined incubation period is one of 3±0.5 hours, 4±0.5 hours, 5±0.5 hours, 6±0.5 hours, 7±0.5 hours, 8±0.5 hours, 9±0.5 hours, 10±0.5 hours, 11±0.5 hours, 12±0.5 hours, 13±0.5 hours and 14±0.5 hours.  
   
   
       13 . The method of  claim 8  wherein in the step of forming an incubation mixture, the iron is provided as ferric citrate or ferrous sulfate.  
   
   
       14 . The method of  claim 8  wherein in the step of forming an incubation mixture, the sulfur is provided as cysteine and thiosulfate.  
   
   
       15 . The method of  claim 8  wherein in the step of estimating the number of sulfide-producing bacteria, the correlation schedule comprises:  
     
       
         
               
               
             
                   
               
                   
               
                 Detection Time (hr) 
                 Number of SPB (CFU/g) 
               
                   
               
                 3-6 
                 5 × 10 6   
               
                  7-10 
                 5 × 10 5 -5 × 10 6   
               
                 11-12 
                 10 3 -5 × 10 5   
               
                 >12 
                 <10 3   
               
                   
               
                   
               
           
              
              
              
              
             
             
              
              
              
              
              
              
             
          
         
       
     
   
   
       16 . A method of detecting and/or enumerating sulfide-producing bacteria in a fish sample, comprising: 
 providing a fish sample which is intact;    providing a growth medium comprising iron and sulfur and disposing the fish sample into the growth medium forming an incubation mixture, and wherein an iron precipitate is formed in the incubation mixture when sulfide-producing bacteria from the fish sample act upon the iron and sulfur in the growth medium;    incubating the incubation mixture at an incubation temperature; and    determining a time to color change at which time the color of the incubation mixture changes to black due to an accumulation of the iron precipitate in the incubation mixture.    
   
   
       17 . The method of  claim 16  wherein in the step of providing the fish sample, the fish sample is selected from the group comprising freshwater fish, saltwater fish, shellfish, and molluscs, and crustaceans, or any product derived therefrom.  
   
   
       18 . The method of  claim 16  comprising the additional step of estimating the number of sulfide-producing bacteria on the fish sample by comparing the time to color change to a correlation schedule.  
   
   
       19 . The method of  claim 16  wherein in the step of incubating the incubation mixture, the incubation temperature is from about 28° C.±0.5° C. to about 35° C.±0.5° C.  
   
   
       20 . The method of  claim 16  wherein in the step of forming an incubation mixture, the iron is provided as ferric citrate or ferrous sulfate.  
   
   
       21 . The method of  claim 16  wherein in the step of forming an incubation mixture, the sulfur is provided as cysteine and thiosulfate.  
   
   
       22 . The method of  claim 16  wherein in the step of estimating the number of sulfide-producing bacteria, the correlation schedule comprises:  
     
       
         
               
               
             
                   
               
                   
               
                 Detection Time (hr) 
                 Number of SPB (CFU/g) 
               
                   
               
                 3-6 
                 5 × 10 6   
               
                  7-10 
                 5 × 10 5 -5 × 10 6   
               
                 11-12 
                 10 3 -5 × 10 5   
               
                 >12 
                 <10 3