Patent Publication Number: US-6037451-A

Title: Method of degreasing bone for gelatin manufacture

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application relates to commonly assigned application Ser. No. 09/186,443 and now U.S. Pat. No. 5,919,906 filed simultaneously herewith. This application relates to commonly assigned copending application Ser. No. 09/185,491, filed simultaneously herewith. This application relates to commonly assigned copending application Ser. No. 09/185,440, filed simultaneously herewith. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the production of gelatin. More, particularly the present invention provides a method of degreasing bone from which high quality gelatin can be extracted. 
     BACKGROUND OF THE INVENTION 
     Rendered bone contains varying amounts of fat or grease depending on the efficiency of the rendering process at removing tallow from the bone. Multiple sourcing of rendered bone results in a wide range of fat content in the starting bone material for gelatin manufacture. Fat contained in rendered bone is an impurity in photographic gelatin that must be removed during the gelatin manufacturing process. It is known that the fat content of bone directly effects photographic gelatin yield. Typically photographic gelatin yields are defined by the amount of gelatin produced with an absorbance at 420 nm of less than 0.150. Analysis of production data comparing low fat and high fat bone suppliers suggests that each 1% decrease in fat content of the starting bone results in a 0.4% increase in photographic gelatin yield. 
     Currently, fat or grease is removed at various stages throughout the gelatin manufacturing process with varying levels of efficiency. Typically, cattle bone used in the manufacture of high grade gelatin, is cleaned of its fats, grease, blood and bone marrow by hot water washing. The bone is then demineralized in a liquid/solid extraction/reaction process using an acid solution. Removal of minerals from the bone particles in the demineraliztion process yields particles of a material known as ossein. The gelatin is extracted from the ossein. 
     Following washing and acidulation, and sometimes liming of the cattle bone, the fat content ranges from 4-6% of the organic content of ossein. Although fat is reduced through each step of the gelatin manufacturing process, fat remaining in the final gelatin product is directly impacted by the fat content of the starting bone. Due to the inability to reproducibly reduce fat content of the bone to a low level, photographic gelatin yield is reduced. Ideally, to minimize photographic gelatin yield losses due to fat impurities a process for producing a uniform low fat rendered bone is required. This invention describes a single purification step specifically designed and optimized for fat removal from rendered bone. 
     SUMMARY OF THE INVENTION 
     The present invention is a process for degreasing bone for the manufacture of gelatin. A lipase solution at a concentration of greater than 0.1 ppm is added to a collagen containing material and treated for a time sufficient to solubilize the fat contained in the collagen containing material to produce degreased collagen containing material with a fat content of less than 1 weight percent. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is a process for degreasing bone for the manufacture of gelatin. A lipase solution at a concentration of at least 0.1 ppm is added to a collagen containing material. The collagen containing material is treated for a time sufficient to solubilize fat contained in the collagen containing material to produce degreased collagen containing material with a fat content of less than 1 weight percent. 
     Collagen containing material is that requires degreasing is obtained from animal bone and hide. Bone is defined as bone material that is essentially freed of attached meat and may contain cartilage, connective tissue, marrow, etc. Bones are obtained from beef, chicken, pork and fish. Preferably cow bones are used as a starting material. 
     Lipases are biological catalysts or enzymes found in living organisms. More specifically, lipases are a group of carboxylesterases, which preferentially hydrolyze emulsified neutral fats to fatty acids and glycerol or monoacylglycerols. The commercially available lipases described herein were produced via biofermentation of genetically modified strains of bacteria 
    
    
     EXAMPLES 
     Lipase effectiveness on grease removal from bone was determined by providing, ground cow bone incubated with 0.5-5% w/w lipase for varied amounts of time at temperatures ranging from 17-70 ° C. The pH was varied from 4.0 to 12.0. Following incubation the enzyme solution containing released fatty acids was decanted from the ground bone. Fat still present in the ground bone was detected by analysis of the fatty acid content of the treated ground bone. Briefly, ground bone (0.2 grams in 50ml water) was boiled for 15 minutes and allowed to cool to room temperature. Following pH adjustment to pH 45 with weak acetic acid the sample was again boiled for 15 minutes. Subsequently the sample was incubated with 0.5% v/w alkaline protease for 30 minutes at 40 ° C., pH 10. This procedure degrades the bone to peptides and allows determination of total fat content as measured by the fatty acid components of the grease. 
     Fatty acid content in a degraded gelatin sample matrix was determined by Gas Chromatography of fatty acid methyl ester derivatives. The fat containing degraded gelatin sample matrix was boiled with NaOH and cooled prior to extraction with petroleum ether at a low pH. The ether extracted fatty acids were converted to their methyl ester derivatives using Borontrifluoride/methanol. The derivatives were extracted into n-hexane and chromatographed on a 30 m×0.32 mm, 0.25 micron loading DB-5 capillary column. The concentration of each fatty acid was calculated from the computer integrated peaks using the response factor of an internal standard. 
     
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LIPASES:                                                                  
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Lipase G-1000       Genencor International                                
                     Lipomax CXT Enzyme Genencor International            
  Lipase BC-100 Genencor International                                    
  Lipolase Novo Nordisk                                                   
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     EXAMPLE 1: 
     Reduction of the fat content of bone from 3.35% to 0.60% following treatment with 1% Novo Lipolase: 
     
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         ppm                                                              
           Myristic Palmitic Oleic Stearic                                
                                          Total                           
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Bone in H.sub.2 O                                                         
           220      1836     3132  1524   6712                            
  1% Novo Lipolase:                                                       
  RT 1 hr. 0 643 1006 674 2023                                            
  RT 5 hr. 0 437 694 468 1599                                             
  RT 8 hr. 0 364 563 355 1282                                             
  50° C. 1 hr. 0 338 514 355 1207                                  
  50° C. 5 hr. 0 214 301 232 747                                   
  50° C. 8 hr. 0 455 753 498 1706                                  
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     In this Example, ground bone was incubated with 1% (volume) Novo Lipase for 1, 5 or 8 hours at room temperature and 50 ° C. Fat content of the bone following removal of the lipase soaks was compared to the untreated bone in water. The results above show that I hour lipase treatment at either temperature was sufficient for fat removal. 
     EXAMPLE 2: 
     Dramatic reduction in the fat content of bone from a very high (28%) fat, bone sample following treatment with Genencore Lipase G1000 or Lipomax. In this study Lipase G-1000 was incubated with ground bone for 5 hours at pH 7.0, 45 C., and Lipomax was incubated with ground bone for 30 minutes at pH 9.0, 55 C. Fat content of the bone following these treatments is shown below. 
     
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        ppm                                                               
          Myristic                                                        
                  Palmitic Oleic  Stearic                                 
                                        total                             
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No Enzyme 1517    15502    26748  12588 56355                             
  1.0% Lipase 160 1171 1730 949 4010                                      
  1.0% Lipomax 0 895 174 1623 2692                                        
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     Both Lipase G- 1000 and Lipomax reduced the fat content of the starting bone to less than 0.5 weight %, although the conditions for optimum reaction (temperature, time, pH and enzyme concentration) are dependent on the lipase being used. 
     EXAMPLE 3: 
     In this example varying concentrations of Lipase BC-100 were incubated with cow bone (3/16&#34; to &gt;3/8&#34;) for 26 hours at pH 7.0, 20.5 C. Following enzyme incubation the enzyme solution was decanted from the bone and the remaining fat content of the bone was determined. Fat content of bone following Lipase BC-100 treatment is shown below. These results show that a water wash does not remove fat from the starting bone. 
     
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            PPM                                                           
              Myristic                                                    
                      Palmitic                                            
                              Oleic                                       
                                   Stearic                                
                                         Total                            
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Starting Bone 362     2592    4008 1902  8864                             
  Water Wash 352 2636 4833 1971 9793                                      
  0.001 ppm Lipase BC-100 371 3003 4777 2200 10350                        
  0.01 ppm Lipase BC-100 272 2412 3969 1776 8429                          
  0.1 ppm Lipase BC-100 334 2850 4988 2123 10294                          
  1 ppm Lipase BC-100 187 1500 2354 1184 5255                             
  30 ppm Lipase BC-100 0 510 597 400 1506                                 
  143 ppm Lipase BC-100 0 455 671 370 1495                                
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     The results above show that for the particular lipase tested, greater than 0.1 ppm (weight) concentration is required to reduce fat content of the bone. The minimum level of lipase depends on the enzyme used. 
     The invention has been described in detail with particular reference to certain preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.