Mucopolysaccharide having flocculating activity of protein and method for producing the same
This invention retates to a newly found mucopolysaccharide having flocculating activity of protein and method for producing the same by microorganisms. 
     Various materials are known to have flocculating activity of protein (flocculant), and are used for separating proteins dissolved or suspended in water. For example, persimmon tannin which is a known flocculant has been used for sedimentating lees in brewages. The other known flocculants are, for example, polyacrylamide, chitosan, calcuim ions, ferric ions, gelatin, and solium alginate. The flocculants became more important recently for precipitating proteins in waste water of food manufacturing, for separating microbial cells for the production of &#34;single cell proteins&#34;. 
     A new flocculant of mucopolysaccaride has been found in a culture broth of microorganism. The mucopolysaccharide contains in the molecule amino acids, amino sugars and organic acids. 
     The newly found mucopolysaccharide is produced by various microorganisms as exemplified below: 
     Aspergillus sojae AJ 7002 (FERM -P 2558) 
     aspergillus flavus AJ 17047 (FERM -P 2559) 
     aspergillus ochraceus AJ 7184 (FERM -P 2560) 
     aspergillus tamarii AJ 7249 (FERM -P 2561) 
     Microorganisms identified by FERM -P numbers are available from the Fermentation Research Institute of the Agency of Industrial Science &amp; Technology, Chiba-shi, Chiba-ken, Japan. 
     When the microorganisms as above are cultured aerobically in conventional aqueous medium for 1 to 5 days preferably with adjusting pH 3 to 8 and 20° to 40° C, substantial amount of the mucopolysaccharide is accumulated in the culture medium. 
     The aqueous culture medium contains carbon sources, nitrogen sources, iorganic ions, and where required minor organic nutrients. Suitable carbon sources are, for example, starch, glucose, sucrose, molasses, glycerine, ethanol and acetic acid. As the nitrogen source, ammonium ions, nitrate ions, urea, proteins, amino acids and amines are preferably used. Minor organic nutrients are usually vitamines, amino acids and purines. 
     The mucropolysaccharide produced in the aqueous culture medium can be precipitated and recovered by adjusting a pH at 7.2 to 7.8, by addition of organic solvent such as ethanol or acetone, to the aqueous culture medium, by addition of inorganic ions such as calcium ions, magnesium ions, or aluminium ions, or by salting out method. When required, the precipitated mucopolysaccharide is purified by various known methods such as gel-filtration or ion-chromatography. The aqueous culture broth, cells and mycerium can be used for flocculating proteins as they are, since they contain enough flocculating activity for the purpose. 
     The mucropolysaccharide thus obtained has the following chemical and physical characteristics: 1. U V spectrum: FIG. 1. 
     2. I R spectrum: FIG. 2. (KBr) 
     3. Elemental analysis: Contains carbon, hydrogen, oxygen and nitrogen, but no phosphorus or sulfur. 
     4. Constituents: 
     Proteins: 27.5% 
     Amino sugars: 
     glucosamine: 0.3% 
     galactosamine: 20.9% 
     Organic acids 
     2-ketogluconic acid: 35.3% 
     5. Molecular weight: 
     more than 2 × 10 5  (gel-filtration method with &#34;Sephadex G-200&#34;) 
     6. Viscosity: 
     400 c.p. (in 1N HCl at 22° C) 
     7. Contents of amino acids: Table 3 
     8. Stability: 
     The flaccutaing activity is inactivated by the treatment of protease or periodic acid. When aqueous solution of the mucopolysaccharide (pH 9.0) is heated at 100° C for 20 minutes, 70% of its activities is lost, while the flocculating activity is not lost by heating at pH 7.5 and 90° C for 20 minutes 
     9. Amino acid contained: Table 1 
     Method of preparation of the mucopolysaccharide: An aqueous culture medium was prepared to contain, per deciliter, 0.1 g KH 2  PO 4 , 0.1 g MgSO 4 .7H 2  O, and 2.0g casein. 
     15 L of the aqueous culture medium was placed in 30 l fermentation vessel, and inoculated with Aspergillus sojae FERM -P 2558. Cultivation was carried out aerobically at 30° C for 48 hours. 
     Culture broth thus obtained was centrifuged to remove mycerium, and adjusted with 6N HCl at pH 7.0. The precipitates of mucopolysaccharide were separated by filtration and washed with 500 ml acetone and subsequently 300 ml chloroform-methanol mixture, and dissolved in 2 l of 1N HCl. After removing impurities by centrifuging, the solution was adjusted to pH 7.0 with 6N NaOH, whereby precipitates of the mucopolysaccharide were formed. This purification procedure was repeated twice. The resultant precipitates were washed with 200 ml of 0.005N NaOH twice and 300 ml of water once, and dissolved in 200 ml of 0.1N HCl. The solution was added with trichloroacetic acid to contain 5 g/d1 in the solution, and heated at 50° C for 10 minutes, centrifuged to remove impurities, and dialyzed against 100 l water for 72 hours. Freeze-dried mucopolysaccharide (1.45 g) was separated from the dialyzed solution. 
     The mucopolysaccharide produced by an Aspergillus microorganism employed in this invention may differ slightly from that produced by another. All mucopolysaccharides within the scope of the invention, however, will have the foregoing chemical and physical characteristics. 
     The mucopolysaccharide of this invention can be applied for flocculating and sedimentating any proteins dissolved or suspended in water, and used for recovering proteins or removing proteins from waste water containing proteins. For example, yeast, bacterial cells and mycerium which are a protein can be easily sedimentated when the mucopolysaccharide is added to the solution containing those microbial cells and therefore the mucopolysaccharide can be applied for &#34;Sincle Cell Protein&#34; production. Waste water from which proteins are removed by the mucopolysaccharide is, for example, fermentation processing water, brewing water, and fish processing water. The mucopolysaccharide can be applied also for sedimentating lees in brewages or in refressing drinks. 
    
    
     EXAMPLE 1 
     An aqueous culture medium (50ml) containing, per deciliter 0.1 g K 2  HPO 4 , 0.1 g MgSO 4 .7H 2  O, and 3.0 g yeast extract, of pH 6.0 was placed in 500 ml flasks and heated with steam. Aspergillus sojae AJ 7002 was inoculated in the aqueous culture media and cultured at 30° C for 70 hours with shaking. 
     The resultant culture broth was filtered with guaze to remove mycerium, and mixed with 50 ml of acetone. Fibrous precipitates were winded on glass rod, washed with ethylalcohol and freeze-dried. It weighed 56 mg. Each protein (microbial cells or proteins) listed in Table 2 (200 mg) was suspended or dissolved in 50 ml water placed in 50 ml graduated cylinder. 
     The mucopolysaccharide mentioned above was dissolved in 50 ml of 0.01N HCl and a portion of the solution was added to the graduated cylinder to contain in the protein suspension 5 ppm or 10 ppm. Sedimention rate of the microbials cells was determined by measuring the height of clear part in the cylinder. Sedimentation rate of proteins was determined by weighing filtered protein precipitates. The results are shown in Table 2. 
     EXAMPLE 2 
     An aqueous culture medium was prepared to contain, per deciliter, 0.1 g KH 2  PO 4 , 0.1 g MgSO 4 .7H 2  O, 0.2 g (NH 4 ) 2  SO 4  and 2.0 g casein, adjusted to pH 6.0. 
     Aspergillus flavus AJ 17047 was cultured and the flocculant accumulated in the culture broth was recovered by the analogus manner as in Example 1. The mucopolysaccaride recovered weighed 73 mg. 
     The flocculation activity of the mucopolysaccharide was determined by the same method as in Example 1 and is shown in Table 3. 
     EXAMPLE 3 
     A half liter of waste water of fish processing (optical density at 520 nm being 0.75, being pH 6.7, being COD 1400 ppm and containing 500 ppm proteins) was added with the mucopolysaccharide shown in Example 1 to contain 20 ppm, and filtered to remove precipitates formed. Optical density of the filtrate at 520 nm was 0.15, pH was 6.7, COD was 120 ppm and proteins concentration was 8 ppm. 
     EXAMPLE 4 
     Bakers&#39; yeast (200 g) was extracted with 1 liter of boiling water of pH 8.0 for an hour, the water solution was centrifuged and the resultant supernatant contained 14.0% residue on evaporation and was 2.8 optical density at 520 nm. The water solution was added with 10 ppm of the mucopolysaccharide shown in Example 1, and centrifuged at 1000 r.p.m. for 30 minutes, whereby the optical density was reduced to 0.1. 
     While, when sodiun alginate or polyacrylamine (each 10 ppm) was used in place of the mucopolysaccharide to the waste water, the optical density of the waste water was not reduced. 
     EXAMPLE 5 
     Activated sladge (200 ml) containing 3.2% residues on evapolation was added with 200, or 400 ppm of the mucopolysaccharide of Example 1 and stirred for 5 minutes, and placed in 200 ml graduated cylinder. 
     For the purpose of comparison, sodium alginate, polyacrylamide or ferric chloride was used as the flocculant. 
     The sedimentation rates are shown in Table 4. 
     
                       Table 1                                                     
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Amino acid          (%)                                                   
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Tryptophan          --                                                    
Lysine              0.9                                                   
Histidine           0.4                                                   
Ammonia             --                                                    
Arginine            0.9                                                   
Cysteic acid        0.1                                                   
Taurine             --                                                    
Aspartic acid       1.9                                                   
Threonine           0.8                                                   
Serine              2.2                                                   
Glutamic acid       4.3                                                   
Proline             0.9                                                   
Glycine             0.8                                                   
Alanine             1.2                                                   
Cystine             0.1                                                   
Valine              2.9                                                   
Methionine          0.4                                                   
Isoleucine          1.4                                                   
Leucine             1.7                                                   
Tysosine            3.5                                                   
Phenylalanine       1.2                                                   
β-Alanine      --                                                    
Glucosamine         --                                                    
γ-Aminobutylic acid                                                 
                    --                                                    
Total               25.6                                                  
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                       Table 2                                                     
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               Sedimentation rate (cm/min)                                
               The amount of flocculant                                   
Proteins         0          5        10ppm                                
______________________________________                                    
Microbial cells                                                           
Candida lipolytica                                                        
                 0.03       1.8      3.2                                  
ATCC 16618                                                                
Saccharomyces cerevisiae                                                  
                 0.01       1.9      3.3                                  
ATCC 15248                                                                
Rhodotorula glutinis                                                      
                 0.04       2.0      3.1                                  
ATCC 4054                                                                 
Endomycopsis burtonii                                                     
                 0.05       1.5      2.6                                  
ATCC 13169                                                                
Hansenula anomala                                                         
                 0.02       1.8      3.2                                  
ATCC 2149                                                                 
Debaryomyces vanriji                                                      
                 0.03       1.8      3.8                                  
ATCC 20125                                                                
Pichia ohmeri    0.02       2.1      3.9                                  
ATCC 9766                                                                 
Trycosporon fermentans                                                    
                 0.03       1.3      2.8                                  
ATCC 10675                                                                
Brevibacterium flavum                                                     
                 less than                                                
ATCC 14067       0.01       1.4      2.5                                  
Brevibacterium lacto-                                                     
                 less than                                                
fermentum ATCC 13869                                                      
                 0.01       1.7      3.2                                  
Bacillus megatherium                                                      
                 less than                                                
ATCC 6455        0.01       0.9      2.3                                  
Bacillus subtilis                                                         
                 less than                                                
ATCC 6051        0.01       1.1      2.4                                  
Escherichia coli less than                                                
ATCC 4157        0.01       0.5      1.3                                  
Oerskovia turbata                                                         
                 less than                                                
ATCC 25835       0.01       0.7      1.8                                  
Chlorella burgaris                                                        
                 0.05       3.5      4.3                                  
ATCC 9765                                                                 
Manaskas anka    0.12       4.6      7.8                                  
ATCC 16360                                                                
Proteins         less than                                                
milk casein      0.01       0.3      1.2                                  
                 less than                                                
bovin albumin    0.01       0.2      1.1                                  
soybean protein  less than                                                
(containing 53.7%                                                         
                 0.01       0.3      1.2                                  
protein)                                                                  
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                       Table 3                                                     
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               Sedimentation rate(cm/min)                                 
               The amount of flocculant                                   
               added                                                      
Proteins         0          5        10ppm                                
______________________________________                                    
Microbial cells                                                           
Candida lipolytica                                                        
                 0.03       1.9      3.3                                  
ATCC 16618                                                                
Saccharomyces cerevisiae                                                  
                 0.02       2.0      3.4                                  
ATCC 15248                                                                
Rhodotorula glutinis                                                      
                 0.03       2.3      3.2                                  
ATCC 4054                                                                 
Endomycopsis burtonii                                                     
                 0.04       1.5      3.0                                  
ATCC 13169                                                                
Hansenula anomala                                                         
                 0.02       1.9      3.2                                  
ATCC 2149                                                                 
Debaryomyces vanriji                                                      
                 0.03       1.8      3.8                                  
ATCC 20125                                                                
Pichia ohmeri    0.02 2.1   3.9                                           
ATCC 9766                                                                 
Trycosporon fermentans                                                    
                 0.03       1.3      3.0                                  
ATCC 10675                                                                
Brevibacterium flavum                                                     
                 less than                                                
ATCC 14067       0.01       1.4      2.5                                  
Brevibacterium lacto-                                                     
                 less than                                                
fermentum ATCC 13869                                                      
                 0.01       1.7      3.2                                  
Bacillus megatherium                                                      
                 less than                                                
ATCC 6458        0.01       0.9      2.3                                  
Bacillus subtilis                                                         
                 less than                                                
ATCC 6051        0.01       1.1      2.4                                  
Escherichia coli less than                                                
ATCC 4157        0.01       0.6      1.6                                  
Oerskovia turbata                                                         
                 less than                                                
ATCC 25835       0.01       0.8      1.9                                  
Chlorella burgaris                                                        
                 0.05       3.5      4.3                                  
ATCC 9765                                                                 
Monaskas anka    0.12       4.6      7.8                                  
ATCC 16360                                                                
Proteins                                                                  
                 less than                                                
milk casein      0.01       0.5      1.2                                  
                 less than                                                
bovin albumin    0.01       0.6      1.3                                  
soybean protein  less than                                                
(containing 53.7%                                                         
                 0.01       0.5      1.3                                  
protein)                                                                  
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                       Table 4                                                     
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               Amount used  Sedimentation                                 
Flocculant     (ppm)        rate (cm/hr)                                  
______________________________________                                    
The mucopolysaccharide                                                    
               200          2.3                                           
of this invention                                                         
               400          4.2                                           
Polyacrylamide 400          1.2                                           
FeCl.sub.3     4000         3.2                                           
none           --           1.2                                           
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