Method for limiting damage due to bacteriophages in fermentation media
United States Patent Yamanaka et al.  
 1451 Apr. 29, 1975 METHOD FOR LIMITING DAMAGE DUE TO BACTERIOPHAGES IN FERMENTATION MEDIA Inventors: Shigeru Yamanaka, Yokohama;  
 Nobukazu Kashima, Kawasaki; Koji Mitsugi, Yokohama, all of Japan Assignee: Ajinomoto Co., Inc., Tokyo, Japan Filed: Sept. 3, 1974 Appl. No.: 503,047  
 Foreign Application Priority Data Sept. 4, 1973 Japan 48-99635 US. Cl 195/122; 195/47 Int. Cl. ..C12b l/24; C12d 13/06 Field of Search 195/100, 101, 102, 110,  
 References Cited FOREIGN PATENTS OR APPLICATIONS 45-32234 10/1970 Japan 195/122 Primary Examiner-A. Louis Monacell Assistan! Examiner-C. A. Fan  
 Attorney, Agent, or Firm-Cooper, Dunham, Clark, Griffin &amp; Moran [57] ABSTRACT 8 Claims, No Drawings METHOD FOR LIMITING DAMAGE DUE TO BACTERIOPI-IAGES IN FERMENTATION MEDIA BACKGROUND OF THE INVENTION The presence of bacteriophages in fermentation media designed to produce important products such as drugs, vitamins, amino acids and the like is a serious problem which has plagued the industrial production of these important materials by limiting the growth of desirable bacteria in growth cultures. Accordingly, much effort has been directed to the problem of controlling these bacterial viruses.  
 THE INVENTION A method has now been discovered for limiting the damage resulting from the presence and growth of bacteriophages in fermentation media by effecting the fermentation in a medium containing an amount of at least one selected N-acylamino derivative of glutamic acid, glutamine or homocysteic acid which is effective to inhibit the production of the bacteriophage. The N- acylamino group is characterized as one which contains an alkyl or alkenyl moiety having from about 16 to 18 carbon atoms. Metallic salts of these compounds may also be employed, the preferred being alkali metal salts, especially sodium or potassium salts.  
  A particular advantage of the invention is that. while the selected agents inhibit propagation of the bacteriophages they have little or no effect on the growth of the bacteria or the production of the desired product of the fermentation.  
  The inhibiting agent can be added to the medium prior to the initiation of bacterial growth, or after such growth is already well underway. Normally, it is added in the initial stages of fermentation or at least before the growth of bacteria reaches the stationary phase. The amount of the selected compound or compounds employed is typically less than 50 u/ml, and there is rarely any advantage in exceeding this amount. It is generally preferred to use more than 2 palm] of N- acylamino acid. It is not necessary to alter the usual methods of bacteia cultivation due to the presence of the inhibiting agent.  
  The advantage of this invention are applicable to fermentation processes including, for example, those designed for the production of antibiotics, enzymes, amino acids, nucleotides and nucleosides.  
  The following non-limiting examples are given by way of illustration only.  
 EXAMPLE 1 An aqueous culture medium was prepared to contain, per deciliter, 3.6 g glucose, 0.1 g KI-I PO 0.1 g MgSO -7H O, 2 mg FeSO &#39;7l-I O, 2 mg MnSO &#39;4I-I O, 24 mg (as nitrogen) soyprotien acid hydrolyzate, 100 pg thiamine-HCl, 0.3 pg biotin. N-acylamino acid shown in Table l (50 #g/ml) was also added to the medium.  
  Brevibacterium lactofermentum ATCC 13869 was inoculated into 30 ml of the aqueous medium placed in 500 ml flask, to a concentration of 2.74 X cells/ml, and the medium was infected with phage L, (Journal of the Agricultural Chemical Society of Japan, Vol. 37, No. 1], 686-689 (1963) at a concentration of 1.16 X 10 P.F.U./ml.  
 LII  
 I Cultivation was carried out at 30C for 24 hours with shaking. Glutamic acid accumulated in the cultivation broth is shown in Table 1.  
 Table 1 Glutamic acid accumulated g/dl N-acyl amino acid added N-acyl moiety. amino acid moiety Lauric acid Myristic acid Palmitic acid Stcaric acid Olcic acid Arachidic acid Lauric acid Myristic acid Palmitic acid Stearic acid Oleic acid Arachidic acid Laurie acid Homocysteic ac id Glutamic acid Myristic acid Palmitic acid Stearic acid Oleic acid As a comparison test, when the fermentation was carried out without adding either N-acyl amino acid or phages, 1.63 g/dl glutamic acid accumulated, while when the fermentation was carried out in the presence of infecting phages but without adding 50 ug/ml N-acyl amino acid, only 0.23 g/dl glutamic acid accumulated.  
 EXAMPLE 2 In the process of glutamic acid fermentation as described in Example 1, N-acylamino acid as shown in Table 2 was used (50 ug/ml), and cultivation was carried out by the same manner as in Example 1. Glutamic acid accumulated in the fermentation broth is shown in Table 2.  
 Table 2 Glutamic acid accumulated g/dl N-acyl amino acid added N-acyl moiety. amino acid moiety N-acylamino acid as shown in Table 3 was added to the medium of Example 1 in the amount shown in Table 3. Cultivation of Brevibacterium lactofermentum ATCC 13869 was carried out by the same manner as in Example 1. The amounts of glutamic acid shown in Table 3 were accumulated.  
 Table 3 Table 5-Continued Amount N-oleoyl Nstcaroyl N-palmitoyl Phage Biotin N-palmitoyl Growth Glutamic acid added glutamic acid glutamine homocystcic glutamic acid accumulated acid 5 (#g/l) (Mg/ml) (g/ (r g/ml) 1.551001) .21 (g/dl) 0.21 (g/dl) 0 a .89 0.99  
  3 50 0.940 1.16 g8 12 12-? {2 Infected 5 50 0.920 1.65 70 1.21 1.18 1.05 l 50 0980 100 1.01 0.98 0.75 0 200 0.70 0.50 0.48  
 Table 6 EXAMPLE 4 Phage Biotin N-palmitoyl Growth Glutamic acid glutamic acid accumulated Brevibacterium lactofermentum ATCC 13869 was (#g/l) (pg/ml) 1/26) (g/dl) inoculated into the medium of Example 1 (2 X 10 O 0 4O cells/ml) which was infected at an initial concentration I 0 8: 8 {:23 of 2 X 10 phages/ml. N-acylamino acid mixtures con- Not 2 0 0.950 1.23 taining 50 percent N-oleoyl glutamic acid and 50 perg g 81 8&#39;32 cent N-palmitoyl glutamic acid were added in the 10 0 1:24 0:04 amount of 20 ug/ml or 50 ug/ml at 0, 5, 8 or 12 hours 7 2 098 l 6 cultivation time. After 12 hours cultivation in the same 3 2 manner as in Example 1, the amounts of glutamic acid 3 -3Z 8%? as shown 1n Table 4 were found In the culture broths. 3 0 0560 0.38 Infected 5 0 0.870 0.61 Table 4 10 0 1.17 0.30 i i 8833 18% 0 5 8 12 Hours Hours Hours Hours I0 2 H5 (g/dl) (g/ (g/ l) tgl n 20 #g/ml 1.69 1.68 1.00 0.80 50 1.55 1.60 1.08 0.81 EXAMPLE 6 An aqueous culture medium was prepared to con- 35 tain, per deciliter, 10 g glucose, 0.1 g KH PO 0.04 g EXAMPLE 5 MgSO &#39;7H O, 0.2 mg ferrous ion, 0.2 mg manganese Brevibacterium lactofermentum ATCC 13869 was 100 mg (as mtrogen) P P acld&#39;hydfolyzatei cultured in the presence of phage L (phages number g (Nlihhsoh 5 thlamme&#39;HCl: and adlusted at cells number 73 x -2 or in the presence of pH 8.0. 20 Ml batches were placed 1n 500 ml flasks, phage S1 (phages number cells number 42 X 102) and to each flask was added the amount of N-stearoyl Phage S is reported in Agricultural and Biological glutamic aCIdFhOWn m Table Chemistry Japan vol 31 N0 7 8614367 (1967) The Brevibacterium lactofermentum FERM-P 1711 was medium ernployed was i Same as in Example 1 inoculated in each flask and cultured at 30C for 72 cept for the amount of biotin (shown in Table 5). N- 45 l Shakmg palmitoyl glutamic acid was added to the medium in Mlcroblal growth and Lflysme accumulated were the amount of O 2 or #g/mL termined and are shown in Table 7.  
  Growth of Brevibacterium lactofermentum ATCC Table 7 13869 was determined by measuring optical density of 26 times dilution at 562 mu. Glutamic acid in the cul- 50 PhagesNoJCells N-stearoyl Growth Lysine ture broth was also determined. The results with phage :322:12 i accumula&#39;ed L, and with phage S are shown in Tables 5 and 6, re- (Lg/m1) (X 1/26) (g/dn spectlvely.  
  0 0 1.10 3.80 Table 5 0.2 1.05 3.92 2 1.08 3.59 20 1.09 3.62 Phage Biotin N-palmitoyl Growth Glutamic acid 200 1.05 3.73 glutamic acid accumulated X -3 0 02 013 ag/1) wig/ml) l 0.2 0.32 0.56 2 0.58 1.20 0 0 0.406 1.11 20 1.10 3.63 I O 0.770 1.63 200 03 3 7 Not 2 0 0.950 1.23 infected 3 0 0.975 0.64 5 0 1.04 0.06 10 0 1.24 0.04  
  EXAMPLE 7 2 50 0.90 1.62 3 50 0.95 0.81 An aqueous culture medium was prepared to con- 28 1% 862 tain, per deciliter, 8 g glucose, 0.02 g KH PO 1.5 g. 3 O 0:300 NH NO 0.2 g CaCl 0.2 mg ferrous ion, 0.2 mg man- 5 0.16 ganese ion, 4 ml/dl soy-protein acid hydrolyzate, and&#39; 0.l g RNA of yeast. 20 Ml batches of the medium were placed in 500 ml flasks, and to each flask was added the amount of N-oleoyl glutamic acid potassium salt shown in Table 8. The flasks were sterilized with steam.  
  Each flask was inoculated with Bacillus subtilis FERM-P 2107, and was held at 34C for 72 hours with shaking. A phage of FERM-P 2107 was added after 12 hours cultivation. Growth of FERM-P 2l07 and guanosine accumulated in the culture broth were determined and are shown in Table 8.  
  An aqueous culture medium was prepared to contain, per deciliter, l g bouillon, l g peptone, 0.5 g soyprotein and 5 g soluble starch, and adjusted at pH 7.0. 50 Ml batches were placed in 500 ml flasks. To each flask was added the amount of N-oleoyl homocysteic acid shown in Table 9. The flasks were sterilized with steam, and inoculated with Bacillus subtilis FERM-P 305. Cultivation was carried out at 3 l .5C for 48 hours with shaking. After 6 hours cultivation l P.F.U. of phages/ml were added to the medium.  
  The protease activities shown in Table 9 were found in the culture broth.  
  6 Table 9 Phage N-olcoyl lrotcase homocysteic acid activity sodium salt (#g/ml) (units/dl) 0 600 0.1 1,340 Added 0 2,500 O.l 2,640 Not added What is claimed is:  
  1. A method for limiting damage resulting from the presence of a bacteriophage in a fermentation medium, which comprises effecting the fermentation in a medium containing an amount of at least one N-acylamino derivative of glutamic acid, glutamine, homocysteic acid, metallic salts thereof or mixtures thereof which is effective to inhibit the production of bacteriophage in the fermentation medium, said acyl radical of said N- acylamino group being characterized by containing an alkyl or alkenyl moiety having from about 16 to 18 carbon atoms.  
  2. A method as in claim 1, wherein the amount of N- acylamino derivative is less than 50 ug/ml.  
  3. A method as in claim 1, wherein the fermentation medium contains a glutamic acid producing strain of the genus Brevibacterium.  
  4. A method as in claim 1, wherein the fermentation medium contains a lysine producing strain of the genus Brevibacterium.  
  5. A method as in claim 1, wherein the fermentation medium contains a guanosine producing strain of the genus Bacillus.  
  6. A method as in claim 1, wherein the fermentation medium contains a protease producing strain of the genus Bacillus.  
  7. A method as in claim 1, wherein the N-acylamino acid is N-stearoylglutamic acid.  
 8. A method as in claim 1, wherein the N-acylamino acid is N-oleoyl glutamic acid.