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Streptomycin production by Streptomyces griseus from fractions of peanuts and soybeans
Streptomycin Production by Stre$fomyces griseus from Fractions of Peanuts and Soybeans H. H. Thornberry
and A. J. Shanahan
From the Department of Horticulture, University of Illinois, Received April 5, 1951
l
Urbana, Illinois
These studies were planned to determine the nutritive value of some fractions of peanut and soybean meal (oil extracted) for the production of streptomycin by Streptomyces griseus (Krainsky) Waksman and Henrici. Some of the preliminary results have been presented (1,2). The purpose of this paper is to report the later results obtained. MATERIALS
AND METHODS
The materials used were oil-extracted (solvent) peanut meal (No. P-136, Southern Regional Research Lab., U. 5. Dept. of Agr.) and soybean flakes (Northern Regional Research Lab., U.S.D.A.). These were fractionated by a procedure of protein extraction based upon the information supplied by personal communications (see acknowledgment) and by some pertinent references for peanut meal (3,4) and soybean meal (5,6,7). The following samples of peanut and soybean meal were used: I. Original meal, oil extracted. II. Residue, consisting of the solid material remaining after extraction of meal in water (100 g./l.) at pH 8.0 for 30 min. at 26°C. and straining through four-ply cheesecloth. III. Extract, materials in the aqueous suspension at pH 8.0 that filtered through four-ply cheesecloth. IV. Protein, substances that settled upon protein precipitation at pH 4.5 and standing for 4 hr. at room temperature. V. Waste liquor, substances remaining in the liquid after protein precipitation and sedimentation. The liquid was filtered through a 5-mm.-thick Celite filter cake to remove suspended particles of insoluble materials in the liquid.
Amount of these fractions referred to herein represent the material originating from that weight of original meal, i.e., 10 g. of waste liquor represents the waste liquor from 10 g. of original meal.
1Present address: erick, Maryland.
Biological
Department, 459
Chemical
Corps, Camp Detrick,
Fred-
460
H.
H.
THORNBERRY
AND
T.%BLE
A.
J.
SHANAHAN
I
Amount qf Streptomycin Produced by Streptomyces griseus from Non-Ashed and Ashed Fractions of Peanut and Soybean Meal in S-day Submerged Culture Soybean COIIC. O./l.=
Fractions
I. Original
II.
III.
meal
Residue
Extract
IV. Protein
5’. Waste liquor
1 5 10 20 30 49 1 5 10 20 30 40 1 5 10 20 30 40 1 5 10 20 30 40 1 5 10 20 30 40
Basal medium
-
Basal and corn steep non-ashed
10
Non-ashed d d
M-55.8* 13-43.5 21-11.6 12-11,7 d d 52-25.4 63-12.4 52-49.8 29-15.6 d d 52-36.0 44-54.6 22-11.1 12-18.6
d d
149-50.6 94-46.4 84-48.8 69-43.1 d d
57-30.3 118-46.0 154-66.9 133-61.1 d
d
123-87.0 92-36.4 97-34.3 172-66.9
d
d
d
d
16-108.9 15-79.5 19-37.2 24-53.2
o-O.0
41-57.0 52-23.8 43-40.9
d
d
d
d
Non-ashed
.%shed
37-22.8 244-60.3 351-25.2 109-37.4 83-52.5 17-22.6
94-36.5 180-24.3 216-51.7 74-32.6 76-28.4 77-21.6
17-30.7 40-26.5 76-18.0 81-21.6 12334.8 261-27.9
86-18.4 110-24.6 285-31.7 222-26.4 146-31.7 107-27.9
30-28.7 127-49.0 241-26.0 168-41.9 74-38.8 56-25.4
46-18.6 130-27.5 264-15.4 206-31.8 82-44.6 79-29.2
18-23.4 25-29.0 25-12.5 12-36.4 11-41.6 16-29.4
41-51.4 73-36.7 128-46.6 181-23.8 76-33.2 52-21.9 31-18.6 55-26.3 99-38.1 102-18.6 162-23.2 164-46.4
58-51.5 87-54.4 93-15.7 105-39.9
88-12.0 225-54.7 240-38.8 119-18.4
20-15.9 69-50.1 103-12.9 110-18.5 127-36.3 142-42.1
29-25.1
16-18.4
17-29.6
19-17.5
222-66.6
195-72.8
429-24.1
326-38.5
a Units of streptomycin/ml. (One unit is equivalent to 1 pg. streptomycin base as evaluated against a standard containing 800 pg. of streptomycin sulfate/mg.). b C.V. = coefficient of variability. c Amounts corresponding to grams of the original mea! from which derived. d Not determined.
461
STREPTOMYCIN PRODUCTION
Ashing of materials was accomplished in a muffle furnace at 600-650°C. The amount of ash from the fractions was not determined. The submerged culture of S. griseus, estimation of streptomycin and growth, growth conditions, and inoculum were similar to those previously stated (8,9). The basal medium consisted of glucose (Cerelose) 10 g., bactopeptone (Difco) 5 g., XaCl 5 g., and distilled water 1000 ml. -4 corn steep control medium for checking growth condit,ions consisted of 10 g. of corn steep added to 1 1. of this basal medium. All media were adjusted with NaOH or HCI to pH 7.0-7.2 before sterilization at 121°C. for 15 min. EXPERIMENTAL
AND RESULTS
Experiments were designed to measure the nutritive value of the peanut and soybean fractions in varied amounts in the basal medium for the production of streptomycin. The results from three runs in the case of peanut fractions (each run with flasks in triplicate) and two runs in the case of soybean fractions (each run with flasks in triplicate) are given in Table I. The results obtained from the spectrographic semiquantitative analysis (see acknowledgment) of peanut waste liquor ash are given by the following groups (the value in parentheses being the percentage of the element necessary to be detected) :
1. Elements present are Al (.OOl), B (.OOOl), Ca (.OOl), Cr (.OOOl), Cu (.OOOl), Fe (.OOOl), K (lO.O), Mg (.OOOl), Mn (.OOOl), P (.Ol), Si (.OOOl), Ti (.OOl), and V (.OOl). 2. Elements questionable are Ba (.Ol), MO (.OOOl), Sr (.Ol), and Zn (.Ol). 3. Elements highly questionable are Bi (.OOOl), Li (.OOl), Ni (.OOOl), Pb (.OOOl), and Rh (.OOl). Growth of the organism was good in media containing all fractions except, the proteins which supported only moderate growth. Growth in the basal medium was poor whereas that in the basal containing corn steep was excellent. The hydrogen-ion concentration of all media decreased as fermentation progressed. It changed from that of the initial values (pH 6.2-6.7) after sterilization
to an alkaline
reaction
(pH 7.9-8.9)
in 3 days.
Since the substances in some of the fractions obviously were in excess for streptomycin production, the effect of varied amounts of corn steep liquor in the medium was also determined. Optimal amounts of corn steep were from 4 to 10 g. of corn steep liquor per lit,er of medium. The
462
H.
H.
THORNBERRY
AND
A.
J.
SHANAHAN
yields of streptomycin obtained from these varied amounts of non-ashed corn steep were 1 g./l. 104 units/ml., 2 g. 130 units, 4 g. 292 units, 6 g. 397 units, 8 g. 370 units, 10 g. 258 units, 20 g. 133 units, 30 g. 80 units; whereas from corn steep ash they were 1 g. 96 units, 5 g. 194 units, and 10 g. 51 units.
DISCUSSION From the non-ashed peanut material, the waste liquor fraction gave the best yield of streptomycin. This indicates that the fractionation procedure providing this waste liquor removes some of the inhibitive substances and allows nutritive substances to remain with this fraction. However, all of the inhibiting substances are not removed by the fractionation because the waste liquor ash outyielded the comparable nonashed sample. Since all ashed materials outyielded their comparable non-ashed samples, minerals appear to be the principal nutrients of peanut meal for the production of streptomycin by this organism. Improper balance of the minerals in the meal ash is offered to explain the low yield of streptomycin from this ash. The relatively low yield of streptomycin from protein ash suggests that these proteins do not contain the nutritive minerals in sufficient concentration or balance for streptomycin production. Spectrographic analysis of peanut waste liquor ash shows the presence of two minor elements (Fe and Mn) of the three elements (Fe, Mn, and Zn) found to influence the metabolism of S. griseus in a chemically defined medium (8,9). Zinc, although questionable in the analysis, must have been present in the ash since growth was stimulated by this ash and zinc is necessary for growth (8,9). Zinc along with Mn, Fe, Co, Cu, B, and F have been reported in peanut kernels (10). However, in surface growth of S. griseus in Belmont medium containing either peptone or “Pronutrin” (casein hydrolyzate), zinc was reported to retard streptomycin production (11). From the non-ashed soybean material, sizeable yields of streptomycin were obt’ained from the original meal and all fractions except the protein one. Since soybean protein ash gave essentially a fourfold increase in production over the non-ashed material, it is evident that the proteins either acted in an inhibitory manner or retained the nutritive materials in an unavailable form. There was no indication of inhibitive substances in the other fractions. However, the residue ash at some of the concentrations employed did outyield the comparable amounts of
STREPTOMYCIN PRODUCTION
463
non-ashed materials. The results show that the nutritive value of soybean meal is associated with the minerals and the waste liquor fraction. The results herein suggest that the nutritive substances in the soybean meal used in commercial production of streptomycin and in the peanut (groundnut) meal in experimental productions (12) are related to the incorporated minerals, their balance for the organism being important. Waste liquors from the acid precipitation of peanut and soybean proteins are important and cheap nutrients for the production of streptomycin. However, their use commercially may await supplementation to achieve yields of streptomycin suitable for commercial processing. ACKNOWLEDGMENTS Acknowledgment and appreciation of assistance are extended to Dr. Allan K. Smith, Northern Regional Research Laboratory, for the soybean meal and valuable information on soybean protein and its extraction; to Dr. John D. Guthrie and Mr. Carrol L. Hoffpauir, Southern Regional Research Laboratory, for the peanut meal and valuable information on peanut protein and its extraction; to Dr. Victor J. Miller (now Department of Horticulture, University of Kansas) for the spectrographic analysis of peanut waste liquor ash during a course in physical methods under the supervision of Dr. D. T. Englis, University of Illinois; and to Mrs. Fredia M. Shannon for technical assistance. SUMMARY
1. Minerals are the principal nutrients in peanut and soybean meals for streptomycin production in the medium used. 2. Substances inhibitory to streptomycin production are present in peanut meal but are not present in soybean meal. 3. In the non-ashed peanut and soybean proteins, the minerals appear to be partially unavailable for the production of streptomycin. 4. In the peanut protein, the mineral nutrients for streptomycin production are either in low concentration or in an unfavorable balance. 5. Waste liquor fractions of the peanut and soybean meals contain the nutrients of these plant products that favor streptomycin production. REFERENCES 1.
THORNBERRY,
H. H., Ph@pathoZogy (Abstracts) 37, 21-2 (1947).
2. THORNBERRY, H. H., Phytopathology (Abstracts) 38, 26 (1948). 3. BURNETT, R. S., AND FONTAINE, T. D., Ind. Eng. Chem. 36, 284-8 (1944). 4. KLAW, T. J., PARKER, E. D., POMES, A. F., AND PORGES,N., Oil & Soap 22,
319-21 (1945).
464
H. H. THORNBERRY
AND
A. J. SHANAHAN
5. SMITH, A. K., AND CIRCLE, S. J., Ind. Eng. Chem. 30, 1414-18 (1938). 6. SMITH, A. K., AND CIRCLE, S. J., Ind. Eng. Chem. 31, 1284-8 (1939). 7. SMITH, A. K., CIRCLE, S. J., AND BROTHER, G. H., J. Am. Chem. Xoc. 60, 1316-20 (1938). 8. THORNBERRY, H. H., Phytopathology (Abstracts) 36, 412 (1946). 9. THORNBERRY, H. H., AND ANDERSON, H. W., Arch. Biochem. 16, 389-97 (1948). 10. GTJ~HRIE, J. D., HOFFPAUIR, C. L., STEINER, E. T., AND STANSBURY, M. F., U. S. Bur. Agr. Ind. Chem., Mimeographed Cir. Ser. AIC 61, 1-86 (1944). 11. SLIPSBURY, J. F., Brit. Mycol. Sot. Trans. 31, 210-28 (1948). 12. RAO, R. R., RAO, S. S., AND VENKATARAMAN, P. R., Nature 158, 23-4 (1946).
and A. J. Shanahan
From the Department of Horticulture, University of Illinois, Received April 5, 1951
l
Urbana, Illinois
These studies were planned to determine the nutritive value of some fractions of peanut and soybean meal (oil extracted) for the production of streptomycin by Streptomyces griseus (Krainsky) Waksman and Henrici. Some of the preliminary results have been presented (1,2). The purpose of this paper is to report the later results obtained. MATERIALS
AND METHODS
The materials used were oil-extracted (solvent) peanut meal (No. P-136, Southern Regional Research Lab., U. 5. Dept. of Agr.) and soybean flakes (Northern Regional Research Lab., U.S.D.A.). These were fractionated by a procedure of protein extraction based upon the information supplied by personal communications (see acknowledgment) and by some pertinent references for peanut meal (3,4) and soybean meal (5,6,7). The following samples of peanut and soybean meal were used: I. Original meal, oil extracted. II. Residue, consisting of the solid material remaining after extraction of meal in water (100 g./l.) at pH 8.0 for 30 min. at 26°C. and straining through four-ply cheesecloth. III. Extract, materials in the aqueous suspension at pH 8.0 that filtered through four-ply cheesecloth. IV. Protein, substances that settled upon protein precipitation at pH 4.5 and standing for 4 hr. at room temperature. V. Waste liquor, substances remaining in the liquid after protein precipitation and sedimentation. The liquid was filtered through a 5-mm.-thick Celite filter cake to remove suspended particles of insoluble materials in the liquid.
Amount of these fractions referred to herein represent the material originating from that weight of original meal, i.e., 10 g. of waste liquor represents the waste liquor from 10 g. of original meal.
1Present address: erick, Maryland.
Biological
Department, 459
Chemical
Corps, Camp Detrick,
Fred-
460
H.
H.
THORNBERRY
AND
T.%BLE
A.
J.
SHANAHAN
I
Amount qf Streptomycin Produced by Streptomyces griseus from Non-Ashed and Ashed Fractions of Peanut and Soybean Meal in S-day Submerged Culture Soybean COIIC. O./l.=
Fractions
I. Original
II.
III.
meal
Residue
Extract
IV. Protein
5’. Waste liquor
1 5 10 20 30 49 1 5 10 20 30 40 1 5 10 20 30 40 1 5 10 20 30 40 1 5 10 20 30 40
Basal medium
-
Basal and corn steep non-ashed
10
Non-ashed d d
M-55.8* 13-43.5 21-11.6 12-11,7 d d 52-25.4 63-12.4 52-49.8 29-15.6 d d 52-36.0 44-54.6 22-11.1 12-18.6
d d
149-50.6 94-46.4 84-48.8 69-43.1 d d
57-30.3 118-46.0 154-66.9 133-61.1 d
d
123-87.0 92-36.4 97-34.3 172-66.9
d
d
d
d
16-108.9 15-79.5 19-37.2 24-53.2
o-O.0
41-57.0 52-23.8 43-40.9
d
d
d
d
Non-ashed
.%shed
37-22.8 244-60.3 351-25.2 109-37.4 83-52.5 17-22.6
94-36.5 180-24.3 216-51.7 74-32.6 76-28.4 77-21.6
17-30.7 40-26.5 76-18.0 81-21.6 12334.8 261-27.9
86-18.4 110-24.6 285-31.7 222-26.4 146-31.7 107-27.9
30-28.7 127-49.0 241-26.0 168-41.9 74-38.8 56-25.4
46-18.6 130-27.5 264-15.4 206-31.8 82-44.6 79-29.2
18-23.4 25-29.0 25-12.5 12-36.4 11-41.6 16-29.4
41-51.4 73-36.7 128-46.6 181-23.8 76-33.2 52-21.9 31-18.6 55-26.3 99-38.1 102-18.6 162-23.2 164-46.4
58-51.5 87-54.4 93-15.7 105-39.9
88-12.0 225-54.7 240-38.8 119-18.4
20-15.9 69-50.1 103-12.9 110-18.5 127-36.3 142-42.1
29-25.1
16-18.4
17-29.6
19-17.5
222-66.6
195-72.8
429-24.1
326-38.5
a Units of streptomycin/ml. (One unit is equivalent to 1 pg. streptomycin base as evaluated against a standard containing 800 pg. of streptomycin sulfate/mg.). b C.V. = coefficient of variability. c Amounts corresponding to grams of the original mea! from which derived. d Not determined.
461
STREPTOMYCIN PRODUCTION
Ashing of materials was accomplished in a muffle furnace at 600-650°C. The amount of ash from the fractions was not determined. The submerged culture of S. griseus, estimation of streptomycin and growth, growth conditions, and inoculum were similar to those previously stated (8,9). The basal medium consisted of glucose (Cerelose) 10 g., bactopeptone (Difco) 5 g., XaCl 5 g., and distilled water 1000 ml. -4 corn steep control medium for checking growth condit,ions consisted of 10 g. of corn steep added to 1 1. of this basal medium. All media were adjusted with NaOH or HCI to pH 7.0-7.2 before sterilization at 121°C. for 15 min. EXPERIMENTAL
AND RESULTS
Experiments were designed to measure the nutritive value of the peanut and soybean fractions in varied amounts in the basal medium for the production of streptomycin. The results from three runs in the case of peanut fractions (each run with flasks in triplicate) and two runs in the case of soybean fractions (each run with flasks in triplicate) are given in Table I. The results obtained from the spectrographic semiquantitative analysis (see acknowledgment) of peanut waste liquor ash are given by the following groups (the value in parentheses being the percentage of the element necessary to be detected) :
1. Elements present are Al (.OOl), B (.OOOl), Ca (.OOl), Cr (.OOOl), Cu (.OOOl), Fe (.OOOl), K (lO.O), Mg (.OOOl), Mn (.OOOl), P (.Ol), Si (.OOOl), Ti (.OOl), and V (.OOl). 2. Elements questionable are Ba (.Ol), MO (.OOOl), Sr (.Ol), and Zn (.Ol). 3. Elements highly questionable are Bi (.OOOl), Li (.OOl), Ni (.OOOl), Pb (.OOOl), and Rh (.OOl). Growth of the organism was good in media containing all fractions except, the proteins which supported only moderate growth. Growth in the basal medium was poor whereas that in the basal containing corn steep was excellent. The hydrogen-ion concentration of all media decreased as fermentation progressed. It changed from that of the initial values (pH 6.2-6.7) after sterilization
to an alkaline
reaction
(pH 7.9-8.9)
in 3 days.
Since the substances in some of the fractions obviously were in excess for streptomycin production, the effect of varied amounts of corn steep liquor in the medium was also determined. Optimal amounts of corn steep were from 4 to 10 g. of corn steep liquor per lit,er of medium. The
462
H.
H.
THORNBERRY
AND
A.
J.
SHANAHAN
yields of streptomycin obtained from these varied amounts of non-ashed corn steep were 1 g./l. 104 units/ml., 2 g. 130 units, 4 g. 292 units, 6 g. 397 units, 8 g. 370 units, 10 g. 258 units, 20 g. 133 units, 30 g. 80 units; whereas from corn steep ash they were 1 g. 96 units, 5 g. 194 units, and 10 g. 51 units.
DISCUSSION From the non-ashed peanut material, the waste liquor fraction gave the best yield of streptomycin. This indicates that the fractionation procedure providing this waste liquor removes some of the inhibitive substances and allows nutritive substances to remain with this fraction. However, all of the inhibiting substances are not removed by the fractionation because the waste liquor ash outyielded the comparable nonashed sample. Since all ashed materials outyielded their comparable non-ashed samples, minerals appear to be the principal nutrients of peanut meal for the production of streptomycin by this organism. Improper balance of the minerals in the meal ash is offered to explain the low yield of streptomycin from this ash. The relatively low yield of streptomycin from protein ash suggests that these proteins do not contain the nutritive minerals in sufficient concentration or balance for streptomycin production. Spectrographic analysis of peanut waste liquor ash shows the presence of two minor elements (Fe and Mn) of the three elements (Fe, Mn, and Zn) found to influence the metabolism of S. griseus in a chemically defined medium (8,9). Zinc, although questionable in the analysis, must have been present in the ash since growth was stimulated by this ash and zinc is necessary for growth (8,9). Zinc along with Mn, Fe, Co, Cu, B, and F have been reported in peanut kernels (10). However, in surface growth of S. griseus in Belmont medium containing either peptone or “Pronutrin” (casein hydrolyzate), zinc was reported to retard streptomycin production (11). From the non-ashed soybean material, sizeable yields of streptomycin were obt’ained from the original meal and all fractions except the protein one. Since soybean protein ash gave essentially a fourfold increase in production over the non-ashed material, it is evident that the proteins either acted in an inhibitory manner or retained the nutritive materials in an unavailable form. There was no indication of inhibitive substances in the other fractions. However, the residue ash at some of the concentrations employed did outyield the comparable amounts of
STREPTOMYCIN PRODUCTION
463
non-ashed materials. The results show that the nutritive value of soybean meal is associated with the minerals and the waste liquor fraction. The results herein suggest that the nutritive substances in the soybean meal used in commercial production of streptomycin and in the peanut (groundnut) meal in experimental productions (12) are related to the incorporated minerals, their balance for the organism being important. Waste liquors from the acid precipitation of peanut and soybean proteins are important and cheap nutrients for the production of streptomycin. However, their use commercially may await supplementation to achieve yields of streptomycin suitable for commercial processing. ACKNOWLEDGMENTS Acknowledgment and appreciation of assistance are extended to Dr. Allan K. Smith, Northern Regional Research Laboratory, for the soybean meal and valuable information on soybean protein and its extraction; to Dr. John D. Guthrie and Mr. Carrol L. Hoffpauir, Southern Regional Research Laboratory, for the peanut meal and valuable information on peanut protein and its extraction; to Dr. Victor J. Miller (now Department of Horticulture, University of Kansas) for the spectrographic analysis of peanut waste liquor ash during a course in physical methods under the supervision of Dr. D. T. Englis, University of Illinois; and to Mrs. Fredia M. Shannon for technical assistance. SUMMARY
1. Minerals are the principal nutrients in peanut and soybean meals for streptomycin production in the medium used. 2. Substances inhibitory to streptomycin production are present in peanut meal but are not present in soybean meal. 3. In the non-ashed peanut and soybean proteins, the minerals appear to be partially unavailable for the production of streptomycin. 4. In the peanut protein, the mineral nutrients for streptomycin production are either in low concentration or in an unfavorable balance. 5. Waste liquor fractions of the peanut and soybean meals contain the nutrients of these plant products that favor streptomycin production. REFERENCES 1.
THORNBERRY,
H. H., Ph@pathoZogy (Abstracts) 37, 21-2 (1947).
2. THORNBERRY, H. H., Phytopathology (Abstracts) 38, 26 (1948). 3. BURNETT, R. S., AND FONTAINE, T. D., Ind. Eng. Chem. 36, 284-8 (1944). 4. KLAW, T. J., PARKER, E. D., POMES, A. F., AND PORGES,N., Oil & Soap 22,
319-21 (1945).
464
H. H. THORNBERRY
AND
A. J. SHANAHAN
5. SMITH, A. K., AND CIRCLE, S. J., Ind. Eng. Chem. 30, 1414-18 (1938). 6. SMITH, A. K., AND CIRCLE, S. J., Ind. Eng. Chem. 31, 1284-8 (1939). 7. SMITH, A. K., CIRCLE, S. J., AND BROTHER, G. H., J. Am. Chem. Xoc. 60, 1316-20 (1938). 8. THORNBERRY, H. H., Phytopathology (Abstracts) 36, 412 (1946). 9. THORNBERRY, H. H., AND ANDERSON, H. W., Arch. Biochem. 16, 389-97 (1948). 10. GTJ~HRIE, J. D., HOFFPAUIR, C. L., STEINER, E. T., AND STANSBURY, M. F., U. S. Bur. Agr. Ind. Chem., Mimeographed Cir. Ser. AIC 61, 1-86 (1944). 11. SLIPSBURY, J. F., Brit. Mycol. Sot. Trans. 31, 210-28 (1948). 12. RAO, R. R., RAO, S. S., AND VENKATARAMAN, P. R., Nature 158, 23-4 (1946).