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Production of oxytetracycline, by streptomyces rimosus 12907, as an animal feed supplement
P R O D U C T I O N O F O X Y T E T R A C Y C L I N E , BY STREPTOMYCES RIMOSUS 12907, AS A N A N I M A L F E E D SUPPLEMENT
A. O. BAGHLAF, A. A. ABOU-ZEID, AHMED I. EL-DIWANY & ABD EL-WAHAB 1. EISSA
Microbiological and Enzyme Chemistry Research Unit, National Research Centre, Dokki, Cairo, Egypt & MAHMOUD FOUAD 8,~ MOHAMED YESSEIN
Organic" Chemical Factories oJ the Egyptian Sugar and Distillation Company, Giza, El-Hawamdia, Egypt
ABSTRACT
Production oJ oxytetracycline by Streptomyces rimosus 12907 as an animal Jeed supplement was achieved in a ./ermentation medium containing the Jollowing ingredients (glitre-l): black-strap molasses, 30; jodder yeast, 20; rice bran, 10, KHzP04, 0-2 and tap water. The dr), mash, containing oxytetracycline, thus produced was 50g litre ~ oJJermentation medium. The amount of oxytetracycline present in the dr)' mash was about 4 °/o. The dr)' mash containing the oxytetracycline was analysed Jor the amino acids L-arginine, L-histidine, L-lysine, L-isoleucine, Lleucine, L-phenylalanine, DL-methionine and L-tryptophan which could he oj nutritional value./or chickens.
INTRODUCTION
Tetracyclines, antibiotics of a broad antimicrobial spectrum, are closely related compounds widely used in the treatment of infectious diseases (Duggar, 1948; Finlay et al., 1950; Stephens et al., 1952; Shapposhnikov et al., 1958; DiMarco & Boretti, 1956: Dulaney & Perlman, 1947; Garner & Koffler, 1953; Miller et al., 1936). The carbon sources have been found to play an important role in the formation of tetracyclines by Streptomyces spp. (McVeigh & Regyes, 1961; Zaitseva & Orlova, 1958; Bitii et al., 1954; Goodman, 1954; Niedercorn, 1952; Katagiri, 1960; Shibata 301 Agricultural Wastes 0141-4607/79/0001-0301/$02.25 © Applied Science Publishers Ltd, England, 1979 Printed in Great Britain
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et al., 1961 ; Zygmunt, 1961). The tetracycline antibiotics display features indicative
of an acetate origin, and the general concept of an acetate-derived precursor in the sense of a polyketomethylene chain applies in the case of oxytetracycline. The nitrogen sources of the medium are also important factors in the biosynthesis of tetracycline antibiotics (Darken et aL, 1959; McGhee & Megna, 1957; Lein & Gourevitch, 1956; Goodman, 1960; McCormick et al., 1960; Barnett, 1954). The economic potential for the production of such antibiotics depends on the availability of cheap substrates. In Egypt, there are many agricultural by-products remaining from industrial production, such as black-strap molasses, fodder yeast, rice bran, corn bran and corn-steep liquor, and these could be used in a medium for antibiotic production. Antibiotics have been used in many countries as growth stimulants for farm animals. The precise way in which the antibiotics exert their beneficial effects on growth and food utilisation is still debatable. Some workers (Biely et al., 1952; Davies & Briggs, 1951; Eyssen & Somer, 1963; MacGregor et aL, 1952) claim that the growth-stimulating effect of antibiotics is most probably due to suppression of Gram-positive intestinal bacteria which interfere with the absorption of nutrients. Other bacteria may be beneficial because they produce nutrients (e.g. vitamins and/or unidentified growth factors) that are not present in the food in sufficient quantity. Titus (1961) claimed that several antibiotics added to the food of growing chickens increase their rate of growth or improve their food utilisation efficiency or do both under certain conditions. Saxena et al. (1953), Silinger et al. (1952) and Silinger et al. (1953) reported that continuous supplementation with diamine penicillin, at a level of 3 ppm in mash, during starting and developing, significantly improved the body weight of turkeys at 24 weeks. A greater response was obtained in males than in females. The present work deals with the fermentative production of oxytetracycline by Streptomyces rimosus 12907 using black-strap molasses, fodder yeast and rice bran in a medium to produce an animal feed supplement.
METHODS
Maintenance o j Streptomyces rimosus 12907 An active strain of Streptomyces rimosus 12907 (Institute for Fermentation,
Juso-Nishino-Cho, Higashiyodogawa-Ku, Osaka, Japan) was maintained on a medium containing the following ingredients (g litre-1): glucose, 10-0; peptone, 5.0; KHzPO 4, 1.0; MgSO4.7H20, 0.5 and agar, 20.0 in distilled water. The initial pH of the medium was adjusted to 6.0-6.5. The ingredients were thoroughly dissolved and the medium apportioned into test tubes. The tubes were plugged with cotton wool and sterilised at 121 °C for 20rain. When the test tubes containing
PRODUCTION OF OXYTETRACYCLINE
303
the maintenance medium attained room temperature they were inoculated with Streptomyces rimosus 12907. The inoculated slants were incubated at 30°C for 10 days to obtain luxuriant growth and sporulation. The slants were then kept at 5 °C in a refrigerator.
Vegetative medium The active strain of Streptomyces rimosus 12907 was grown in a "vegetative medium' which contained the following ingredients (glitre 1): glucose, 10.0: peptone, 5.0; K H z P O 4, 1.0 and MgSO 4 . 7 H 2 0 , 0.5; in distilled water. The initial pH of this vegetative medium was adjusted to 7. The medium was apportioned into Erlenmeyer flasks (250 ml capacity), each containing 50 ml. The flasks were plugged with cotton wool and sterilised at 121 °C for 20 min. When the flasks attained room !emperature they were inoculated with a standard spore suspension of Streptomyces rimosus under aseptic conditions. The inoculated flasks were incubated on a rotary shaker (200rpm) at 27°C for 48h. The vegetative medium was used for the inoculation of the fermentation medium.
Fermentation medium The defined fermentation medium used for the fermentative production of oxytetracycline by Streptomyces rimosus 12907 contained the following ingredients (glitre- 1): citric acid, 12.8; sucrose, 40.0; (NH,~)2SO 4, 6-0; MgSO 4 . 7 H 2 0 , 0.25: K H z P O 4, 0.15; C a C O 3, 1.0; MnSO4. H 2 0 , 0.01; ZnSO 4 . 7 H 2 0 , 0.4. The initial pH of the medium was adjusted to 7. The carbon source of the defined fermentation medium was replaced by black-strap molasses (50 ')o sugar content). The nitrogen source of the fermentation medium was also replaced by fodder yeast (40 ° 0 crude protein) and undefatted rice bran. With molasses, fodder yeast and rice bran replacing carbon and nitrogen sources, citric acid, C a C O 3, MnSO 4 . 4 H 2 0 and ZnSO 4. 7 H 2 0 were removed from the fermentation medium. The fermentative production of oxytetracycline by Streptomyces rimosus 12907 was further tested using fodder yeast, corn bran and rice bran in the presence and absence of K H 2 P O ~ and MgSO 4 . 7H20. The fermentation medium was apportioned into Erlenmeyer flasks (500 ml capacity), each containing 100 ml. The flasks were plugged with cotton wool and sterilised at 121 °C for 20 rain. When the flasks attained room temperature, they were inoculated under aseptic conditions with the vegetative medium (5 '~/0v/v) containing growing cells of Streptomyces rimosus 12907. The inoculated flasks were incubated on a rotary shaker (200rpm) at 27°C for 120h. At the end of the fermentation process the final pH of the fermented medium, the suspended dry matter and the amount of oxytetracycline produced were determined.
Biological determination oJ oxytetracycline A biological-standard curve was drawn between the logarithm of different concentrations of oxytetracycline and the inhibition zones of Bacillus subtilis N R R L B-543 (Abou-Zeid & Shehata, 1969).
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Production of oxytetracycline in a fermenter (1200 litre) The amounts of the most suitable ingredients for the fermentation medium were scaled up to be used in the fermentative production of oxytetracycline in a fermenter (1200 litre). The fermentation medium contained the following ingredients in tap water (g litre-i): molasses, 30.0; fodder yeast, 20.0; rice bran, 10-0 and K H 2 P O 4, 0.2. The initial pH of the fermentation medium was adjusted to 6-5. Before charging the fermenter, it was thoroughly washed and sterilised by steam. Seven-hundred litres of the fermentation medium were introduced into the fermenter. The medium was sterilised at 121 °C for 40 min by steam and when the fermenter attained room temperature it was inoculated with Streptomyces rimosus 12907 grown on the vegetative medium mentioned above. The percentage of inoculum was 5 ~o (v/v). To the fermentation medium was added cotton-seed oil anti-foam (0.5~o). The fermentation medium was stirred and sparged with sterile air. The process was conducted at 27°C for 96h. Every 24h a sample was taken to determine the pH of the fermented medium, suspended dry matter and antibiotic yield. The whole fermented mass was taken off and, after the pH had been adjusted to 4.0 4.5 with HCI, poured into trays (stainless steel, 75 x 40 × 7cm), each tray containing 15 litres. The trays containing fermented mash were placed on tables in a room, the temperature of which was controlled at 40 °-45 °C by means of a contact thermometer. Hot air was directed on to the surfaces of the trays while a suction pump was attached to the room to evacuate air and water and other vapours. The process was continued until the fermented mash was completely dried, when the mash was removed and ground. The amount of oxytetracycline present in the dry, fermented mash was tested as before. Determination oJamino acids present in Jermented mash Quantitative determination of amino acids present in the fermented mash was carried out according to the techniques of Block et al., 1958 and Blauth et al., 1963. The amino acids L-arginine, L-histidine, L-lysine, L-isoleucine, L-leucine, Lphenylalanine, DL-methionine, L-threonine and L-tryptophan were determined. Anti-microbial spectrum of oxytetracycline present in the dr),' mash The inhibitory effects of oxytetracycline present in the dry mash were biologically assayed on the following micro-organisms: Bacillus subtilis, Staphylococcus aureus, Sarcina lutea, Klebsiella pneumoniae, Salmonella schottmuelleri, Escherichia coli, Proteus vulgaris and Mycobacterium avium.
RESULTS A N D DISCUSSION
The results obtained (Table l) show that the amount of oxytetracycline produced by Streptomyces rimosus 12907 increased with increasing incubation period, reaching
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TABLE 1 AMOUNTS OF OXYTETRACYCLINE PRODUCED BY Streptomyces rimosus 12907 AT DIFFERENT PERIODS OF THE FERMENTATION PROCESS
Incubation period (h )
Final pH
Suspended dry matter (rag millilitre - l )
O xytetracycline (#g millilitre- t)
24 48 72 96 120 144
5,5 6,8 7.5 8.3 8.5 8-5
5.8 6.5 7.5 9.0 9.2 9-1
-300 450 600 500 475
D e f i n e d m e d i u m , see " M e t h o d s ' .
an optimum at 96 h, after which there was a decrease. A drop in pH was obtained during 48h of the fermentation process and this may be correlated with accumulation of organic acids. These were then further utilised by the microorganism so that at the end of the fermentation process the final pH was moved to the alkaline side (8.5). The defined medium used in the fermentative production of oxytetracycline contained costly ingredients; therefore certain local ingredients such as black-strap molasses, fodder yeast and rice bran were tested for the production of oxytetracycline by Streptomyces rimosus 12907. The results obtained (Table 2(a)) show that when sucrose was replaced by different concentrations of black-strap molasses the amount of oxytetracycline produced increased with the increase of black-strap molasses and reached an optimum at 30-40 g litre-~, above which a decline in yield occurred. The final pH was variable, depending to some extent on the amount of molasses added. Molasses (30 g litre ~) was used instead of sucrose and (NH4)2SO 4 was replaced by different concentrations of fodder yeast (40'~0 crude protein), while the other ingredients of the medium remained unchanged. The results obtained (Table 2(b)) show that the amount of oxytetracycline produced increased with the increase of fodder yeast concentration, reaching an optimum at 20 g litre- ~, above which a decrease in the antibiotic yield occurred. Fodder yeast used in the medium gave a better yield of the antibiotic than (N H4)2SO4 . The final pH of the fermented medium was 7.5-8.5. J~lolasses (30 g litre- l) was used instead of sucrose and (NH4)2SO 4 was removed and replaced by different concentrations of rice bran, while the other ingredients of the medium remained unchanged. The results obtained (Table 2(c)) show that the amount of oxytetracycline excreted in the medium increased with the increase of rice bran concentration, reaching an optimum at 30-40 g litre- i. Different concentrations of rice bran in the presence of molasses ( 3 0 g l i t r e - l ) and fodder yeast (20glitre t) were incorporated into the medium. The results obtained (Table 2(d)) indicated that addition of rice bran to fodder yeast favoured a high antibiotic yield. The amount of oxytetracycline
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TABLE 2 A M O U N T S O F O X T E T R A C Y C L I N E P R O D U C E D BY Streptomyces rimosus 12907 W H E N C O N T A I N I N G MOLASSES, F O D D E R YEAST A N D RICE BRAN
Concentrations
10 15 20 25 30 40 50 60 70
Final pH
Suspended dry matter (mg millilitre ~ 1)
(a) Different concentrations of molasses (g litre-~) 7.5 6-5 8.0 7.0 8.0 7.0 8.5 7.5 8-5 8.0 8.0 8.5 8.0 8-5 7.0 8-5 7-0 8.5
GROWN
IN MEDIA a
Oxytetrac)'cline (tag millilitre ~)
350 400 450 500 550 500 300 200 150
(b) Molasses (30 g litre- 1) + different concentrations of fodder yeast (g litre- a) 2-5 8.0 6.5 500 5.0 8-0 7.0 550 10.0 8-0 7.5 650 15.0 8.0 7.5 750 20-0 8-5 8-5 800 25.0 8.0 9.5 700 30.0 7.5 10.0 650 (c) Molasses (30 g litre-1) + different concentrations of rice bran 10 7.5 7.0 450 20 7-5 8-0 450 30 8.5 9.5 600 40 8.5 10.0 600 50 8.0 10.5 500 (d) Molasses (30 g litre- l) + fodder yeast (20 g litre - ~) + different concentrations of rice bran (g litre - ~) 5 7-5 9.0 650 I0 7-5 10-0 850 15 8.0 10-5 750 20 8-5 11-9 700 25 8.5 11-5 600 30 8.0 12.0 600 40 7.0 12.0 600 (e) Molasses (30 g litre- 1) + rice bran (10 g litre- 1) + different concentrations of fodder yeast (g litre- ~) 5 7.0 10.0 800 10 7.5 10.5 850 15 8.0 11.0 950 20 8-0 ~ 12-0 1000 25 8-0 12.5 950 30 7-5 12.5 800 35 7.5 13.5 750 40 7.5 14.5 750 45 7-0 15.0 700 50 7.0 15.0 700 ° For other constituents of media see 'Methods' and 'Results'.
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PRODUCTION OF OXYTETRACYCLINE
excreted in the m e d i u m increased with the initial increase o f rice b r a n c o n c e n t r a t i o n , reaching an o p t i m u m at 10 g l i t r e - 1 and then decreasing. M o l a s s e s ( 3 0 g l i t r e 1), rice b r a n ( 1 0 g l i t r e -1) a n d different c o n c e n t r a t i o n s of f o d d e r yeast were i n c o r p o r a t e d into the m e d i u m w i t h o u t ( N H 4 ) 2 S O 4, while the o t h e r ingredients o f the m e d i u m r e m a i n e d u n c h a n g e d . T h e results o b t a i n e d (Table 2(e)) show t h a t a suitable c o n c e n t r a t i o n o f f o d d e r yeast in the presence o f molasses a n d rice b r a n was 20 g l i t r e - 1. T h e best a m o u n t s o f molasses, f o d d e r yeast and rice b r a n for a n t i b i o t i c p r o d u c t i o n were 30, 20 a n d 1 0 g l i t r e -~, respectively. These a m o u n t s of ingredients were then used in a simplified m e d i u m with different
TABLE 3 AMOUNTS OF O X Y T E T R A C Y C L I N E P R O D U C E D BY
Streptomyces rimosus 12907 W H E N
G R O W N ON A MEDII~ M
CONTMNINGKH2PO 4 AND MgSO 4 . 7H20 Concentrations
Final p H
Suspended dry matter (mg millilitre- l )
Oxytetracycline (l~g millilitre ~)
(0 Molasses (30glitre 1)+ fodder yeast (20glitre 1)+ rice bran (10glitre ~)+different concentrations of KH2PO 4 (glitre t) 0.00 7.5 9-5 1000 0-10 7.5 9.5 1200 0.20 7.5 10.5 1250 0.30 8.5 11-5 1000 0.40 8.5 12.5 1000 0.50 8.5 12.5 950 0-75 8.0 11.5 800 1.00 8-0 12.0 800 1-50 8.0 12.0 750 2.00 8-0 t2.0 700 (g) Molasses (30glitre-t) + fodder yeast (20glitre 1) + rice bran (10glitre 1) + KHzPO~ (0.2glitre 1) + different concentrations of MgSO 4. 7H20 (glitre ~) 7.0 8.5 1000 0.00 7"0 8.5 950 0.10 7.0 9.5 950 0.20 7-5 10"5 900 0.30 8"0 I IO 800 0.40 8"5 12.0 75O 0.50 8.0 11.0 700 0.75 8"0 11"0 700 1-00
c o n c e n t r a t i o n s o f K H 2 P O 4 or MgSO~. T h e results r e c o r d e d (Table 3) show that the a m o u n t o f o x y t e t r a c y c l i n e p r o d u c e d increased with increase o f K H 2 P O 4 c o n c e n t r a t i o n reaching an o p t i m u m at 0.2 g l i t r e 1 but at this c o n c e n t r a t i o n of K H 2 P O ~ a d d i n g M g S O 4 at different c o n c e n t r a t i o n s d i d n o t i m p r o v e the a n t i b i o t i c yield. A m e d i u m c o n t a i n i n g molasses, f o d d e r yeast, rice b r a n , K H 2 P O 4 a n d t a p water was then used for large-scale a n t i b i o t i c p r o d u c t i o n . T h e initial p H o f the f e r m e n t a t i o n m e d i u m was 6.5 a n d at the end o f the f e r m e n t a t i o n process it was 8-5
308
A. O. BAGHLAF ET AL. TABLE 4 OXYTETRACYCLINEa PRODUCED IN A FERMENTER (1200 LITRE)
Initial pH
Final pH
Dry mash (%)b
Oxytetracycline (rag per gramme dry mash)
6-5
8.5
5.0
40
a Fermentation medium used contained the following ingredients (g litre-1): black-strap molasses 30, fodder yeast (40% crude protein) 20, rice bran 10, K H 2 P O 4 0.2 and tap water. b % (w/v) of fermentation medium.
(Table 4). At the end of the fermentation process the whole fermented mash was hotair dried under reduced pressure at 40 ° to 45 °C. The percentage of dry suspended matter which included microbial cells, unutilised medium constituents, oxytetracycline and other microbial metabolites, was about 5 ~o. The amount of oxytetracycline present in this dry mash was 4 ~o by weight. The results (Table 5) show the action of the unpurified oxytetracycline on certain types of bacteria: Bacillus subtilis, Staphylococcus aureus, Sarcina lutea, Klebsiella pneumoniae, Salmonella schottmuelleri, Escherichia coli, Proteus vulgaris and
Mycobacterum avium. TABLE 5 INHIBITORY ACTION ON BACTERIA OF OXYTETRACYCLINE PRESENT IN DRY MASHMADE INTO AN AQUEOUSSOLUTION (2mg millilitre- ~) AND USING 6"35 mm DIPPED DISCS
Bacteria Bacillus subtilis Staphylococcus aureus Sarcina lutea Klebsiella pneumoniae Salmonella schot tmueller i Escherichia coli Proteus vulgaris Mycobacterium avium
Inhibition zone (mm) 17 30 35 24
31 25 16 35
For commercial use it would not be necessary to extract the antibiotic from the fermentation mash as it would be produced for use in poultry rearing. The antibiotic mash could be used in poultry feed or in drinking water. The aim of its addition is to prevent infectious diseases and to accelerate the growth of poultry; therefore, the presence of the microbial cells and metabolites is not harmful. On the contrary, these microbial cells and metabolites may be useful as micro-nutrients in poultry diets. Analyses showed that the hydrolysate of the dry mash contained the amino acids Larginine, L-histidine, L-lysine, L-isoleucine, L-leucine, L-phenylalanine, DLmethionine, L-threonine and L-tryptophan in the amounts shown in Table 6.
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TABLE 6 AMOUNTS OF AMINO ACIDS PRESENT IN DRY MASH CONTAINING OXYTETRACYCLINE
Amino acid
Dry mash (g kilogramme- 1)
L-Arginine L-Histidine L-Lysine L-lsoleucine L-Leucine L-Phenylalanine DL-Methionine L-Threonine c-Tryptophan
9-21 3.25 6.73 6.95 8.74 5.39 0"82 4.70 1.50
These amino acids are beneficial in the growth of poultry. The utilisation of oxytetracycline present in dry mash is also more economic than when it is extracted from the fermentation mash for use in poultry feeds.
ACKNOWLEDGEMENT
This work was carried out in the laboratories of the Organic Chemical Factories of the Egyptian Sugar and Distillation Company, Giza, El-Hawamdia, in collaboration with the National Research Centre, Dokki, Cairo, Egypt.
REFERENCES ABOU-ZEID, A. A. & SHEHATA, Y. M. (1969). A simple technique for assaying antibiotics using a methylene blue as an indicator. Indian Journal oJ Pharmacy, 31, 72-5. BARNETT, A. J. G. (1954). SilageJermentation. Academic Press Inc., New York. BILLY, J., MARCH, B. E. & SMITh, D. E. (1952). Effect of feeding aureomycin on the bacterial content of chick feces. Poul. Sci., 31, 863. BIFF1, G., BORETTI, G., D1MARCO, A. & PENELLA, P. (1954). Metabolic behaviour and chrotetracycline production by Streptomyces aureojaciens in liquid culture. Appl. Microbiol., 2, 288. BLAUTH, O. J., CHARESINSKI, M. • BERBEC,H. (1963). A new method for determining tryptophane. Anal. Biochem,, 6, 69-70. BLOCK, R. J., DRUM, E. L. & ZWEIG, G. A. (1958). A manual of paper chromatography andpaper electrophoresis. (2nd ed.) Academic Press Inc., New York. DAVIES, R. L. & BRIGGS, G. M. (1951). Vitamin B12 requirement of the chick. Poul. Sci., 30, 767. DIMARCO, A. & BORETTI,G. (1956). Complexes formed by streptomycin and basic dyes with ribonucleic acid-interference by salts. Gironal di Microbiologia, 2, 1. DUGGER, B. M. (1948). Aureomycin. Ann. N.Y. Academy of Science, 51, 177. DULANEY, E. & PERLMAN, D. (1947). Bull. Torry Botany Club, 74(6), 504. EVSSEN, H. L. & SOMER, P. D. E. (1963). Mode of action of antibiotic in stimulating growth of chicks. Poul. Sci., 42, 1373. FINLAY, A. C., HABOY, G. L., P'AN, S. Y., REGNA, P. O., ROUTIN, J. B., SEELY, D. B., SHULL, G, M., SOLOMONS, I. A., VINSON, J. W. & KANE, J. H. (1950). Terramycin, a new antibiotic. Science, 11, 85.
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GARNER, H. & KOFFLER, M. (1953). Terramycin. Amer. J. Botany, 40(5), 286. GOODMAN,J. J. (1954). Process for production of chlortetracycline. Canadian Patent 499, 649. GOODMAN,J. J. (1960). Terramycin. US Patent 3,05,446. KATAGIRI,K. (1960). Study on the chlorotetracycline. Improvement of Chlorotetracycline producing strain by several kinds of methods. Journal of Antibiotics (Japan), Ser. A, 7, 45-52. LEIN, J. & GOUREVITCH,A. (1956). Tetracyclines. US Patent 2,739,924. MACGREGOR,H. I., BLACKELY,R. M. & ANDERSON,R. W. (1952). Antibiotic in the diet of turkey poults. Poul. Sci., 31,924. MCCORMICK, J. R. D., SJOLANDER,N. O. & HIRSCH, U. (1960). Biological conversion of 5a (lla) dehydrochlortetracycline to broad spectrum antibiotics. US Patent 2, 965-546. McGHEE, W. J. & MEGNA, J. C. (1957). Tetracycline. US Patent 2,776,342. McVEIGH, 1. & REGYES,R. C. (1961). Tetracycline. Antibiotics and Chemotherapy, 10, 312. MILLER, P. A., MCCORMICK, J. R. D. & DOERSCHUK,A. P. (1956). Studies of chlortetracycline biosynthesis and the preparation of chlortetracycline-C t4. Science, 123, 1030. NIEDERCORN,J. G. (1952). Aureomycin. US Patent 2,609,329. SAXENA,H. C., BLAYLOCK,L. G., STADELMAN,W. J., CARVER,J. S. & McGINNIS, J. (1953). The effect of penicillin on growth feed efficiency and fattening of turkeys. Poul. Sci., 32, 721-6. SHAPPOSHNIKOV,V. N., ZAITSEVA,Z. M. & ORLOVA,N. V. (1958). Synthetic medium for biosynthesis of oxytetracycline by cultures of" Actinomyces rimosus. Dokkle Akad. Nauk. Chem. Soc., 121, 2. SHIBATA,M., HIGASHIDE,E., KANZKI,T., YAMAMATO,H. & NAKAZAWA,K, (1961). Streptomycetes. I. Streptomycespulvevaceus producing antibiotics zygomycins A and B. Agr. Biol. Chem. (Tokyo), 25, 171-5. SIL1NGER,S. J., MORPHET,A. M., GARTLEY,K. M. & ARTHUR, D. (1952). Effect of penicillin on the reproductive performance of turkey. Poul. Sci., 31,881. SILINGER,S. J., PEPPER, W. F., MORPHET,A. M. & EVANS,E. V. (1953). Effect of penicillin on the niacin requirement of turkey and a carry over effect of penicillin. Poul. Sci., 32, 754. STEPHENS,C. R., CONVER, L. H., HOSTEIN, F. A., REGNA, P. P., PILGRIM, F. J., BRUNINGS,K. J. & WOODWARD,R. B. J. (1952). Terramycin XII. Structure of aureomycin and terramycin. J. Amer. Chem. Soc., 74, 4976. TITUS,H. W. (1961). The scientificJeeding of chickens. (4th ed.) The Interstate Printer and Publisher Inc., Danville, Illinois, USA. ZAITSEVA, Z. i . & ORLOVA, N. V. (1958). Organic acids for biosynthesis of oxytetracycline. Microbiology, 228, 202. ZYGMUNT, W. A. (1961). Oxytetracycline formation by Slreptomyces rimosus in chemically defined media. Appl. Microbiol., 9, 502 7.
A. O. BAGHLAF, A. A. ABOU-ZEID, AHMED I. EL-DIWANY & ABD EL-WAHAB 1. EISSA
Microbiological and Enzyme Chemistry Research Unit, National Research Centre, Dokki, Cairo, Egypt & MAHMOUD FOUAD 8,~ MOHAMED YESSEIN
Organic" Chemical Factories oJ the Egyptian Sugar and Distillation Company, Giza, El-Hawamdia, Egypt
ABSTRACT
Production oJ oxytetracycline by Streptomyces rimosus 12907 as an animal Jeed supplement was achieved in a ./ermentation medium containing the Jollowing ingredients (glitre-l): black-strap molasses, 30; jodder yeast, 20; rice bran, 10, KHzP04, 0-2 and tap water. The dr), mash, containing oxytetracycline, thus produced was 50g litre ~ oJJermentation medium. The amount of oxytetracycline present in the dr)' mash was about 4 °/o. The dr)' mash containing the oxytetracycline was analysed Jor the amino acids L-arginine, L-histidine, L-lysine, L-isoleucine, Lleucine, L-phenylalanine, DL-methionine and L-tryptophan which could he oj nutritional value./or chickens.
INTRODUCTION
Tetracyclines, antibiotics of a broad antimicrobial spectrum, are closely related compounds widely used in the treatment of infectious diseases (Duggar, 1948; Finlay et al., 1950; Stephens et al., 1952; Shapposhnikov et al., 1958; DiMarco & Boretti, 1956: Dulaney & Perlman, 1947; Garner & Koffler, 1953; Miller et al., 1936). The carbon sources have been found to play an important role in the formation of tetracyclines by Streptomyces spp. (McVeigh & Regyes, 1961; Zaitseva & Orlova, 1958; Bitii et al., 1954; Goodman, 1954; Niedercorn, 1952; Katagiri, 1960; Shibata 301 Agricultural Wastes 0141-4607/79/0001-0301/$02.25 © Applied Science Publishers Ltd, England, 1979 Printed in Great Britain
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A.O.
B A G H L A F ET A L .
et al., 1961 ; Zygmunt, 1961). The tetracycline antibiotics display features indicative
of an acetate origin, and the general concept of an acetate-derived precursor in the sense of a polyketomethylene chain applies in the case of oxytetracycline. The nitrogen sources of the medium are also important factors in the biosynthesis of tetracycline antibiotics (Darken et aL, 1959; McGhee & Megna, 1957; Lein & Gourevitch, 1956; Goodman, 1960; McCormick et al., 1960; Barnett, 1954). The economic potential for the production of such antibiotics depends on the availability of cheap substrates. In Egypt, there are many agricultural by-products remaining from industrial production, such as black-strap molasses, fodder yeast, rice bran, corn bran and corn-steep liquor, and these could be used in a medium for antibiotic production. Antibiotics have been used in many countries as growth stimulants for farm animals. The precise way in which the antibiotics exert their beneficial effects on growth and food utilisation is still debatable. Some workers (Biely et al., 1952; Davies & Briggs, 1951; Eyssen & Somer, 1963; MacGregor et aL, 1952) claim that the growth-stimulating effect of antibiotics is most probably due to suppression of Gram-positive intestinal bacteria which interfere with the absorption of nutrients. Other bacteria may be beneficial because they produce nutrients (e.g. vitamins and/or unidentified growth factors) that are not present in the food in sufficient quantity. Titus (1961) claimed that several antibiotics added to the food of growing chickens increase their rate of growth or improve their food utilisation efficiency or do both under certain conditions. Saxena et al. (1953), Silinger et al. (1952) and Silinger et al. (1953) reported that continuous supplementation with diamine penicillin, at a level of 3 ppm in mash, during starting and developing, significantly improved the body weight of turkeys at 24 weeks. A greater response was obtained in males than in females. The present work deals with the fermentative production of oxytetracycline by Streptomyces rimosus 12907 using black-strap molasses, fodder yeast and rice bran in a medium to produce an animal feed supplement.
METHODS
Maintenance o j Streptomyces rimosus 12907 An active strain of Streptomyces rimosus 12907 (Institute for Fermentation,
Juso-Nishino-Cho, Higashiyodogawa-Ku, Osaka, Japan) was maintained on a medium containing the following ingredients (g litre-1): glucose, 10-0; peptone, 5.0; KHzPO 4, 1.0; MgSO4.7H20, 0.5 and agar, 20.0 in distilled water. The initial pH of the medium was adjusted to 6.0-6.5. The ingredients were thoroughly dissolved and the medium apportioned into test tubes. The tubes were plugged with cotton wool and sterilised at 121 °C for 20rain. When the test tubes containing
PRODUCTION OF OXYTETRACYCLINE
303
the maintenance medium attained room temperature they were inoculated with Streptomyces rimosus 12907. The inoculated slants were incubated at 30°C for 10 days to obtain luxuriant growth and sporulation. The slants were then kept at 5 °C in a refrigerator.
Vegetative medium The active strain of Streptomyces rimosus 12907 was grown in a "vegetative medium' which contained the following ingredients (glitre 1): glucose, 10.0: peptone, 5.0; K H z P O 4, 1.0 and MgSO 4 . 7 H 2 0 , 0.5; in distilled water. The initial pH of this vegetative medium was adjusted to 7. The medium was apportioned into Erlenmeyer flasks (250 ml capacity), each containing 50 ml. The flasks were plugged with cotton wool and sterilised at 121 °C for 20 min. When the flasks attained room !emperature they were inoculated with a standard spore suspension of Streptomyces rimosus under aseptic conditions. The inoculated flasks were incubated on a rotary shaker (200rpm) at 27°C for 48h. The vegetative medium was used for the inoculation of the fermentation medium.
Fermentation medium The defined fermentation medium used for the fermentative production of oxytetracycline by Streptomyces rimosus 12907 contained the following ingredients (glitre- 1): citric acid, 12.8; sucrose, 40.0; (NH,~)2SO 4, 6-0; MgSO 4 . 7 H 2 0 , 0.25: K H z P O 4, 0.15; C a C O 3, 1.0; MnSO4. H 2 0 , 0.01; ZnSO 4 . 7 H 2 0 , 0.4. The initial pH of the medium was adjusted to 7. The carbon source of the defined fermentation medium was replaced by black-strap molasses (50 ')o sugar content). The nitrogen source of the fermentation medium was also replaced by fodder yeast (40 ° 0 crude protein) and undefatted rice bran. With molasses, fodder yeast and rice bran replacing carbon and nitrogen sources, citric acid, C a C O 3, MnSO 4 . 4 H 2 0 and ZnSO 4. 7 H 2 0 were removed from the fermentation medium. The fermentative production of oxytetracycline by Streptomyces rimosus 12907 was further tested using fodder yeast, corn bran and rice bran in the presence and absence of K H 2 P O ~ and MgSO 4 . 7H20. The fermentation medium was apportioned into Erlenmeyer flasks (500 ml capacity), each containing 100 ml. The flasks were plugged with cotton wool and sterilised at 121 °C for 20 rain. When the flasks attained room temperature, they were inoculated under aseptic conditions with the vegetative medium (5 '~/0v/v) containing growing cells of Streptomyces rimosus 12907. The inoculated flasks were incubated on a rotary shaker (200rpm) at 27°C for 120h. At the end of the fermentation process the final pH of the fermented medium, the suspended dry matter and the amount of oxytetracycline produced were determined.
Biological determination oJ oxytetracycline A biological-standard curve was drawn between the logarithm of different concentrations of oxytetracycline and the inhibition zones of Bacillus subtilis N R R L B-543 (Abou-Zeid & Shehata, 1969).
304
A. O. B A G H L A F E]" A L .
Production of oxytetracycline in a fermenter (1200 litre) The amounts of the most suitable ingredients for the fermentation medium were scaled up to be used in the fermentative production of oxytetracycline in a fermenter (1200 litre). The fermentation medium contained the following ingredients in tap water (g litre-i): molasses, 30.0; fodder yeast, 20.0; rice bran, 10-0 and K H 2 P O 4, 0.2. The initial pH of the fermentation medium was adjusted to 6-5. Before charging the fermenter, it was thoroughly washed and sterilised by steam. Seven-hundred litres of the fermentation medium were introduced into the fermenter. The medium was sterilised at 121 °C for 40 min by steam and when the fermenter attained room temperature it was inoculated with Streptomyces rimosus 12907 grown on the vegetative medium mentioned above. The percentage of inoculum was 5 ~o (v/v). To the fermentation medium was added cotton-seed oil anti-foam (0.5~o). The fermentation medium was stirred and sparged with sterile air. The process was conducted at 27°C for 96h. Every 24h a sample was taken to determine the pH of the fermented medium, suspended dry matter and antibiotic yield. The whole fermented mass was taken off and, after the pH had been adjusted to 4.0 4.5 with HCI, poured into trays (stainless steel, 75 x 40 × 7cm), each tray containing 15 litres. The trays containing fermented mash were placed on tables in a room, the temperature of which was controlled at 40 °-45 °C by means of a contact thermometer. Hot air was directed on to the surfaces of the trays while a suction pump was attached to the room to evacuate air and water and other vapours. The process was continued until the fermented mash was completely dried, when the mash was removed and ground. The amount of oxytetracycline present in the dry, fermented mash was tested as before. Determination oJamino acids present in Jermented mash Quantitative determination of amino acids present in the fermented mash was carried out according to the techniques of Block et al., 1958 and Blauth et al., 1963. The amino acids L-arginine, L-histidine, L-lysine, L-isoleucine, L-leucine, Lphenylalanine, DL-methionine, L-threonine and L-tryptophan were determined. Anti-microbial spectrum of oxytetracycline present in the dr),' mash The inhibitory effects of oxytetracycline present in the dry mash were biologically assayed on the following micro-organisms: Bacillus subtilis, Staphylococcus aureus, Sarcina lutea, Klebsiella pneumoniae, Salmonella schottmuelleri, Escherichia coli, Proteus vulgaris and Mycobacterium avium.
RESULTS A N D DISCUSSION
The results obtained (Table l) show that the amount of oxytetracycline produced by Streptomyces rimosus 12907 increased with increasing incubation period, reaching
305
PRODUCTION OF OXYTETRACYCLINE
TABLE 1 AMOUNTS OF OXYTETRACYCLINE PRODUCED BY Streptomyces rimosus 12907 AT DIFFERENT PERIODS OF THE FERMENTATION PROCESS
Incubation period (h )
Final pH
Suspended dry matter (rag millilitre - l )
O xytetracycline (#g millilitre- t)
24 48 72 96 120 144
5,5 6,8 7.5 8.3 8.5 8-5
5.8 6.5 7.5 9.0 9.2 9-1
-300 450 600 500 475
D e f i n e d m e d i u m , see " M e t h o d s ' .
an optimum at 96 h, after which there was a decrease. A drop in pH was obtained during 48h of the fermentation process and this may be correlated with accumulation of organic acids. These were then further utilised by the microorganism so that at the end of the fermentation process the final pH was moved to the alkaline side (8.5). The defined medium used in the fermentative production of oxytetracycline contained costly ingredients; therefore certain local ingredients such as black-strap molasses, fodder yeast and rice bran were tested for the production of oxytetracycline by Streptomyces rimosus 12907. The results obtained (Table 2(a)) show that when sucrose was replaced by different concentrations of black-strap molasses the amount of oxytetracycline produced increased with the increase of black-strap molasses and reached an optimum at 30-40 g litre-~, above which a decline in yield occurred. The final pH was variable, depending to some extent on the amount of molasses added. Molasses (30 g litre ~) was used instead of sucrose and (NH4)2SO 4 was replaced by different concentrations of fodder yeast (40'~0 crude protein), while the other ingredients of the medium remained unchanged. The results obtained (Table 2(b)) show that the amount of oxytetracycline produced increased with the increase of fodder yeast concentration, reaching an optimum at 20 g litre- ~, above which a decrease in the antibiotic yield occurred. Fodder yeast used in the medium gave a better yield of the antibiotic than (N H4)2SO4 . The final pH of the fermented medium was 7.5-8.5. J~lolasses (30 g litre- l) was used instead of sucrose and (NH4)2SO 4 was removed and replaced by different concentrations of rice bran, while the other ingredients of the medium remained unchanged. The results obtained (Table 2(c)) show that the amount of oxytetracycline excreted in the medium increased with the increase of rice bran concentration, reaching an optimum at 30-40 g litre- i. Different concentrations of rice bran in the presence of molasses ( 3 0 g l i t r e - l ) and fodder yeast (20glitre t) were incorporated into the medium. The results obtained (Table 2(d)) indicated that addition of rice bran to fodder yeast favoured a high antibiotic yield. The amount of oxytetracycline
306
A. O. BAGHLAF ET AL.
TABLE 2 A M O U N T S O F O X T E T R A C Y C L I N E P R O D U C E D BY Streptomyces rimosus 12907 W H E N C O N T A I N I N G MOLASSES, F O D D E R YEAST A N D RICE BRAN
Concentrations
10 15 20 25 30 40 50 60 70
Final pH
Suspended dry matter (mg millilitre ~ 1)
(a) Different concentrations of molasses (g litre-~) 7.5 6-5 8.0 7.0 8.0 7.0 8.5 7.5 8-5 8.0 8.0 8.5 8.0 8-5 7.0 8-5 7-0 8.5
GROWN
IN MEDIA a
Oxytetrac)'cline (tag millilitre ~)
350 400 450 500 550 500 300 200 150
(b) Molasses (30 g litre- 1) + different concentrations of fodder yeast (g litre- a) 2-5 8.0 6.5 500 5.0 8-0 7.0 550 10.0 8-0 7.5 650 15.0 8.0 7.5 750 20-0 8-5 8-5 800 25.0 8.0 9.5 700 30.0 7.5 10.0 650 (c) Molasses (30 g litre-1) + different concentrations of rice bran 10 7.5 7.0 450 20 7-5 8-0 450 30 8.5 9.5 600 40 8.5 10.0 600 50 8.0 10.5 500 (d) Molasses (30 g litre- l) + fodder yeast (20 g litre - ~) + different concentrations of rice bran (g litre - ~) 5 7-5 9.0 650 I0 7-5 10-0 850 15 8.0 10-5 750 20 8-5 11-9 700 25 8.5 11-5 600 30 8.0 12.0 600 40 7.0 12.0 600 (e) Molasses (30 g litre- 1) + rice bran (10 g litre- 1) + different concentrations of fodder yeast (g litre- ~) 5 7.0 10.0 800 10 7.5 10.5 850 15 8.0 11.0 950 20 8-0 ~ 12-0 1000 25 8-0 12.5 950 30 7-5 12.5 800 35 7.5 13.5 750 40 7.5 14.5 750 45 7-0 15.0 700 50 7.0 15.0 700 ° For other constituents of media see 'Methods' and 'Results'.
307
PRODUCTION OF OXYTETRACYCLINE
excreted in the m e d i u m increased with the initial increase o f rice b r a n c o n c e n t r a t i o n , reaching an o p t i m u m at 10 g l i t r e - 1 and then decreasing. M o l a s s e s ( 3 0 g l i t r e 1), rice b r a n ( 1 0 g l i t r e -1) a n d different c o n c e n t r a t i o n s of f o d d e r yeast were i n c o r p o r a t e d into the m e d i u m w i t h o u t ( N H 4 ) 2 S O 4, while the o t h e r ingredients o f the m e d i u m r e m a i n e d u n c h a n g e d . T h e results o b t a i n e d (Table 2(e)) show t h a t a suitable c o n c e n t r a t i o n o f f o d d e r yeast in the presence o f molasses a n d rice b r a n was 20 g l i t r e - 1. T h e best a m o u n t s o f molasses, f o d d e r yeast and rice b r a n for a n t i b i o t i c p r o d u c t i o n were 30, 20 a n d 1 0 g l i t r e -~, respectively. These a m o u n t s of ingredients were then used in a simplified m e d i u m with different
TABLE 3 AMOUNTS OF O X Y T E T R A C Y C L I N E P R O D U C E D BY
Streptomyces rimosus 12907 W H E N
G R O W N ON A MEDII~ M
CONTMNINGKH2PO 4 AND MgSO 4 . 7H20 Concentrations
Final p H
Suspended dry matter (mg millilitre- l )
Oxytetracycline (l~g millilitre ~)
(0 Molasses (30glitre 1)+ fodder yeast (20glitre 1)+ rice bran (10glitre ~)+different concentrations of KH2PO 4 (glitre t) 0.00 7.5 9-5 1000 0-10 7.5 9.5 1200 0.20 7.5 10.5 1250 0.30 8.5 11-5 1000 0.40 8.5 12.5 1000 0.50 8.5 12.5 950 0-75 8.0 11.5 800 1.00 8-0 12.0 800 1-50 8.0 12.0 750 2.00 8-0 t2.0 700 (g) Molasses (30glitre-t) + fodder yeast (20glitre 1) + rice bran (10glitre 1) + KHzPO~ (0.2glitre 1) + different concentrations of MgSO 4. 7H20 (glitre ~) 7.0 8.5 1000 0.00 7"0 8.5 950 0.10 7.0 9.5 950 0.20 7-5 10"5 900 0.30 8"0 I IO 800 0.40 8"5 12.0 75O 0.50 8.0 11.0 700 0.75 8"0 11"0 700 1-00
c o n c e n t r a t i o n s o f K H 2 P O 4 or MgSO~. T h e results r e c o r d e d (Table 3) show that the a m o u n t o f o x y t e t r a c y c l i n e p r o d u c e d increased with increase o f K H 2 P O 4 c o n c e n t r a t i o n reaching an o p t i m u m at 0.2 g l i t r e 1 but at this c o n c e n t r a t i o n of K H 2 P O ~ a d d i n g M g S O 4 at different c o n c e n t r a t i o n s d i d n o t i m p r o v e the a n t i b i o t i c yield. A m e d i u m c o n t a i n i n g molasses, f o d d e r yeast, rice b r a n , K H 2 P O 4 a n d t a p water was then used for large-scale a n t i b i o t i c p r o d u c t i o n . T h e initial p H o f the f e r m e n t a t i o n m e d i u m was 6.5 a n d at the end o f the f e r m e n t a t i o n process it was 8-5
308
A. O. BAGHLAF ET AL. TABLE 4 OXYTETRACYCLINEa PRODUCED IN A FERMENTER (1200 LITRE)
Initial pH
Final pH
Dry mash (%)b
Oxytetracycline (rag per gramme dry mash)
6-5
8.5
5.0
40
a Fermentation medium used contained the following ingredients (g litre-1): black-strap molasses 30, fodder yeast (40% crude protein) 20, rice bran 10, K H 2 P O 4 0.2 and tap water. b % (w/v) of fermentation medium.
(Table 4). At the end of the fermentation process the whole fermented mash was hotair dried under reduced pressure at 40 ° to 45 °C. The percentage of dry suspended matter which included microbial cells, unutilised medium constituents, oxytetracycline and other microbial metabolites, was about 5 ~o. The amount of oxytetracycline present in this dry mash was 4 ~o by weight. The results (Table 5) show the action of the unpurified oxytetracycline on certain types of bacteria: Bacillus subtilis, Staphylococcus aureus, Sarcina lutea, Klebsiella pneumoniae, Salmonella schottmuelleri, Escherichia coli, Proteus vulgaris and
Mycobacterum avium. TABLE 5 INHIBITORY ACTION ON BACTERIA OF OXYTETRACYCLINE PRESENT IN DRY MASHMADE INTO AN AQUEOUSSOLUTION (2mg millilitre- ~) AND USING 6"35 mm DIPPED DISCS
Bacteria Bacillus subtilis Staphylococcus aureus Sarcina lutea Klebsiella pneumoniae Salmonella schot tmueller i Escherichia coli Proteus vulgaris Mycobacterium avium
Inhibition zone (mm) 17 30 35 24
31 25 16 35
For commercial use it would not be necessary to extract the antibiotic from the fermentation mash as it would be produced for use in poultry rearing. The antibiotic mash could be used in poultry feed or in drinking water. The aim of its addition is to prevent infectious diseases and to accelerate the growth of poultry; therefore, the presence of the microbial cells and metabolites is not harmful. On the contrary, these microbial cells and metabolites may be useful as micro-nutrients in poultry diets. Analyses showed that the hydrolysate of the dry mash contained the amino acids Larginine, L-histidine, L-lysine, L-isoleucine, L-leucine, L-phenylalanine, DLmethionine, L-threonine and L-tryptophan in the amounts shown in Table 6.
PRODUCTION OF OXYTETRACYCLINE
309
TABLE 6 AMOUNTS OF AMINO ACIDS PRESENT IN DRY MASH CONTAINING OXYTETRACYCLINE
Amino acid
Dry mash (g kilogramme- 1)
L-Arginine L-Histidine L-Lysine L-lsoleucine L-Leucine L-Phenylalanine DL-Methionine L-Threonine c-Tryptophan
9-21 3.25 6.73 6.95 8.74 5.39 0"82 4.70 1.50
These amino acids are beneficial in the growth of poultry. The utilisation of oxytetracycline present in dry mash is also more economic than when it is extracted from the fermentation mash for use in poultry feeds.
ACKNOWLEDGEMENT
This work was carried out in the laboratories of the Organic Chemical Factories of the Egyptian Sugar and Distillation Company, Giza, El-Hawamdia, in collaboration with the National Research Centre, Dokki, Cairo, Egypt.
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