AgrwulturalWa~te~2(1980) 293 301
U T I L I S A T I O N OF F O O D I N D U S T R Y B Y - P R O D U C T S IN THE P R O D U C T I O N OF O X Y T E T R A C Y C L I N E BY S T R E P T O M Y C E S R I M O S U S 93060
ABOLI-ZIqD A. ABOU-ZE1D. AHMED I. EL-DIWAI',"r & HOSSAM EL-DEFY SHAKER
Microhio]ogical amt Enzyme Chemist~3' Research Unit, National Re.~eurch Centre, Dokki. Cairo. Eg)pl &
HASSAN M. SALEM
D~Tmrtment o[ Biochemistry, Faculty of Agriculture, Cairo Unit,ersity~ Gi:--a, Cairo, Egypt
Di[Ji, rent carbon sources were utilised in the biosynthesis o/ ox)'tetraeyeline hv S. rimosus 93060. Glucose was the best earhon source Jot antibiotic produetion in a medium with peptone as the nitrogen source. Two agricultural wastes were used as eheap partial replaeements f o r glucose. The first was Egyptian black-strap molasses. The in~uence of Egyptian black-strap tnolasses obtainedJi'om d([J'erent sugar cane manuJztcturers was tested. A bou-Korkas molasses was the best j o r the production o/ oxytetrac.vcline. Potassium ferroeyanide, EDTA and methylene blue were used in the chelation or reduction of inorganic salts present in Abou-Korkas molasses. ?Ttese materials did not give high titres q/oxytetra~3'eline. The suitability oJ A bou- Kork as molasses was due to its micronutrients. A second substrate tested Jor antihiotie production was theJ)'eeze-dried olive water obtainedji'ont the pressing and scparat ion of olive oil. The chemical composition of olive water was int,estigated. Olit,e water alone was a poor substrate Jor antibiotic production, hut with glucose it was a.s eective as molasses.
Antibiotic metabolites of various microorganisms are compounds of widely differing chemical structures but their most important property is their inhibitive action against pathogenic microorganisms. 293 Agricultural Wastes 0141-4607/80/0002-0293/'$02"25 :! Applied Science Publishers Ltd, England, 1980 Printed in Great Britain
ABOU-ZEID A. ABOU-ZE1D e t
The tetracyclines are a group of broad-spectrum antibiotics. Several structurally related antibiotics are elaborated by various strains of the genus Streptomyces. Three have received most attention, Chlortetracycline (CTC) was isolated by Duggar (1948) from submerged, aerated culture of Streptomyces aureofaciens. Finlay et al. (1950) discovered oxytetracycline (OTC) which was formed by Streptomyces rimosus. The tetracycline (TC) produced by Streptomyces viridifaciens was investigated by Boothe et al. (1953) and by Conover et al. (1953). Other tetracycline type antibiotics which have been isolated are bromotetracycline (Sensi et al., 1955; Doerschuk et al., 1955) and demethyl-tetracycline (McCormick et al., 1957). Reedy et al. (1955) studied the activity of the three antibiotics--CTC, OTC and TC---currently employed in therapy against some bacterial species. The authors also concluded that oxytetracycline shows even higher antibiotic activities against some other microorganisms such as amoebae. Koch (1955) reported that, generally speaking, Gram-positive organisms showed a different susceptibility to the three antibiotics according to the following succession: CTC, TC and OTC. This phenomenon does not occur with the Gramnegative organisms. Weinberg (1957) stated that the antibacterial actions of the tetracycline antibiotics should be ascribed to the chelating activity of the substances leading to the formation of complexes with metallic cations which are essential components of the co-enzymes catalysing various reactions of the bacterial metabolic system. In the fermentative preparation of oxytetracycline, the work of Zygmunt (1961) on biosynthesis by a strain of Streptomyces rimosus has shown that organic compounds are an important source of nitrogen for the production of this antibiotic. Short-chain organic acids are not utilised by the microorganism and do not increase the growth of the mycelium although Birch et al. (1962) reported that acetate can serve as a precursor of the tetracycline ring system. Borenstajn & Wolf (1955) reported that oil has a beneficial effect on the pH of the culture, in addition to its action as an antifoam. Niedercorn (1952) reported that calcium or magnesium salts, or both, had the ability to reduce the toxicity of the antibiotic towards the producing organism. Orlova et al. (1961) described the use of oils as sole carbon sources for oxytetracycline production, using 3-4 ~o lipid instead of 3 ~o starch. Borenstajn & Wolf (1955) reported that for oxytetracycline production a pH value of 7.0 was suitable. Boghlof et al. (1979) reported on the production of a dried Streptomyces mash, containing oxytetracycline, as a poultry-feed supplement. The economic potential of the production of such antibiotics depends on the availability of cheap substrates (Abou-Zeid et al., 1976), The work reported in the present paper dealt with the fermentative production of oxytetracycline using some food industry by-products in the fermentation medium. The chemical composition of dried olive water was also investigated.
F O O D B Y - P R O D U C T S FOR G R O W T H
Maintenance O[ Streptomyces rimosus 93060 Streptomyces rimosus 93060 was maintained on a medium containing (g litre 1): glucose, 10.0; peptone, 5-0; K H 2 P O 4, 1.0; KCI, 0-5: agar (DIFCO), 20.0; distilled water to 1000 ml. The initial pH of the medium was adjusted to 7.0. The medium was dispensed in 5 ml amounts into test tubes and the tubes were plugged with cotton wool and sterilised at 120°C for 15min. The slants were inoculated with the experimental organism under aseptic conditions and incubated at 27 °C for 10 days to obtain luxuriant growth and sporulation, and then kept in a refrigerator at 5 °C. l~k,rmentation media The media contained peptone (5g) in distilled water (1000ml) plus glucose. glucose and molasses, dried olive water or glucose and olive water in the concentrations given below. The ingredients were thoroughly mixed and the initial pH of the medium was adjusted to 7.0. For further studies in the fermentative production of oxytetracycline, molasses was treated with different concentrations of chelating agents to see if this increased its efficiency and potential for antibiotic production. The fermentation medium was apportioned into 250 ml Erlenmeycr flasks, each containing 50ml. The flasks were plugged with cotton wool and sterilised at 120 °C for 15 rain. The flasks were inoculated with a standard inoculum of the Streptomyces rimosus 93060 and incubated at 27 °C for 5 days. Treatment o[ molasses Black-strap molasses from the sugar industry contains variable constituents. Some of the ingredients are essential for the growth of Streptomyces rimosu,s and antibiotic production, others are inhibitors for microbial growth and antibiotic production. The inhibitory ingredients are restricted to the muddy precipitates in the commercial molasses. Therefore, molasses was diluted (2 volumes molasses:l volume distilled water) and the solution was thoroughly mixed and centrifuged at 4000rpm for 20rain to precipitate muds and other insoluble substances. The supernatant solution was taken and the required amount was added to the fermentation medium. The volume added was such as to give the stated weight of undiluted molasses. The molasses contained 50 ~, of reducing sugars by weight. Determination of oxytetracycline A biological standard curve was drawn between the logarithm of different concentrations of oxytetracycline and the inhibition zones of the susceptible bacterium Bacillus subtilis N R R L B-543, using a cup-plate method (Abou-Zeid & Shehata, 1969).
ABOU-ZEID A. ABOU-ZEID el
Determination oJpH value A pH M62 standard pH meter (Radiometer)was used for the adjustment of media pH values and the determination of the final pH values of the different cultures.
Determination of sugars Sugars were determined according to the colorimetric methods of Somogyi (1945) and Nelson (1944).
Determination oJ total nitrogen Total nitrogen of dried olive water was determined by the method of Miller & Houghton (1945).
Paper chromatographic' determination oJamino acids The separated amino acids were determined according to the method of Whitehead (1964).
Determination of total Jats Lipids were isolated by subjecting the material to Soxhlet extraction with ethyl acetate, benzene or chloroform/methanol (1:1 v/v) for 16 h according to the method of Abdel-Fattah & Edress (1971). Thereafter, the filtered extract was evaporated under vacuum at 40°C.
RESULTS AND DISCUSSION
Preliminary experiments showed that molasses alone was a relatively poor substrate for growth of the Streptomyces and antibiotic production, compared with glucose. The aim of the present work was to see if molasses could replace part of the glucose in a high-sugar medium to provide a cheaper medium and yet one which would give good antibiotic production and a reasonably short fermentation period. The results obtained (Table 1) show that different cane sugar molasses can act TABLE 1 UT1LISATIONOF BLACK-STRAPMOLASSESIN THE PRODUCTIONOF OXYTETRACYCLINEBYStreptomyces rimosus 93060
A b o u - K o r k a s molasses N a g a - H a m a d i molasses K o m o m b o molasses Edfo molasses
Final p H value
Suspended dry matter (g lit re- 1)
Oxytetraeycline (g litre 1)
8-75 8.70 8.90 8.65
3-24 3.65 3.90 2.50
1-844 1.516 1.262 1.135
Medium. Molasses, 15 g; glucose, 10 g; peptone, 5 g.
FOOD BY-PRODUCTS FOR G R O W T H OF
with glucose as the carbon source in the fermentation medium in oxytetracycline production. Different types of cane sugar molasses obtained from sugar cane manufacturers gave different antibiotic productions. Different techniques are used in different factories for obtaining sucrose; therefore, the chemical constituents of cane sugar molasses are variable. A b o u - K o r k a s molasses was the best carbon source for the production of oxytetracycline by S. r i m o s u s . The results given in Table 2 TABLE 2 EFFECT OF DIFFERENT C O N C E N T R A T I O N S OF MOLASSES ON THE BIOSYNTHESIS OF O X Y T E T R A C Y C L I N E B'~
Streptomyces rimosus 93060 Concentration (g litre ~)
Final pH value
Su.~pended dr 1" matter (g litre 1)
Oxytetracvcline (g litre i)
0.5 5.00 10.00 15.00 18.00 25-00 30.00 Control
9.00 9-00 8.90 8.78 8-75 8.55 8-37 9.00
0.832 0.955 1-450 1-700 1.900 2.00 2.77 2.14
0.752 1.352 1-664 1.664 1.844 1.664 1.516 0.892
The control medium contained (g litre- ~): glucose, 10.0; peptone, 5-0. The different concentrations of molasses were added to this medium. show that different concentrations of A b o u - K o r k a s molasses influenced the biosynthesis of oxytetracycline. The yield of the antibiotic was increased with the increase of molasses concentrations, reaching its optimum at 18 g.ltre- i above which the yield ofoxytetracycline decreased. The results obtained (Table 3) show the effect of chelation and the reduction of the inorganic salts present in A b o u - K o r k a s molasses on the production of oxytetracycline by S. r i m o s u s 93060 using different concentrations of" EDTA, potassium ferrocyanide and methylene blue. Methylene blue is reducing, but can also act as a chelating agent (Holzbuher & Divis, 1976: Abou-Zeid & Eissa, 1971). Cane sugar molasses contains carbohydrates (mainly sucrose, glucose and fructose), nitrogen sources, magnesium, calcium and trace elements (Mn, Fe and Zn). Chelating agents can remove some constituents, especially divalent metals, which may retard antibiotic production fi'om the fermentation medium. However, all of these reagents decreased antibiotic production. The olive water (aqueous layer) was obtained fiom the Egyptian Vineyards and Distilleries Co. (Gabaklis plant, Alexandria) as a by-prodtlct remaining after the extraction of olive oil by pressure. After pressing the olive fruits (with their seeds) the ,juice passes through the separation (centrifugation) process where two layers are usually separated and collected--i.e, the oil layer and the aqueous layer. The latter contains all the components of the juice except the oil.
ABOU-ZE1D A. ABOU-ZEID et a].
TABLE 3 EFFECT OF REDUCTION OF INORGANIC SALTS PRESENT IN ABOU-KORKAS MOLASSES ON THE PRODUCTION OF OXYTETRACYCLINE BY Streptomyces rimosus 93060
Concentrations (g litre- l )
Final p H value
Suspended dry matter
Oxytetracycline (g litre - 1)
0.10 0.50 1.00 1.50 2-00
8.24 8.27 8.22 8.21 7.23
2.86 1.83 1.55 1-24 1.31
1.664 1.516 1.352 1.262 1.138
Potassium ferrocyanide 0.10 0.50 1-00 1.50 2.00
8-46 8.48 8.69 8.48 8.63
1-25 1.54 1.72 1.95 2.43
1.664 0-363 0-224 nil nil
Methylene blue 0.10 0.50 1.00 1.50 2.00 Control
8-00 7-00 5-85 5.60 5-60 8-20
0.91 1.04 0.27 0.38 0-58 2.61
0.224 0.036 nil nil nil 1-844
The control medium contained (g litre-~): glucose, 10; peptone, 5; molasses, 18.
TABLE 4 CHEMICAL COMPOSITION OF DRIED OLIVE WATER
Composition Amino acids Alanine Lysine + histidine Serine + glycine Threonine Leucine + iso-leucine Glutamic acid Valine + methionine Aspartic acid Tryptophan Total sugars Before hydrolysis After hydrolysis TotalJats Extraction, chloroform:methanol (I .1 v/v) Ethyl acetate Benzene
Concentration (rag gramme- 1) 2-59 2-55 2-17 2-00 1-50 0.95 0.57 0.44 0" 14 (mg g r a m m e - 1) 16-43 18.83 (g gramme 1) 0.36 0-075 0.064
TABLE 5 UTILISATION OF
OXYTETRACYCLINE Dried olive water (g litre- l )
Final p H value
Suspended dr), matter (g litre- 1)
Oxytetracycline (g litre- ~)
Control ~ Control b 0.50 1.00 2.00 4-00 8.00 10.00 15.00 25-00 30.00
7-65 9.10 9.00 9.10 0.10 9.10 9.10 9.00 8-80 8.55 8.35
2.81 2.00 0.49 0.70 0-90 1.00 2-10 3-00 2.95 3-56 4-03
1.516 0.418 0-448 0.448 0.532 0.532 0.670 0.752 0.814 0.794 0.752
"The control contained (g litre 1): glucose, 10.0; molasses, 15.0; peptone, 5-0. bThe control contained (g litre- 1): peptone, 5.0. To t his was added the dried olive water. For preparation of olive water solids see 'Methods'.
From the aqueous layer the Total Solids were separated by freeze drying. The dry powder represented 10 ~o(W/V) from the total aqueous layer. The olive water is one of the by-products which have never been utilised. Ten litres of olive water were freeze dried and the chemical composition was investigated. The results are shown in Table 4. The amounts of total sugar and total fat were determined and the results indicated that chloroform:methanol (1 "1 v/v) extracted more lipids than ethylacetate or benzene. Table 5 shows the utilisation of different concentrations of dried olive water as a carbon source in the production of oxytetracycline; the basal fermentation medium which was used contained peptone TABLE 6 UTILISATIONOF DRIEDOLIVEWATERAND GLUCOSEAS CARBONSOURCESIN THE FERMENTATIONMEDIUMiOF Streptomyces rimosus 93060 Dried olive water (g litre-- i )
Final p H value
Suspended dry matter (g litre - 1)
Oxytetracycline (g litre- l )
0.5 1-5 4.0 7.0 10.0 15.0 Control Q Control b
8-00 7-8 7.56 7.53 7.53 7-52 7.65 8.32
1.20 1"68 3"00 3.85 3"98 4"20 1.88 2-45
0-956 1.138 1"516 1-352 1.138 1.138 1.516 1.062
The control comparison fermentation medium contained (g litre ~): molasses, 15-0; glucose, 10-0 and peptone 5.0. b The control contained (g litre 1): glucose 10"0 and peptone 5'0. To this was added the dried olive water.
ABOU-ZEID A. ABOU-ZEID el a/.
5 - 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 o f d r i e d olive w a t e r . T h e best c o n c e n t r a t i o n was 10 to 15-0 g l i t r e - l ; h o w e v e r , the a n t i b i o t i c p r o d u c t i o n was less t h a n the c o m p a n i o n f e r m e n t a t i o n m e d i u m c o n t a i n i n g ( g l i t r e - 1 ) : g l u c o s e , 10.0; m o l a s s e s , 15.0 a n d p e p t o n e , 5.0. T h e final p H v a l u e o f the f e r m e n t a t i o n m e d i u m was shifted t o w a r d s the a l k a l i n e side b u t the s u s p e n d e d d r y m a t t e r i n c r e a s e d w i t h the i n c r e a s e o f the c o n c e n t r a t i o n o f d r i e d olive w a t e r . O n t h e o t h e r h a n d , as s h o w n in T a b l e 6, g l u c o s e was a d d e d to the f e r m e n t a t i o n m e d i u m at a c o n c e n t r a t i o n o f 10.0 g l i t r e - 1 w i t h d i f f e r e n t c o n c e n t r a t i o n s o f d r i e d olive w a t e r r a n g i n g f r o m 0-5 to 15 O g l i t r e - 1. T h e best c o n c e n t r a t i o n was 4-0 g l i t r e - 1 o f d r i e d olive w a t e r , a b o v e w h i c h a d e c r e a s e in t h e a n t i b i o t i c yield w a s r e c o r d e d . T h e a n t i b i o t i c p r o d u c t i o n in this m e d i u m w a s as g o o d as t h a t in the m o l a s s e s m e d i u m , so t h a t olive w a t e r c o u l d be u s e d i n s t e a d o f m o l a s s e s as a c o n s t i t u e n t o f a m e d i u m for o x y t e t r a c y c l i n e production.
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FOOD BY-PRODUCTS FOR GROWTH OF S. rimosus
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