PRODUCTION OF STREPTOMYCIN FROM STREPTOMYCES

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Mamatha et al.

World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue 8, 907-922.

Research Article

ISSN 2278 – 4357

PRODUCTION OF STREPTOMYCIN FROM STREPTOMYCES GRISEUS UNDER SOLID STATE FERMENTATION, & ITS PRODUCTION ENHANCEMENT BY MUTATION AND ANALYSIS BY HPLC Mamatha J*1, Sudipa Bhadra1, and Mahesh M2 1

P.G. Department of Biotechnology, The Oxford College of Science, Bangalore 560102, Karnataka, India. 2

Article Received on 11 May 2014, Revised on 09 June 2014, Accepted on 15 July 2014

Azyme Biosciences Pvt Ltd, Bangalore Karnataka, India ABSTRACT The actinomycete strain Streptomyces griseus isolated from soil is used for the production of streptomycin, a potent antibiotic drug which is of great

commercial

importance.

The strain

was

identified

by

morphological features and several biochemical tests. Further they *Correspondence for Author Mamatha J P.G. Department of

were grown as a pure culture in starch casein agar media. Mutation was done both by physical and chemical methods and it was found that

Biotechnology, The Oxford

the antimicrobial activity was increased by 10% for the mutated strain

College of Science, Bangalore

kept under UV light for 5mins. Production was achieved by solid state

560102, Karnataka, India

fermentation using oranges, pineapple and sugarcane bagasse, as the substrate which are cheaply available. Purification was done by

filtration with charcoal and acidified methanol followed by evaporation to produce antibiotic in powder form. High purity was obtained during analysis by High Pressure Liquid Chromatography (HPLC) and it was shown that the production increased by 61.97% with oranges and 4.88% increase with pineapple while there was an 11.64% increase in production with sugarcane bagasse using the physically mutated sample. Hence it was concluded that mutation with UV light for 5mins and solid state fermentation using oranges as substrate yielded the best results. This paper also reviews the possible methods of increasing the production, efficiency and cost effectiveness of streptomycin at the industrial scale. Keywords: Streptomyces griseus, Mutation, antimicrobial activity, solid state fermentation, High Pressure Liquid Chromatography (HPLC).

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INTRODUCTION Antibiotics are low-molecular-mass (1500kDa) products of secondary metabolism, usually produced during the late growth phase (idiophase) of a relatively small group of microorganisms. Antibiotics are not essential for the growth of producing cultures but serve diverse survival functions in nature

[1]

. In addition, antibiotics are very important for the

health, nutrition and economics of our society

[2].

Owing to the use of antibiotics and other

secondary metabolites, the average life expectancy in the United States increased from 47 years in 1900–1974 (males) to 80 years (females) in the year 2000

[3]

. Probably, the most

important use of secondary metabolites has been as anti infective drugs. In the year 2000, anti-infective secondary metabolites marketed 55 billion dollars [4], but in the year 2007, the market for antibiotics increased to 66 billion dollars [5]. Streptomycin is an antibiotic (anti-mycobacterial) drug, the first of a class of drugs called amino glycosides to be discovered, and it was the first antibiotic remedy for tuberculosis

[6]

. It is derived from the action bacterium Streptomyces griseus.

Streptomycin is a bactericidal antibiotic. Streptomycetes are the source of several useful antibiotics that are used not only in the treatment of various human and animal diseases but also in agriculture and biochemistry as metabolic poisons. At least 70 of the approximately 100 marketed antibiotics used for the treatment of infections in humans are derived from substances produced by Streptomyces spp [7]. The genus Streptomvces belongs to order Actinomycetales. This bacteria is filamentous, aerobic, gram positive and spread mainly in soil and considered as a good source for more than half of all antibiotic

[8]

. Also it is known to produce many other products like extra

cellular enzymes and inhibitors [9, 10, 11, 12, 13, and 14]. Streptomycin is an antibiotic that inhibits both Gram-positive and Gram-negative bacteria, and is therefore a useful broad-spectrum antibiotic. It cannot be given orally it has to be administered by regular intramuscular injections.

[9, 10, and 11]

. There are no major and

substantial research has engaged in India.

The present study deals with the isolation of Streptomyces griseus colonies, production of streptomyces under solid state fermentation using various substrates such as sugarcane bagasse, orange peel, pineapple peel and strain improvement for increased production of Streptomvces and analysis by HPLC. www.wjpps.com

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MATERIALS AND METHOD Collection of Soil Sample: Five different soil samples were collected from different regions around Bangalore. Using pre-sterilized zip lock cover and sterile spatula. Precautionary measures were taken to minimize the contamination. The soil sample was mixed well and processed on the same day. Exactly 0.2g of each of the soil samples were weighed and kept in a paper wrapped tightly. Isolation of Organism in Specific Media: Five test tubes containing 10 ml of 1% saline in each, standard casein media, and 5 petriplates were sterilized in autoclave at 121° C for 15 min and after cooling 0.2 g of each soil samples were transferred in each of the 5 test tubes containing 10 ml of the 1% saline. Casein was taken separately and pasteurized at 72°C for half an hour. Then the casein transferred to the media before it was solidified and mixed properly. Media was poured into the petriplates and allowed to solidify. 100 µl of the soil sample from the test tubes is transferred to the petriplates and spreaded using sterile looped spreader. The petriplates were kept for incubation in the incubator for 48hrs. Pure Culture in Slants and Inoculation in Broth Media: One of the colonies from the previous incubated culture from soil is inoculated into the starch casein broth and incubated at 37°C for 48hrs in an orbital shaker incubator. Slants were prepared from starch casein media and allowed to solidify. Streaking was done on the slants which are prepared to get pure cultures. Slants were kept in an incubator at 37°C for 48hrs. Pure cultures were stored in freezer for further use. Identifying the Morphological Features Using Gram Staining: The bacterial isolates obtained were maintained on starch casein slants and were refrigerated at 4°C for further use. Simple gram staining was performed and the slides were observed under microscope and the morphological features of the bacteria were observed. Screening for the Production of Antibiotics by Crowded Plate Technique: The inhibition of microbial growth under standardized conditions may be utilized for demonstrating the therapeutic efficiency of any antibiotics on L B agar (Hi Media, India). The microbiological assay is based upon comparisons of the inhibition of growth of bacteria which was performed using crowded plate technique.

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Biochemical Test for Identification- Catalase test, Starch hydrolyses test, Oxidase test, Casein hydrolysis test, Methyl red / Voges Proskauer test, Citrate test, Indole test, Urease test, Gelatin liquifaction test were carried out on the pure sample. ENHANCING THE PRODUCTION BY MUTATION a) Physical mutation To each of five starch casein media petriplates 100 µl of the pure culture is pipette out and then swabbed over the surface. All 5 petriplates are then kept under UV light in the LAF chamber for 5, 10, 15, 20, 25 minutes of time interval respectively and incubated at 37° C for 48hrs. After 48 hrs they were stained and were examined under the microscope to see which culture among the different time intervals gave the similar structure as that of the pure culture. Other plates which did not resemble the pure culture were discarded. An increase for the inhibition zone was tested. b) Chemical mutation The materials required for this protocol are ethidium bromide, 6 test tubes, LB broth, and physically mutated culture. The LB broth and test tubes are autoclaved. After autoclaving the 1st test tube is marked as blank, the other test tubes are marked as 1, 2, 3, 4, and 5 respectively. 50 µl of ethidium bromide is added to the blank test tube. To the other 5 test tubes ethidium bromide is added in the order as 10, 20, 30, 40, 50 µl respectively. 100 µl of physically mutated culture was added to all the tubes except blank and kept for incubation for 48hrs. After 48 hrs they were stained and were examined under the microscope to see which culture among the different time intervals gave the similar structure as that of the pure culture. Other plates which did not resemble the pure culture were discarded. An increase for the inhibition zone was tested. Selection of the Solid Substrate: Sugarcane bagasse, oranges and pineapple procured from the local market were used as solid substrates and their effect on the production of streptomycin was determined. 25 grams of these dried powders of solid substrates were weighed individually and placed in culture bottles. 50 ml of minimal essential media was added to the solid substrates to maintain the moisture; bottles were wrapped in aluminum foils and autoclaved.

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Bottles were allowed to cool and 5 ml of the mutated culture was added to each bottle. The entire content of the bottles were mixed uniformly with the help of sterile spatulas for uniform distribution of the suspension. Bottles were incubated at 37°C for 72hrs. After every 24hrs the culture was checked for antibiotic production. The crowded plate technique was done and the MIC was checked for whether the antimicrobial activity is increased or not. Purification by filtration with charcoal and acidified methanol Solid state fermented cultures were mixed with phosphate buffer to make the pH neutral. Then all the cultures- pure, chemically mutated, physically mutated and solid state fermented sugarcane culture, orange culture, and pineapple culture were all centrifuged in a cooling centrifuge at 10,000 rpm for 10 minutes. The supernatant of all the 6 cultures were collected in 6 clean tissue culture bottles. After centrifugation 2% charcoal was added to each bottle according to the amount of supernatant collected. Pure culture was found to be 76ml, chemically mutated to be 72ml, physically mutated to be 74ml, sugarcane to be 35ml, orange to 10.5ml, pineapple to be 22ml. Hence 1.52g, 1.44g, 1.48g, 0.7g, 0.21g, 0.44g of charcoal was added to each bottle respectively. Next all the bottles were kept in shaker for 1hr for incubation, followed by filtration was done by passing the charcoal and supernatant mixture over filter paper kept in funnels. The antibiotics gets adhered to the filter paper and when acidified methanol is passed over the filter paper the antibiotics gets filtered out in new clean culture bottles. After filtration all the bottles were kept open for 48hrs for complete evaporation to takes place. Quantification using high pressure liquid chromatography After evaporation the antibiotic was in the form of powder at the bottom of the culture bottles. They were dissolved in 40ml phosphate buffer and antibiotics was extracted and kept in vials for HPLC analysis. Mobile phase used in HPLC is acetonitrile: water in the ratio of 7:3. Then pH was adjusted to 3.5 by adding orthophosphoric acid, using a pH meter. Filtration was done using a membrane filter unit having a 0.45 micron nylon membrane. The solvent was collected in a brown bottle and kept in ultra-sonicator for degassing for 15mins.

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Standard for streptomycin was prepared using the powder which is already available in the laboratory. 10mg of the standard is weighed and dissolved in 25ml of the mobile phase. 1st the HPLC is washed for half an hour using the distilled water which is membrane filtered at a rate of 1ml/min then the HPLC was run with the mobile phase for half an hour at the rate of 1ml/min, this is done to get a straight base line. If the base line is not straight then there are some impurities and the flow of the mobile phase has to be done for more time till we get a straight base line. The flow rate was set at 1ml/min and wavelength at 235nm and all the 6 samples were injected one by one by using a syringe and the retention time and area were recorded and saved. RESULT AND DISCUSSION a) Isolation and identification of Streptomyces griseus Isolation of Streptomycin species from the samples:

After the incubation the minimal

agar plates were examined for the presence of colonies of Streptomycin species. The numerous colonies were observed in the petriplates, which were further isolated and pure culture plates were then prepared. Pure culture of Streptomyces griseus: The pure culture of Streptomyces griseus was obtained both as slant as well as by streaking in petriplates. No contamination was there and only the pure culture of this strain grew. Growth was seen after 48hrs of incubation but for broth it took 168hrs to produce the antibiotics. Hence we can conclude that minimum 48hrs are required for this culture to grow. Gram staining Gram staining was done with the pure culture and viewed under microscope. It was seen that the bacteria were having hyphae which proved that it belonged to the order of actinomycetes. It was observed that culture from one of the slant had a lot of hyphae and they were gram positive hence we can conclude that the bacteria are pure culture of Streptomyces griseus, biochemical tests were conducted for further confirmation. Antimicrobial activity of pure culture

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The antimicrobial activities were conducted on 2 broths of the pure culture. Tests were conducted continuously for every 24hrs to check for antimicrobial activity. After 168hrs, the antimicrobial activity was seen for both the broth culture as zone of inhibition was formed. Biochemical tests Biochemical tests

Result

Catalase test

Positive

Starch hydrolysis test

Positive

Oxidase test

Positive

Casein hydrolysis test

Negative

MR/VP test

MR-positive, VP-negative

Citrate test

Positive

Urease test

Positive

Gelatine test

Negative

Indole test

Negative

b) Antimicrobial activity after mutation 1) Physical mutation Reports on strain improvements, however, have been very scanty. During the past three years slightly more than 3,700 isolates of Streptomyces griseus have been screened, following irradiation with either ultraviolet light or X-rays [15]. Physical mutation was done for the pure culture, which was swabbed on the casein agar media and kept under UV in LAF for 5,10,15,20 mins and then incubated. After that all the mutated cultures were gram stained and observed under microscope. 5min mutated culture had morphological features of the pure culture i.e., they had hyphae structure and was choosen for further production. Every 24hrs the antimicrobial activity of the 5mins mutated culture was checked and found out that after 96hrs the antimicrobial activity had increased by 2% for every concentration of the culture. Hence we can say physical mutation has increased the production as well as increased the antimicrobial activity.

2) Chemical mutation

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Chemical mutation was done on physically mutated culture which was exposed to UV for 5mins. This physically mutated culture was inoculated to starch casein broth and 0.2µl of EtBr was added to it. The culture was allowed to grow for 72hrs. Next the culture was centrifuged and the pellet was dissolved in 1ml saline which was added to the starch casein broth to allow it to grow. Next the antimicrobial activity was checked for every 24hrs. Zone of inhibition was found after 216 hrs i.e., on 9th day. And there was a decrement of the antimicrobial activity by almost 10%. c) Solid State fermentation(SSF) Solid state fermentation was used earlier in many countries in Asia like Japan and China in the production of many kinds of food such as soy souse

[16]

. These methods used in

production many antibacterial from S. halstedii, S .hydgrcophca and S. grise

[17]

.The

development of antibacterial. Production depended to use cheep raw material or neutral culture media, as solid state fermentation technical instead of liquid fermentation. This technique used for cephalosporin production from S. clavuligerus [18] and tetracycline from sweet potatoes [19]. The Solid state fermentation has several advantage including absence of free water

[18]

, reduced volume of production media[19] utilized for high products and the

relatively low costs of production [20,21,22 ]. Solid state fermentation was done using waste of sugarcane, orange, and pineapple. Since the fermentation was done laboratory scale only 25g of each was taken in tissue culture bottles instead of fermentors which can handle large volumes at the industrial scale. SSF constitutes an interesting alternative since the metabolites so produced are concentrated and purification procedures are less costly [25, 26, 27] In the earlier studies sugarcane bagasse has been reported to produce promising results [28, 29]. In the present study reveals orange peels, has proved to be the best solid substratum might be because of the high moisture content in the fibres and also presence of satisfactory amount of residual sugars. The antimicrobial activity was checked for all sugarcane, orange and pineapple for every 24hrs. Production started after 48hrs and antimicrobial activity was shown in the test. Solid state fermentation helped in reducing the time of production as compared to the normal

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production using media. Overall all the 3 media sugarcane, orange and pineapple were equally efficient in producing antibiotics. d) Purification and quantification by high pressure liquid chromatography(HPLC) 1) Purification by filtration with charcoal and acidified methanol Filtration was done by using charcoal and acidified methanol. It took about 5-6 hrs for filtration of 76, 72, 74, 35, 10.5, and 22ml of the 6 cultures (pure, chemically mutated, physically mutated, sugarcane, orange, pineapple respectively) using 2% charcoal. It took 72hrs for complete evaporation and thus only the antibiotic remained at the bottom of the bottle in powder form. Hence the antibiotics were purified. 2) Quantification using high pressure liquid chromatography High-performance liquid chromatography (HPLC) is a type of liquid chromatography used to separate and quantify compounds that have been dissolved in solution. HPLC is used to determine the amount of a specific compound in a solution. For example, HPLC can be used to determine the amount of morphine in a compounded solution. In HPLC and liquid chromatography, where the sample solution is in contact with a second solid or liquid phase, the different solutes in the sample solution will interact with the stationary phase as described. The differences in interaction with the column can help separate different sample components from each other

[23]

. One of the main areas in which HPLC is used is in

therapeutic drug monitoring. Monitoring is beneficial under a variety of circumstances-for example, when the therapeutic dose is close to the toxic dose, when signs of toxicity are difficult to detect clinically, when the rate of metabolism varies widely between patients, or when drug metabolism is impaired owing to organ dysfunction or altered by other drugs. Monitoring when rates of metabolism might vary is especially important if the drug metabolite is the therapeutically active form or the toxic form

[24]

. The antibiotics were

dissolved by adding 1000µl of Phosphate buffer to the powders in each of the 6 bottles and mixed well and collected in 2 vials for each culture. The mobile phase used here is acetonitrile: water at a ratio of 7:3. Wavelength is 235 nm and flow rate is 1ml/min. 20 µl of standard (0.4mg/ml) was injected and the retention time was found out to be 3.737min and the area was found out to be 70.115m Vs. The peak was very resolved and there were no impurities found. Similarly 20 µl of different cultures samples were then injected into the HPLC injector and there retention time and area were noted.

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"Fig. 1": The result of streptomycin standard sample is as follows-

"Fig. 2": The result of streptomycin produced by pure culture is as follows

"Fig. 3": The result of streptomycin produced by physically mutated culture is as follows

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"Fig. 4": The result of streptomycin produced by chemically mutated culture is as follows

Fig. 5": The result of streptomycin produced by solid state fermentation of sugarcane bagasse is as follow

"Fig. 6": The result of streptomycin produced by solid state fermentation of orange is as follow

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"Fig. 7": The result of streptomycin produced by solid state fermentation of pineapple is as follow From the above result HPLC analysis it was calculated that the antibiotics produced for 75ml of pure culture is 104mg. The antibiotics produced per 25mg of solid state fermented culture of sugarcane , oranges and pineapple is 241mg, 409mg, 179mg respectively. Hence we can conclude that the production has increased by 61% when mutated culture are grown over oranges then the production has also increased by 11% and 4% when mutated culture are grown over sugarcane and pineapple. Hence we can conclude that oranges is a better substrate as compared to sugarcane and pineapple to produce streptomycin by solid state fermentation. CONCLUSION From the above HPLC chromatograms the concentration of streptomycin produced from different mutated cultures and substrates are found to be as followsSamples

Retention time

Concentration of Streptomycin (mg/ml)

Standard

3.737

0.400

Pure culture (control)

3.467

0.118

Physically mutated culture

3.490

0.381

Chemically mutated culture

3.410

0.212

Mutated culture grown on sugarcane

3.607

0.132

Mutated culture grown on orange

3.547

0.310

Mutated culture grown on pineapple

3.523

0.124

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From the above data an increment in the production of streptomycin with respect to the control was also found out according to the formulaSamples

Increment of production* (%)

Physically mutated culture

69.04

Chemically mutated culture

44.55

Mutated culture grown on sugarcane bagasse

11.64

Mutated culture grown on orange peel

61.97

Mutated culture grown on pineapple

4.88

Increment of production= [(Area of the sample loaded - area of the control loaded) / area of the sample] * 100 The above data reveals that the concentration of streptomycin produced can be directly concluded from the area of the chromatograms of different samples, where the physically mutated sample has the highest concentration of 0.381mg/ml which is very much close to the standard streptomycin concentration of 0.4mg/ml. Production of streptomycin has increased the greatest with physically mutated culture with 69.04% which was more than chemically mutated culture having a production of only 44.55% more than that of control (pure culture). Among the substrates used for production of antibiotic, orange peels had much better production with 61.97% but it was less than that of physically mutated culture production. The first antibiotic ever reported from a bacterium comes from strains of S. griseus. The interest towards these strains was sought because of its ability to produce streptomycin, a compound which demonstrated significant bactericidal activity against organisms such as Yersinia pestis (the causative agent of plague) and Mycobacterium tuberculosis (the causative agent of tuberculosis). With the increase in its application spectrum, the demand for the antibiotic with specificity is increased. Commercially most of the production of streptomycin is carried out in submerged fermentation, but solid-state fermentation is being looked at as a potential tool for its production, especially applying agro-industrial residues as substrate. The strain can be easily isolated from soil and production of antibiotic streptomycin from the pure as well as mutated cultures was efficiently done in laboratory conditions by www.wjpps.com

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SSF. These mutated cultures were also grown on different substrates of pineapple, orange peels and sugarcane bagasse, all of which gave positive result. But among all, physically mutated culture grown in LB broth as well as when grown on orange peel as substrate gave the most effective result with 0.381mg/ml and 0.310mg/ml production. The production of streptomycin by SSF has several advantages: stability in a range of temperature and pH being some of them. These, along with the economics of the production make SSF the ideal choice for the production of streptomycin. Further studies aiming in this direction should be undertaken. ACKNOWLEDGEMENT We are sincerely grateful to The Oxford College of Science and Azyme Biosciences Pvt Ltd, Bangalore, Karnataka, India, for allowing us to use all facilities for our work, and their encouragement and support. We are also thankful to Deepthi, trainer of the Azyme Biosciences Pvt Ltd, who constantly supported us during our research work. REFERENCES 1.Demain, A. L. & Fang, A. (The natural functions of secondary metabolites). in Advances in Biochemical Engineering/Biotechnology: History of Modern Biotechnology I (ed. Fietcher, A.) , Springer, Berlin, 2000; 69, 2–39. 2. Berdy, J. (Bioactive microbial metabolites. A personal view). J. Antibiot, 2005; 58, 1–26. 3. Lederberg, J. (Infectious history). Science, 2000; 288, 287–293. 4. Barber, M., Giesecke, U., Reichert, A. & Minas, W. (Industrial enzymatic production of Cephalosporin- based b-lactams). Adv. Biochem. Eng. /Biotechnol, 2004; 88, 179–215. 5. Demain, A. L. & Sanchez, S. (Microbial drug discovery: 80 years of progress). J. Antibiot, 2009; 62, 5–16. 6. Hinshaw and Feldman (1945) Hinshaw, Feldman and Pfuetze(1946), Keefer et al(1946) . (lois dickinson-effect of streptomycin on experimental tuberculosis in guinea-pigs). Brit. J. Pharsmaol, 1947; 2, 23. 7. Maha A. Hassan, Moustafa Y. El-Naggar and Wafa Y. Said, Physiological factors affecting the production of an antimicrobial substance by Streptomyces violatus in batch cultures Egyptian Journal of Biology, 2001, Vol. 3, pp 1-10. 8. Vaurnakis, J.N. and Blander, R.P. (Genetic Manipulation on of Antibiotic Producing Microorganisms). Science, 1983; 219, 703-709.

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9. Mohammed I. Nader Production of antibacterial agent from Streptomyces griseus by using Semi Solid Fermentation Fac Med Baghdad 2009; Vol.51, No1 10. Chater, K.F. and Merrick, M.J. (Streptomyces in: Development Biology of Prokaryotes) (ed. J. H. Parish). Blackwell Scientific Publication oxford, 1979; 93-114. 11. Yang, S and Yi ang, J. (Protease and amylase production of Streptomyces rimosus in submerged and Solid State cultivation). bot.bull.acad.sin, 1999, 40; 259-265 12. Kuzhadhaivel, S; Vetrivel and Kuppamutha. (Isolation of a chitinase overproducing mutant of Strepomyces peucetius defective in daunorubicin biosynthesis). J. Microbio., 2000, 46(10); 959-960 13. Nicole K; Susan, E; Dylan, C and Brenda, k. (The positive activator of cephamycin C and clavulanic acid production in Streptomyces clavuliqerus is mistranslated in a bldA mutant). Microbiology, 2002, 148; 643-656 14. Mohammed I. Nader PhD. (Production of antibacterial agent from Streptomyces griseus by using Semi Solid Fermentation). 15. George M. Savage. (Improvement in streptomycin-producing strains of Streptomyces griseus by ultraviolet and x-ray energy). 16. Cannel, E. and Moo-Young, M. (Solid state Fermentation systems). Process Biochem, 1980, 15(5); 2-7. 17. Manufacturing of Drug and Medical appliances (in Arabic). 18. Demain. A.L. and Jermini, M.F.G. (Solid state fermentation for cephalosporin production by Streptomyces clavuligerus and Cephalosporium acremoninm). Experiential, 1889, 45; 1061-1065. 19. Berovic, M. and Loger -derenin, M. (Solid State Fermentation of pectinolitic Enzyames by Aspergillus nigar). J. Chem .Tech Biotchnol, 1993, 58; 209-211. 20. Yang, S and Yi ang, J. (Protease and amylase production of Streptomyces rimosus in submerged and Solid State cultivation). Bot. bull. Acad .sin, 1999, 40; 259-265. Bhattacharyya,B ,k.Pal,S,Cand Sen,S,K. (Antibiotic production by Steeptomyces hygroscopcus D1.5: cultural effect REV). Microbiol, 1998, 29. Auli,P ;raimo,M and Timo S. (Inhibition of human NK cell function by Valinomycin , a toxin from Streptomyces qriseus in indoor air). Infection and Immunity, 2000, 68(1); 165-169. 21. Tom Kupiec, PhD, Analytical Research Laboratories Oklahoma City, Oklahoma. (Quality-Control Analytical Methods: High-Performance Liquid Chromatography). International Journal of Pharmaceutical Compounding Vol. 12 No. 1 January/February 2008. www.wjpps.com

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22. Ian M Bird. (High performance liquid chromatography: principles and clinical applications). BMJ, 1989, 299, 783-787. 23. Kunamneni A, Singh S. Response surface optimization of enzyme atichydrolysis of maize starch for higher glucose production. Biochem Eng J 2005; 27: 179-190. 24. Arima K. Microbial Enzyme Production. In: Global Impacts of Applied Microbiology, Starr, M.P. (Ed.). John Wiley and Sons, New York; 1964; J. Appl.Microbiol. Biotechnol. 12: 113. 25. Miller GL. Use of dinitro salicylic acid reagent for determination of reducing sugar. Anal Chem 1959; 31: 426-429. 26. Lowry OH, Rosenbrough PJ, Farr AL, Randell RJ. Protein measurement with Folinphenol. J Biol Chem 1951; 193: 265-275. 27. Pandey A. Recent process developments in solid-state fermentation. Proc Biochem 1992; 27: 109-117.

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