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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
Azyme Biosciences Pvt Ltd, Bangalore Karnataka, India

ABSTRACT
Article Received on
11 May 2014, The actinomycete strain Streptomyces griseus isolated from soil is used
Revised on 09 June
2014, for the production of streptomycin, a potent antibiotic drug which is of
Accepted on 15 July 2014
great commercial importance. The strain was identified by
morphological features and several biochemical tests. Further they
*Correspondence for Author were grown as a pure culture in starch casein agar media. Mutation
Mamatha J
was done both by physical and chemical methods and it was found that
P.G. Department of
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
[1]
diverse survival functions in nature . In addition, antibiotics are very important for the
[2].
health, nutrition and economics of our society Owing to the use of antibiotics and other
secondary metabolites, the average life expectancy in the United States increased from 47
[3]
years in 19001974 (males) to 80 years (females) in the year 2000 . 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
[6]
for tuberculosis . 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
[8]
than half of all antibiotic . 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
[9, 10, and 11]
administered by regular intramuscular injections. . 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.

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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 72C 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 l 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 37C 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 37C 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 4C 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 l 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 l 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 l respectively.
100 l 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 37C 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 0.2l 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
[16]
the production of many kinds of food such as soy souse . These methods used in
[17]
production many antibacterial from S. halstedii, S .hydgrcophca and S. grise .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
[18]
free water , 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
[23]
components from each other . 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
[24]
metabolite is the therapeutically active form or the toxic form . The antibiotics were
dissolved by adding 1000l 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 l 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 l 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 follows-

Concentration of
Retention
Samples Streptomycin
time
(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 formula-

Samples 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

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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.

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