Pak. J. Biotechnol. Vol.

14 (2) 251- 256 (20177) ISSN Print: 1812-1837

www.pjbt.org ISSN Online: 2312-7791

OPTIMIZATION OF CULTURE CONDITIONS TO PRODUCE SECONDARY METABOLITES

BY PLEUROTUS OSTREATUS
Maryam Rana1,2 and M. Umar Dahot1
1
Enzyme and Fermentation Research Laboratory, Institute of Biotechnology and Genetics Engineering,
University of Sindh, Jamshoro, Pakistan. 2Key Laboratory of Molecular Epigenetics of the Ministry of
Education (MOE), School of Life Science, Northeast Normal University, Changchun, China.
E.mail: maryamrana@yahoo.com, Ruin870@nenu.edu.cn
Article received 15.12.2016, Revised 8.6.2017, Accepted 15.6.2017
ABSTRACT
The specie of mushroom Pleurotus ostreatus grown on the Potato Dextrose Agar (PDA) medium for the
activation and then inoculated in the fermentation medium to obtain maximum production of metabolite. Maximum
production of metabolites produced by Pleurotus ostreatus at different incubation time periods. The effect of different
carbon sources glucose, galactose, fructose, sucrose and starch was checked on the growth and production of
metabolite by Pleurotus ostreatus and the higher production was achieved with glucose. The effect of different
nitrogen sources like potassium nitrate, sodium nitrate, ammonium sulphate, casein and urea were checked on the
growth and production of metabolites by Pleurotus ostreatus. Among the different nitrogen sources maximum
metabolite were obtained with casein. The effect of pH on the growth and production of metabolites by Pleurotus
ostreatus was studied in the range of 4.5-8.5 and maximum production was noted with 4.5 pH. The effect of
temperature was checked on the growth and production of metabolite by Pleurotus ostreatus in range of 25 ̊C-35 ̊C
and higher production of metabolites were achieved at 30 ̊C.
Key words secondary metabolites and Pleurotus ostreatus
INTRODUCTION
All organism needs to convert and transform a flavanols possess antioxidant, anticancer, anti-
large variety of organic compounds to enable their mutagenic, antimicrobial and antiradical properties
selves to reproduce and grow. They need to provide (Sahu and Green, 1997, Amarowicz, et al., 2004).
energy to themselves in the form of ATP and to Growth, reproduction and protection to the plants
construct their own, tissues also have a need of against pathogens and predators is facilitated by
supply of building blocks. For this purpose, an Phenolics compounds (Bravo, 1998). Due to the
integrated network of enzyme mediated and chemi- presence of antioxidant properties of flavonoids
cally regulated reactions are used which is referred and phenolics, lipid peroxidation and shelf life of
as intermediary metabolism. The pathways invol- the product is increased by adding them in food,
ved are called metabolic pathways primary meta- which consists of lipids and its associated foods
bolic pathways which can synthesize, degrade and (Yasoubi, 2007). Several physio-chemical factors
interconvert compounds are commonly found in all such as nutrient supply, oxygenation, temperature
organisms whereas another area of metabolism and pH have profound influence on the production
whose compound have limited distribution in nat- process of secondary metabolites. To enhance the
ure is called secondary metabolites (Berdy, 2005, production of metabolites these factors have tradi-
Martin, 1978). Secondary metabolites specifically tionally optimized and controlled in industrial
special metabolites, which are not consumed by the fermentation. Additionally, different pharmaceu-
producing micro-organisms but they are harmful to tical industries used traditional mutagenesis pro-
other microorganisms. They are not necessarily grams to improve strain and yield production
required by the cell for its growth, however second- (Carlos, et al., 2008). Some of the secondary meta-
dary metabolites are considered as important bolites such as digitalis, morphine and quinine are
survival factor in nature (Juan, 1980). Secondary considered as plant metabolites while penicillin,
metabolites are also called as idiolites (Walkers, cephalosporin, ergot rate and the statins are known
1974). Because they are synthesized during the as fungal secondary metabolites. Although few
idiophase (Berdy, 1974). It has been seen that common biosynthetic pathways are followed by the
secondary metabolism is regulated by overall chemically diverse secondary metabolites for their
regulatory controls which operate as functions of production in collabo-ration with morphological
growth rate and Specific regulatory effects on development. Recently using modern techniques
individual pathways (Martin, 1978). such as molecular biology, bioinformatics and
Growth phase of secondary metabolite is termed comparative genomics, it has been revealed that
as “tropophase” whereas, production phase is ter- genes which are involve in production of fungal
med as “idiophase” (Bu'Lock, 1975). Phenolics and
 252 Rana and Dahot Pak. J. Biotechnol.

metabolites are clustered and found near telomeres mixed with 2.0ml of dinitrosalicylic acid in test
(Nancy, et al., 2005). tube. The mixture was heated in boiling water bath
In this paper, we have tried to investigate the for 5 minutes. The tubes were cooled in tap water
optimum culture conditions to produce metabolites and color intensity was read against blank at
from Pleurotus ostreatus. 540nmon Spectro-UV-Vis Double PC spectropho-
MATERIALS AND METHODS tometer, LaboMed, USA 11DV-60Hz or 220V-
Culture collection: P. ostreatus was gifted by 50Hz Serion Number 001151. Instead of sample
Department of Biotechnology, Sindh Agriculture distilled water was used for the preparation of
University Tando Jam. blank. The concentration of reducing sugar was
Preparation of stock culture: Stock culture was calculated from the standard graph that was
prepared by activating the culture on PDA media prepared in same manner as test sample by using
(potato 200g dextrose 20g agar 1.5g in 1L). The pH different concentration of the glucose.
of media at 6.5 was adjusted by using 1N NaOH Reducing Power: The reducing power of ethanol
and 1N HCl. The sterilized media was inoculated extracts of plants was checked as by the scheme of
with P. ostreatus culture. After inoculation, the Oyaizu [13]. Took 0.5 ml sample solution, 102.5
petri plates were covered and incubated at 25̊C for ml phosphate buffer (6.6pH) and 2.5 ml 1% potass-
one week in growth room. ium ferricyanide. The reaction mixture was kept in
Preparation of spore suspension solution: Spore water bath at 50°C for 20 minutes. Then 2.5ml of
suspension solution was prepared by using 50 ml 10% T.C.A. was mixed and centrifuged at 1000
of sterilized distilled water. Sterilized distilled rpm for 10 minutes. The supernatant (2.5 ml)
water was added into a culture test tube and gently was added in 2.5 ml distilled water and 0.5 ml of
rub the surface of test tube in such a way that it gets 0.1% of ferric chloride. Absorbance was read at
mixed with sterilized distilled water and prepare 700 nm on Spectro-UV-Vis Double PC spectro-
spore suspension solution. photometer, LaboMed, USA 11DV-60Hz or 220V-
Preparation of culture media: Starter culture was 50Hz Serion Number 001151. Absorbance of the
prepared for fermentation broth media containing sample is directly proportional to the reducing
25g glucose, 1.5g corn steep liquor, 5g peptone, power of sample. Higher will be the reducing
0.5g NH4NO3 in 500ml distilled water and the pH power with the increase of absorbance.
of media was adjusted at 6.5 by using 1N NaOH Total Antioxidants Activity: Total antioxi-
and 1N HCl. The media was sterilized in autoclave dant’s capacity was measured by modifying phos-
at 121̊C for 20 minutes at 15 psi. 50 ml sterilized phomolybdate method as reported by Prieto et al.,
media was taken in conical flasks. The sterilized (1999) using α- tocopherol as a standard. An
media was inoculated with 5 ml of spore suspen- aliquot of 0.4 ml of plant extracts was mixed
sion solution in each flask. After inoculation flasks with 4 ml of reagent (0.6M sulphuric acid, 28 mM
were covered and incubated at 25̊C for 12 days in sodium phosphate and 4mM of ammonium moly-
growth room. bdate). All tubes were capped and kept in boiling
Analysis of secondary metabolite: The fermen- water bath at 95˚C for 90 minutes. Then the sam-
tation media was filtered through Wattman filter ples were cooled to room temperature and the
paper and filtrate was used for analysis of flava- absorbance was read at 695 nm on Spectro-UV-
nols, reducing sugar, tannin contents, reducing Vis Double PC spectrophotometer, LaboMed,
power, antioxidant activity, and ascorbic acid USA 11DV-60Hz or 220V-50Hz Serion Number
contents under optimized culture conditions. 001151 against a blank solution prepared in the
Determination of total flavanol: Total flavanol same conditions by replacing the sample with 0.1
concentration was determined by spectrophoto- ml of methanol. The total antioxidants activity was
metric method as reported by Kumaran and expressed as mg/ml equivalents of α- tocopherol.
Karunakan (2007) with slight modification and The standard error was calculated by using Micro-
results were calculated from standard graph using soft excel.
quercetin. Absorbance = 0.4544 (alpha tocopherol) + 0.0131
Determination of tannin content: The tannin Determination of Ascorbic acid: Ascorbic acid
content from the culture broth was determined by was determined by spectrophotometric method as
Tamilselvi et al., (2012) using gallic acid as reported by Bajaj and Kaur (1981). The concen-
standard. tration of ascorbic acid was calculated from the
Reducing Sugar Determination: Reducing sugar standard graph that was prepared in same manner
content was determined by the method reported by as test sample by using different concentration of
Miller (1959). According to this method, 2.0ml ascorbic acid.
(0.2ml sample + 1.8d.H2O) of test solution was
Vol. 14 (2) 2017 Optimization of culture conditions ..… 253

Optimization of culture conditions: Optimiza- tation media having best time of incubation, best
tion of culture conditions includes time of incubi- carbon and nitrogen source for effective production
tion, carbon sources, nitrogen sources, pH and of secondary metabolite.
temperatures for effective production of secondary Effect of temperature: The effect of different
metabolite. temperatures (̊C) was analyzed such as 25, 30, and
Effect of time of incubation: The effect of time of 35in fermentation media which containing best
incubation was analyzed to find best time of time of incubation, best carbon and nitrogen source
incubation for production of secondary metabolite along with best pH to obtain best production of
after interval of 3days, 6 days and 12 days. secondary metabolite.
Effect of carbon source: The carbon source was RESULTS AND DISCUSSION
optimized separately by using same concentrations The effect of incubation time: The effect of
of galactose, starch, sucrose and fructose along incubation time (6 days, 9 days, 12 days) on growth
with glucose to find effective production of secon- and production of metabolite is shown in figure 1.
dary metabolite for optimized time of incubation. It shows that higher amount of tannin production
Effect of nitrogen source: The effect of different obtained at 12 days whereas flavanol and antioxi-
nitrogen sources such as ammonium sulphate, dant production was maximum after 9 days. The
sodium nitrate, potassium nitrate, casein and urea present results are consistent the reported results of
instead of sodium sulphate were incorporated in Gökhan, et al., (2015) using edible mushrooms
fermentation media separately which contain best including P. ostreatus consist of higher contents of
time of incubation and best carbon source to find flavanol and phenols after incubation of 8 days to
maximum production of secondary metabolite. 60 days.
Effect of pH: The effect of different pH was
analyzed between the range of 4.5 to 8.5 in fermen-

1.6

1.4
METABOLITES MG/ML

1.2

0.8

0.6

0.4

0.2

0
Total flavonol Total tannin Total reducing sugar Antioxidant Reducing power Ascorbic acid

6 days 9 days 12 days

Figure 1: Metabolites production by P. ostreatus grown at different time using glucose as carbon source and
incubated at 25̊C with pH 6.5.

The effect of carbon sources: The effect of carbon different carbon sources were used by Chang
sources (glucose, galactose, fructose, sucrose and (1989), Wang (1993) and Yang (1996) for maxi-
starch) on the growth and production of metabo- mum production of mycelium and glucose referred
lites were checked as shown in fig 2, and higher as suitable source for growth and mycelium growth
amount of tannin flavanol 0.0501 mg/ml were of various mushrooms.
achieved with glucose when P. ostreatus grown in
a fermentation media at 25 ̊ C, 6.5 pH. Similarly,
 254 Rana and Dahot Pak. J. Biotechnol.

1.2

METABOLITES MG/ML
1
0.8
0.6
0.4
0.2
0
Total flavonol Total tannin Reducing sugar Antioxidant Reducing power Ascorbic acid

Glucose Galactose Fructose Starch Sucrose

Figure 2: Metabolites production by P. ostreatus grown on different carbon source at 25 ̊ C with pH 6.5

The effect of nitrogen sources: The effect of source at 25 ̊ C and at 6.5 pH. The result is in
nitrogen sources (potassium nitrate. ammonium contrast with the experiment performed by Hoa and
sulphate, sodium nitrate, casein and urea) was Wang (2015) using different nitrogen sources and
checked to investigate the growth and production among them ammonium chloride and ammonium
of flavanol as shown in figure 3, among all these sulphate appeared as best nitrogen source for
nitrogen sources casein gave higher amount of growth and mycelium production by P. ostreatus
tannin 0.9 mg/ml when P. ostreatus grown in and P. cystidiosus.
fermentation media containing glucose as carbon

1
0.9
0.8
METABOLITES MG/ML

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Total flavonol Total tannin Reducing sugar Antioxidant Reducing power Ascorbic acid
casien urea NH₄S0₄ NaNO₃ KNO₃

Figure 3: Metabolites production by P. ostreatus grown with different nitrogen sources with glucose as carbon
source and 6.5 pH

The effect of pH: The effect of pH from 4.5 to 8.5 mg/ml in a fermentation medium containing
was checked to determine the growth and glucose as carbon source and casein as nitrogen
production of metabolites as shown in figure 4, source at 25 ̊C and most of the organisms produce
among different ranges 5.5 pH appeared as best for various compounds at different pH ranges and their
growth and maximum production of tannin 0.225 production rate is also affected by pH.
Vol. 14 (2) 2017 Optimization of culture conditions ..… 255

2.5

METABOLITES MG/ML 2

1.5

0.5

0
Total flavonol Total tannin Total reducing sugar Antioxidant Reducing power Ascorbic acid

4.5 5.5 6.5 7.5 8.5

Figure 4: Metabolites production by P. ostreatus grown with different ranges of pH.

The effect of temperature: The effect of tempera- at 30 ̊C were achieved when P. ostreatus grown in
ture like 25 ̊C, 30 ̊C and 35 ̊C on the growth and fermentation media with glucose as carbon source
maximum production of metabolites was checked and casein as nitrogen source at 8.5 pH. These
as shown in figure 5 and the maximum production results are nearly similar to the results reported by
of tannin 1.1mg/ml and reducing power 0.5mg/ml Hoa and Wang (2015) where highest mycelium
achieved at 25 ̊C, whereas flavanol 0.584 mg/ml, growth of P. ostreatus was obtained at 28 ̊C.
antioxidant 0.42mg/ml and ascorbic acid 0.6mg/ml

1.2
METABOLITES MG/ML

1
0.8
0.6
0.4
0.2
0
Total flavonol Total tannin Total Antioxidant Reducing Ascorbic acid
reducing power
sugar

25 ̊C 30 ̊C 35 C
̊

Figure 5: Effect of temperature on the growth of P. ostreatus and production of metabolites

CONCLUSION Berdy, J., Bioactive microbial metabolites. J.
On the light of the results obtained from this Antibiot. 58(1): 1–26 (2005)
study, it could be concluded that Pleurotus Berdy, J., Recent developments of antibiotic
ostreatus is capable to produce different metabolite research and classification of antibiotics accor-
but higher amount was found of tannin in ding to chemical structure. Adv. Appl. Micro-
comparison to other constituents. biol. 18: 309-406 (1974)
Bravo, L., Polyphenols: chemistry, dietary sources,
REFERENCES metabolism, and nutritional significance.
Amarowicz, R., R.B. Pegg, P.R. Moghaddam, B. Nutrition Rev. 56: 317–333 (1998)
Barl and J.A. Weil, Free-radical scavenging Bu'Lock, J.D., D. Hamilton, M.A. Hulme, A.J.
capacity and antioxidant activity of selected Powell, H.M. Smalley, D. Shepherd, and G.N.
plant species from the Canadian prairies. Food Smith, Metabolic development and secondary
Chemistry 84: 551–562 (2004) biosynthesis in Penicillium urticae. Can. J.
Bajaj, K.L. and G. Kaur, Spectrophotometric Microbiol. 11: 765-778 (1975)
Determination of L. Ascorbic Acid in Vege- Carlos, Lombo, Méndez and J.A. Salas, Improving
tables and Fruits. Analyst 106: 117-120 (1981) production of bioactive secondary metabolites
 256 Rana and Dahot Pak. J. Biotechnol.

in actinomycetes by metabolic engineering. Nancy, P., Turner and W. Bennett, Fungal secon-
Metab. Eng. 10 (5): 281-292 (2008) dary metabolism from biochemistry to geno-
Chang, S.T., Edible Mushroom and their Cultiva- mics. Nature Rev. Microbiol. 3: 937–947 (2005)
tion. Boca Raton, Florida: CRC Press (1989) Oyaizu, M., Studies on product of browning reac-
Gökhan. Sadi, Buğrahan. Emsen, Abdullah. Kaya, tion prepared from glucose amine. Japanese
Aytaç. Kocabaş, Seval. Çınar and D.I. Kartal, Journal of Nutrition 44: 307-315 (1996).
Cyto-toxicity of some edible mushrooms ext- Prieto, P.M., Pineda and M. Aguilar, Spectropho-
racts over liver hepatocellular carcinoma cells tometric quantitation of antioxidant capacity
in conjunction with their antioxidant and anti- through the formation of a phosphomolybde-
bacterial properties. Pharmacogn. Mag. (Suppl num Complex Specific application to the
1): S6– S18 (2015) determination of vitamin E. Analyst. Biochem.
Hoa and Wang, The effects of temperature and 269: 337-341 (1999)
nutritional Conditions on mycelium growth of Sahu, SC. and S. Green. Their dual role in carcno-
two oyster mushrooms (Pleurotus ostreatus genesis. Food Aos. 11: 329–330 (1997)
and Pleurotus cystidiosus). Mycobiology Tamilslvi, N.P.R., P. Krishnamoorthy, E. Damo-
43(1): 14-20 (2015) tharan, Arumugam and E. Sagadevan, Analysis
Juan, F., Martin and A.L. Demain, Control of Anti- of total phenols, total tannins and screening of
biotic Biosynthesis. Microbiol. Rev. 44(2): phytocomponents in Indigofera aspalathoides
230-251 (1980) (Shivanar Vembu) Vahl EX DC. J. Chem.
Kumaran, A. and R.J. Karunakaran, In vitro Pharm. Res. 4: 3259-3262 (2012)
antioxidant activities of methanol extracts of Walkers, J.B., Biosynthesis of the monoguani-
Phyllantus species from India. Lebensm Wiss dinated inositol moiety of blue neomycin, a
Technol. 40: 344–352 (2007) possible evolutionary precursor of strepto-
Martin, J. F., Manipulation of gene expression in mycin. J. Biol. Chem. 249: 2397-2404 (1974)
the development of antibiotic production. IN: Wang, N.L., Edible Fungi Cyclopedia of China.
Antibiotics and other secondary metabolites. Beijing Chinese Agricul. Publ. House (1993)
Biosynthesis and production. Editors R. Yang, X.M., Cultivation of Edible Mushroom in
Hutter, T. Leisinger, J. Nuesch, and W. Wehrli, China. Beijing Agricul. Printing House (1996)
Academic Press Inc., New York, Pp. 19-37 Yasoubi, P., M. Barzegar, M.A.Sahari and M.H.
(1978) Azizi, Total Phenolic Contents and Antioxi-
Miller, G.L., (1959) use of dinitrosalicylic acid dant Activity of Pomegranate (Punica grana-
reagent for determination of reducing sugar. tum L.) Peel Extracts. Journal of Agric. Sci.
Anal. Chem. 31(3): 426–428 (1959) Technol. 9: 35-42 (2007)