Advances in Bioscience and Biotechnology, 2012, 3, 643-647 ABB
http://dx.doi.org/10.4236/abb.2012.35083 Published Online September 2012 (http://www.SciRP.org/journal/abb/)
Solid state fermentation of pomegranate seed for lovastatin
production: A bioprocessing approach
Azza S. Naik, Smita S. Lele*
Food Engineering and Technology Department, Institute of Chemical Technology, Mumbai, India
Received 20 June 2012; revised 28 July 2012; accepted 16 August 2012
ABSTRACT
This study reports lovastatin production by solid state
fermentation using pomegranate seeds as a substrate.
Six different fungal strains and several agro-industrial
wastes were selected and screened. Various physico-
chemical parameters were optimized to improve lovas-
tatin productivity. Moreover, chemical mutation was
system-atically employed to enhance lovastatin yield
on selected strains. Productivity of 3 ± 0.06 mg lovas-
tatin/gm dfm was obtained prior to optimization. One
factor a time followed by Response Surface Method-
ology (RSM) gave 4.2 ± 0.10 mg lovastatin/gm dfm
yield in an optimized setup with pomegranate seed
powder (5 gms), KH2PO4 (0.1% w/v), glucose (5%
w/v), moisture (60% w/w), pH 5 in a 15 days fermen-
tation cycle. The production was further increased to
6.5 ± 0.08 mg lovastatin/gm dfm through chemical
mutation of the strain. This process is simple and re-
producible for the production of lovastatin using
pomegranate seed as an agro-industrial waste.
Keywords: Lovastatin, Pomegranate Seeds; Solid
Substrate Fermentation; Aspergillus terreus
1. INTRODUCTION
Pomegranate (Punica granatum) is a tropical fruit com-
monly grown in Indian subcontinent. Recently, the fruit
gained public interest due to its remarkable antioxidant
properties [1]. A number of pomegranate processing in-
dustries have been set up in India for last few years. This
has led to a boost in the number of processed products
like jams, jellies, juices, carbonated beverages, syrups,
liqueurs and as osmotically dehydrated bits in fruit bars
[2]. The edible part of pomegranate represents 52% of
total fruit weight, comprising of 78% juice and 22% seeds
[3]. Processing of this fruit is such that the peels and
seeds get automaticall y separated resulting in tons of eas-
ily utilisable segregated fruit waste. In spite of high pro-
duction of fruits in general and pomegranate in particular,
India like other developing countries lacks adequate post
harvest facilities. Inadvertently, an agro-industrial project
faces economic inviability. Fruit waste utilisation by fer-
mentation for value added products instead of conven-
tional applications such as cattle fodder or compost gen-
eration can positively change the current scenario.
Lovastatin, a fungal secondary metabolite acts as a
competitive inhibitor of HMG-CoA reductase enzyme
that catalyzes the rate limiting step of cholesterol bio-
synthesis [4]. This is due to the structural similarity of its
acid form with HMG-CoA (the natural substrate of re-
ductase), thus finding an application as a potent hyper-
cholesterolemia drug [5]. Although many fungal genera
like Penicillium, Monascus, Trichoderma, Pleurotus, Do-
ratomyces have been reported to produce lovastatin; As-
pergillus is the most commonly used one for its robust
nature [6]. Several papers have also reported the effect of
major nutrients like carbon and nitrogen on productivity
of this biomolecule from the aforementioned fungus.
However, most of these studies have been performed
under submerged conditions utilising complex media
[7-9]. Solid Substrate fermentation on the contrary in-
volves solid support with little or no water. It is termed
as the “low-technology” system; ideal for production of
high cost-low bulk pharmaceuticals [10]. Here we report
solid substrate fermentation of pomegranate seeds using
Aspergillus terreus IFO 7078 strain, as an alternative
process strategy to increase the productivity, simplify the
downstream processing and utilize the waste as well.
2. MATERIALS AND METHODS
2.1. Chemicals
All media components were purchased from Himedia,
Mumbai, India unless and otherwise mentioned. High
performance liquid chromatography (HPLC) grade ace-
tonitrile was obtained from SD-Fine chemicals along
with other chemicals like Ammonium carbonate, Ammo-
nium sulphate, Ammonium chloride, Ammonium nitrate,
Magnesium sulphate (MgSO4), Sodium nitrate (NaNO3),
*Corresponding a uthor.

A. S. Naik, S. S. Lele / Advances in Bioscience and Biotechnology 3 (2012) 643-647 644
and Potassium dihydrogen phosphate (KH2PO4). Stan-
dard sample of lovastatin was gifted by Biocon Ltd.
Pomegranate seeds & Mango peel powder were obtained
from Jain Irrigation Ltd., India while other substrates
were obtained from a local market.
2.2. Screening Studies
The Six fungal strains (IFO7078, IFO 4520, Isolate 3A,
NRRL 680, NRRL 1841 & NRRL 1596) were screened
for lovastatin production under submerged condition
using media previously reported in literature (Table 1).
IFO7078 strain of A. terreus was selected for further
studies. Once organism was selected, screening of vari-
ous substrates like Mango peel powder, Ashgourd peel
powder, Pomegranate peel powder and Passion fruit peel
powder was done. Thereafter, the best results were com-
pared with the substrate of interest i.e. Pomegranate seed
powder. In addition, proximate analysis of the substrate
was done. Comparative study on the effect of defatted
and non-defatted substrate was also carried out.
2.3. Culture Maintenance and Inoculum
Development
IFO7078 strain of A. terreus was maintained on Potato
dextrose agar slants. It was periodically subcultured ever y
15 days. Inoculum preparation was done by suspending
spores in a sterile solution of 0.1% (v/v) tween 80. This
solution was centrifuged and spores were re-suspended
in saline. O.D of the spores was adjusted to 3 × 108
spores per ml. This spore suspension was then used to
inoculate pre-autoclaved medium.
Table 1. Screening of strains for lovastatin production under
submerged conditions.
Strain Organism Media Lovastatin
yield mg/L
IFO 7078 A. terreus SF1 Gyorgy Szakacs
et al. (1998) 200
IFO 4520 M. pilosus GGP Tsuyoshi
Miyake et al. (2006) 150
Isolate 3A A. terreus SF1 Gyorgy
Szakacs et al. (1998) -
NRRL 680 A. terrues SF1 Gyorgy
Szakacs et al. (1998) -
NRRL 1841 P. citrinum Malt, glucose
and peptone. -
NRRL 1596 M. purpureus Dextrose containing media
Sadik Sayyad et al. (2007) 90
Based on the reported literature appropriate media was used for respective
strain. Uniform condition maintained during screening of different strains.
Temperature was 28 ˚C and the experiment was carried out under shake flask
condition.
2.4. Substrate Preparation
Pomegranate seeds were oven dried at 50˚C for 24 h or
till a constant weight was achieved. Seeds were then
ground to a uniform particle size and passed through a
sieve to separate coarse particles.
2.5. Substrate Defatting
Defatting of pomegranate seeds was achieved by Soxlet
apparatus using petroleum ether as the preferred solvent
for fat extraction.
2.6. Fermentation Optimisation
5 gms of dried pomegranate seed powder having particle
size in the range of 250 - 800 microns was autoclaved at
121˚C for 20 min in a 250 ml flask. Separately auto-
claved solutions o f carbon , nitrogen and salt supplements
were used to wet the substrate, adjusting the moisture
content at appropriate levels. Several physic-chemical
parameters were optimised using one factor at a time
method (Figure 1).
Moisture was adjusted to levels 50%, 60%, 70 %, 80%
and 90% (w/w). Optimisation studies for temperature as
a parameter were carried out at 22˚C, 25˚C, 28˚C, and
30˚C. Production profile was made on the basis of lovas-
tatin content every 3 days for 18 days. Carbon sources
such as Glucose, Fructose, Sucrose, Lactose, Maltose,
Galactose, and Glycerol were screened. Various nitrogen
sources were screened (at 1% w/v) as supplements like
(organic sources) Tryptone, Urea, Malt extract, Beef ex-
tract, Yeast extract, Soyabean meal, Peptone and (inor-
ganic sources); Ammonium carbonate, Ammonium Sul-
phate, Ammonium chloride, Ammonium nitrate. Miner-
als like Mg, Na and K were used as MgSO4, NaNO3, and
KH2PO4, for optimisation studies.
Figure 1. Comparison of lovastatin productivity on defatted
versus non defatted substrate.

A. S. Naik, S. S. Lele / Advances in Bioscience and Biotechnology 3 (2012) 643-647 645
2.7. Extraction
A mixture containing Acetonitrile:Water (1:1 v/v) was
added to the flask containing fermented matter and was
sonicated for 5 min. The flask was then placed on Rotary
shaker for 12 hrs. The broth was centrifuged at 10,000
rpm for 10 min followed by filtering it through 0.22 mi-
cron filter. The extracted sample was then subjected to
HPLC analysis.
2.8. Analytical Method
Lovastatin was quantitatively analysed using HPLC
(JASCO, India) with Hemilton C18 column (250 × 4.6
mm I.D) and solvent system of HPLC Grade acetonitrile
containing 0.1% orthophosphoric acid (60:40 v/v) as the
mobile phase (1 ml/min). Peaks were detected at 238 nm
using U. V detector (Figur e 2).
2.9. Response Surface Methodology
Response surface methodology (RSM) by central com-
posite design using DESIGN EXPERT 7.0 was imple-
mented to batch cultures of A. te rreus, for identifying the
effects of three independent process parameters i.e
KH2PO4, glucose and pH of medium on production of
lovastatin. Each factor was varied at three levels. The
experimental design consisted of 20 runs with six repli-
cates of the central point to determine the experimental
error. Regression analysis was performed on the data
obtained from the design experiments.
2.10. Strain Improvement Studies
Ethyl methyl sulphonate (EMS), an alkylating agent was
used at concentrations of 0.15 M, 0.3 M and 0.5 M re-
spectively and each concentration was incubated with
spores for time periods of 30, 60, 90 and 120 minutes.
The procedure involved treating loopful of spores with
the above concentrations of EMS for respective time
Figure 2. Flask level SSF batch on pomegranate seed powder.
duration and subsequently curbing the reaction by addi-
tion of stopping agent sodium thiosulphate. The spores
were then serially diluted and inoculated onto PDA
plates. Random colonies were picked, about 10 colonies
per plate and subcultured. The resultant colonies were
checked for lovastati n production.
3. RESULTS AND DISCUSSION
In this study we report utilization of pomegranate seeds
for the production of lovastatin by solid state fermenta-
tion. Preliminary experiment was conducted on standard
reported media under submerged condition to select
suitable fungal strain. Of the six strains screened; A. ter-
re us IFO7078 was found to give maximum yield of
lovastatin (Table 1). Hence this strain was selected for
further studies.
3.1. Defatted versus Non-Defatted Substrate
The use of defatted substrate has been reported to give
better productivity of statins by filamentous fungi [5].
Proximate analysis showed that pomegranate seed is rich
in fat content (Table 2). Hence we carried out a com-
parative study on defatted and non-defatted substrate.
Defatted substrate was less suitable compared to non-
defatted for lovastatin productivity which is in agreement
with previous report on soya bean substrate [4]. Com-
parative profile of lovastatin production on defatted and
non defatted pomegranate seed substrate is shown in
Figure 1. Absence of lovastatin production on pome-
granate peels, mango peels and passion fruit peels might
be due to lesser amount of fats (Tabl e 2 ) and consider-
able amount of phenolics (data not shown) which pre-
vents the growth of micro-organisms [11]. Thus it can be
concluded that Pomegranate seeds are favourable choice
for lovastatin production.
Ta bl e 2. Fat content of substrate and corresponding lovastatin
yield on it.
Substrate Fat content Lovastatin yield
mg/gm DFM
Mango peel
powder 2.22 ± 0.06 -
Pomegranate
peel powder 0.13 ± 0. 02 -
Passion fruit
peel powder 1.08 ± 0. 04 -
Ashgourd
peel powder 5.0 ± 0.10 2.8 ± 0.04
Pomegranate
seed powder 32 ± 0.11 3.0 ± 0.06

A. S. Naik, S. S. Lele / Advances in Bioscience and Biotechnology 3 (2012) 643-647 646
3.2. Optimisation of Lovastatin Yield
Different physical and chemical parameters were consid-
ered in our study. Primary batches of SSF proved that
thorough mixing of substrate after inoculation and mois-
ture addition was crucial for enhanced mycelial devel-
opment (Figure 2) and eventual lovastatin production.
Maximum productivity (3.42 mg/gm dfm) was obtained
at 60% moisture level. Incubation temperature (25˚C) and
pH 5 was found to be optimum for lov astatin production
(3.60 mg/gm dfm). From the growth curve the maximum
yield of 3.68 mg/gm dfm was obtained on 15th day (Fig-
ure 3). Hence, physical factors did not affect lovastatin
production to great extent, indicating the robustness of
SSF as compared to submerged fermentation which is
known to be highly sensitive to such parameters. The
various chemical parameters considered in our study
were carbon, nitrogen and mineral sources. Contrary to
popular belief about glucose repression, the present study
found it to be the best carbon sour ce (at 5% w/v) to give
highest productivity of lovastatin (3.72 mg/gm dfm). This
could be attributed to the fact that pomegranate seeds
have a high content of slow metabolising sugars and in
combination with glucos e supplement (fast metabolisin g)
would result in better productivity [12]. Nitrogen repres-
sion of lovastatin productivity is an established pheno-
menon [8] and was further backed by our results wherein
highest productivity was seen in control batches having
no added nitrogen content. Thus supplementation with
nitrogen sources was seen to repress lovastatin produc-
tion in our study. Minerals were seen to play a crucial
role in enhancing productivity and this could be due to
the trace element requirement of specific strain. Of the
minerals screened KH2PO4 gave a defin ite incr ease in the
yield (3.90 mg/gm dfm).
Though RSM was performed not much increase in
productivity was seen after one factor at a time optimisa-
tion. Maximum production of lovastatin obtained using
optimized medium was 4.2 ± 0.03 (mg/g of dfm) post
RSM.
Figure 3. Production profile along with biomass concentration.
3.3. Mutation Studies
Beyond a certain point lovastatin yield failed to increase
and this was observed after RSM application to our sys-
tem. Thus further increase would need changes at the
genetic level. Hence mutation studies were carried out to
further increase lovastatin yields (Table 3). Of the sev-
eral colonies picked after exposure to EMS (Ethyl
Methyl Sulphonate), about 6 colonies showed lovastatin
yield (highest 6.5 ± 0.07 mg/gm dfm) higher than the
control/untreated colonies (Figure 4). These colonies
were subcultured for three generations on PDA and
checked for lovastatin yield under both solid state fer-
mentation and submerged conditions. High lovastatin
productivity obtained consistently for three generations
indicated positive effect of mutagen treatment. Enhanced
productivity could be a result of alteration in the related
metabolic pathway with the probable result of yet again
relieving feedback inhibition commonly seen in lovas-
tatin producing cultures [13]. Yield of lovastatin by dif-
ferent wild and mutant strains on varied solid substrates
in our study is shown in Table 4.
4. CONCLUSION
Various fruit-vegetable peels and seeds were tried as
substrates for lovastatin production by fungal cultures.
Table 3. Methodology for Mutant clones with increased yield.
Nomenclature EMS concentration Exposure time
M2-30a 0.3 M 30 min
M1-30b 0.15 M 30 min
M2-60a 0.3 M 60 min
M1-60b 0.15 M 60 min
M1-60a 0.15 M 60 min
M3-60b 0.5 M 60 min
Figure 4. Effect of EMS exposure on lovastatin productivity.

A. S. Naik, S. S. Lele / Advances in Bioscience and Biotechnology 3 (2012) 643-647 647
Table 4. Lovastatin yield of IFO 7078 compared with other strains.
Strain Productivity mg/gm dfm Substrate Reference
A. terreus
IFO 7078 6.50 ± 0.08 (mutant) 4.20 ± 0.10 (wild) Pomegranate seed Present work
A. terreus UV 1718 3.72 (mutant) Wheat bran Pansuriya and Singhal, 2010 [14]
A. terreus ATCC 20542 2.90 Rice or wheat bran Wei, 2007
A. terreus TUB F-514 1.50 Extracted sweet sorghum pulp
supplemented with cheese whey Szakács et al., 1998
Monascus purpureus MTCC 369 3.42 Rice based solid medium Sayyad, S. A., et al., 2009 [15]
M. pilosus M12-69 2.52 Red fermented rice Chen and Hu, 2005 [16]
[7] Szakács, G., Morovján, G. and Tengerdy, R.P. (1998)
Production of lovastatin by a wild strain of Aspergillus
terreus. Biotechnology Letters, 20, 411-415.
doi:10.1023/A:1005391716830
Pomegranate seeds were found to be the best substrate
and with 0.1% w/v Potassium dihydrogen phosphate
(KH2PO4), 5% w/v glucose, 60% w/w moisture, pH 5
with 15 days fermentation cycle under optimized solid
state fermentation conditions, 4.2 ± 0.03 mg lovasta-
tin/gm dfm was produced. Suitability of substrate was
found to depend on presence of fats in the substrate.
Further increase in productivity was obtained by muta-
tion studies using Ethyl Methyl Suplphonate (EMS) to
give a maximum lovastatin yield of 6.5 ± 0.07 mg/gm
dfm. Thus a SSF process for pomegranate seeds utiliza-
tion was developed with the aim to make pomegranate
processing industry a truly profitable venture.
[8] Jia, Z., Zhang, X., Zhao, Y. and Cao, X. (2010) En-
hancement of lovastatin production by supplementing
polyketide antibiotics to the submerged culture of Asper-
gillus terreus. Applied Biochemistry and Biotechnology,
160, 2014-2025. doi:10.1007/s12010-009-8762-1
[9] Kim, H., Moon, J.Y., Kim, H., Lee, D.S., Cho, M., Choi,
H.K., Kim, Y.S., Mosaddik, A. and Cho, S.K. (2010) An-
tioxidant and antiproliferative activities of mango (Mangif-
era indica L.) flesh and peel. Food Chemistry, 12 1, 429-
436. doi:10.1016/j.foodchem.2009.12.060
[10] Pandey, A. (2003) Solid-state fermentation. Biochemical
Engineering Journal, 13, 81-84.
doi:10.1016/S1369-703X(02)00121-3
5. ACKNOWLEDGEMENTS
The authors of this study would like to express their gratitude to DBT,
India for sponsoring the above study through allotment of contingency
and fellowship.
[11] Wei, P., Xu, Z. and Cen, P. (2007) Lovastatin production
by Aspergillus terreus in solid state fermentation. Journal
of Zhejiang, 8, 1521-1526. doi:10.1631/jzus.2007.A1521
[12] Vilches Ferron, M.A., Casas Lopez, J.L., Sanchez Perez,
J.A., Fernandez Sevilla, J.M. and Chisti, Y. (2005) Rapid
screening of Aspergillus terreus mutants for overproduc-
tion of lovastatin. World Journal of Microbiology & Bio-
technology, 21, 123-125. doi:10.1007/s11274-004-3045-z
REFERENCES
[1] Jurenka, J. (2008) Therapeutic applications of pomegra
nate (Punica granatum L.): A review. Alternative Medi-
cine Review, 13, 128-144. [13] Valera, H.R., Gomes, J., Lakshmi, S., Gururaja, R., Sury-
anarayan, S. and Kumar, D. (2005) Lovastatin production
by solid state fermentation using Aspergillus flavipes.
Enzyme and Microbial Technology, 37, 521-526.
doi:10.1016/j.enzmictec.2005.03.009
[2] Aghsaghali, A.M. (2011) Evaluating potential nutritive
value of pomegranate processing by-products for rumi-
nants using in vitro gas productio n technique. ARPN Jou r-
nal of Agricultural and Biological Science, 6, 45-51. [14] Chen, F. and Hu, X. (2005) Study on red fermented rice
with high concentration of monacolin K and low concen-
tration of citrinin. International Journal of Food Micro-
biology, 103, 331-337.
doi:10.1016/j.ijfoodmicro.2005.03.002
[3] EL-Nemr, S.E., Ismail, I.A. and Raga, M. (1990) Chemi-
cal composition of juice and seeds of pomegranate fruit.
Die Nahrung, 34, 601-606.
doi:10.1002/food.19900340706
[4] Xu, B., Wang, Q., Jia, X. and Sung, C. (2005) Enhanced
lovastatin production by solid substrate fermentation of
Monascus rubber. Biotechnology and Bioprocess Engi-
neering, 10, 78-84.
[15] Pansuriya, R.C. and Singhal, R.S. (2010) Response sur-
face methodology for optimisation of production of lovas-
tatin by solid state fermentation. Brazilian Journal of Mi-
crobiology, 41, 164-172.
doi:10.1590/S1517-83822010000100024
[5] Manzoni, M., Bergomi, S., Rollini, M. and Cavazzoni, V.
(1999) Production of statins by filamentous fungi. Bio-
technology Letters, 21, 253-257.
doi:10.1023/A:1005495714248
[16] Sayyad, S.A., Panda, B.P., Javed, S. and Ali, M. (2007)
Optimization of nutrient parameters for lovastatin pro-
duction by Monascus purpureus MTCC 369 under sub-
merged fermentation using response surface methodology.
Applied Microbiology and Biotechnology, 73, 1054-1058.
doi:10.1007/s00253-006-0577-1
[6] Samiee, S.M., Moazami, N., Haghighi, S., Mohseni, F.A.,
Mirdamadi, S. and Bakhtiari, M.R. (2003) Screening of
lovastatin production by filamentous fungi. Iranian Bio-
medical Journal, 7, 29-33.