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OPTIMIZATION OF SUBMERGED STATE FERMENTATION PROCESS FOR TERREIC ACID PRODUCTION APPLYING BIOSTATISTICAL TOOLS

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A quinine epoxide called terreic acid a secondary metabolite produced by the fungus Aspergillus terreus, is considered to be the “next generation antibiotic” due to its broad range antibiotic specificity. The antibiotic property of terreic acid was recognized more than 60 years ago. Previously many researchers were attempted different techniques and conditions for obtaining terreic acid. Very scanty research has been carried out on the optimization of SmF process for the production of terreic acid. Mathematical designs and biostatistical tools were never been used in the process optimization studies. In the present investigation, the focus is on optimization of various nutrients factors of Aspergillus terreus MTCC-11395; SmF cultures, namely Dextrose, Sucrose, Starch, Mannitol, Yeast extract, Dried yeast, L-tyrosine, Acetic acid, Malt extract, Sodium nitrate and process parameters such as pH, Agitation (Rpm), Temperature, Inoculum volume, Fermentation time considering, Agar (dummy1), Agarose (dummy2), Mineral oil (dummy3) and Water (dummy4) as a four dummy variables, for enhanced production of terreic acid applying Plackett-Burman design (PBD) and Response Surface Methodology (RSM). The terreic acid in the fermented broth was confirmed by bioassay and estimated through UV spectrophotometry (214nm). PBD identified Sucrose, L-tyrosine, Agitation (rpm) and Inoculum volume were the principal factor influencing the production of terreic acid (0.463 mg/ml). Further, PBD identified principle factors were optimized applying Central Composite Design (CCD) of Response Surface Methodology (RSM). An optimized medium containing 65 g/L of sucrose, 1 g/L of L-tyrosine, Agitation 180 RPM and 15% of inoculum volume was found to support high yield (0.620 mg/L) of terreic acid under SmF process. Keywords: Aspergillus terreus, terreic acid, Submerged fermentation process, Plackett-Burmann design, Response Surface Methodology, Bioassay
Plackett-Burmann experimental design of design expert (8.0.7.1) showing the 19 selected variables. Optimization by Response Surface Methodology design: The screened and identified factors of PBD; Sucrose, L-tyrosine, Agitation (rpm) and Inoculum volume, were optimized using Response Surface Methodology for the enhanced yield of terreic acid in the SmF culture of Aspergillus terreus MTCC-11395. A four factor RSM design was generated with the Design-Expert 8.0.1.7 software. The model applied was central composite design (CCD) and a second order polynomial response equation gives the yield of terreic acid. By default, the high levels of the factors are coded as +1 and the low levels of the factors are coded as-1. The coded equation is useful for identifying the relative impact of the factors by comparing the factor coefficients. Optimization studies were carried out as a batch experiments based on CCD of RSM as shown in Table 2 within the 250 ml conical flask with the volume of 100 ml as a production media 27,28. Downstream processing of terreic acid: At the end of fermentation process, pH of the final fermentation broth was measured and adjusted to 2.0 using dilute HCl (1N). Acidified broth was filtered using Whatman no 1filter paper. Filtered broth was extracted separately with the volumes of 1:2 ratios ethyl acetate in a shaker incubator (220 rpm) at 36 o C for 2 hours. Solvent layer (organic phase) was separated by centrifugation at 45000 rpm for 8 min. The separated organic layer was subjected to evaporation at 45˚C for 2hrs. The crystals (pale yellow) obtained were dissolved in acetonitrile (5ml) and storage at-20˚C 29. Qualitative analysis of Terreic acid by Bioassay: Bacillus subtilis and E.coli cultures were uniformly (carpet culture) grown on LB agar plates. Discs dipped in extracted samples were placed on the LB agar plates; ethyl acetate was used as a negative control. These plates were incubated at 37°C, for 18-24 hours. The zone of inhibition was observed and diameter measured 15,30. UV Spectrophotometric analysis of lovastatin: The presence of terreic acid in prepared sample were qualitatively analyzed using UV Spectrometric scan (Shimadzu, Model no UV-2450 and Software UV-probe 2.21) between 200nm–320nm and subsequently estimated at 214 nm, using pure terreic acid (sigma-Aldrich) as a standard 31. Results Screening of selected microorganism: Morphological and microscopic screening of the selected fungal cultures is an important parameter to assess the growth stability and purity of the revived fungal cultures. The morphological properties of newly identified strain; Aspergillus terreus-MTCC 11395 on PDA medium was shown in Fig 2. The microscopic studies of the selected fungi explained that the Aspergillus terreus 11395 exhibits hyaline, septate hyphae and smooth, elliptical conidia form long chains with biseriate phialides. The
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OPTIMIZATION OF SUBMERGED
STATE FERMENTATION PROCESS
FOR TERREIC ACID PRODUCTION
APPLYING BIOSTATISTICAL TOOLS
*R.S.UPENDRA1, PRATIMA KHANDELWAL1, AKSHITHA N2, ISHITA CHAKRABORTY2,
SANDRA AVEENA MONIS2, SHRUTHI NA2
1 Sr. Assistant Professor, 1Prof & Head, Dept. of Biotechnology, New Horizon College of Engineering,
Outer Ring Road, Bellandur Post, Marathahalli, Bangalore -560103, Karnataka, India
2BE BT, NHCE alumni (2008-2013),
*E-mail: rsupendra.nhce@gmail.com
Tel: 91-80-6629 7777, Fax: 91-80-28440770.
ABSTRACT
A quinine epoxide called terreic acid a secondary metabolite produced by the fungus Aspergillus terreus, is
considered to be the “next generation antibiotic” due to its broad range antibiotic specificity. The antibiotic
property of terreic acid was recognized more than 60 years ago. Previously many researchers were attempted
different techniques and conditions for obtaining terreic acid. Very scanty research has been carried out on the
optimization of SmF process for the production of terreic acid. Mathematical designs and biostatistical tools
were never been used in the process optimization studies. In the present investigation, the focus is on
optimization of various nutrients factors of Aspergillus terreus MTCC-11395; SmF cultures, namely Dextrose,
Sucrose, Starch, Mannitol, Yeast extract, Dried yeast, L-tyrosine, Acetic acid, Malt extract, Sodium nitrate and
process parameters such as pH, Agitation (Rpm), Temperature, Inoculum volume, Fermentation time
considering, Agar (dummy1), Agarose (dummy2), Mineral oil (dummy3) and Water (dummy4) as a four
dummy variables, for enhanced production of terreic acid applying Plackett-Burman design (PBD) and
Response Surface Methodology (RSM). The terreic acid in the fermented broth was confirmed by bioassay and
estimated through UV spectrophotometry (214nm). PBD identified Sucrose, L-tyrosine, Agitation (rpm) and
Inoculum volume were the principal factor influencing the production of terreic acid (0.463 mg/ml). Further,
PBD identified principle factors were optimized applying Central Composite Design (CCD) of Response
Surface Methodology (RSM). An optimized medium containing 65 g/L of sucrose, 1 g/L of L-tyrosine,
Agitation 180 RPM and 15% of inoculum volume was found to support high yield (0.620 mg/L) of terreic acid
under SmF process.
Keywords: Aspergillus terreus, terreic acid, Submerged fermentation process, Plackett-Burmann design,
Response Surface Methodology, Bioassay INTRODUCTION
Antibiotics are a class of drugs used to prevent the growth and proliferation of microorganisms1,2,3. Antibiotics
can be classified based on their source of origin, structure and mechanism of action4. Antibiotics form an
important class of metabolites, can be obtained in several forms and are used for the treatment and diagnosis of
several types of diseases5. Several types of acids, phenols and aromatic compounds can also be used as
antibiotics6. Recently bacteriocines produced by lactic acid bacteria were identified with brand range of
antimicrobial activity7,8. Antimicrobial resistance is an increasingly problematic issue that leads to millions of
deaths every year9. A few infections become completely untreatable due to the antibiotic resistance6. A World
Health Organization (WHO) report released April 2014 stated, "this serious threat is no longer a prediction for
the future, it is happening right now in every region of the world and has the potential to affect anyone, of any
age, in any country10, stating the urge of finding new antibiotics. Fungi are accomplished chemists that produce
a wide range of complex organic molecules with important applications in the pharmaceutical industry11. The
biological properties of these compounds and the availability of 13C labeling make fungal metabolites good
candidates for studying biosynthetic pathways exploitations using NMR12.
The fungus Aspergillus terreus has a worldwide distribution in different soils and known to produces several
metabolites such β-lactam antibiotics penicillin, cephalosporin, aniticholesterol drug lovastatin13,14, antifungal
antibiotic griseofulvin, antibacterial compounds terrinol and terreic acid15. Terreic acid is a metabolite with
antibiotic properties, produced by the fungus Aspergillus terreus16. The antibiotic properties of terreic acid were
recognized more than 60 years ago, but its cellular and molecular modes of action remained obscure.
Chemically, terreic acid is a quinine epoxide, therefore sharing with fosfomycin a potential reactivity towards
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nucleophiles17. Terreic acid found to inhibit the enzymatic activity of Bruton’s tyrosine kinase (Btk) in mast
cells, cell-free assays and functions as an immunomodulator and known to show the properties of
antinflammation both invivo and invitro and in its combined form is being used as a component for several
antinflammatory drugs18. Terreic acid inhibits the growth of C. albicans AS2 at low concentration and has
fungicidal action at high concentration19. Terreic acid is shown to be a potent and irreversible inhibitor of
acetylcholinesterase (AChE)20. The molecular formula for terreic acid is found to be C7H6O4. It comprises of
two oxygenated methyne protons and one methyl group attached to a quarternary carbon. The fifth and sixth
carbon atoms are attached to an oxygen atom forming cis epoxide21. Terreic acid had higher potency of
hydrogen peroxide radical scavenging activity than terremutin and similar with ascorbic acid. The antioxidant
property of terreic acid is studied using a lipophillic free radical, derived from the DPPH compound22.
Very scanty research has been carried out on the isolation and extraction of terreic acid. Previously many
techniques and conditions were used for obtaining terreic acid. Mathematical designs and statistical tools were
never used in the process optimization of terreic acid production. Optimization techniques like Placket Burman
Design and Response Surface methodology gives a detailed study of several factors and their dependence on the
yield of terreic acid. This study further helps in the design of the media with equal distribution of several
physical and process parameters, which aids in achieving the enhanced yield. Media optimization experiments
with the Aspergillus species showed that glucose and sucrose are the best carbon source for terreic acid
production.
In the present investigation, the focus is on optimization of various nutrients factors of Aspergillus terreus
MTCC-11395; SmF cultures, namely Dextrose, Sucrose, Starch, Mannitol, Yeast extract, Dried yeast, L-
tyrosine, Acetic acid, Malt extract, Sodium nitrate and process parameters such as pH, Agitation (Rpm),
Temperature, Inoculum volume, Fermentation time considering, Agar (dummy1), Agarose (dummy2), Mineral
oil (dummy3) and Water (dummy4) as a four dummy variables, for enhanced production of terreic acid applying
Plackett-Burman design (PBD) and Response Surface Methodology (RSM). The terreic acid in the fermented
broth was confirmed by bioassay and estimated through UV spectrophotometry (214nm). PBD identified
Sucrose, L-tyrosine, Agitation (rpm) and Inoculum volume were the principal factor influencing the production
of terreic acid (0.463 mg/ml). Further, PBD identified principle factors were optimized applying Central
Composite Design (CCD) of Response Surface Methodology (RSM). An optimized medium containing 65 g/L
of sucrose, 1 g/L of L-tyrosine, Agitation 180 RPM and 15% of inoculum volume was found to support high
yield (0.62 mg/L) of terreic acid under SmF process.
MATERIALS AND METHODS
All the chemicals and reagents used in this study were of analytical grade (Merck, India).
Screening of selected microorganism: The culture of newly identified strain23; Aspergillus terreus-MTCC
11395 was obtained from Microbial Type Culture Collection (IMTech-Chandigarh) and host culture was
revived on the potato dextrose agar (PDA) slants and stored at 4°C. Further revived fungal isolate was cultured
on PDA plates. The fully grown culture of the selected fungal isolate was studied morphologically (Form,
Margin, Elevation, Surface Texture and Color) and microscopically (hyphae type, conidia size, shape and color)
using light microscopy at 100 X magnification applying fungal specific lacto phenol cotton blue staining24.
Plackett-Burman experimental design: Dextrose, Sucrose, Starch, Mannitol, Yeast extract, Dried yeast, L-
tyrosine, Acetic acid, Malt extract, Sodium nitrate were the ten medium constituents, pH, Agitation (Rpm),
Temperature, Inoculum volume, Fermentation time were the five process parameters, considering, Agar
(dummy1), Agarose (dummy2), Mineral oil (dummy3) and Water (dummy4) as a four dummy variables a total
of 19 parameters were selected for the study. The Plackett-Burman experimental design for 19 variables (Fig 1),
i.e. ten nutritional components, five process parameters and four dummy variables, were used to evaluate the
impact on the enhanced production of terreic acid. Data analysis was carried out by the standard procedure of
Plackett-Burman experimental design along with the design expert software (8.0.7.1)25, 26.
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145
Fig.1. Plackett-Burmann experimental design of design expert (8.0.7.1) showing the 19 selected variables.
Optimization by Response Surface Methodology design: The screened and identified factors of PBD;
Sucrose, L-tyrosine, Agitation (rpm) and Inoculum volume, were optimized using Response Surface
Methodology for the enhanced yield of terreic acid in the SmF culture of Aspergillus terreus MTCC-11395. A
four factor RSM design was generated with the Design- Expert 8.0.1.7 software. The model applied was central
composite design (CCD) and a second order polynomial response equation gives the yield of terreic acid. By
default, the high levels of the factors are coded as +1 and the low levels of the factors are coded as-1. The coded
equation is useful for identifying the relative impact of the factors by comparing the factor coefficients.
Optimization studies were carried out as a batch experiments based on CCD of RSM as shown in Table 2 within
the 250 ml conical flask with the volume of 100 ml as a production media27,28.
Downstream processing of terreic acid: At the end of fermentation process, pH of the final fermentation
broth was measured and adjusted to 2.0 using dilute HCl (1N). Acidified broth was filtered using Whatman no
1filter paper. Filtered broth was extracted separately with the volumes of 1:2 ratios ethyl acetate in a shaker
incubator (220 rpm) at 36oC for 2 hours. Solvent layer (organic phase) was separated by centrifugation at 45000
rpm for 8 min. The separated organic layer was subjected to evaporation at 45˚C for 2hrs. The crystals (pale
yellow) obtained were dissolved in acetonitrile (5ml) and storage at -20˚C 29.
Qualitative analysis of Terreic acid by Bioassay: Bacillus subtilis and E.coli cultures were uniformly (carpet
culture) grown on LB agar plates. Discs dipped in extracted samples were placed on the LB agar plates; ethyl
acetate was used as a negative control. These plates were incubated at 37°C, for 18-24 hours. The zone of
inhibition was observed and diameter measured15,30.
UV Spectrophotometric analysis of lovastatin: The presence of terreic acid in prepared sample were
qualitatively analyzed using UV Spectrometric scan (Shimadzu, Model no UV-2450 and Software UV-probe
2.21) between 200nm–320nm and subsequently estimated at 214 nm, using pure terreic acid (sigma-Aldrich) as
a standard 31 . Results
Screening of selected microorganism: Morphological and microscopic screening of the selected fungal
cultures is an important parameter to assess the growth stability and purity of the revived fungal cultures. The
morphological properties of newly identified strain; Aspergillus terreus-MTCC 11395 on PDA medium was
shown in Fig 2. The microscopic studies of the selected fungi explained that the Aspergillus terreus 11395
exhibits hyaline, septate hyphae and smooth, elliptical conidia form long chains with biseriate phialides. The
R.S.Upendra et al. / International Journal of Pharma Sciences and Research (IJPSR)
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146
results explained that the revived culture selected for the strain improvement methodologies was of pure in
culture and study in growth
Fig.2. Morphological and microscopic observation of Aspergillus terreus MTCC-11395 culture. 2A) Fungal culture on PDA plates showing
cinnamon brown colour colonies. 2B) septate hyphae with biseriate phialides (100X)
Plackett-Burman experimental design: The Plackett Burman design identified Sucrose, L-tyrosine, Agitation
(rpm) and Inoculum volume, were the principal factor influencing the production of terreic acid. The effects of
various nutritional factors and process parameters on terreic acid production based on the observations of
Plackett- Burman design experiments were shown in (Table 1). Results showed that the main parameters
influencing the production of the terreic were found to be Sucrose, L-tyrosine, Agitation (rpm) and Inoculum
volume with high F values (0.180, 0.577, 1.137 and 5.1648 resp.).
Table 1. Plackett-Burman experimental design observations of terreic production.
Slno Factor E (H) E (L) Mean
square E value F test % of
contribution
1. Sucrose 2.461 2.821 0.00065 -0.036 0.180 2.00
2. Dextrose 2.701 2.4919 0.00021 0.02 0.06 0.70
3. Starch 2.6439 2.639 0.00003 0.0004 0.006 0.07
4. Mannitol 2.6859 2.598 0.00004 0.0003 0.010 0.11
5. Dummy 1 2.6832 2.426 0.0005 0.025 0.013 0.15
6. Dried Yeast 2.4339 2.386 0.000014 0.0004 0.0003 0.003
7. Malt Extract 2.569 2.7139 0.00014 -0.014 0.029 0.33
8. Acetic acid 2.7559 2.527 0.000002 0.02 0.0004 0.004
9. L-tyrosine 3.021 2.3762 0.0200 0.06 0.577 6.7
10. Dummy 2 3.418 2.858 0.0156 0.056 0.43 5.03
11. Yeast
Extract 2.328 2.9549 0.000019 -0.06 0.000045 0.0005
12. pH 2.315 2.9679 0.021 -0.065 0.592 6.9
13. RPM 2.189 3.0939 0.04 -0.09 1.137 13.30
14. Dummy 3 2.984 2.2929 0.023 0.069 0.011 0.128
15. Temperature 2.8919 2.411 0.011 0.04 0.321 3.757
16. Inoculum
volume 2.3498 2.781 -0.043 -0.043 5.1648 60.45
17. Sodium
nitrate 2.702 2.8449 0.0009 -0.013 0.026 0.30
18. Fermentation
time 2.5889 2.694 0.0005 -0.10 0.015 0.1755
19. Dummy 4 2.4789 2.789 0.0004 -0.031 0.011 0.128
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Optimization by Response Surface Methodology design: Based on the experimental results of CCD in Table
2, a quadratic polynomial was established to identify the relationship between adsorption capacity and process
variables. Final Equation in terms of coded factors represents the yield of terreic acid Fig.3.
Fig.3. Final Equation in terms of coded factors representing the yield of terreic acid
Table 2. CCD RSM desing table representing the observation of the procces terreic acid yield
Std Run Factor 1(SU) Factor 2 (LT) Factor 3 (RPM) Factor 4(IV) Yield g/l
4 1 65.00 1.00 140.00 5.00 0.41
3 2 45.00 1.00 140.00 5.00 0.325
15 3 45.00 1.00 180.00 15.00 0.5
20 4 55.00 1.45 160.00 10.00 0.511
17 5 35.00 0.55 160.00 10.00 0.315
10 6 65.00 0.10 140.00 15.00 0.421
22 7 55.00 0.55 200.00 10.00 0.456
18 8 75.00 0.55 160.00 10.00 0.3
11 9 45.00 1.00 140.00 15.00 0.435
24 10 55.00 0.55 160.00 20.00 0.335
6 11 65.00 0.10 180.00 5.00 0.398
23 12 55.00 0.55 160.00 0.00 0
29 13 55.00 0.55 160.00 10.00 0.413
21 14 55.00 0.55 120.00 10.00 0.378
27 15 55.00 0.55 160.00 10.00 0.413
26 16 55.00 0.55 160.00 10.00 0.413
2 17 65.00 0.10 140.00 5.00 0.345
19 18 55.00 -0.35 160.00 10.00 0.175
13 19 45.00 0.10 180.00 15.00 0.254
30 20 55.00 0.55 160.00 10.00 0.413
7 21 45.00 1.00 180.00 5.00 0.289
1 22 45.00 0.10 140.00 5.00 0.002
16 23 65.00 1.00 180.00 15.00 0.62
9 24 45.00 0.10 140.00 15.00 2.57
28 25 55.00 0.55 160.00 10.00 0.413
14 26 65.00 0.10 180.00 15.00 0.435
5 27 45.00 0.10 180.00 5.00 0.157
12 28 65.00 1.00 140.00 15.00 0.523
8 29 65.00 1.00 180.00 5.00 0.512
25 30 55.00 0.55 160.00 10.00 0.413
Final Equation in Terms of Actual Factors:
Yields =
+17.81994
-0.25347 * Sucrose
-7.92394 * L-Tyrosine
-0.097181 * RPM
+0.10680 * Inoculum volume
+0.054139 * Sucrose * L-Tyrosine
+1.37375E-003 * Sucrose * RPM
-8.17500E-004 * Sucrose * Inoculum volume
+0.030000 * L-Tyrosine * RPM
-6.61111E-003 * L-Tyrosine * Inoculum volume
-2.60000E-004 * RPM * Inoculum volume
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The experimental results and the predicted values obtained from the model equation were compared. The Model
F-value of 3.28 implies the model is significant. There is only a 1.25% chance that a "Model F-Value" this large
could occur due to noise. Values of "Prob > F" (less than 0.0500) indicate; A, B ,C, D, AB, AC, AD, BC, BD,
and CD were significant model terms. The "Lack of Fit F-value" of 0.19 implies the Lack of Fit is not
significant relative to the pure error (0.000) Table 3.
Table 3. ANOVA for Response Surface 2FI Model of CCD RSM of terreic acid production
Analysis of variance table [Partial sum of squares - Type III]
Source Sum of
Squares df Mean
Square F
Value p-value
Prob > F
Model 4.67 10 0.47 3.28 0.0125 Significant
A-Sucrose 0.35 1 0.35 2.45 0.1338
B-L-Tyrosine 0.22 1 0.22 1.54 0.2299
C-RPM 0.57 1 0.57 4.02 0.0594
D-Inoculum volume 0.17 1 0.17 1.16 0.2949
AB 0.95 1 0.95 6.66 0.0183
AC 1.21 1 1.21 8.48 0.0090
AD 0.027 1 0.027 0.19 0.6698
BC 1.17 1 1.17 8.19 0.0100
BD 3.540E-003 1 3.540E-003 0.025 0.8764
CD 0.011 1 0.011 0.076 0.7859
Residual 2.71 19 0.14
Lack of Fit 2.71 14 0.19
Pure Error 0.000 5 0.000 Insignificant
Cor Total 7.38 29
Qualitative analysis of Terreic acid by Bioassay: Bioassay of SmF extracted samples (terreic acid) on
Bacillus subtilis and E.coli (LB agar) plate cultures were shown on Fig 4.
Fig.4. Bioassay of SmF extracted samples (SP) exhibiting zone on inhibition, NC represents negative control
4A). Bacillus subtilis culture plate showing zone on inhibition. 4B). E.coli culture plate showing zone on inhibition
UV Spectrophotometric analysis of lovastatin: The concentration of terreic acid was calculated
spectrophotometrically (three replicates). The samples and the standard exhibited a peak at 214 nm in the
spectrophotometer scanning (200nm-320nm). Terreic acid in the sample was subsequently estimated at 214nm
using terreic acid standard obtained from (Sigma Aldrich-India). The results explained that the yield of terreic
acid after PBD (Fig.5) was found to be (0.463 mg/l) and after optimization of PBD screened principle factors
through CCD RSM the terreic acid yield was enhanced (0.62 mg/l), reporting the 1.3 fold raise compare to the
yield of PBD.
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Fig.5. Bar charts representing the UV quantitative analysis of PBD samples
DISCUSSION
Terreic acid a secondary metabolite produced by the fungus Aspergillus terreus, is considered to be the “next
generation antibiotic” due to its broad range antibiotic specificity. In the present investigation, the focus is on
optimization of various nutrients factors of Aspergillus terreus MTCC-11395; SmF cultures, namely Dextrose,
Sucrose, Starch, Mannitol, Yeast extract, Dried yeast, L-tyrosine, Acetic acid, Malt extract, Sodium nitrate and
process parameters such as pH, Agitation (Rpm), Temperature, Inoculum volume, Fermentation time
considering, Agar (dummy1), Agarose (dummy2), Mineral oil (dummy3) and Water (dummy4) as a four
dummy variables, for enhanced production of terreic acid applying Plackett-Burman design (PBD) and
Response Surface Methodology (RSM). The terreic acid in the fermented broth was confirmed by bioassay and
estimated through UV spectrophotometry (214nm). PBD identified Sucrose, L-tyrosine, Agitation (rpm) and
Inoculum volume were the principal factor influencing the production of terreic acid (463 mg/l). Further, PBD
identified principle factors were optimized applying Central Composite Design (CCD) of Response Surface
Methodology (RSM). An optimized medium containing 65 g/L of sucrose, 1 g/L of L-tyrosine, Agitation 180
RPM and 15% of inoculum volume was found to support high yield (620 mg/L) of terreic acid under SmF
process. Temperature as a factor in the elaboration of mycotoxins by two fungi in groundnut fodder was
investigated and the study reported 77 mg/l of terreic acid yield, which was 8, fold lesser than the yield of
present optimization study. Isolation of Antioxidant compounds from Aspergillus terreus LS01 cultures was
investigated and the study reported 150 mg/l of terreic ac id yield22, compared present study reported
approximately 4 fold higher terreic acid (620 mg/L).
CONCLUSION
Considering these references as discussed above, it could be concluded that Aspergillus terreus MTCC 11395
SmF culture was optimized successfully and reported enhanced terreic acid yield. It can be also seen that the
present investigation led to explore the application of mathematical design (PBD) and biostatistical tools (RSM)
on bioprocess optimization for biomolecule production.
ACKNOWLEDGEMENT
We wish to express our sincere gratitude to Chairman and Principal, New Horizon College of Engineering,
Bangalore for providing us with all facilities to undertake research work on “optimization of submerged state
fermentation process for terreic acid production applying biostatistical tools”. We extended our sincere thanks to
Dept Biocon, Bangalore, India for their help. We also wish to express our gratitude to the officials and other
staff members who rendered their help during the period of this research work.
0.429 0.438
0.13
0.135
0.392
0.4119
0.158
0.257
0.125
0.142
0.382
0.151
0.285
0.45
0.113
0.241
0.463
0.099
0.243 0.238
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
1 2 3 4 5 6 7 8 9 1011121314151617181920
Concentration of TA (mg/ml)
SmF-PB Trial Codes
UV Quantitative Analysis
Yields
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ISSN : 0975-9492
Vol 7 No 03 Mar 2016
150
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International Journal for Biotechnology and Molecular Biology Research
R.S.Upendra et al. / International Journal of Pharma Sciences and Research (IJPSR)
ISSN : 0975-9492
Vol 7 No 03 Mar 2016
151
Article
he preset study aimed to optimize downstream process (DSP) conditions of fungal derived secondary metabolite named lovastatin for the submerged state fermentation (SmF) cultures of strain Aspergillus terreus-11045. A five factorial central composite design methodology (CCD) of Response Surface design Methodology (RSM) employed to enhance impactful factors of fungal derived lovastatin biosynthesis, i.e Ethyl acetate (200–1000 mL) pH (2.0–10.0), Temperature (30–38oC), Agitation (120–200 rpm), Incubation Time (0.5–2.5 h). The optimized trail of RSM design was validated with artificial neural network (ANN). RSM trail of 750 mL of ethyl acetate, pH 2.0, temperature 38 oC, agitation speed-160 rpm and incubation time-2 h, has conferred higher yield (3.453 mg/g dry matter). ANN validated yield (3.447 mg/g dry mass) was in congruence with the experimental values (3.453 mg/g dry mass) and more than the predicted value-CCD-RSM (3.406 mg/g dry mass). The standardized conditions of the present study reported the lovastatin yield (3.453 mg/g dry mass) approximately by 3.5 times compared to that of (952.7?mg/L) lovastatin reported earlier, similarly the lovastatin yield is 3.5 times higher than the lovastatin yield (0.997 mg/g dry matter) of suboptimal SmF DSP of Aspergillus terreus MTCC-11045. Keyword: Aspergillus terreus; Lovastatin; Downstream process; Response Surface Methodology; Artificial Neural Network.
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Lactic Acid Bacteria (LAB) known to produce bacteriocins; a small, ribosomally synthesized antimicrobial peptides considered as next generation antibiotics due to the broad spectrum antibiotic nature. Scanty research was reported on the isolation and screening of different LAB species with high bacteriocin yield. With this lacuna the present study aimed to screen various isolated LAB cultures for their bacteriocin yield abilities and subsequently indentifies the elite LAB species representing higher bacteriocin yield. Initially isolated and preserved LAB cultures were sub-cultured and further screened for their bacteriocin yield abilities on CM media as a production media under submerged state fermentation (SmF) conditions. Based on the bacteriocin yield an elite species was identified, and studied morphologically by growing on different media namely Nutrient agar (NA) media, Acetate agarmedia and MRSmedia. Pediococcus pentosaceus strain 2269 obtained from culture collection centre NCIM, Pune, India was used as a standard culture. Microscopic study was performed to screen the visible similarities between the isolate and the standard strain. Further biochemical characterization i.e. Indole, Methyl red, Vogues Proskaeur, Catalase and Carbohydrate fermentation; studies were carried to confirm the culture at Genus level. Finally molecular characterization was done using 16s RNA sequencing followed by phylogenetic analysis (MEGA V-6)to confirm the isolate at species level. Results reported that the culture isolated from the Sanna batter (Sanna 14) was identified as an elite species i.e. Pediococcus pentosaceus with higher bacteriocin yield (0.4mg/l) among the three bacteriocin positives cultures reported in the present study. The 16s RNA sequence of the Pediococcus pentosaceus culture was submitted to NCBI GenBank with an issued accession number MF183113.
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Traditional oriental fermented foods namely soy sauce, miso, vinegar, red rice vinegar, pickled tofu and Ang-kak were becoming increasingly popular in different parts of India and shall be used for isolation of hypolipidemic agent, lovastatin producing representative fungi. Existing food grade (GRAS) fungi, Monascus sp were known to produce lovastatin through SSF. Further exploration of food grade fungi from indigenous and traditional oriental fermented food products shall augment such sources and lead to better and safe food product development. Such a strategy shall give benefits to the elderly and the concept of preventive health care shall take envisaged shape. With this objective the present investigation focus on isolation and microbiological characterization of the lovastatin producing GRAS filamentous fungi from various traditional oriental fermented food samples obtained from India and China. Initially food grade fungal cultures were isolated by serial dilution technique and morphological characteristic of the isolated fungal cultures was studied on Potato dextrose agar (PDA) medium. Microscopic confirmation of isolated fungal cultures were performed using light microscopy. Further, microbiologically characterized fungal cultures were tested on a novel substrate combination of rice and barley using Solid state fermentation (SSF) process for the production of lovastatin. At the end of the SSF process the presence of lovastatin in the prepared samples from the end product was analysed qualitatively using Thin Layer Chromatography and ATR-FTIR. The yield of lovastatin was quantified using UV spectroscopic analysis at 238 nm using pure drug lovastatin as a standard (Biocon, India). Present study isolated one Monascus purpureus and three Monascus ruber fungal cultures and concluded that all the four characterized fungal cultures were found to be positive for lovastatin production.
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Submerged cultures (SmF) of filamentous fungi ('Aspergillus terreus, Aspergillus flavus') are widely used to produce commercially important metabolite lovastatin. Limited investigations have been made on optimization of the different nutrients and process parameters using standard optimization methods. No previous work has used statistical analysis in documenting the interactions between nutritional and process parameters in lovastatin production. With this lacunae, Plackett-Burman experimental design was used for the first time to screen and investigate the effects of the nine factors -i.e. Source (lactose, glycerol and honey) and concentrations of carbon (40-80 g/L), source (mycological peptone and yeast extract) and concentrations of Nitrogen (5-25 g/L); pH of the fermentation process (6.0-7.6); Temperature of the fermentation process (24 degreesC -32 degreesC) ; Agitation (120 - 200 rpm) and Fermentation time (5-13 days) -on the concentrations of lovastatin produced in batch cultures (SmF) of 'Aspergillus terreus' (newly discovered strains (nhceup) MTCC-11045, 'Aspergillus terreus' (NHCEUPBT) MTCC-11395, 'Aspergillus flavus' (NHCEUPBTE) MTCC-11396 and 'Aspergillus terreus' MTCC-1782. Lovastatin in the sample was confirmed and estimated by UV Spectrophotometry, HPLC and FTIR analysis; Plackett-Burman design identified the "source and concentrations of C, N, pH and incubation period" were the principal factor influencing the production of lovastatin. Temperature and agitation were found to have least impact on lovastatin production. Botha a limitation and excess of carbon and nitrogen reduced lovastatin titers. A medium containing 70 g/L carbon supplied as lactose, 20 g/L nitrogen supplied Yeast extract, 6.8pH, and 10 days of incubation period were shown to support high titers (2990 mg/L) of lovastatin production in submerged fermentation process. The above optimized fermentation conditions raised the lovastatin titer by 1.7-fold compared with the yield (1761.6 mg/L) of lovastatins by using Dox-rice medium as a carbon. Optimization by using Response Surface Methodology is under study.
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Fluoride is a naturally occurring element in water systems and enters food chain mostly through drinking water. The WHO permissible limit of fluoride in water is 1.0 mg/l. At < 1.0 mg/l, it inhibits dental caries, at > 1.0 mg/l causes molting of teeth, lesion of endocrine glands, thyroid, liver and other organs. At still higher concentration (3-6 mg/l), it causes skeletal fluorosis. Existing fluoride removal techniques haven’t been very effective as these remove fluoride only up to 2 mg/l. Therefore, an economically viable, eco-friendly and easy method for defluoridation of drinking water is highly desirable. In the present investigation, Ocimum sp. leaves along with ragi seed husk was used as natural fluoride adsorbents and the process parameters such as absorbent dosage (1-10 g/l), pH (3-12) and contact time (10-150 min) were optimized using Central Composite Design (CCD) of Response Surface Methodology (RSM). The fluoride content in the water was quantitatively determined by UV spectrophotometric analysis and the presence of fluoride in the treated Ocimum sp. leaves were identified with EDAX analysis. RSM design optimized conditions i.e - 5.5 g/l each of Ocimum sp. leaves and ragi seed husk, 6.0 pH and 50 min contact time gave the end values of 0.43 mg/l of fluoride. The optimized values of RSM with respect to the end fluoride content (0.43 mg/l) after treatment process were validated using feed forward model of Artificial Neural Network (ANN). ANN predicted value (0.4250 mg/l) was very close to the optimized experimental value of RSM design (0.43 mg/l) and the error was 0.049. In conclusion, an optimized process was developed for the removal of fluoride from the drinking water using Ocimum sp. leaves and Ragi seed husk as natural fluoride adsorbents. Final concentration of 0.43 mg/l of fluoride was achieved from initial concentration of 10mg/l. Key Words : Fluoride removal, Adsorption, Ocimum sp. leaves and Ragi seed husk, Response Surface Methodology (RSM), Artificial Neural Network (ANN), EDAX analysis
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Lactic acid bacteria (LAB) are a group of Gram-positive; non-spore forming, non-motile, non-respiring, bacteria produces variety of antimicrobial compounds such as lactic acid, acetic acid, ethanol, formic acid, fatty acids, hydrogen peroxide and bacteriocins. Among them bacteriocines (a small molecular weight proteins) are in prime important due to their antimicrobial nature with food preservative abilities. Bacteriocins have gained a lot of attentions as bio-preservatives because of its GRAS status without causing any adverse effects on food. Nisin has been approved by US-FDA as a food preservative and is being used commercially worldwide by food industries. With these rationales, the aim of the present study is to produce bacteriocin (Nisin) from lactic acid bacteria isolated from selected fermented food sources, such as Curd, Mayonnaise and Jelly. Initially preserved lactic acid bacterial cultures were sub-cultured and their growth characters were studied on four different media namely MRS media, HJ media, KT media and DO media. Further seed culture of the selected bacterial species was prepared on the MRS broth (24hrs) and used as an inoculum for the production of bacteriocins. Later the 10% of the seed culture was inoculated to the 100 ml production media (CM media). After the 72 hrs of batch fermentation process, a crude extract of the fermentation broth was screened for the presence of Bacteriocins using agar well diffusion assay technique on E.coli and Kleibshella sp culture plates. All the three cultures of lactic acid bacteria showed antagonistic activity on the tested bacterial sp. Partial purification of the bacteriocin was done using ammonium sulphate precipitation and molecular weight characterisation of the obtained bacteriocins was done using Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The results exhibited that, the bands representing the molecular weight less than 14 KDa and also the absorbance peak at 225 nm in UV spectra confirmed the presence of bacteriocin production. In conclusion in the present study attempt were made successfully in producing bacteriocine for indigenous cultures of LAB isolated from selected fermented foods samples. Keywords: Lactic acid bacteria (LAB), Bacteriocins, Antimicrobial Activity, E.coli, Kleibshella sp and SDS- PAGE.
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Epiphyllum oxypetalum is a species of cactus and one of the most commonly grown of the Epiphyllum species. It is one of the under-utilized resources available in the tropical regions of the globe and can be used as a substitute for digitalis. The Shoshone Indian tribe calls the night blooming Cereus "Pain in the heart" and used it for heart pain. Scanty work was reported on the phytochemical properties of leaf extract and no documented research work was reported on its leaf and flower for assessment of nutritive value and antimicrobial properties. Thus, the present investigation was carried out to access the nutritive values, phytochemical constituents and antimicrobial potentials of leaf extracts. The nutritive values of plant showed significant presence of proteins (14 mg/g), fatty acids (4.6 mg/g), and vitamins (0.18 mg/g), while carbohydrates were found to be absent. Phytochemical analysis of Epiphyllum oxypetalum showed the presence of saponins, phenolic compounds, steroids, glycosides, tannins, terpenoids, and resins while reducing sugars, alkaloids, flavanoids, sterols, phlobatanins and acidic compounds were absent. Since reducing sugars are absent, plant leaf can be used as a diet supplement to diabetic patients. Assessment of antimicrobial properties was done by using three solvent systems, (Petroleum ether, Acetone and Ethanol). All solvent systems at different concentrations were evaluated for antibacterial and antifungal capacity against selected bacterial and fungal pathogens; Maximum zone of inhibition was exhibited by acetone and petroleum ether (14mm) leaf extracts for Escherichia coli, acetone (14mm) for Staphylococcus aureus, acetone (11mm) and ethanol (10mm) for Klebsiella pneumonia and petroleum ether (12mm) for Bacillus subtilis. All the three leaf extracts were found to be ineffective against fungal strains (Aspergillus niger, Aspergillus terreus, Aspergillus oryzae and Rhizopus oryzae) tested.
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Fungi is somewhere in between the micro and macro organisms which is a good source of producing biologically active secondary metabolites. Fungi have been used as tool for producing different types of secondary metabolites by providing different nutrients at different laboratory conditions. The fungi have been engineered for the desired secondary metabolites by using different laboratory techniques, for example, homologous and heterologous expressions. This review reported how the fungi are used as chemical industry for the production of secondary metabolites and how they are engineered in laboratory for the production of desirable metabolites; also the biosynthetic pathways of the bio-organic-molecules were reported.
Conference Paper
Statins are competitive inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A reductase, the rate limiting enzyme of cholesterol biosynthesis pathway (Alberts et al., 1980). Aspergillus terreus, Aspergillus flavus are known to produce cholesterol reducing drug- Lovastatin (Novak et al., 1997). Lovastatin isolated from Aspergillus terreus was the first statin to be approved by FDA in 1987 for therapeutic use (Devi et al., 2011). Submerged cultures of filamentous fungi (Aspergillus terreus, Aspergillus flavus) are widely used to produce commercially important metabolites such as lovastatin (C24H36O5), Mevinolin, Monacolin K and Mevacor TM. (Lopez et al., 2005). Optimization of the different parameters of submerged fermentation process (Smf) has been done by the various researchers (Lai et al, 2007, Atalla et al., 2008, Osman et al., 2011,). Limited investigations have been made on optimization of the different nutrients and process parameters using standard optimization methods (Lopez et al., 2004, Sayyad et al., 2007). No previous work has used statistical analysis in documenting the interactions between nutrient concentrations and process parameters in lovastatin production. With this lacunae, Plackett Burman experimental design was used for the first time to investigate the effects of the six factors —i.e. Source and concentrations of Carbon, source and concentrations of Nitrogen; pH of the fermentation process; Temperature of the fermentation process; Agitation and fermentation time —on the concentrations of biomass and lovastatin produced in batch cultures (Smf) of (newly discovered strains) Aspergillus terreus (nhceup) MTCC-11045, Aspergillus terreus (nhceupBT) MTCC-11395, Aspergillus flavus (nhceupBTE) MTCC-11396 and Aspergillus terreus MTCC-1782. The values of the various factors in the experiment ranged widely, as follows: Three different carbon sources namely lactose, glycerol and honey were tested at different concentration ranging from 40–80 g/L. Two different nitrogen sources: mycological peptone and yeast extract were tested at different concentration ranging from 5–25 g/L, with pH of the fermentation process varying in 6.0-7.6 and temperature being 24 oC –32 oC. Agitation of the media was set in between 120 – 200 rpm and incubation period being 5–13 days. The Plackett Burman design identified the “source and concentrations of C, N, pH, and incubation period” were the principal factor influencing the production of lovastatin. Temperature and agitation were found to have least impact on lovastatin production. Both a limitation and excess of carbon and nitrogen reduced lovastatin titers. A medium containing 70 g/L carbon supplied as lactose, 20 g/L nitrogen supplied Yeast extract, 6.8 pH, 10 days of incubation period was shown to support high titers (2990 mg/L) of lovastatin production in submerged fermentation process. The above optimized fermentation conditions raised the lovastatin titer by 1.7-fold compared with the yield (1761.6 mg/L) of lovastatins by using Dox-rice medium as a carbon source (Atalla et al., 2007), 15.3 – fold compared with the yield (188.3 mg/L) using Oat meal as a carbon source (Osman et al., 2011), 14.3 –fold compared with the yield (202.8mgdm−3) using lactose as a carbon source (Lopez et al., 2004). Optimization of Solid State Fermentation (SSF) process by using Response Surface Methodology is under study. Key words: Aspergillus terreus, Lovastatin, Plackett Burman design, RP-HPLC, FTIR, Lactose