1
PRODUCTION OF NOVEL LIPASE INHIBITOR
FROM STREPTOMYCES SP
A thesis submitted to the University of Mysore for the award of the degree of
DOCTOR OF PHILOSOPHY
IN
BIOTECHNOLOGY
By
NAVEEN BABU KILARU
Department of Fermentation Technology and Bioengineering
Central Food Technological Research Institute Mysore - 570 020, India
July 2005
2
Dr. A. P. Sattur, Date:
Scientist,
Fermentation Technology and
Bioengineering Department,
CERTIFICATE I hereby certify that the thesis entitled “ PRODUCTION OF NOVEL LIPASE
INHIBITOR FROM STREPTOMYCES SP” submitted to the University of Mysore for the
award of the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY by Mr.
NAVEEN BABU KILARU, is the result of the research work carried out by him in the
Department of Fermentation Technology and Bioengineering, Central Food Technological
Research Institute, Mysore, India, under my guidance during the period 2001-2005.
(A. P. Sattur)
3
NAVEEN BABU KILARU, Date: Senior Research Fellow, Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute, Mysore – 570 020.
DECLARATION
I hereby declare that the thesis entitled “PRODUCTION OF NOVEL LIPASE INHIBITOR
FROM STREPTOMYCES SP” submitted to the University of Mysore for the award of the
degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY is the result of the
research work carried out by me in the Department of Fermentation Technology and
Bioengineering, Central Food Technological Research Institute, Mysore, India, under the
guidance of Dr. Avinash P Sattur during the period 2001-2005.
I further declare that the work embodied in this thesis had not been submitted for the award of
degree, diploma or any other similar title.
(NAVEEN BABU KILARU)
4
CONTENTS
Page No.
List of Tables VIII
List of Figures XII
CHAPTER 1. INTRODUCTION 1.1. Actinomycetes 1
1.2. Lipases 2
1.2.1. Reaction mechanism 3
1.2.2. Specificity of lipases 5
1.2.3. Pancreatic lipase 6
1.3. Obesity a medical complication caused by lipase 7
1.3.1.a. Reduction of food intake 8
1.3.1.a.1. Noradrenergic receptors 8
1.3.1.a.2. Serotonergic receptors 8
1.3.1. b. Increased energy expenditure 9
1.3.1.c. Altered metabolism 9
1.4. Sources of lipase inhibitors 10
1.4. a. Lipase inhibitors from microbial origin 10
1.4.b. Lipase inhibitors from plant sources 12
1.5. Scope of the present investigation 15
CHAPTER 2. MATERIALS AND METHODS
2.1. Screening of actinomycetes 17
2.1.A. Collection of different terrestrial substrates for selective
isolation of actinomycetes 17
2.1.B. Pretreatment of soil samples 17
2.1.B.1. Calcium carbonate treatment 17
2.1.B.2. Dry heat treatment 17
2.1.B.3. Phenol treatment 18
2.1.B.4. Centrifugation method 18
2.1.B.5. Control (no pretreatment) 18
5
2.2. Media composition 18
2.2.A. Actinomycetes 18
2.2.A.1. Medium for screening of actinomycetes for pancreatic
lipase inhibitor 19
2.2.A.2. Culture maintenance medium 20
2.2.A.3. Morphology and pigmentation 20
2.2.A.4. Cultural Characteristics media 21
2.2.A.4.1. Enzyme activity test media 27
2.2.A.4.2. Degradation tests media 29
2.2.A.4.3.Test for resistance to antibiotics 33
2.2.A.4.4.Effect of temperature on growth of isolate N2 33
2.2.A.4.5. Effect of pH on growth of isolate N2 34
2.2.A.4.6.Growth in the presence of inhibitory compounds 34
2.2.A.4.7.Test for carbon utilization medium 34
2.2.A.4.8.Test for nitrogen utilization medium 35
2.2.A.4.9.Test for production of acid and gas 36
2.2.A.4.10. Media for cultivation of cells for determination of cell
wall composition 36
2.2.A.5. Primary screening medium 37
2.2.A.6.Secondary screening media 37
2.2.A.7. Selection of inoculum media for production of streptolipin 38
2.2.A.8. Standard media for screening streptolipin production 39
2.2.B. Fungi 43
2.2.B.1. Culture maintenance medium for fungi 43
2.2.B.2. Screening of fungal cultures for the production of lipase
inhibitor 43
2.2.B.3. Medium for submerged fermentation (SmF) of fungi 44
2.2.B.4. Medium for solid state fermentation (SSF) of fungi 44
2.3. General fermentation conditions
2.3.1. Inoculum development for screening 44
6
2.3.2. Submerged fermentation for actinomycetes cultures 45
2.3.3. Solid state fermentation for fungal cultures (SSF) 45
2.3.4. Submerged fermentation for fungal cultures (SmF) 45
2.4. General extraction conditions
2.4. 1. Extraction of inhibitor from SmF broth 46
2.4.2. Extraction of inhibitor from SSF bran 46
2.5. Analytical methods
2.5.1. Lipase assays 46
2.5.2. Tests for cell wall composition of isolate N2 48
2.5.2.1. Amino acid analysis 48
2.5.2.2. Sugar analysis 49
2.5.2.3. Analysis of menaquinones 49
2.5.2.4. Analysis of fatty acids 50
2.5.2.5. Analysis of phospholipids 50
2.5.2.6. Analysis of mycolic acids 51
2.5.2.7. Phylogenetic analysis of isolate N2 51
2.5.3.1. Column chromatography 52
2.5.3.2. Thin layer chromatography (TLC) 52
2.5.3.3. High pressure liquid chromatography (HPLC) method 53
2.5.3.4. Melting point determination 53
2.5.3.5. Elemental analysis 53
2.5.3.6. Infrared spectroscopy 54
2.5.3.7. Nuclear magnetic resonance spectroscopy (NMR) 54
2.5.3.8. Liquid chromatography mass spectroscopy (LC-MS) 54
2.5.3.9. Detection of elements by chemical method 55
2.6. Microorganisms used for comparative studies 56
2.7. In vivo efficacy of streptolipin on experimental animal models 56
2.7.1. Effect of single and multiple doses of streptolipin on fat
absorption 56
2.7.2. Faecal triglycerides 57
2.7.3. Lipid peroxides 58
2.7.4. Antioxidant enzymes 58
7
2.7.4.1. Catalase assay 58
2.7.4.2. Estimation of Superoxide dimutase 59
2.7.4.3. Estimation of Glutathione peroxidase 59
2.7.5. Plasma non specific enzymes 59
2.7.6. Lipid analysis 60
CHAPTER. 3. RESULTS AND DISCUSSION
CHAPTER. 3, SECTION A. Screening and selection of actinomycetes for the
production isolation of pancreatic lipase inhibitor
3.A.1.Screening of different terrestrial substrates for selective isolation
of actinomycetes 61
3.A.2.Techniques for the isolation of actinomycetes 61
3.A.3. Screening of microorganisms for pancreatic lipase inhibitor
production 70
CHAPTER 3, SECTION B. Culture characterization and identification of isolate N2 as
Streptomyces vayuensis
3.B.1. Morphological characters of isolate N2 87
3.B.2. Cultural characteristics of isolate N2 on different media 87
3.B.3. Antimicrobial activity of isolate N2 92
3.B.4. Enzyme activity tests for isolate N2 93
3.B.5. Degradation tests of isolate N2 94
3.B.6. Antibiotics resistance of isolate N2 94
3.B.7. Effect of temperature and pH on growth of isolate N2 94
3.B.8. Growth of isolate N2 in the presence of inhibitory compounds 94
3.B.9. Test for carbon source utilization by isolate N2 95
3.B.10. Test for nitrogen utilization by isolate N2 101
3.B.11. Test for production of acid and gas by isolate N2 101
3.B.12. Chemotaxonomic characteristics of isolate N2 101
3.B.12.1. Test for sugars and amino acids 101
3.B.12.2. Test for menaquinones 102
3.B.12.3. Test for mycolic acid 102
8
3.B.12.4.Test for fatty acids 102
3.B.12.5. Test for phospholipids 102
3.B.13. Phylogenetic analysis of isolate N2 105
3.B.14. Comparative studies of isolate N2 112
3.B.15. Final and brief description of isolate N2 as Streptomyces
vayuensis sp.nov 112
CHAPTER. 3, SECTION C. Isolation, purification and structure elucidation of Streptolipin, a
pancreatic lipase inhibitor from Streptomyces vayuensis
3.C.1.Isolation and purification of pancreatic lipase inhibitor 117
3.C.2. Adaptation of the PNPB spectrophotometric assay to
TLC system 117
3.C.3. Physico-chemical properties of streptolipin 119
3.C.4.Structure elucidation of the inhibitor 125
3.C.5. Kinetic studies on inhibition of streptolipin against pancreatic
lipase 127
3.C.5.1. Lineweaver-Bulk (LB) plot of pancreatic lipase inhibition
by streptolipin 127
3.C.5.2. Determination of irreversibility of the pancreatic lipase
enzyme inhibition 131
3.C.6. Other biological activities of streptolipin 131
CHAPTER. 3, SECTION D. Studies on media optimization for streptolipin production
3.D.1. Standard HPLC curve for streptolipin 136
3.D.2.Time course fermentation for streptolipin production 136
3.D.3. Optimization of physical parameters for production of streptolipin
3.D.3.1. Selection of inoculum media for production of streptolipin 137
3.D.3.2. Effect of temperature on production of streptolipin 139
3.D.3.3. Effect of initial pH of the medium on production of streptolipin 139
3.D.3.4. Effect of aeration on production of streptolipin 142
3.D.4. Optimization of nutritional parameters for the production
of streptolipin
9
3.D.4.1. Screening of standard media for the production of streptolipin 142
3.D.4.2. Effect of different carbon sources on production of streptolipin 144
3.D.4.3. Effect of inorganic nitrogen sources on production of streptolipin 149
3.D.4.4. Effect of organic nitrogen sources on production of streptolipin 149
3.D.4.5.Effect of trace elements on production of streptolipin 151
3.D.4.6. Effect of lipids on production of streptolipin 154
3.D.4.7. Effect of molasses on production of streptolipin 156
3.D.4.8. Formulation of medium for the production of streptolipin 156
3.D.4.9. Effect of different glucose concentrations on the kinetics 157
of streptolipin production
3.D.4.10. Effect of different concentrations of yeast extract on
of streptolipin production 169
3.D.4.11. Production of streptolipin with optimum conditions
on shake flask 171
3.D.4.12. 10 L Fermentor study 176
3.D.5. Optimization of down stream processing conditions for
streptolipin extraction
3.D.5.1. Choice of the extraction solvent 179
3.D.5.2. Biomass to solvent ratio 180
3.D.5.3. Effect of static and agitated conditions during extraction 180
3.D.5.4. Repeated extraction 181
3.D.5.5. Sequential extraction 181
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CHAPTER. 3, SECTION E. In vivo efficacy of streptolipin on experimental animal
models
3.E.1. Influence on lipase activity and dietary triglyceride absorption 185
3.E.2. Influence on blood and liver lipid profile 192
3.E.3. Influence on antioxidant status 199
CHAPTER .4. Summary and highlights of the investigation 203
Recommendation for further work 206
References 207
List of publications 225
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LIST OF TABLES
Table No. Title Page No.
Table 3.1: Different soil samples for screening of actinomycetes 62
Table 3.2: Different pretreatments of various soil samples 64
Table 3.3: Isolation of actinomycetes on different media 65
Table 3.4: Distribution of actinomycete isolates in various soil samples 68
Table 3.5: Distribution of actinomycete isolates from various samples 69
Table 3.6: Primary screening of fungal cultures from CFTRI Culture
Collection Center for pancreatic lipase inhibition 71
Table 3.7: Primary screening of actinomycetes isolates for pancreatic
lipase inhibition 75
Table 3.8: Classification of actinomycete isolates based on inhibition
(%) of pancreatic lipase 82
Table 3.9: Distribution of actinomycetes isolates showing inhibition
greater than 20% on soil sample type 83
Table 3.10: Distribution of actinomycetes isolates showing inhibition
greater than 20% on soil pretreatment basis 83
Table 3.11: Distribution of active actinomycetes isolates on the basis
of colour 84
Table 3.12: Secondary screening of actinomycetes cultures for
reproducibility and consistent production of lipase inhibitor 86
Table 3.13: Morphological characters of isolate N2 89
Table 3.14: Cultural characteristics of isolate N2 on different media 90
Table 3.15: Antimicrobial activity of isolate N2 92
Table 3.16: Enzyme activities of isolate N2 93
Table 3.17: Degradation of various compounds by isolate N2 96
Table 3.18: Resistance of isolate N2 to antibiotics 97
Table 3.19: Growth of isolate N2 at different temperature 98
Table 3.20: Growth of isolate N2 at different pH 98
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Table 3.21: Growth of isolate N2 in the presence of inhibitory compounds 99
Table 3.22: Growth of isolate N2 on different carbon sources 100
Table 3.23: Growth of isolate N2 on nitrogen source 103
Table 3.24: Production of acid and gas by isolate N2 104
Table 3.25: rRNA gene sequence similarity of Streptomyces vayuensis
sp. nov. (strain N2) with species of Streptomyces exhibiting
95% similarity at the rRNA gene level as determined by
BLAST 110
Table 3.26: Phenotypic and chemotaxonomic characteristics that
differentiate Streptomyces vayuensis sp. nov. (strain N2)
from the closely related species of the genus Streptomyces 114
Table 3.27: Differences in the phenotypic and chemotaxonomic
features of isolate N2 and S. violaceusniger, the
phylogenetically nearest neighbour 116
Table 3.28.A: 1H and 13C NMR chemical shifts of compound 128
Table 3.28.B: Mass spectrometry; fragmentation of inhibitor in EI-MS (m/z) 129
Table 3.29: Physico-chemical properties of the inhibitor 130
Table 3.30: Selection of inoculum media for production of streptolipin 139
Table 3.31: A list of standard media for the production of antibiotics 145
Table 3.32: Screening of standard media for the production of
streptolipin 146
Table 3.33: Effect of different carbon sources on production of
streptolipin 147
Table 3.34: Effect of inorganic nitrogen sources on production
of streptolipin 150
Table 3.35: Effect of organic nitrogen sources on production of
streptolipin 150
Table 3.36: Effect of different amino acids on production of streptolipin 150
Table 3.37: Effect of trace elements on production of streptolipin 151
Table 3.38: Effect of lipids on production of streptolipin 154
Table 3.39: Production of streptolipin on different nitrogen sources in
presence of 15 g/L Galactose 156
13
Table 3.40: Production of streptolipin on different nitrogen sources in
presence of 15 g/L sodium pyruvate 157
Table 3.41: Effect of initial glucose concentration on biomass synthesis 158
Table 3.42: Effect of initial glucose concentration on streptolipin synthesis 166
Table 3.43: Effect of initial glucose concentration on glucose utilization pattern 166
Table 3.44: Effect of initial yeast concentration on biomass formation 170
Table 3.45: Effect of initial yeast extract concentration on streptolipin synthesis 170
Table 3.46: Comparison of medium studies before and after
optimization in shake flask 174
Table 3.47: Comparison between shake flask and fermentor studies
after optimization of media 176
Table 3.48: Choice of the extraction solvent 180
Table 3.49: Effect of streptolipin on faecal excretion of triglycerides
(Single dose study) 187
Table 3.50: Effect of streptolipin on faecal excretion of triglycerides
(Multiple dose study) 188
Table 3.51: Effect of streptolipin on serum lipid profile
(Single dose study) 193
Table 3.52: Effect of streptolipin on serum lipid profile
(Multiple dose study) 194
Table 3.53: Effect of streptolipin on liver lipid profile
(Single dose study) 195
Table 3.54: Effect of streptolipin on liver lipid profile
(Multiple dose study) 196
Table 3.55: Effect of streptolipin on liver and perirenal fat weight
(Single dose study) 197
Table 3.56: Effect of streptolipin on liver and perirenal fat weight
(Multiple dose study) 198
Table 3.57: Effect of streptolipin on activities of hepatic antioxidant
enzymes (Multiple dose study) 201
Table 3.58: Effect of streptolipin on the activities of plasma non-specific enzymes (Multiple dose study). 202
14
LIST OF FIGURES
Figure No. Title Page No.
Figure 1.1: Lipase-catalyzed hydrolysis of p-nitrophenyl acetate. 4
Figure 1.2: Proposed reaction mechanism showing acylation and
deacylation via the formation of an acylenzyme intermediate. 5
Figure 2.1: Sample preparation for scanning electron microscope 22
Figure 3.1: Screening of actinomycetes on starch casein agar 66
Figure 3.2: Isolated culture slants of actinomycetes 67
Figure 3.3: Isolation and screening of actinomycetes isolates 73
Figure 3.4: Scanning electron micrograph showing N2 warty spores
and spiral spore chains 88
Figure 3.5: Culture growth on glycerol aspargine agar 91
Figure 3.6: Mass spectra of menaquinones isolated from N2 106
Figure 3.7: Cellular fatty acids of the isolate N2 107
Figure 3.8: Neighbour-joining tree based on 16S rRNA gene sequences
showing the phylogenetic relationship between Streptomyces
vayuensis sp. nov. and other species of the genus Streptomyces
and related reference microorganisms. Bootstrap values
(expressed as percentages of 1000 replications) greater than
50% are given at the nodes 111
Figure 3.9: Purification protocol of lipase inhibitor 118
Figure 3.10: Adaptation of the PNPB (p-nitrophenylbutyrate) spectrophotometric
assay to TLC system 120
Figure 3.11: IR-spectrum of inhibitor, isolated from Streptomyces vayuensis 121
Figure 3.12: 1H NMR spectrum of inhibitor from Streptomyces vayuensis 122
Figure 3.13: 13C NMR spectrum of inhibitor from Streptomyces vayuensis 123
Figure 3.14: LCMS of inhibitor from Streptomyces vayuensis 124
Figure 3.15: Chemical structure of streptolipin 127
Figure 3.16: Concentration dependent inhibition of streptolipin on
pancreatic lipase 132
Figure 3.17: [V] versus [S] plot in the presence of different fixed
15
concentrations of streptolipin 133
Figure 3.18: L-B plot of 1/[V] vs1/[S] in the presence of different fixed
concentrations of streptolipin 134
Figure 3.19: The slope (km/Vmax) of the lines described from the double
reciprocal plot are plotted against the streptolipin concentration
in order to derive the Ki value for the inhibitor 135
Figure 3.20: Standard curve for streptolipin 137
Figure 3.21: Time course fermentation of streptolipin production 138
Figure 3.22: Effect of temperature on production of streptolipin 140
Figure 3.23: Effect of initial pH on production of streptolipin 141
Figure 3.24: Effect of aeration on production of streptolipin 143
Figure 3.25: Effect of dipotassium hydrogen sulphate on production of
streptolipin 152
Figure 3.26: Effect of zinc sulphate on production of streptolipin 153
Figure 3.27: Effect of molasses on production of streptolipin 155
Figure 3.28: Variation of total biomass with initial glucose concentration 160
Figure 3.29: Semilogarithmic plot of biomass with time 161
Figure 3.30: Monod's plot of initial specific growth rates vs substrate
concentration 162
Figure 3.31: Double reciprocal plot of specific growth rate against the initial
glucose concentration 163
Figure 3.32: Variation of total streptolipin with initial glucose concentration 164
Figure 3.33: Rates of streptolipin formation at different initial glucose
concentration 165
Figure 3.34: Time course of initial glucose consumption during fermentation 167
Figure 3.35: Rates of glucose utilization at different initial glucose concentration 168
Figure 3.36: Influence of initial yeast extract concentration on cell growth 172
Figure 3.37: Influence of initial yeast extract concentration on streptolipin
production 173
Figure 3.38: Production of streptolipin with optimum conditions on shake flask 175
Figure 3.39A: Production of streptolipin in 10L laboratory fermentor 177
Figure 3.39.B: 10 L Fermentor study 178
16
Figure 3.40: Effect of biomass to ethyl acetate ratio on extraction efficiency of
streptolipin 182
Figure 3.41: Effect of the extraction time of streptolipin during static and
agitated conditions 183
Figure 3.42: Effect of sequential extraction on streptolipin recovery from
the biomass 184
Figure 3.43: Effect of streptolipin on absorption of dietary triglyceride
(Single dose) 189
Figure 3.44: Effect of streptolipin on absorption of dietary Triglyceride
(Multiple dose) 190
Figure 3.45: Effect of streptolipin on activity of intestinal lipase 191
Figure 3.46: Effect of streptolipin in serum lipid peroxides in serum
(circulation) and liver (Multiple dose study) 200
17
1.1. Actinomycetes:
Microorganisms are among man’s best friends and worst enemies. Exploitation of
microorganisms for useful purpose is known since 6000 B.C. History reveals many
applications of microbial processes for the production of desirable materials. The harnessing of
the activities of microorganisms represent one of the most fascinating aspects of man’s
scientific and technological development. From the stand point of industrial microbiology,
microorganisms can be considered as chemical factories in miniature. They have the potential
to produce novel and new therapeutic agents and to convert relatively inexpensive raw
materials into end products of value for human use, thus becoming attractive for commercial
exploitation.
Actinomycetes are that group of intracellular branching organisms which reproduce
either by fission or by means of spores or conidia. From an ecological point of view,
actinomycetes stand in an intermediate position between the fungi and the bacteria in terms of
numerical frequency of occurrence in various biotypes. They are closely related to the true
bacteria and frequently they are considered as higher filamentous bacteria. The outstanding
fungal characteristic of actinomycetes is morphological- possession of a true branching
mycelium. In addition actinomycetes may also show strong parallels with the true fungi in their
production of sporangia and motile spores. However, mycelium diameter and spore size is of a
lower order of magnitude in actinomycetes averaging 1µm only as compared with fungi. They
usually form a mycelium, which may be of a single kind designated as substrate (vegetative),
or of two kinds, substrate (vegetative) and aerial mycelium. They produce a wide variety of
spore types which includes the endospore, long regarded as the typical spore structure of
eubacterials. Some genera such as Streptomyces and Micromonospora form an extensive
branched mycelium composed of individual hyphae, subdivided by frequent cross walls. The
actinomycetes are predominantly aerobic, heterotropic and saprophytic. They have a cell wall
structure characteristic of bacteria and frequently show the presence of lytic viruses
(actinophages).
It is estimated that 85% of the antibiotics from microbial sources are being produced by
actinomycetes. Since the discovery of streptomycin by Waksman in 1943, interest of many
18
microbiologists was switched over to explore naturally occurring substrates for isolation of
novel actinomycete strains and novel metabolites with resounding commercial success. This is
the main reason for prominence of conventional screening programmes even in this age.
Many biological molecules are known to inhibit specific enzymes. As several diseases
are associated with abnormal enzyme activities, this concept has yielded valuable
pharmaceutical compounds. They can be called also as “target-enzyme” inhibitors and display
distinct pharmaceutical action depending on the target enzyme. The screening of microbial
culture filtrates for low molecular mass enzyme inhibitors was initiated by Hamao Umezawa in
1966. The first inhibitor, leupeptin, was discovered in 1969 as a metabolite of Streptomyces
strain. Since then many inhibitors have been discovered in culture filtrates of actinomycetes,
bacteria and fungi (Aoyagi and Takeuchi (1989) and some have been introduced commercially
(Umezawa, 1982).
1.2. Lipases:
Lipases (EC 3.1.1.3, triacylglycerol hydrolases) are ubiquitous enzymes playing a
pivotal role in all aspects of fat and lipid metabolism in variety of organisms. In humans and
other vertebrates, a variety of lipases control the digestion, absorption and reconstitution of fat
as well as lipoprotein metabolism (Desnuelle, 1986). In plants, during post germination, the
metabolism of oil reserves provide energy and carbon skeleton for embryonic growth and is
controlled by the action of lipases (Huang, 1987). Microorganisms such as bacteria and fungi
are also known to produce a wide spectrum of extracellular lipid degrading enzymes to
breakdown the insoluble lipid into soluble polar components to facilitate absorption (Lie et al,
1991).
Lipases belong to the hydrolase group of enzymes, which catalyze the hydrolysis of
glyceride ester bonds. They are also termed as acylglycerolases, acyl hydrolases or
triacylglycerol hydrolases. Their substrate specificities are wide and compounds other than
acylglycerols are also hydrolyzed. These enzymes can also be considered as transferases since
the fatty acids released is transformed to water or some other compound having a free hydroxyl
group related moiety (nucleophile).
19
One major characteristic of lipases is that they are more active with insoluble ester
substrates as against esterases that act only on soluble ester substrates. Lipases also hydrolyze
water soluble substrates which is characteristic of esterases. Thus lipases can be considered as
a special class of esterases, that is, esterases with high activity towards water insoluble
substances (Sarda and Desnuelle, 1958; Entressangles and Desnuelle, 1968). Until recently,
little was known about the molecular basis of lipid hydrolysis. A number of hypotheses have
been put forward with respect to the mechanism of interfacial activation of lipases. These
involve-(a) increased substrate availability by displacement of water shell around the ester
molecules (Brockerhof, 1968): (b) increase in substrate concentration at the interface
(Brockman et al, 1973): (c) better orientation of the ester bond to be cleaved (Wells, 1974;
Sarda and Desnuelle, 1958): (d) conformational change in the enzyme could be responsible for
the enhancement of activity at the oil-water interface.
1.2.1. Reaction mechanism:
Lipases and esterases contain an active Ser in a consensus sequence G-X-S-X-G which is
reminiscent of the pentapeptides in serine proteases (Gly-Asp-Ser-Gly-Gly in the trypsin
family, and Gly-X-Ser-X-Ala in subtilisin) (Brenner, 1988). Most lipases are susceptible to
inactivation with classic serine potease inhibitors, such as diisopropylphosphofluoridate and
diethyl-p-nitrophenyl phosphate indicating that lipases belong to the mechanistic class of serine
proteases. Recent structural analyses have been shown conclusively that lipases contain the
same constellation of the catalytic triad, Ser … His… Asp, present in all serine proteases,
although the topological position of the individual residues side chain varies (Winkler et al,
1990; Brady et al, 1990). It has also been demonstrated that the hydrolysis of dissolved p-
nitrophenyl acetate by pancreatic lipase proceeds via an acylenzyme intermediate (Figure 1.1).
20
O2N OCOCH3k-1
k1 O2N OCOCH3E------E +
[Enzyme-Substrate] Complex
O2N O-Acylation k2
O
CH3E
Acyl-enzyme
k3H2O
Deacylation
E+CH3COOH
Figure 1.1: Lipase-catalyzed hydrolysis of p-nitrophenyl acetate
The following mechanism is suggested to occur based on the catalytic activity pathway of
serine proteases (Figure 1.2).
1. The enzyme first binds the substrate to form a Michaelis-Menten adsorption complex.
2. Nucleophilic attack by the essential Ser-OH on the acyl carbon of the substrate yields a
covalent tetrahedral intermediate. This step is facilitated by general base catalysis by
the His in the triad.
3. A colipase of the intermediate to an acylenzyme involves the His-catalyzed protonation
of the ester oxygen of the leaving group.
4. In the following step, deacylation of the acylenzyme occurs with H2O assisted by
general base catalysis involving the His, resulting in the formation of tetrahedral
intermediate.
5. Finally protonation of the Ser causes the break down of the intermediate,
resulting in liberation of the product as a carboxylic acid.
21
coo-
Asp
......... NH N+
His
.......H-O:
Ser
coo-
Asp
........ NH N+
His
.....H.....R1
O
O
R2
R1O
:O-
R2
O
Ser R1OH
coo-
Asp
........ NH N+
His
R2
O
O
Ser
ACYLATION
R2
O
O
Ser
coo-
Asp
......... NH N+
His
OH
H
:
DEACYLATION....
NH NH+
His
.....H.....
HO
:O-
R2
O
Ser R2 COOH
.... coo-
Asp
NH N
His
......coo-
Asp
............OH
Ser
. . .. ..
..... .
ACYL ENZYME
Figure 1.2: Proposed reaction mechanism showing acylation and deacylation via the formation of an acylenzyme intermediate
1.2.2. Specificity of lipases:
The substrate specificity of lipase is defined by its positional specificity or
stereospecificity or its preference for short or long chain saturated or unsaturated fatty acids
(Brockerhoff and Jensen, 1975). Some lipases have affinity for short chain fatty acids (acetic,
butyric, capric, caproic, caprylic etc), some for unsaturated fatty acids (oleic, linoleic, linolenic
etc), while many others are non specific and randomly split the fatty acids from the
triglycerides. From the glycerol side of the triglycerides, the lipases often show positional
specificity and attack the fatty acids at 1 or 3 carbon position of glycerol or at both the
positions, but not the fatty acid at the 2 position of the glycerol molecule. However, through
random acyl migration, the 2 fatty acid monoglyceride undergoes rearrangement pushing the
fatty acid to the 1 or 3 position of the glycerol molecule: as acyl migration is a slow process
and as the available lipases do not act on glycerol 2 mono fatty acid esters, the hydrolysis
22
slows down and awaits the acyl migration to complete for enabling the lipase to attack the
glyceride at 1 or the 3 position (Saxena et al, 1999).
1.2.3. Pancreatic lipase:
Pancreatic lipase (PL) hydrolyses the water insoluble triacylglycerols in the intestinal
lumen and thereby plays an important role in the dietary fat absorption. Pancreatic lipase has a
molecular weight of approximately 50 kDa and has been isolated from a number of
mammalian species. Triglyceride hydrolysis by PL is inhibited by physiological concentrations
of bile salts. This inhibition can be overcome by the addition of co-lipase, a small pancreatic
protein that binds to the lipase and to lipid micelles. The crystal structure of human pancreatic
lipase has been refined to a resolution of 2.3 A0 (Winkler et al, 1990; Gubernator et al, 1991).
Its amino-acid sequence, comprises 449 residues for the mature enzyme. The protein is folded
into two domains, a larger N-terminal domain (N domain) comprising residues 1-335 and a
smaller C-terminal domain (C domain). The core of the N domain is formed by nine-stranded
β-pleated sheet in which most of the strands run parallel to one another. Seven α-helical
segments of varying length occur in the strand connections and six of them pack against the
two faces of the core sheet. The C domain is formed by two layers of antiparallel sheet, the
strands of which are connected by loops of varying length. The N domain contains the active
site, a glycosylation site a Ca2+- binding site and possibly a heparin-binding site. In the
crystalline form, the active site is buried beneath a surface loop, termed the flap. In this form
the enzyme cannot be enzymically active.
The enzyme consists of six disulfide bridges, Cys4-Cys10, Cys91-Cys102, Cys238-
Cys262, Cys286-Cys297, Cys300-Cys305 and Cys434-Cys450 with two free cysteines, Cys
104 and Cys 182. The porcine pancreatic enzyme is N-glycosylated at Asn167. The
glycosylation site Asn167-Gly168-Thr 169 in the porcine enzyme is conserved in the human
and canine lipases, and the glycan structure of porcine pancreatic lipase has been elucidated
(Caro et al, 1983; Benkouka et al, 1982, Hermoso et al, 1996; Bourne et al, 1994; Fournet at al,
1987). In contrast to the human pancreatic lipase, porcine and canine ovine and bovine
enzymes are not glycosylated. Unlike most of the pancreatic enzymes which are secreted as
proenzymes and further activated by proteolytic cleavage in the small intestine, pancreatic
23
lipase is directly secreted as a 50 kDa active enzyme. More information is available in reviews
on lipases (Rovery et al, 1978; Brady et al, 1990; Brazozowski et al, 1991; Uppenberg et al,
1994; Grochulski et al, 1994; Derewenda et al, 1994a; van Tilbeurgh et al, 1993; Bourne et al,
1994; Lawson et al, 1992).
1.3. Obesity a medical complication caused by lipase:
Excessive intake of dietary fat contributes to the development and maintenance of both
obesity and hyperlipidemia. It is generally accepted that obesity is not merely a cosmetic
problem, but that it also represents an unhealthy state, currently accepted as an illness. Obesity
is defined as an abnormal increase in body fat and is associated with a high basal metabolic
rate, low levels of physical activities, low rate of fat oxidation, increased insulin sensitivity,
low sympathetic nervous system activity and low plasma leptin concentration, heart disease
and stroke leading to an increased rate of mortality (Guzelhan et al, 1991; Drent and
Vanderveen, 1993; Guzelhan et al, 1994; Drent and Vanderveen, 1995; Drent et al, 1995).
Although obesity may be advantageous during starvation, excessive body fat is
associated with a long list of serious medical complications. The evidence surrounding this
concept reinforces the notion that effective obesity therapy must be directed towards limiting
the intake or absorption of dietary energy, whether through behavioral modification of eating
habits, effective pharmaceutical suppression of appetite or selective disruption of the normal
processes of digestion, whereas it is nearly axiomatic that behavioral modification is effective
only for those few whose time, motivation and financial resources permit intensive training in
eating and exercise habits and where as anorectic drugs pose unacceptable or potential risks,
the focus now turns to selective blockade of dietary fat absorption through inhibition of the
pancreatic enzyme lipase.
Conventional treatment for obesity has focused largely on strategies to control energy
intake. As obesity results from an imbalance between energy intake and energy expenditure,
methods to treat obesity can be divided into three groups; (a) reducing food intake (b) altering
metabolism and (c) increasing thermogenesis or increase energy expenditure.
24
1.3.1.a. Reduction of food intake:
1.3.1.a.1. Noradrenergic receptors:
A number of monoamines and neuropeptides are known to modulate food intake. Both
noradrenergic receptors and serotonergic receptors have been used as the site for clinically
useful compounds to decrease food intake (Bray, 1997; Bray et al, 1995; Bray and Inoue, 1992;
Bray, 1998; Frankish et al, 1995; Arch et al, 1981). Activation of the α1- and β2- adrenoceptors
decreases food intake. In animals however, stimulation of the α2-adrenoceptor increases food
intake. Direct agonists and drugs that release or block norepinephrine is released.
Phenylpropanolamine is an α1-agonist that decreases food intake by acting on α1-adrenergic
receptors in the paraventricular nucleus. The weight gain observed in patients treated for
hypertension or prostatic hypertrophy with α1 adrenergic antagonist indicates that the α1
adrenoceptor is clinically important in regulation of body weight. Stimulation of the β2-
adrenoxceptor by agonists such as terbutaline, clenbuterol or salbutamol reduces food intake.
1.3.1.a.2. Serotonergic receptors:
The serotonin 5-HT receptor system consists of seven families of receptors. Stimulation
of receptors in the hydroxytryptamine 5-HT1 and 5-HT2 families have the major effects on
feeding. Activation of the 5-HT1A receptor increases food intake but this acute effect is rapidly
down-regulated and is not clinically significant in regulation of body weight. Activation of 5-
HT2C and possibily 5-HT1B receptor increases food intake. Direct aganosits (quipazine) or
drugs that block serotinin reuptake (fluxetine, sibutramine, sertraline, fenfluramine) will reduce
food intake by acting on these receptors or by providing serotinin that modulates these
receptors (Toubro and Astrup, 1997; Davis and Faulds, 1996; Levitrsky and Troiano, 1992;
Mayer and Walsh, 1998).
1.3.1. b. Increased energy expenditure:
Increased energy expenditure through exercise would be an ideal approach to treating
obesity. Drugs that have same physiologic consequences as exercise could provide useful
25
pharmaceutical ways of treating obesity (Astrup et al, 1990; Dulloo and Miller, 1986; Vansal
and Feller, 1999; Colker et al, 1999).
1.3.1.c. Altered metabolism:
Excess fat is the visible sign of obesity. Metabolite strategies have been directed to pre
absorptive and post absorptive mechanisms of modifying fat absorption or metabolism (Zhi et
al, 1994). Pre absorptive mechanisms that influence digestion and absorption of macronutirents
have been studied which inhibit intestinal digestion of fat and lowers body weight. The second
strategy is to effect intermediary metabolism such as enhancing lipolysis, inhibiting
lipogenesis, and affecting fat distribution between subcutaneous and visceral sites.
Pancreatic lipase is a key enzyme for lipid breakdown that leads to the absorption of
fatty acids. Pancreatic lipase, one of the exocrine enzymes of pancreatic juice, catalyzes the
hydrolysis of emulsified esters of glycerol and long chain fatty acids. Short chain fatty acids
can be directly absorbed into the blood, while long-chain fatty acids and monoglycerides
combine with bile salts to form water soluble micelles. The micelles are absorbed into the
mucosal cells of the intestine and the fatty acids and monoglycerides are resynthesized into
triglycerides. Dietary triglyceride is usually stored in the adipose tissue. Pharmacological
agents that reduce the absorption of dietary triglycerides, reduce the probability of the
formation of atherosclerotic plaque.
26
Potent and specific lipase inhibitors are of special interest for four reasons:
1. They find applications as antiobesity agents.
2. They contribute to a better understanding of the mechanisms of lipase action.
3. They help to understand the non-catalytic functions of lipases.
4. They may find applications for the treatment of infectious diseases.
1.4. Sources of lipase inhibitors:
1.4.a. Lipase inhibitors of microbial origin:
1.4.a. 1. Lipstatin:
Lipstatin, a novel and very potent inhibitor of pancreatic lipase has been isolated from
Streptomyces toxytricini. Its hydrogenated analogue, tetrahydolipstatin (THL) ((s)-1-{[(1s, 2s,
3s)-3-hexyl-4-oxo-2-oxetanyl] methyl} dodecyl-(s)-1-fomamido-4-methylvalerate) has
selective inhibitory action for pancreatic lipase, where as phospholipase A2, amylase and
trypsin activity was not altered by THL. Lipstatin contains beta lactone structure carrying two
aliphatic residues with a chain length of 6 – 13 carbon, that probably accounts for the
irreversible lipase inhibition. The inhibition is due to covalent binding of THL to ser 152,
which is one of the residues in the catalytic trial of this enzyme (Hadvary et al, 1991). Lipstatin
is closely related to the esterase inhibitor esterastin, which contains a n-acetyl asparagine side
chain instead of N-formyl leucine. Lipoprotein lipase was rapidly inactivated by low
concentration of the inhibitor tetrahydrolipstatin (Weibel et al, 1987).
o ooo
NH CHO
Tetrahydrolipstatin
27
1.4.a.2. Panclicins:
Panclicins A, B, C, D and E are novel pancreatic lipase analogues of tetrahydrolipstatin,
which contain a β - lactone and a N- formyl leucine ester, isolated from Streptomyces sp. The
potency of the inhibitory activity of each compound is attributed to the amino acid moiety of
each structure. The panclicins are either glycine- type compounds such as Panclicin A, C, D, E
which are two or three fold more potent than THL or alanine type compounds such as
Panclicin A and B, which are less potent than the glycine type compounds. They irreversibly
inhibit pancreatic lipase. However, the compounds do not irreversibly inhibit the enzyme as
strongly as THL (Mutoh et al, 1994).
1.4.a.3. Marine algae:
The presence of an inhibitor of pancreatic lipase (triacylglycerol acylhydrolase) was
screened in 54 marine algae. An active inhibitor, caulerpenyne, was purified from an extract
Caulerpa taxifolia using ethylacetate extraction. Caulerpenyne competitively inhibited lipase
activities using emulsified tirolein and dispersed 4-methylumbelliferyl oleate (4-MU oleate) as
substrates. The concentrations producing 50% inhibition against triolein and 4-MU oleate
hydrolysis were 2mM and 13 µM respectively (Bitou et al, 1999).
1.4.a.4. Ebelactone B:
NHCHO
CO
O OO
CH3(CH2)6 (CH2)7CH(CH 3)2
3HC
Panclicin A
AcO
OAcOAcCaulerpenyne
28
Ebelactone A and B, natural products from Streptomyces aburaviensis are potent
inhibitors of pancreatic lipase. Ebelactone B inhibited, in a dose dependent manner, the
intestinal absorption of fat in animals. The most effective inhibition was observed when the
inhibitor was administered 60 min prior to fat feeding. When ebelactone B was administered
at 10mg/kg, the serum level of TG and cholesterol were decreased by 58 and 35 %
respectively. Since ebelactone B is effective inhibitor for fat absorption, it may be a promising
molecule for therapy of hyperlipidemia and obesity (Umezawa, 1980).
1.4.b. Lipase inhibitors from plant sources:
1.4.b.1. Grape seed extracts:
Grape seed extract, rich in bioactive phytochemicals, showed inhibitory activity on the
fat-metabolizing enzymes pancreatic lipase and lipoprotein lipase, thus suggesting that grape
seed extracts might be useful as a treatment to limit dietary fat absorption and the accumulation
of fat in adipose tissue. The reduction in intracellular lipolytic activity of cultured 3T3-L1
adipocytes indicated reduced levels of circulating free fatty acids linked to insulin resistance in
obese patients (Moreno et al, 2003).
1.4.b.2. Carnosic acid:
The methanolic extract from the levels of Salvia officinalis L (sage) showed inhibitory
effect on serum triglyceride elevation in olive oil fed mice (500 and 1000 mg/kg) and
inhibitory activity (IC50 94 µg/mL) against pancreatic lipase. Through bioassay-guided
separation using inhibitory activity against pancreatic lipase activity, 4
OH OO
O
R
Ebelactone A(R=Me)B(R=Et)
29
abietan-type diterpenes (carnosic acid, carnosol, royleanonic acid, 7-methoxyrosmanol) and a
triterpene (oleanolic acid) were isolated from the active fraction. Among these compounds,
carnosic acid and carnosol substantially inhibited pancreatic lipase activity with IC50 values of
36 µM and 13 µM respectively. Carnosic acid significantly inhibited triglyceride elevation in
olive oil fed mice at doses of 5-20 mg/kg. However, other constituents (carnosol, rorleanonic
acid, oleanoic acid) did not show any effects even at a dose of 200 mg/kg. Furthermore,
carnosic acid (20 mg/kg/day) reduced the gain of body weight and the accumulation of
epididymal fat weight in high fat diet-fed mice after 14 days (Ninomiya, et al, 2004).
1.4.b.3. Flavan dimmers:
Flavan dimmers which showed lipase-inhibiting effects were isolated from fruits of
Cassia nomame (Leguminosae). Structures of two new compounds among them were
determined to be (2S)-31, 41, 7-trihydroxyflavan-(4-8)-catechin. Four flavan dimers structurally
related to these two compounds were also synthesized for spectral comparision. Among 10
flavan dimmers tested for lipase-inhibitory activity, (2S)-31, 41, 7-trihydroxyflavan-(2S)-
catechin showed the most potent inhibitory effect (Hatano et al, 1997).
1.4.b.4. Dioscorea nipponica:
COOHHO
OH
Carnosic acid
OH
OH
OH
OH
O HO
HO O
OH H
H H OH
Flavan dimmer
30
The methanol extract of Dioscorea nipponica Makino powder appeared to have potent
inhibitory activity against pancreatic lipase with an IC50 value 5-10 µg/ml. Further purification
of active components present in the herb generated dioscin that belongs to the saponin family.
Dioscin and its aglycone, diosgenin, both suppressed the time dependent increase of blood
triacylglycerol level when orally injected with corn oil to mice, suggesting their inhibitory
potential against fat absorption. Sorauge-Dawley rats fed on a high-fat diet containing 5%
Dioscorea nipponica Makino and 40% beef tallow gained significantly less body weight and
adipose tissue. The IC50 of the dioscin (20 µg/ml), diosgenin (28), gracillin (28.9), pro-
sapogenin A (1.8), pro-sapogenin C (42.2) were reported (Kwon et al, 2003).
O
Me
Me
Me
O
O
Me
α-D-Rha(1 4)
α-L-Rha(1 2)
β-D-Glc
Dioscin
1.4.b.5. 5-hydroxy-7-(41-hydroxy-31-methoxyphenyl)-1-phenyl-3-heptone:
A pancreatic lipase inhibitor, 5-hydroxy-7-(41-hydroxy-31-methoxyphenyl)-1-phenyl-3-
heptone (HPH), from the rhizome of Alpinia officinarum was isolated and its
antihyperlipidemic activity was measured. HPH inhibited pancreatic lipase with an IC50 value
of 1.5 mg/ml. HPH significantly lowered the serum triglyceride in corn oil feeding-induced
triglyceridemic mice and reduced serum triglyceride and cholesterol in Triton-WR-1339-
induced hyperlipidemic mice. However, HPH did not show hypolipidemic activity in high
cholesterol diet-induced hyperlipidemic mice (Shin et al, 2004).
OCH
OH5-hydroxy-7-(4 1-hydroxy-3 1-methoxyphenyl)-1-phenyl-3-heptanone
31
1.4.b.6. 3-methylethergalangin:
The pancreatic lipase inhibitory activity of rhizome of Alpinia officinarum and its
antihyperlipidemic activity were measured. 3-methylethergalangin was isolated as an inhibitor
of pancreatic lipase with an IC50 of 1.3 mg/ml in the mice. It inhibited the serum triglyceride
level in corn oil feeding-induced triglyceridemic mice and serum triglyceride and cholesterol in
triton WR-1339-induced hyperlipidemic mice. However, it did not show hypolipidemic
activity in cholesterol diet-induced hyperlipidemic mice (Shin et al, 2003).
1.5. Scope of the present investigation:
In the area of microbial metabolites, a new horizon was opened about 3 decades ago
when the systematic search for small molecular enzyme inhibitors and other bioactive
metabolites from fermentation broths started.
Although a number of investigations have been carried out on the isolation of
pancreatic lipase inhibitors from synthetic, plant and microbial sources, only a few compounds
were reported from actinomycetes. Moreover, there are only a few systematic studies on the
screening, isolation, characterization and media optimization for the production of pancreatic
lipase inhibitors.
O
O
OCH3
3-methylethergalangin
32
In spite of the development of synthetic compounds for pancreatic lipase inhibition,
only a few compounds have gone to the marketing stage. Further, there is a need for the
isolation of new and novel metabolites from the available natural sources, thus leading to the
development of powerful inhibitors.
The present work is an attempt to screen, isolate and characterize pancreatic lipase
inhibitor from actinomycetes. The work carried out in this investigation has been presented in
four chapters. The first chapter surveys the existing information on natural sources.
Actinomycetes and their importance in producing bioactive metabolites and various aspects of
the role of pancreatic lipase and its inhibitors. The second chapter deals with various
experimental methods and analytical procedures used in the investigation. The third chapter
deals with the experimental results and discussion, is further sub-divided into five sections.
Section 1 deals with the screening and selection of actinomycetes for the production of
pancreatic lipase inhibitor. Section 2 deals with characterization of the selected culture. Section
3 deals with isolation and characterization of a new inhibitor designated as streptolipin along
with its biological activities and kinetic parameters. Section 4 deals with studies on
optimization of; physical, nutritional and downstream process parameters for the maximum
production of streptolipin. Section 5 deals with the invitro studies of streptolipin on pancreatic
lipase inhibition along with the hepatoprotective activity and comparative studies with a
commercially available inhibitor, Orlistat. The last chapter gives the summary and highlights
of the present investigation.
33
2.1. Screening of actinomycetes:
2.1.A. Collection of different terrestrial substrates for selective isolation of
actinomycetes:
Fifteen soil samples from different ecosystems like tea plantations, forest, lake, garden,
desert, hill station, cow barn yard, sugarcane, coconut and rice fields areas were collected and
air dried.
2.1.B. Pretreatment of soil samples:
The terrestrial soil samples were pretreated by the following methods:
2.1.B.1. Calcium carbonate treatment:
10 g of the soil sample was ground in a mortar with calcium carbonate in the ratio (1:1)
and incubated for 10 days at 28oC in a closed inverted sterile petri dish with water saturated
filter paper discs (Tsao et al, 1960). From this 2 g of sample was mixed with 100 ml of sterile
distilled water and agitated by shaking on a rotary shaker at 220 rpm for 30 minutes at 280C
and the supernatant was then serially diluted with sterile distilled water.
2.1.B.2. Dry heat treatment:
10g of soil sample was treated at 100oC for one hour (Nonomura and Ohara, 1969). Of
this, 1 g of sample was then mixed with 100 ml of sterile distilled water, agitated by shaking on
a rotary shaker at 220 rpm for 30 minutes at 280C and the supernatant was then serially diluted
with sterile distilled water.
2.1.B.3. Phenol treatment:
34
10 g soil was mixed with 100 ml of sterile distilled water in a 500 ml Erlenmeyer flask
and agitated on a rotary shaker at 220 rpm for 5 minutes at 280C. This suspension was then
mixed with 100 ml of 1.4 % (w/v) phenol solution and incubated for 10 minutes. The
supernatant was serially diluted with sterile distilled water (Lawrence 1956).
2.1.B.4. Centrifugation method:
10 g of the sample was mixed with 100 ml of sterile distilled water in a 500 ml
Erlenmeyer flask and agitated on a rotary shaker for 5 min at 280C. The soil suspension was
centrifuged for 20 minutes at 6000 rpm (Rehacek 1959). The supernatant was then serial
diluted with sterile distilled water.
2.1.B.5. Control (no pretreatment):
1 g of soil sample was mixed with 100 ml of sterile distilled water, and agitated on a
rotary shaker at 220 rpm for 30 minutes at 280C and the supernatant was then serial diluted
with sterile distilled water.
2.2. Media Composition:
2.2.A. Actinomycetes:
Soil samples were serially diluted up to 10-6 level. Of this, one ml was added to each of
50 ml of the sterile molten media maintained at 37 to 400C, thoroughly mixed and plated on
Starch Casein Agar (SCA) isolation medium and incubated at 280C for two weeks (Kuster and
Williams. 1964).
2.2.A.1. Medium for screening of actinomycetes for pancreatic lipase inhibitor:
Starch Casein Agar (g/L)
Soluble starch 10.0
Casein 0.03
35
Potassium nitrate 2.0
Di-potassium hydrogen ortho phosphate 2.0
MgSO4 7H2O 0.05
Calcium carbonate 0.02
Ferrous sulphate 0.01
Bacto agar 20.0
pH 7.0
The media was supplemented with rifampicin (antibacterial) 2.5µg/ml and fluconazole
(antifungal) 75µg/mL.
Potassium tellurite agar medium (g/L):
Potassium tellurite 0.1
Peptone 5.0
Yeast extract 2.5
Bacto-agar 20.0
pH 6.4
Half-strength nutrient agar medium (g/L):
Oxoid nutrient agar nutrients 14.0
Bacto-agar 10.0
pH 7.0
Oat meal agar (g/L):
Oat meal 20.0
Bacto-agar 20.0
20 g oatmeal was cooked or steamed in 1000 mL of distilled water for 20 minutes, filtered
through cheesecloth. Distilled water was added to make the volume of filtrate to 1000 mL.
Trace salt solution 1.0 mL
Bacto-agar 20.0
pH 7.2
All the above media were autoclaved at 1210C for 15 minutes
36
2.2.A.2. Culture maintenance medium:
Glycerol aspargine agar medium (g/L):
L-aspargine (anhydrous) 1.0
Glycerol 10.0
K2HPO4 (anhydrous) 1.0
Trace salt solution 1 mL
Bacto-agar 20
pH 7.0-7.4
Autoclaved at 1210C for 15 minutes. The cultures were subcultured on glycerol
aspargine agar slants. The slants incubated at 280C for two weeks were used for further
experiments.
2.2.A.3. Morphology and pigmentation:
For scanning electron microscopy studies, 14 day old culture of strain N2 grown on ISP
4 medium was used. Spore chain morphology, the presence of sclerotia, substrate spores and
fragmentation of substrate mycelium was examined by scanning electron microscopy LV 435
VP (Lieo Electron Microscopy, England). Sample preparation for scanning electron
microscope is given in Figure 2.1(Groth et al, 1997).
The macro and micro morphological features of the colonies developed on various
media and the colour determinations of the aerial mycelium, substrate mycelium and soluble
pigment were examined after 14 days of incubation. Macro morphology was noted by the
naked eye. The colours of aerial mycelium and substrate mycelium and soluble pigment when
grown on different media were observed and recorded.
Micromorphology was observed by placing the sterile cover slips at an angle of 450C in
solidified agar medium in a petri dish such that half of the cover slip was dipped in medium.
Inoculum was spread along the line where the upper surface of the cover clips meets the
medium. After complete sporulation, the cover slips were removed and examined directly
under the phase contrast microscope (Dietz and Mathews, 1971). The cell morphology of strain
37
N2 was examined by phase contrast microscope with a model BH-2 microscope (Olympus,
Tokyo, Japan). Colours were matched with one of the seven colour wheels of Tresner and
Backus (1963). Motility was studied by hanging drop method.
2.2.A.4. Cultural characteristics media:
The following media were used for studying the cultural characteristics of isolate N2
(Shirling and Gottlieb, 1966):
ISP 1: Tryptone- yeast extract broth
Bacto-Tryptone 5.0 g
Bacto-Yeast extract 3.0 g
Distilled water 1.0 liter
pH 7.0 - 7.2
ISP 2: Yeast extract-malt extract agar
Yeast extract 4.0 g
Malt extract 10.0 g
Dextrose 4.0 g
Agar 20.0 g
Distilled water 1.0 liter
pH 7.3 - 7.5
Figure 2.1: Sample preparation for scanning electron microscope: (Groth et al, 1997)
Biomass Treated with 2.5% glutaraldehyde in 0.1 M
acetate buffer (pH 4-5) Centrifuge
Pellet washed with 0.1 M phosphate buffer pH 7.0 For 15 minutes, at 40C (4 times)
Centrifuge Wash pellet with 50% acetone
Wash pellet with 70% acetone
38
Wash pellet with 80% acetone
Wash pellet with 90% acetone
Wash pellet with 95% acetone
Wash pellet with 100% acetone
Dry the sample under vacuum
Mounted on the SEM stumps
ISP 3: Oatmeal Agar
Oatmeal 20.0 g
Agar 18.0 g
Distilled water 1.0 liter
pH 7.2.
Cook or steam 20.0 g of oatmeal in 1.0 liter distilled water for 20 min. Filter through cheese
cloth. Add 18.0 g agar and make up to 1.0 liter. Add 1 ml of trace salts solution.
ISP 4: Inorganic salts-starch agar
39
Solution I: Soluble starch, 10.0 g. Make a paste of the starch with a small amount of cold
distilled water and bring to a volume of 500 ml.
Solution II:
CaCO3 2.0 g
K2HPO4 (anhydrous) 1.0 g
MgSO4. 7 H2O 1.0 g
NaCl 1.0 g
(NH4)2SO4 2.0 g
Distilled water 500.0 ml
Trace salt solution 1.0 ml
Agar 20.0 g
The pH should be between 7.0 and 7.4. Do not adjust if it is within this range. Mix solutions II
and I together. Add 20.0 g agar. Liquefy agar by steaming at 100°C for 10 to 20 min.
Trace salt solution:
CuSO4.5H2O 0.0064 g FeSO4.7H2O 0.0011 g MnCl2.4H2O 0.0079 g ZnSO4.7H2O 0.0015 g Distilled water 1.0 liter
ISP 5: Glycerol-aspargine agar
L-aspargine (anhydrous basis) 1.0 g
Glycerol 10.0 g
K2HPO4 (anhydrous basis) 1.0 g
Distilled water 1.0 liter
Trace salts solution 1.0 ml
Agar 20.0 g
The pH should be between 7.0 and 7.4. Do not adjust if it is within this range. Liquefy agar by
steaming at 100°C for 15-20 minutes. Sterilize in flasks for pouring into petri dishes.
40
ISP 6: Peptone-yeast extract-iron agar
Bacto-peptone iron agar 36.0 g
Bacto-yeast extract 1.0 g
Distilled water 1.0 liter
36.58 g of the dehydrated Bacto-peptone-iron agar was reconstituted into 1 L of distilled water,
sterilized at 1210C for 20 minutes and poured onto plates. The dehydrated media contained the
following ingredients.
Bacto-peptone 15.0 g
Protease peptone 5.0 g
Ferric ammonium citrate 0.5 g
Dipotassium phosphate 1.0 g
Sodium thiosulphate 0.08 g
Bacto-agar 15.0 g
pH 7.0
ISP 7: Tyrosine agar
Glycerol 15 g
L- tyrosine 0.5 g
L- aspargine 1.0 g
K2HPO4 (anhydrous) 5.0 g
MgSO4. 7H2O 5.0 g
NaCl 5 g
FeSO4. 7H2O 0.1 g
Trace salt solution 1 ml
Agar 20 g
Distilled water 1.0 liter
pH 7.0
Bennett’s agar
Beef extract 1.0 g
41
Glucose 10.0 g
N-Z amine A (enzymatic digest of casein) 2.0 g
Yeast extract 1.0 g
Agar 15.0 g
Distilled water 1.0 liter
pH 7.3
Nutrient agar
Peptone 5.0 g
Meat extract 3.0 g
Agar 15.0 g
Distilled water 1.0 liter
pH 7.0.
Czapek-dox agar
Sucrose 30.0 g
NaNO3 3.0 g
MgSO4.7 H2O 0.5 g
KCl 0.5 g
FeSO4.7 H2O 0.01 g
K2HPO4 1.0 g
Agar 15.0 g
Distilled water 1.0 liter
pH 7.2.
Modified Bennets Agar
Glycerol 10.0 g
Yeast extract 1.0 g
Beef extract 1.0 g
NZ- Amine 2.0 g
Agar 20.0 g
Distilled water 1.0 liter
42
pH 7.3
Glycerol-arginine agar:
Glycerol 12.5 g
Arginine 1.0 g
Sodium chloride 1.0 g
K2HPO4 1.0 g
MgSO4.7H2O 0.5 g
Fe2(SO4)3. 6H2O 0.01 g
CuSO4.5H2O 0.001 g
ZnSO4.7H2O 0.001 g
MnSO4.H2O 0.001 g
Distilled water 1.0 liter
Agar 20.0 g
pH 7.3
All the media were sterilized at 1210C for 20 minutes.
2.2.A.4.1. Enzyme activity test media:
Test for lipolysis and lecithinase: (Nitsch and Kutzner, 1969)
Modified Egg yolk medium (EY)
Bacteriological peptone 10.0 g
Glucose 1.0 g
NaCl 10.0 g
Yeast extract 5.0 g
Agar 12.0 g
Egg yolk emulsion 50.0 g
pH 7.0
Autoclaved at 1210C for 15 minutes
43
Spores were streaked in the center of the plate (white precipitate) to get a band like growth and
incubated at 280C for 14 days. Clearance of the precipitate was the measure of the activity.
Test for proteolysis: (Shirling and Gottlieb, 1966)
Milk casein agar:
Peptone 1.0 g
Agar 20.0 g
Sterile skimmed milk (10 %) 100ml
Distilled water 1.0 liter
pH 7.6
Autoclaved at 1210C for 15 minutes
The proteolytic activity was studied with milk-casein agar by clearing of the precipitate after
incubating the inoculated plates at 280C for 7 days.
Test for pectinolytic activity: (Hankin et al, 1971)
Pectinolytic activity was determined by using the following medium.
KH2PO4 4.0 g
Na2HPO4 6.0 g
Pectin 5.0 g
(NH4)2 SO4 2.0 g
Yeast extract 1.0 g
MgSO4. 7H2O 2.0 g
FeSO4.7H2O 0.001 g
CaCl2 0.001 g
Agar 10.0 g
pH 7.4
Autoclaved at 1210C for 15 minutes.
44
Spores were streaked in the center of the plate to get a band like growth and incubated
at 280C for 6 days. Hydrolysis zones were detected after 6 days by flooding plates with an
aqueous solution of hexadecyltrimethylammonium bromide (1% w/v).
Test for coagulation and peptonization of milk: (Shirling and Gottlieb, 1966)
Milk coagulation and peptonization test was carried out using 10% (w/v) skimmed
milk. The skimmed milk tubes were inoculated and incubated at 280C. The extent of
coagulation and peptonization was recorded on 3rd and 8th day.
Test for catalase production: (Williams et al, 1983)
Catalase production was detected by adding a few drops of 20% (v/v) H2O2 on to 7 day
old colonies grown on modified Bennets agar. Evolution of oxygen was detected under a
binocular microscope.
2.2.A.4.2. Degradation tests media:
Test for nitrate reduction: (Shirling and Gottlieb, 1966)
Nitrate broth:
Meat extract 3.0 g
Peptone 5.0 g
Potassium nitrate 1.0 g
Distilled water 1.0 liter
pH 7.2
Autoclaved at 1210C for 15 minutes
Reagents:
α-naphthylamine test solution:
α-naphthylamine 5.0 g
Conc.H2SO4 8.0 ml
Distilled water 1.0 liter
To the diluted sulphuric acid, α-naphthylamine was added and stirred until solution was mixed.
45
Sulphanilic acid test solution:
Sulphonic acid 8.0 g
Conc.H2SO4 48 ml
Distilled water 1.0 liter
Sulphuric acid was added to 500 ml of water. Then sulphanilic acid was added followed by
water to make up the volume.
Procedure:
5ml of nitrate broth medium was inoculated with a loopful of spores and incubated at
280C for seven days. Controls were also run without inoculation. On 7th day, the broth was
tested for the presence of nitrite. To 1 ml of the broth or control, two drops of sulphanilic acid
solution was added followed by two drops of α-naphthylamine solution were added. The
presence of nitrite was indicated by a pink, red or orange colour and absence of colour change
was considered as nitrite negative. In latter case presence or absence of nitrate in the broth
under examination was confirmed by adding a pinch of zinc dust after the addition of the
reagents, when the unreduced nitrate, if present, gave a pink, red or orange colour.
Test for H2S production: (Shirling and Gottlieb, 1966)
The inoculated peptone-yeast extract-iron agar (ISP 6) slants were incubated for 15
days at 280C. The slant was observed every 12 hrs upto 4 days and thereafter at 24 hrs
intervals, up to 15 days. Observation for the presence of the characteristic greenish brown,
brown, blackish brown, bluish black or black colour of the substrate was indicative of H2S
production.
Test for starch hydrolysis: (Cowan, 1974)
The organism was grown for seven days on 1% (w/v) starch agar plates. At the end of
the incubation period, the plates were flooded with iodine solution. Hydrolyzed zone around
the growth was observed.
46
Test for melanin formation: (Shirling and Gottlieb, 1966)
This test was carried out in tryrptone–yeast extract broth (ISP 1), yeast extract-iron agar
(ISP 6) and tyrosine agar (ISP 7) media. The inoculated tubes were observed every 12 hrs for 4
days. The inoculated tubes were compared with uninoculated controls. Deep brown, greenish
brown, greenish black or black colours were recorded as melanin positive. Absence of brown
to black colours or total absence of diffusible pigment was considered as negative for melanoid
pigment production.
Test for urea decomposition: (Gordon, 1968)
Media composition:
Peptone 20.0 g
NaCl 5.0 g
Mono potassium phosphate 2.0 g
Phenol red 0.012 g
Agar 20.0 g
Distilled water 1.0 liter
pH 7.0
Autoclaved at 1210C for 15 minutes
The media were inoculated heavily by spreading spores over the medium. The decomposition
was observed by characteristic red- violet colour on the plate
Test for arbutin degradation: (Kutzner, 1976)
Media composition:
Yeast extract 3.0 g
Arbutin 1.0 g
Ferric ammonium citrate 0.5 g
Agar 7.5 g
47
Distilled water 1.0 liter
pH 7.2
Autoclaved at 1210C for 15 minutes
Spores were spread over the medium and observed for brown to black pigment after 21 days.
Absence of brown-black pigment was taken as a negative test. A comparison with controls was
essential to avoid confusion with melanin production.
Test for allantoin degradation: (Gordon, 1966)
Media composition:
Yeast extract 0.1 g
KH2PO4 9.1 g
NaHPO4 9.5 g
Allantoin 3.3 g
Phenol red 0.01 g
Agar 7.5 g
Distilled water 1.0 liter
pH 6.8
Autoclaved at 1210C for 15 minutes
The organism was inoculated on the above media. The results were recorded after 28 days and
observed for orange yellow to pink or purple colour.
Test for gelatin hydrolysis: (Waksman, 1961)
Media composition:
Peptone 5.0 g
Beef extract 3.0 g
Gelatin powder 4.0 g
Agar 15.0 g
Distilled water 1.0 liter
48
pH 7.0
Autoclaved at 1210C for 15 minutes
Reagent:
Mercuric chloride 15 mg
Conc. HCl 20 ml
Distilled water 100 ml
The isolate was grown on gelatin agar plated for six days at 280C. After the incubation period,
the plates were flooded with 1 ml of the reagent. Presence of hydrolyzed zones was observed
after 6 days around the culture.
Test for tyrosine reaction: (Williams et al, 1983)
This test was carried out on tyrosine agar (ISP 7) medium slants. The inoculated slants
were incubated at 280C and observations were made at every 12 hrs up to 4 days and at 24 hrs
thereafter up to 15 days. The results were recorded after 15 days and observed for bluish black
or black diffusible pigment.
Other degradation tests: (Williams et al, 1983)
Hypoxanthine, xanthine, guanine, elastin, adenine and xylan (all at 0.5 %, w/v)
degradation tests were carried out on modified Bennets agar medium. Clearing of the insoluble
compounds around areas of growth was observed after 14 days.
2.2.A.4.3. Test for resistance to antibiotics: (Goodfellow and Orchard, 1974)
The isolate was tested for its ability to grow in the presence of antibiotics, using the
freeze-dried filter paper disc method. Discs previously soaked and air dried in ampicillin
(10µg), chloramphenicol (30µg), erythromycin (15µg), neomycin (30µg), oxytetracycline
(30µg), penicillin G (10 IU), rifamycin (10µg), gentamycin (10µg), streptomycin (10µg)and
kanamycin (30µg) were placed on modified Bennets agar inoculated with 0.1 ml of glycerol
spore suspension and incubated at 280C. Growth was noted on 2, 3, 4 and 7 days for all strains
49
and the resistance was scored as positive result. The first readable results were scored for
computation.
2.2.A.4.4. Effect of temperature on growth of isolate N2: (Williams et al, 1983)
Ability of the isolate to grow at different temperatures was studied at temperatures in
the range of 4 to 450C. The organism was inoculated on modified Bennets agar slants and
incubated at the different temperatures as mentioned above. Results were recorded on 7th and
14th day.
2.2.A.4.5. Effect of pH on growth of isolate N2: (Williams et al, 1983)
The pH of modified Bennet’s agar was adjusted to pH in the range of 2.0 to 11.0 and
spores were inoculated and incubated for 15 days. Then the tubes were examined for the extent
of growth of the organism.
2.2.A.4.6. Growth in the presence of inhibitory compounds: (Shirling and Gottlieb,
1966)
A range of potential inhibitors were added to Bennet’s agar medium and their effect on
growth of selected isolates was studied. (%, w/v): Crystal violet (0.00001), phenol (0.1),
thallous acetate (0.001 and 0.01), sodium chloride 1-13), thallous acetate (0.01), sodium azide
(0.01and 0.02) and potassium tellurite (0.001 and 0.01).
15 ml of sterile molten Bennet’s agar medium was cooled to 450C and each of the
above inhibitory compounds was added to petri plate. The isolate was streaked onto the surface
of the medium, incubated at 280C for 7 days and the presence or absence of growth was
recorded.
2.2.A.4.7. Test for carbon utilization medium: (Pridham and Gottlieb, 1948)
50
ISP 9: Basal medium
Carbon source 10.0 g
(NH4)2SO4 2.64 g
KH2PO4 2.38 g
K2HPO4 5.65 g
MgSO4.7H2O 1.0 g
CuSO4.5H2O 0.1 g
FeSO4.7H2O 0.1 g
MnCl2.4H2O 0.1 g
ZnSO4.7H2O 0.0015 g
Agar 15.0 g
Distilled water 1.0 liter
pH 6.8 – 7.0
Autoclaved at 1210C for 15 minutes
The pH of the medium was adjusted to 7.0 and 5 ml of media was dispensed into test
tubes and autoclaved. After cooling to about 450C, sterile aqueous solutions of the carbon
compounds were added at desired concentration. The carbohydrates, polyhydric alcohols, DL-
inositol and salicin were added such that the final concentration was 1 % (w/v), the phenols at
0.1% (w/v) and the sodium salts of organic acids at 0.15% (w/v). Those materials sufficiently
soluble in water were sterilized by filtration through Sietz EK filter pads. Some compounds
(dextrin, starch, DL-inositol and salicin) that were relatively insoluble or did not filter well
were added directly to the basal medium in the proper concentration prior to tubing and
sterilization. After addition of the carbon sources, the tubes were slanted, allowed to solidify
and incubated to determine sterility.
2.2.A.4.8. Test for nitrogen utilization medium: (Williams et al, 1983)
Nitrogen source 1.0 g
D-glucose 10.0 g
MgSO4. 7H2O 0.5 g
51
NaCl 0.5 g
FeSO4. 7H2O 0.01 g
K2HPO4 1.0 g
Agar 20.0 g
Distilled water 1.0 liter
pH 7.0
Autoclaved at 1210C for 15 minutes
52
2.2.A.4.9. Test for production of acid and gas: (Hugh and Lieifson, 1953)
Media composition:
Bacto peptone 2.0 g
Bacto meat extract 1.0 g
Bacto agar 3.0 g
Bromothymol blue solution (0.2% w/v) 15 ml
pH 7.2
Autoclaved at 1210C for 15 minutes
The ability of isolate to produce acid and gas was tested on the different carbohydrates
by adding them to a final concentration of 1% (w/v) to the acid gas medium. The carbohydrates
tested were L-arabinose, cellulose, sucrose, starch, D-xylose, meso-inositol, D-fructose, D-
glucose, L-rhamnose, maltose, D-mannose, D-lactose, inulin, D-melibiose, D-galactose,
melibiose, mannitol, inulin and xylitol (1.0 % w/v) and sodium acetate, sodium citrate (0.1%
w/v). Durham’s tubes were kept in the inverted position for testing gas production. Both the
tests were carried out in the same media. Presence of acid was determined by change in
medium colour to green.
2.2.A.4.10. Media for cultivation of cells for determination of cell wall composition:
Yeast extract glucose broth:
Yeast extract 10.0 g
Glucose 10.0 g
Distilled water 1.0 liter
pH 7.0
Autoclaved at 1210C for 15 minutes
Cells used for chemotaxonomic analysis were obtained after incubation at 30ºC for 3 days in
yeast extract glucose broth.
2.2.A.5. Primary screening medium:
53
For development of inoculum, well sporulated, 7 to 10 days old glycerol aspargine slant
was used for the production of streptolipin. 5 ml of 2% v/v tween 20 was added to the slant and
spore suspension was prepared. This spore suspension was transferred to 95 ml of sterile
production medium (composition of the medium given below) in 500 ml Erlenmeyer flask. The
culture flasks were incubated at 220 rpm, 300C for 7 days.
Media components:
Soybean meal 1.0 g
Corn steep liquor 0.5 g
Soluble starch 1.0 g
Dextrose 0.5 g
Calcium carbonate 0.7 g
The media components were added to distilled water and pH was adjusted to 7.2 with 0.1 N
HCl or 0.1 N NaOH and final volume made up to 95 mL with distilled water. 95 mL of this
medium was taken in 500 mL Erlenmeyer flasks and autoclaved for 15 minutes at 1210C.
2.2.A.6. Secondary screening media:
Production Medium 1:
Yeast extract 0.5 g
Dextrose 1.0 g
Starch 2.0
Calcium carbonate 0.4 g
The media components were added to distilled water and pH was adjusted to 7.2 with 0.1 N
HCl or 0.1 N NaOH and final volume made up to 100 mL with distilled water. 100 mL of this
medium was taken in 500 mL Erlenmeyer flasks and autoclaved for 15 minutes at 1210C.
Production Medium II:
Soybean meal 1.0 g
Corn steep liquor 0.5 g
Soluble starch 1.0 g
54
Dextrose 0.5 g
Calcium carbonate 0.7 g
The above media components were added to the distilled water and pH was adjusted to 7.2
with 0.1 N HCl or 0.1 N NaOH and final volume was made up to 100 mL with distilled water.
100 mL of this medium was taken in 500 mL Erlenmeyer flasks and autoclaved for 15 minutes
at 1210C.
2.2.A.7. Selection of inoculum media for production of streptolipin
In order to minimize the time lag in fermentation process, inocula are raised in media
with a composition similar to that of fermentation medium.
Composition of the two inoculum media are given below:
Inoculum medium I (g/L):
Soyabean meal 10.0
Corn steep liquor 10.0
Glucose 10.0
Calcium carbonate 5.0
pH 7.0
Inoculum medium II:
Soyabean meal 15.0
Glycerol 5.0
NaCl 5.0
Calcium carbonate 1.0
pH 7.0
The media was sterilized at 1210C for 15 minutes.
Spore suspension from maintenance slant was transferred to the above mentioned
inoculum media and incubated on rotary shaker (220 rpm) at 280C for 48 hrs. After 48 hrs
inoculum level (10% v/v) was transferred to the selected production medium (ISP VIII) and
incubated on rotary shaker (220 rpm) at 280C. Samples were withdrawn at the end of the
fermentation cycle (168 hrs) and HPLC analysis for the streptolipin estimation was carried out.
55
2.2.A.8. Standard media for screening streptolipin production:
Streptolipin production of the isolate was carried out in twenty three different
production media. The composition of media are given below (g/L).
M.1. Lindenberg synthetic media M.2. Hobb’s medium
Glycerol 30.0 Glucose 20.0
NaNO3 2.0 NaCl 5.0
K2HPO4 1.0 Na2SO4 5.0
MgSO4.7H2O 0.5 NaNO3 4.5
FeSO4.7H2O 0.4 K2HPO4 1.2
pH 7.0 Trisbase 1.2
MgSO4.7H2O 1.0
ZnSO4 0.01
pH 7.0
M.3. Czepek-Dox broth M.4. O-Brien synthetic media
Sucrose 30.0 Glucose 20.0
NaNO3 3.0 Glycine 2.6
K2HPO4 1.0 Sodium acetate 1.36
MgSO4.7H2O 0.5 (NH4)2SO4 0.54
KCl 0.5 K2HPO4.3H2O 0.05
FeSO4.7H2O 0.01 ZnSO4.7H2O 0.03
pH 7.0 FeSO4.7H2O 0.025
CuSO4.5H2O 0.016
MnSO4.4H2O 0.012
CaCl2. 2H2O 0.05
MgSO4.7H2O 0.5
pH 6.8-7.0
M.5. Dulaney’s medium M.6. Thornberry’s medium
Glucose 10.0 Glucose 10.0
NaCl 5.0 KH2PO4 2.38
56
K2HPO4 2.0 K2HPO4 5.65
MgSO4.7H2O 0.4 NH4NO3 4.0
CaCl2 0.4 MgSO4.7H2O 0.25
FeSO4.7H2O 0.02 Sodium lactate 11.2
ZnSO4.7H2O 0.01 ZnSO4.7H2O 0.14
(NH4)2HPO4 4.0 FeSO4.7H2O 0.014
pH 7.0 MnSO4.4H2O 0.084
CuSO4.5H2O 0.0016
pH 8.4
M.7. Baron’smedium M.8. Numerof’s medium
Glucose 15.0 Glucose 20.0
NH4NO3 4.0 Glycine 2.6
MgSO4.7H2O 0.25 Sodium acetate 1.36
NaCl 5.0 (NH4)2SO4 0.54
Sodium citrate 1.0 FeSO4.7H2O 0.03
KH2PO4 0.1 CuSO4.5H2O 0.5
K2HPO4 0.1 K2HPO4 0.5
CaCO3 3.0 CaCl2 0.05
pH 8.2 pH 5.5
M.9. Complex organic media M.10. Lumb’s medium
Glucose 25.0 Glucose 20.0
Soya bean flour 25.0 MgSO4.7H2O 10.0
Yeast extract 3.0 Sodium citrate 1.0
(NH4)2SO4 2.0 NaCl 2.5
CaCO3 2.0 CaCl2 0.87
NaCl 2.0 KH2PO4 0.5
KH2PO4 0.15 Glycine 5.0
pH 8.4 FeSO4.7H2O 0.0075
MnSO4.4H2O 0.008
CuSO4.5H2O 0.001
57
ZnSO4.7H2O 0.0014
(NH4)2MO4.4H2O 0.0018
pH 8.8
M.11. Corn meal salt medium M.12. Carbohydrate carcode medium
Cornmeal 50.0 Potato starch 5.0
Na2HPO4 1.15 Glucose 5.0
KH2PO4 0.25 Ribose 5.0
KCl 0.2 Glycerol 5.0
MgSO4.7H2O 0.2 Soyaflour 20.0
pH 7.0 (NH4)2SO4 0.2
Yeast extract 2.0
Bactopeptone 2.0
pH 7.0
M.13. ISP Production media I: M.14. ISP Production media II:
Soyabean meal 25.0 Soluble starch 25.0
Glucose 25.0 Corn steep liquor 10.0
NaNO3 4.0 (NH4)2SO4 5.0
K2HPO4 0.05 CaCO3 5.0
NaCl 2.5 ZnSO4 0.04
CaCO3 0.4
pH 7.0
pH 7.0
M.15. ISP Production media III: M.16. ISP Production media IV:
Glycerol 20.0 Glucose 10.0
Peptone 5.0 Soluble starch 10.0
Yeast extract 3.0 Peptone 7.5
Meat extract 3.0 Meat extract 7.5
CaCO3 2.5 NaCl 3.0
pH 7.0 pH 7.0
58
M.17. ISP Production media V: M.18. ISP Production media VI:
Soyabean meal 15.0 Glucose 33.0
Glucose 15.0 Soluble starch 33.0
Glycerol 2.5 Soyabean meal 34.0
Sodium Chloride 5.0 (NH4)2SO4 13.0
CaCO3 3.0 K2HPO4 13.0
pH 7.0 NaCl 2.5
CaCO3 12.5
pH 7.0
M.19. ISP Production media VII: M.20. ISP Production media VIII:
Soyabean meal 20.0 Soyabean meal 10.0
(NH4)2SO4 5.0 Dextrose 5.0
Meat extract 4.0 Corn steep liquor 5.0
Yeast extract 2.5 Soluble starch 1.0
Glucose 6.0 CaCO3 7.0
KCl 4.0
CaCO3 0.1 pH 7.2
K2HPO4 0.1
pH 7.0
2.2.B. Fungi
2.2.B.1. Culture maintenance medium for fungi:
24 g of the potato dextrose medium (containing potatoes infusion from 200 g/L and 20
g dextrose/L) obtained from Himedia, India, was dissolved in one liter of distilled water and
pH was adjusted to 5.5 with 0.1 N HCl. For preparation of agar slants (PDA), 2% w/v bacto-
agar was used. Autoclaved at 1210C for 15 minutes.
59
2.2.B.2. Screening of fungal cultures for the production of lipase inhibitor:
Fungal cultures available at CFTRI Culture Collection Center were used for selective
screening studies for lipase inhibitor. All the cultures were sub cultured on potato dextrose agar
slants. A three day old slant of each fungal culture was used in the experiments. The following
strains were screened for the production of lipase inhibitor.
1. Aspergillus awamori – 1042
2. Aspergillus carbonarius – 1047
3. Aspergillus flavus – 1058
4. Aspergillus niger – 1038
5. Aspergillus niger – 18
6. Aspergillus niger – CFR-W-105
7. Aspergillus niger – 1046
8. Aspergillus oryzae – 1120
9. Aspergillus sp. CFR-H-105
10. Aspergillus sp. CFR-J-105
11. Fusarium sp – 1128
12. Penicillium sp. 1062
13. Polyporus squamosus – 1134
2.2.B.3. Medium for submerged fermentation (SmF) of fungi:
1000 mL distilled water containing 200 g of peeled potato slices was boiled for 30
minutes and filtered. To the cooled filtrate 20 g of dextrose was added and pH was adjusted to
5.5 with 0.1 N HCl and final volume was made up to 1000 mL with distilled water. 100 mL of
this medium was taken in 500 mL Erlenmeyer flasks and autoclaved for 15 minutes at 1210C.
2.2.B.4. Medium for solid state fermentation (SSF) of fungi:
The medium used for basal wheat bran: Each 250 mL Erlenmeyer flask contained, 10 g
dry wheat bran, moistened with 10 mL of 0.2 N HCl comprising 2.1 mg each of ferrous
60
sulphate, zinc sulphate and copper sulphate and 5 mL of distilled water. This solid medium was
autoclaved at 1210C for 60 minutes and cooled to room temperature before inoculation.
2.3. General fermentation conditions
2.3.1. Inoculum development for screening
The spores were subcultured on glycerol aspargine agar media, incubated at 300C for 7
to 10 days. To the well sporulated slant, sterile 5 ml of 2% v/v tween 20 was added to the slant
and spore suspension was prepared for each media. This spore suspension was transferred to a
sterile 95 ml of the production medium in 500 ml Erlenmeyer flask, which were previously
sterilized at 1210C for 15 minutes and incubated at 280C on a rotary shaker at 220 rpm.
2.3.2. Submerged fermentation for actinomycetes cultures
Media components:
Soybean meal 1.0 g
Corn steep liquor 0.5 g
Soluble starch 1.0 g
Dextrose 0.5 g
Calcium carbonate 0.7 g
The above media components were added to 95 ml of distilled water and pH was
adjusted to 7.2 with 0.1 N HCl or 0.1 N NaOH and final volume was made up to 100 mL with
distilled water. 100 mL of this medium was taken in 500 mL Erlenmeyer flasks and autoclaved
for 15 minutes at 1210C.
2.3.3. Solid state fermentation for fungal cultures (SSF)
For development of inoculum, a loopful of spores from a three day old PDA slant was
transferred to a sterile 100 ml PDB in 500 ml Erlenmeyer flask. The culture flasks were
incubated at 220 rpm, 300C for 3 days. 2-3 pellets from a day old broth of each culture were
transferred to a sterile wheat bran medium.
61
After inoculation, the bran was spread as a thin layer and left in slanting position in an
incubator maintained at 300C for one day. After a one day growth, the bed was disturbed in
each flask by mixing thoroughly for a few min and was left for 7 days without any further
disturbance.
2.3.4. Submerged fermentation for fungal cultures (SmF)
A loopful of spores from a three day old PDA slant was transferred to a sterile 100 ml
PDB in 500 ml Erlenmeyer flask. The culture flask were incubated on a rotary shaker operating
at 220 rpm at 300C.
62
2.4. General extraction conditions
2.4.1. Extraction of inhibitor from SmF broth:
After fermentation, each flask containing 100 mL fermented medium was extracted into
50mL ethyl acetate and kept on shaker for 60 minutes at 220 rpm. The organic layer was
separated, dried using anhydrous sodium sulphate and distilled under reduced pressure. The
crude extract was redissolved in DMSO to give a stock of 50mg/mL for fungal cultures and 7.5
mg/mL for actinomycetes cultures. 10 µL of this solution was tested for pancreatic lipase
inhibition assay.
2.4.2. Extraction of inhibitor from SSF bran:
After 8 days fermentation, each culture was treated with 100 mL ethyl acetate and kept
on shaker for 1 hour at 220 rpm. The bran was filtered on a muslin cloth, ethyl acetate layer
was separated, the moisture was removed by using anhydrous sodium sulphate and distilled
under reduced pressure. For screening the crude extract was dissolved in DMSO at a
concentration 50 mg/mL and 10 µL of this solution was tested for pancreatic lipase inhibition
assay.
2.5. Analytical methods
2.5.1. Lipase assays:
Method I: Spectrophotometric assay (For primary screening):
Buffer preparation:
The buffer was prepared by adding 100 mM sodium phosphate monobasic, 150 mM of
anhydrous sodium chloride and 0.5 % (v/v) triton x-100 in distilled water and the final pH was
adjusted to pH 7.4.
63
Assay of pancreatic lipase for preliminary studies:
The assay was carried out by monitoring the appearance of paranitro phenol at 400 nm
with a micro molar extinction coefficient of 0.0148. The assay mixture contained 50 mM
paranitrophenyl butyrate (30 µL stock) and 10 µL of inhibitor sample dissolved in DMSO,
sodium phosphate buffer (pH 7.4). The reaction was initiated by addition of enzyme solution
(<0.15 unit). One unit of the enzyme activity is defined as the enzyme required forming 1 nM
of the product per minute at 370C under assay conditions. The fraction containing the inhibitor
was dissolved in a minimum quantity of DMSO, which was found to have no effect on enzyme
activity at less than 0.1 % concentration. The assay was initiated by adding substrate to the
reaction mixture (Shirai and Jackson, 1982).
Inhibition was expressed as a percentage relative to solvent control. All activities were
carried out in triplicates and the average has been reported. The relative activity was expressed
as percentage ratio of enzyme activity in the presence of inhibitors to the enzyme activity in the
absence of enzyme inhibitors at the end 3 minutes of the enzyme reaction time.
Method I1: pH stat assay (For secondary screening):
The crude extract that remained after distillation of solvent was taken as a source of
inhibitor and dissolved in DMSO to achieve a concentration 75 µg/ml. The determination of
lipase activity and the hydrolysis of fatty acids from trioleate was followed at pH 8 for 10
minutes at room temperature, using a recording pH-stat. The substrate emulsion was prepared
by ultrasonication of trioleate (30 mg/ml) in a solution containing taurodeoxycholate 1mM,
taurocholate 9 mM, cholestrol 0.1 mM, lecithin 1 mM, bovine serum albumin 15 mg/ml, Tris-
HCl 2 mM, NaCl 100 mM and CaCl2 1 mM. This composition of the test emulsion was chosen
to mimic as closely as possible the in vivo conditions. After addition of test compound, or
vehicle solution alone in to 8 mM Tris-HCl, the pH was set to 8.0 and the reaction was started
by the addition of porcine pancreatic lipase. The release of fatty acid was estimated by 0.1 N
NaOH (Weibel et al, 1987). Secondary screening was carried out by this method.
Normality of NaOH: To potassium hydrogen thalate (0.1N), 3 drops of phenolphthalein was
added as an indicator and titrated against 0.1N NaOH.
64
N1V1=N2V2
N1= Normality of potassium hydrogen thalate (0.1N)
V1= Volume of potassium hydrogen thalate consumed
N2= Normality of NaOH
V1= Volume of NaOH
Specific activity = (Test-Blank) x 1000 x N NaOH / A x Incubation time
N NaOH=Normality of NaOH
A=Protein concentration
2.5.2. Tests for cell wall composition of isolate N2:
2.5.2.1. Amino acid analysis: (Becker et al, 1965 and Hasegawa et al, 1983)
3 mg of dried cells are hydrolyzed with 1 ml of 6 N HCl in a screw capped vial at
1000C for 12 hours. After cooling, the hydrolysate is filtered and 1 ml of water was added. It
was concentrated under reduced pressure. The residue was dissolved in 1 ml of water, dried
again and redissolved in 0.3 ml of water.
2 µl of the sample along with the standards at a concentration of 0.1M (meso-
diaminopimelic acid and levo- diaminopimelic acid) was applied at the base line of the
cellulose TLC sheet. Ascending TLC was performed with the solvent system containing
methanol, water, 6 N HCl and pyridine in the ratio of 80: 26: 4: 10 (v/v). After the
chromatogram was air dried, spots were visualized by spraying 0.2% (w/v) ninhydrin in
acetone and heating at 1000C for 3 min.
2.5.2.2. Sugar analysis: (Becker et al, 1965)
50 mg of dried biomass in a reaction tube with a polytetrafluoroethylene cap and 1 ml
of 1 N H2SO4 was added. It was heated at 1000C for 2 hours, cooled and 0.5 mg of barium
hydroxide to neutralize the solution. The cell debris was separated by centrifugation and the
65
supernatant was evaporated on a rotary evaporator under reduced pressure. The residue was
dissolved in 400 µl of water.
1µl of the sample along with the standard solutions (galactose, arabinose and xylose,
rhamnose, mannose and ribose each at 1% concentration) was applied at the base line of the
cellulose TLC sheet. Ascending TLC was performed with the solvent system containing n-
butanol: water: pyridine: toluene (10: 6: 6: 1, v/v). After the chromatogram was air dried, spots
were visualized by spraying the chromatogram with aniline acid phthalate reagent (3.3 g of
phthalic acid dissolved in 100 ml of water saturated n-butanol with 2.0 ml of aniline) and
heated at 1000C for 4 min. Hexoses appear as yellowish, brown spots and pentoses appear as
maroon spots.
2.5.2.3. Analysis of menaquinones: (Collins et al, 1977)
100 mg dried biomass was mixed with 20 ml chloroform/methanol (2:1, v/v) and the
suspension was stirred continuously over night. The biomass was then removed by filtration
and the extract was evaporated to dryness under reduced pressure 370C. Analytical thin layer
chromatography of quinones was performed using 0.5 mm layers of Merck Kiesegel-gel HF254
and developing solvent system consisting of petroleum ether and acetone (85: 15, v/v).
Menaquinones were detected on thin-layer chromatography with short wave ultraviolet light
(254 nm). Chloroform was used to elute the quinone from the silica.
Ultraviolet spectra of menaquinones were recorded in hexane solution. The GC/EIMS
data was generated using VG Auto Spec M mass spectrometer equipped with HP 5890 series II
gas chromatograph under GC/EIMS conditions. For GC, an HP-5 capillary column was used
with the following temperature program: 1000C for 5 min; 100C/min; 2300C, 15 min. injection
temperature was at 2100C. For MS conditions, the source temperature was 2500 C at an
electrical energy of 70 eV.
2.5.2.4. Analysis of fatty acids: (Tamaoka et al, 1983)
50 mg of dried biomass was taken in an 8.5 ml tube fitted with a
polytetrafluoroethylene seal with 3 ml methanol: toluene: concentrated H2SO4 (30:15:1, v/v)
66
and heated for 12 hours. It was then cooled to room temperature and 2 ml of hexane was added
and centrifuged at 100 rpm. A small column of prewashed ammonium hydrogen carbonate in a
Pasteur pipette plugged with cotton wool was prepared and washed with methanol: chloroform
(1:2, v/v). The supernatant hexane was chromatographed to the column and eluted with
methanol: chloroform (1:2, v/v).
The residue was dissolved in hexane and chromatographed on silica gel sheet (Merck
5735) and developed by using hexane: diethyl ether (85:15 , v/v). the sheet was air dried in the
fume hood and sprayed with 0.01% (w/v) rhodamine 6G in 95 % ethanol and visualized under
366 nm. The purple colour band was cut and extracted with 1ml of diethyl ether. This was
further chromatographed on neutral aluminium oxide column and eluted with diethyl ether.
The extraction was repeated and dried by flushing with nitrogen.
The resultant methyl esters were separated on a gas chromatograph (model 5890;
Hewlett-Packard) fitted with a model of HP-5 capillary column (0.53 mm by 30 m; Hewlett-
Packard). The column was conditioned at 1500C for 2 min and then programmed from 150 to
2500C at the rate of 100C/min, with helium as a carrier gas.
2.5.2.5. Analysis of phospholipids: (Minnikin et al, 1977)
The phospholipids were extracted from frozen cells with a solution containing
chloroform: methanol: 0.3% aqueous sodium chloride (9: 10: 3, v/v). The polar lipid extract
was dissolved in 100 µl of chloroform: methanol (2:1, v/v). 5 µl of the sample was spotted on
silica-gel plates (Merck 5554) along with the standard samples (phosphatidylglycerol,
phosphatidylethanolamine, phosphatidylmethylethanolamine, phospholipids containing
glucosamine) and the plate was developed by ascending TLC with a mobile phase containing
chloroform: methanol: water (65:25:4, v/v). The plate was air dried to remove the residual
traces of the solvent and further developed with a solvent system containing chloroform: acetic
acid: methanol: water in the ratio of 40:7.5:6:2 in the second direction. The plates were dried
and the different components were detected by spraying with reagents. One plate was sprayed
with molybdophosphoric acid (5%, w/v) and heated at 1800C for 15 minutes. All polar lipids
appeared as dark spots on a light green ground. Ninhydrin (2% w/v) in water saturated butanol
67
was sprayed lightly and heated at 1000C for 5 minutes. Lipids containing amino groups
appeared as pink-red spots. The spots were marked and sprayed with Zinzadze reagent.
Phosphorus containing lipids appeared as blue spots on a white back ground. The third plate
was sprayed with metaperiodate and left aside for 10 minutes for oxidation. Decolourized with
sulphurdioxide gas and sprayed with schiff reagent for the detection of lipids containing vicinal
diol groups. The fourth plate was lightly sprayed with α-naphthol reagent heated at 1000C for
10 minutes for the detection of glycolipids. Glycolipids appear as brown spots.
2.5.2.6. Analysis of mycolic acids: (Tomiyasu, 1982)
The extraction and purification of mycolic acids was the same as carried out for fatty
acids. Methyl esters of mycolic acids were detected and purified using chromatographic system
described for menaquinones above. Conditions for the analysis of mycolic esters by mass
spectroscopy were the same as for the menaquinone samples.
2.5.2.7. Phylogenetic analysis of isolate N2:
Genomic DNA was prepared as described by Shivaji et al (1992). The 16S rDNA was
amplified (Shivaji et al, 2000; 2004), the PCR product purified using QIA quick PCR
purification kit (Qiagen) and sequenced using an ABI PRISM model 3700 automatic DNA
sequencer using the Big Dye Terminator cycle sequencing kit (Applied Biosystems). The
almost complete sequence of 16S rDNA containing 1464 bp was aligned with closely related
sequences that were deposited in EMBL using CLUSTAL W (Thompson et al, 1994). The
pair-wise evolutionary distances were computed using the DNADIST program with the
Kimura 2-parameter model (Kimura, 1980). Phylogenetic trees were constructed using
NEIGHBOR JOINING and DNAPARS of the PHYLIP package (Felsenstein, 1993). The
stability among the clades of a phylogenetic tree was assessed by taking 1000 replicates of the
data set and was analyzed using the programs SEQBOOT, DNADIST, NEIGHBOR and
CONSENSE of the PHYLIP package.
2.5.3.1. Column chromatography:
68
Silica gel (60-120 mesh) was dried in an oven for one hour at 1000C. 30 gm of this was
packed on to a glass column (15 x 3 cm) fitted with a G0 filter, in hexane with a flow rate of 1
mL/min. Elution of the crude extract was carried out using hexane, chloroform, ethyl acetate,
methanol and various combinations of these solvents. Two bed volumes were taken as a
fraction. Each fraction was analyzed by qualitative TLC and enzyme assay.
2.5.3.2. Thin layer chromatography (TLC):
Qualitative TLC plates preparation:
Qualitative TLC plates were prepared by making slurry of 2 gm of silica gel-G with 5
mL of water and spread over the plate manually on a 5 X 20 cm glass plate followed by air-
drying. The plates were then activated in oven for one hour at 1000C. After activation, the TLC
plates spotted with crude extracts or purified compounds and run using a benzene : methanol (9
: 1) mobile phase.
Preparative TLC plates preparation:
For preparative TLC plates, the slurry was prepared by mixing 8 gm of silica gel-G
with 20 mL of water and spread over the plate manually on a 20 X 80 cm glass plate followed
by air-drying. The plates were then activated in oven for one hour at 1000C. After activation,
the TLC plates spotted with partially purified compound and run using a suitable mobile phase.
2.5.3.3. High pressure liquid chromatography (HPLC) method:
The HPLC method for streptolipin was developed on RP-C18 column [5µm, 250 mm x
4.6 mm, Shimpack CLC-ODS (L1) column (Shimadzu, Japan)] using LC-10A (Shimadzu,
Japan) with a gradient mobile phase of acetonitrile and water. The programme was as follows:
69
0.01 min acetonitrile 10%, 20.0 min acetonitrile 70%, 40.0 min acetonitrile 100% operating at
a flow rate of 1 ml/ minute and the UV detector fixed at 210 nm.
2.5.3.4. Melting point determination:
2 mg of compound was packed into a capillary tube sealed at one end. This tube was
placed on SELACO-650 hot stage apparatus and the melting point were determined and are
uncorrected in a 230V PEW-thermal block and temperature was read manually using a
mercury bulb thermometer.
2.5.3.5. Elemental analysis:
Elemental analysis was carried out on Elementar Vario EL III. Oxygen was used for
combustion and Helium as the mobile phase. The combustion chamber temperature was 1150 0C and the reduction chamber temperature was at 8500C. Detector used for thermal
conductivity and the liberated sulphur dioxide was detected at 1400C. The carbon dioxide,
nitrogen and water were detected at room temperature. The oxygen and helium flow were 1.25
and 2–3 Kg per square cm respectively. Desiccants used were sodium hydroxide and
phosphorus pentaoxide to remove the moisture content.
2.5.3.6. Infrared spectroscopy:
IR absorption spectra were obtained with a Perkin Elmer model 2000 Infrared Fourier-
transform spectrophotometer using an attenuated total reflectance cell on a thin layer of the
sample (1 mg/mL) dissolved in nujol.
2.5.3.7. Nuclear magnetic resonance spectroscopy (NMR):
NMR spectra were recorded at 500 MHz on a Bruker DRX-500 MHZ spectrometer
(500.13 MHz proton and 125 MHz carbon frequencies) at 27 0C. Proton and carbon 900 pulse
widths were 11.2 and 8.8 µs respectively. About 20 mg of the sample-dissolved in CDCl3 was
used for recording spectra.
70
Two-dimensional heteronuclear multiple quantum coherence transfer spectra (2D-
HMQCT) were recorded in magnitude mode with sinusoidal shaped z gradients of strength
25.7, 15.42 and 20.56 G/cm in the ratio of 5:3:4 were applied for duration of 1 ms each with a
gradient recovery delay of 100 µs to defocus unwanted coherence. The t1 was incremented in
256 steps. The size of the computer memory used to accumulate the 2D data was 4K. The
spectra were processed using unshifted and π/4 shifted sine bell window function in F1 and F2.
2.5.3.8. Liquid chromatography mass spectroscopy (LC-MS):
Mass spectral data were achieved by LCMS (waters 2690, HPLC system connected to
micro mass LCZ mass spectrometer) with electro spray ionization in positive mode [ESP+].
The following ion optics were used: capillary 3 Kv conc 30 volt and 60 volt and extra per 7
volts. The source block temperature was 1200C and the desolvation temperature 2500C. The
electrospray probe flow was adjusted to 100 µl/min. continuous mass spectra were recorded
over the range M/z 110 to 800 with scan time 1 second and inter scan delay 0.1 sec. For LC
conditions used were same as HPLC.
2.5.3.9. Detection of elements by chemical method:
A small quantity of test sample with a piece of sodium in a sodium fusion tube was
heated gradually at first and then strongly till the evolution of vapours and smoke ceased. The
red-hot tube was plunged in to 10 ml of water taken in a mortar. The glass tube was crushed
and transferred in to a 100 mL beaker, boiled and filtered and the filterate was collected. The
filterate was used for both nitrogen and sulphur test.
Nitrogen test:
To the filterate, ferrous sulphate was added and boiled. After cooling dil. H2SO4 was added till
a blue precipitate was formed, which confirmed the presence of nitrogen.
Sulphur test:
71
i. To the filterate, sodium nitroprusside and sodium hydroxide were added. The
appearance of purple colour confirmed the presence of sulphur.
ii. To the filterate, excess of acetic acid and lead acetate were added. Black precipitate
confirmed the presence of sulphur.
Test for ester: This was carried out by two methods.
i. Phenolphthalein test:
The compound was dissolved in alcohol, added 2 to 3 drops of alcoholic KOH solution
and one drop of phenolphthalein. The solution was heated gently. Pink colour
discharged showed the presence of ester group.
ii. Ferric hydroxymate test:
To the test compound, 0.5 ml 1N hydroxylamine chloride in rectified spirit was added
along with few drops of NaOH solution. The solution was heated to boiling, gradually
cooled, acidified and two drops of FeCl3 was added. The appearance of purple colour
confirms the presence of Ester group in the compound.
2.6. Microorganisms used for comparative studies:
Reference actinomycete strains used in this study are Streptomyces erumpens (NRRL
B-3163), Streptomyces yatensis (NRRL B-24116), Streptomyces hygroscopicus (NRRL 2387),
Streptomyces malaysiensis (DSM 41697), Streptomyces rimosus (NRRL 2234), Streptomyces
violaceusniger (NRRL-B 1476), Streptomyces sparsogenes (ATCC 25498), Streptomyces
melanosporofaciens (ATCC 25473), Streptomyces platensis (NRRL 8035), Streptomyces
thermodiastaticus (ATCC 27472), Streptomyces rutgersensis (NRRL B-1256), Streptomyces
olivaceiscleroticus (ATCC 15722) and Streptomyces kasugaensis (ATCC 1574).
2.7. In vivo efficacy of streptolipin on experimental animal models: 2.7.1. Effect of single and multiple doses of streptolipin on fat absorption:
72
Mice were used to assess the effect of single and multiple administration of streptolipin
on intestinal fat absorption. Female Swiss albino mice weighing 30-35 g were maintained on
mouse diet. The diet contained (w/w) casein 20%, sucrose 69.8%, refined groundnut oil 5%,
salt mixture 4%, vitamin mixture 1%, choline chloride 0.2%, fat- vitamin mix per kg diet
(retinyl acetate) 600 International Unit (I.U), calciferol 6000 I.U, α-tocopheryl acetate 100 mg.
Two sets of experiments were carried to evaluate the single and multiple dose effect of
streptolipin on faecal triglyceride excretion. One set for single dose was observed for three
days and the other set for multiple dose for seven days. In the multiple dose study, three doses
were administered with each dose on every alternate day. Each set contained five groups, 3
groups for streptolipin, one group for commercially available compound (Orlistat) and one
group for the vehicle control, where each group consisted of ten mice. Streptolipin was
administered orally at 5, 10 and 20 mg/kg body weight and Orlistat was administered at 10
mg/kg body weight. Overnight fasted mice were given streptolipin and Orlistat in a suspension
of 5% gum arabic and 5% defatted milk powder or vehicle solution alone (0.2 ml/animal) and
return to ad-libitum eating. Faeces were collected every day and triglyceride was estimated.
At the end of the feeding period, the animals were starved overnight and sacrificed by
heart puncture under ether anaesthesia. Blood was collected by heart puncture, allowed to clot
and centrifuged for 10 minutes at 2500 rpm to obtain serum. The liver was quickly excised,
washed with ice cold saline, wiped with filter paper, cleaned and weighed. Serum and liver
samples were stored at – 800 C until use for analysis. Small intestine (20-25 cm long segment
between jejunum and caecum leaving about 5 cm on either side) was immediately excised and
flushed with ice cold 0.9% saline. The intestinal segments were then cut open longitudinally
and mucosa was scraped with microscopic slide. The mucosal scrapings were homogenized in
0.9% saline used for lipase assay (Platel and Srinivasan, 1996), washed with ice cold saline and
stored at 40C with saline until use. The intestinal mucosa was scrapped with 2 mM Tris HCl
buffer (pH 8.0) and further analysis was carried out.
2.7.2. Faecal triglycerides:
73
Faeces samples (1 g) were mixed with 1 ml of ethanol and 2 ml of aqueous solution of
gum arabic (20 % w/v) and were homogenized for 5 min and homogenates were used for
estimation of triglyceride.
For the determination of total faecal triglyceride, a 5g aliquot of the homogenate was
boiled with 22 ml of acidified sodium chloride (4.28 mole/litre) for 1 min. after cooling, 40 ml
of amyl alcohol and ethanol (v/v= 1/250) was added and fat extracted with 50 ml of petroleum
ether. A 25 ml aliquot of the petroleum ether layer was evaporated and the residue was
redissolved in 2 ml of ethanol. After adding alcoholic potassium hydroxide, 8 ml (0.11
mol/litre) and 5 drops of thymol blue (15 mmol/ litre), the solution was boiled at 1100C for 20
min. Immediately afterwards, 10 ml of ethanol was added and the amount of potassium
hydroxide not used for saponification was titrated with 0.1 mole/litre hydrochloric acid
(Hartmann et al, 1993).
2.7.3. Lipid Peroxides:
Plasma lipid peroxides were estimated by the fluorimetric measurement of Thiobarbituric
acid complex by the method of Yagi (1984). The fluorimetric measurement was carried out at
an excitation wavelength of 515 nm and emission wavelength of 553 nm and compared with
the standards prepared by reacting 0.5 n mole 1,1,3,3-tetraethoxypropane with TBA reagent.
Lipid peroxides in liver were determined by the method described by Ohkawa et al (1979)
involving photometric measurement of Thiobarbituric acid complex extracted into butanol.
Absorbance of butanol extract was measured at 532 nm. Values were compared with the
tetraethoxypropane, which is used as standard treated similarly.
2.7.4. Antioxidant enzymes:
Five percent liver homogenate was prepared with 0.15 M KCl and centrifuged at 500 X
g for 10 min. The cell-free supernatant was used for the assay of activities of glutathione
peroxidase, catalase and Superoxide dismutase (SOD).
2.7.4.1. Catalase Assay:
74
The catalase assay was carried out according to the method of Aebi (1984). One
milliliter of liver homogenate was added to 1.9 ml of phosphate buffer (50mM, pH 7.4). The
reaction was initiated by the addition of 1 ml of hydrogen peroxide (30mM). Blank without
liver homogenate was prepared with 2.9 ml of phosphate buffer and 1 ml of hydrogen
peroxide. The decrease in optical density due to decomposition of hydrogen peroxide was
measured at the end of 1 min against the blank at 240 nm. Units of catalase were expressed as
the amount of enzyme that decomposes 1µM H2O2 per milligram of protein.
2.7.4.2. Estimation of Superoxide dimutase:
The assay of SOD was based on the reduction of nitro blue tetrazolium (NBT) to water
insoluble blue formazan measured by the method of Beauchamp and Fridovish (1971). Liver
homogenate (0.5 ml) was mixed with 1 ml of 50 mM sodium carbonate, 0.4 ml of 24 µM NBT,
and 0.2 ml of 0.1 mM EDTA. The reaction was initiated by adding 0.4 ml of 1 mM
hydroxylamine hydrochloride. Zero time absorbance was recorded at 560 nm followed by
recording the absorbance after 5 min at 250 C. The control was simultaneously run without
liver homogenate. Units of SOD activity were expressed as the amount of enzyme required to
inhibit the reduction of N—50%. The specific activity was expressed in terms of units per
milligram of protein.
2.7.4.3. Estimation of Glutathione peroxidase:
Glutathione peroxidase assay was carried out according to the method of Nicholos
(1962). Liver homogenate (0.5 ml) was mixed with 1 ml of 10 mM KI solution and 1 ml of
40mM sodium acetate solution. The absorbance of potassium periodide was read at 353 nm,
which indicates the amount of peroxidase. Twenty microliters of hydrogen peroxide (15mM)
was added and the change in the absorbance in 5 min was recorded. Units of peroxidase
activity were expressed as the amount of enzyme required to change the OD by 1 unit per
minute. The specific activity was expressed in terms of units per milligram of protein.
2.7.5. Plasma non-specific enzymes:
75
Alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase
activities were determined in serum by using commercially available test kits (Span Biotech,
India).
2.7.6. Lipid analysis:
Cholesterol in the lipid extracts from plasma and liver was estimated as described by
Searcy and Bergquist (1960). Plasma cholesterol associated with HDL fraction was
determined after precipitation of apolipoprotein-B containing lipoproteins with heparin-
manganese reagent according to the method of Warnick and Albers (1978). Triglycerides in
plasma and liver were determined by the method described by Fletcher (1968) using
triglyceride purifier (Sigma Chemical Co., USA) to remove phospholipids. Phospholipids
were estimated by the ammonium ferrothiocyanate method of Charles and Stewart (1980).
76
3.A.1. Screening of different terrestrial substrates for selective isolation of actinomycetes:
Identification of the area for the collection of soil samples was done randomly.
However, while choosing the terrestrial samples sufficient care was taken to see that the points
of collection had, as widely varying characteristics as possible, with regard to the organic
matter content, moisture content, particle size, color of soil and geographical distribution. Six
samples from Karnataka state, two samples from Andhra Pradesh state, five samples from
Tamilnadu state from India and two soil samples from the desert of Muscat Saudi Arabia, were
collected for the selective isolation of actinomycetes. All the samples were collected into
sterile containers. A brief account of the different samples collected is given in Table 3.1.
3.A.2. Techniques for the isolation of actinomycetes:
Isolation and screening of actinomycetes cultures is being intensively pursued for the
discovery and commercialization of a number of novel antibiotics as they have the capability to
synthesize secondary metabolites (Jong-Gwan et al, 2002). Several methods have been
suggested in literature for the preferential growth and isolation of actinomycetes, of which the
soil dilution plate and soil plate technique (Warcup, 1950; Corke and Chase, 1956; Corbaz et
al, 1963; Porter, 1960; Williams, 1965) are the most widely used methods. Many differential
media for suppressing the growth of fungi and bacteria have been described. By following
these methods, it may be possible to miss some important strains as the bacteriostatic and
fungistatic agents incorporated in the media may also suppress some actinomycetes. However,
the simplest, traditional and the best method of isolation of actinomycetes is the crowded plate
technique and some of the best antibiotic producers have been isolated in this way. Hence, this
method was employed in the present study.
Table 3.1: Different soil samples for screening of actinomycetes:
Sample Nature of the Type of the sample Place of collection
77
code sample
B Grayish free
flowing
Forest soil Coonoor, Tamilnadu
C Shiny, moist
reddish brown
Lake front Ooty, Tamilnadu
V Reddish free
flowing
Tea plantations Ooty, Tamilnadu
S Greenish muddy Sugarcane field soil Manali, Tamilnadu
E Reddish granular Tea plantations Ooty, Tamilnadu
M Brownish
granular
Coconut field soil Nanjangud, Karnataka
T Reddish brown
granular
Sugarcane Mandya, Karnataka
F Reddish, free
flowing
Lalbagh botanical
garden
Bangalore, Karnataka
N Black dry, lumpy Cow barn yard Mysore, Karnataka
K Grayish, muddy Kukkarahalli lake Mysore, Karnataka
G Black, sticky Rice field Naganahalli, Karnataka
A Blackish brown
granular
Hill station Tirumala, Andhra Pradesh
P Black dry, lumpy Sugarcane Tirupathi, Andhra Pradesh
D Light brown
sandy
Desert Muscat, Saudi Arabia
H Light brown
sandy
Desert Muscat, Saudi Arabia
Tsao et al (1960) and Lawrence (1956) have reported that bacterial and fungal
contamination could be reduced by physical treatment (centrifugation and heat treatment) of
the soil sample. Reduction in contamination of bacteria and fungi through chemical treatment
78
(phenol and calcium carbonate) of the soil sample was reported by Nonomura and Ohara
(1969) and Rehacek (1959).
In the present study, both physical and chemical pretreatments were employed for the
isolation of actinomycetes, to decrease the bacterial and fungal contaminants. It was observed
that the soil sediments treated with phenol resulted in complete elimination of fungal growth
but was ineffective against bacterial contamination. The control plates, which were not
pretreated, exhibited overgrowth by bacteria and fungi. The use of heat treatment reduced the
number of bacteria and fungi in isolation plates and number of actinomycetes colonies (Table
3.2).
These results were in concurrence with the results reported by Nonomura and Ohara
(1969), where the heat treatment effectively reduced the outgrowth of the soil sediments.
Pretreatment with calcium carbonate decreased the contamination of bacteria and fungi and the
number of actinomycetes colonies significantly increased, whereas in centrifugation method
the bacterial and fungal contamination was more with significantly less actinomycetes. Also,
there was not much difference between the centrifugation method and control, as the
actinomycetes colonies and contaminants were similar (Table 3.2).
Actinomycetes can be isolated from soil samples and other natural substrates by plating
them on suitable agar media after diluting them appropriately. Actinomycetes colonies, when
grown on suitable media are usually compact and have leathery and dry surface which makes
them to be distinguished from those of fungi and bacteria on dilution plates. Many media,
which preferentially encourage the growth of actinomycetes, have been suggested such as
czapeck dox agar (Lawrence, 1956), egg albumin agar, glucose aspargine agar, half strength
nutrient agar (Lacey and Goodfellow,
Table 3.2: Different pretreatments of various soil samples.
79
Soil Pretreatment Actinomycetes (% of
total microbial
population)
Actinomycetes per
gram of soil
(x 103)
Control 37 27.5
Phenol 56 22
Heat treatment 20 10
Calcium carbonate 96 200
Cow barn yard
Centrifugation 38 32.5
Control 33 32
Phenol 58 24
Heat treatment 25 11
Calcium carbonate 91 185
Forest
Centrifugation 34 36
Control 34 39
Phenol 55 28
Heat treatment 26 8.6
Calcium carbonate 88 120
Tea plantation
Centrifugation 36 42.5
Control 32 34
Phenol 60 30
Heat treatment 27 9.2
Calcium carbonate 82 160
Lake
Centrifugation 38 35
1975), starch casein agar (Kuster and Williams, 1964), nutrient agar, raffinose-histidine
agar, czapeck dox yeast extract casamino acid agar (CYC), Gauze agarized medium No
1(Kuznetsova et al, 1988), Benedict’s modification of Lindenbein’s medium, Humic
acid vitamin agar (Hayakawa and Nonomura, 1987; Hayakawa et al 1991), Potassium
80
tellurite, AV agar (Nonomura and Ohara, 1969) and oatmeal agar (Williams and Cross,
1971).
In the present work four media were employed for selective isolation of actinomycetes.
1. Starch casein agar medium.
2. Oatmeal-agar medium.
3. Potassium tellurite agar medium.
4. Half strength nutrient agar medium.
Out of four media, starch casein agar medium was found to be the best media for isolation
of actinomycetes (Table 3.3) and further work was carried out using the same medium.
Table 3.3: Isolation of actinomycetes on different media:
Medium Actinomycete colonies (x 104)/g soil
Starch casein agar medium 3.2
Oatmeal-agar medium. 2.4
Potassium tellurite agar medium 2.1
Half strength nutrient agar medium. 1.8
The soil samples, following calcium carbonate treatment were plated on starch casein agar
medium. At the end of incubation, the plates were observed for actinomycetes and the selected
colonies were subcultured on the glycerol aspargine agar slants depending up on the colour of
aerial mycelium, reverse colour, soluble pigment and colony texture to the naked eye. Doubtful
colonies were confirmed by microscopic examination. The distribution of actinomycetes in
different samples is shown in Table3.4. Screening of
Figure 3.1: Screening of actinomycetes on starch casein agar:
81
Figure 3.2: Isolated culture slants of actinomycetes:
82
Table 3.4: Distribution of actinomycete isolates in various soil samples:
Samples code Total number of isolated actinomycetes
83
B 20
C 18
V 16
S 2
E 14
M 4
T 3
F 17
N 30
K 39
G 3
A 25
P 7
D 17
H 15
Total isolated: 230
actinomycetes on starch casein agar was shown in Figure 3.1 and some of the isolated culture
slants actinomycetes were shown in Figure 3.2.
84
Table 3.5: Distribution of actinomycete isolates from various samples.
Number of isolates Colour of aerial mycelium
Source
Gray White Red Blue Green Yellow Cream Polymorphic (Pink or orange )
No aerial mycelium
Total Soluble pigment
Desert 15 9 1 3 0 2 0 1 2 33 8 Hill station 13 3 1 0 2 1 0 2 3 25 10 Cow barn yard
3 19 2 1 0 4 0 0 1 30 9
Forest 7 2 3 3 0 2 3 0 0 20 12 Garden 8 7 1 1 17 Lake 30 16 4 2 0 2 0 1 2 57 16 Fields 14 5 0 0 0 0 0 0 0 19 Tea plantations
16 8 0 0 0 2 0 2 1 29 13
Total 106 69 11 9 2 14 4 6 9 230 68 Percentage of total
46.0 30.0 4.78 3.91 0.86 6.08 1.7 2.6 3.9 100 29.56
85
230 isolates recovered from terrestrial samples were distributed in to series
according to color of the mature sporulated aerial mycelium (Table 3.5). Members of the
gray series were found to represent 46 percent of the total number of isolates and the
lowest occurrence
was noted for the green series (0.86%). The highest occurrence of isolates of the gray
series is in agreement with that reported by Ndonde and Semu (2000).
Masmeh (1992) in his study on distribution of Streptomyces flora in Jordan
reported that the white color class dominated the soil samples (43.6%). Table 3.5 shows
that, out of 230 isolates, 68 produced soluble pigment, representing 29.56%. The
differences in color of aerial mycelia of the isolates as well as those of the pigments
produced, may be an indication of the diversity of actinomycetes in the sites investigated.
Almost all the isolates showed vegetative mycelium and aerial hyphae, with a few (4%)
with only the substrate mycelium, which were red and cream colored.
3.A.3. Screening of microorganisms for pancreatic lipase inhibitor production:
Fermentation of the cultures was done by solid state and submerged methods
using wheat bran and potato dextrose broth as media, respectively for fungi. These media
were selected, as earlier reports in our laboratory showed fungal cultures had the ability
to produce novel and new enzyme inhibitors (Rao et al, 2001; 2002a; 2002b; 2002c; 2002d;
2003). The quantity of crude extract from solid state fermentation was more as compared
to submerged fermentation. This might be due the solubility of the components from
wheat bran to ethyl acetate during extraction. However, the concentration of the inhibitor
in solid state fermentation was less, when compared to submerged fermentation. Taking
this into consideration, the crude extract from solid state fermentation was used at higher
concentration for enzyme inhibition studies.
15 fungal cultures from CFTRI culture collection center were screened for lipase
inhibition. Among the 15 fungal isolates screened, the highest activity was shown by
Penicillium sp-1062 followed by Polyporus squamosus-1134 in submerged fermentation,
86
Table 3.6: Primary screening of fungal cultures from CFTRI Culture Collection
Center
for pancreatic lipase inhibition:
%Inhibition of pancreatic lipase
S.No
Culture SmF (at 0.5 mg
crude)
SSF (at 1.0 mg
crude)
1. Aspergillus niger-1038 0.15 0.31
2. Aspergillus niger-1105 1.36 1.24
3. Aspergillus niger-1037 0.98 1.96
4. Aspergillus niger-18 1.25 2.47
5. Aspergillus niger-CFR-W-
105
2.34 2.16
6. Aspergillus flavus-1058 1.23 1.85
7. Aspergillus carbonarius-1047 5.21 4.55
8. Aspergillus oryzae-1120 1.11 2.35
9. Aspergillus awamori-1042 2.34 5.67
10. Aspergillus niger-1046 3.79 6.62
11. Aspergillus sp-CFR-H-105 3.21 2.98
12. Aspergillus sp-CFR-J-105 4.22 7.96
13. Penicillium sp-1062 9.82 8.76
14. Polyporus squamosus-1134 7.97 9.03
15. Fusarium sp-1128 5.34 7.67
87
where as in solid state fermentation, the highest activity was shown by Polyporus
squamosus-1134 followed by Penicillium sp-1062. However, the culture extracts
obtained from both solid state and submerged fermentation showed less than 10%
inhibition (Table 3.6). The results obtained were not convincing. Hence further studies
using these cultures were discontinued.
Primary screening allows the detection and isolation of new microorganisms
exhibiting desired property and remove the unrequired microorganisms on the basis of
relatively simple and fundamental criteria. Secondary screening is strictly essential in
systematic screening programmes intended to select highly potent isolate of the desired
activity. It is very useful in sorting out microorganisms that have real commercial value
from many isolates obtained during primary screening. At the same time, microorganisms
that have poor applicability in a fermentation process are discarded. It provides
information pertaining to the effect of different components of a medium and product
yield potentials of different isolates. It detects gross genetic stability in microbial
cultures. This type of information is very important, since microorganisms tending to
undergo mutation or alteration in some way may lose their capability of maximum
accumulation.
Two hundred and thirty actinomycetes cultures, which were isolated were grown
for seven days and at the end of fermentation each culture was extracted with ethyl
acetate. Ethyl acetate was chosen as extraction solvent because of its immiscible nature
with aqueous phase and its partitioning ability of hydrophobic and hydrophilic
compounds. For sake of brevity, a flow chart is given below for the process of isolation
and screening of actinomycetes (Figure 3.3).
In submerged fermentation, as ethyl acetate extraction alone does not remove all
the metabolites from the fermented broth, the aqueous broth filtrates were also tested for
lipase inhibition. As none of the culture filtrates showed inhibition, further studies were
focused only on inhibitions shown by the ethyl acetate extracts. The biomass was
separated by cheese cloth, the aqueous layer and solvent layer were separated by
separating funnel. The solvent
88
Figure3.3: Isolation and screening of actinomycetes isolates
Soil samples Pretreatment with CaCO3 for 10 days
1g soil sample with 50ml of sterile water Serial dilution
Plating with Starch Casein Agar Medium
Incubated for 3 weeks
Isolation of actinomycetes colonies
Sub-culturing
Fermentation for 8 days in ISP medium
Solvent extraction (Ethyl acetate)
Crude extract dissolved in DMSO 75µg
Inhibition of the target pancreatic lipase enzyme
89
was distilled under reduced pressure and the residue remained after complete distillation
was used as a source of inhibitor. The crude residue was dissolved in DMSO to give an
extract of 7.5 mg/mL and 75µg each of these crude extracts were then tested for lipase
inhibition. The control experiment contained the ethyl acetate extract of the unfermented
broth at the same concentration as the sample.
From a total of 230 strains of actinomycetes isolated from various soil samples,
84 cultures did not exhibit any inhibition against pancreatic lipase and 146 isolates were
found to produce pancreatic lipase enzyme inhibitors. The results indicate that K12, V22,
N62, B80, V41, A56, N2, N18, A37, K44, B18, A21, D4, D7, N21, H5, N46, K10, N32, C4, C18, N35
strains are effective against pancreatic lipase enzyme (Table 3.7). The isolate N2 was
found to be the most potent with 91.8% inhibition followed by A37. Of the 146 active
isolates, 124 cultures exhibited inhibition below 20% and 22 isolates have shown activity
against pancreatic lipase above 20%. Among the 22 strains, 7 cultures were found to have
high potency to inhibit pancreatic lipase (Table 3.8). Isolates showing more than 50%
inhibition were further selected and subjected to secondary screening.
From a total of 230 isolates, 22 isolates were found to produce pancreatic lipase
inhibitors. Among these, 7 were recovered from the soil sediment collected from cow
barnyard, 5 from lakes, 2 from desert, 2 from tea plantations, 3 from hill station, 2 from
forest and 1 from coconut field (Table 3.9). Table 3.10 shows that, only one active
culture was obtained from heat treated sediments, three from sediments pretreated with
phenol and most of the cultures were from calcium carbonate pretreatment. Further, the
greatest number of active strains were isolated from samples, which were pretreated with
calcium carbonate followed by phenol.
The percentage of active isolates also varied within each colour series (Table
3.11). 9.5% of the isolates were active against the target enzyme. The highest number of
isolates exhibiting activity against the target enzyme was from gray series (12) followed
by white series (7) and the percentage inhibitions were 11.3 and 10.1 respectively.
Saadoun et al (1998), studying the antimicrobial activity of isolates from northern Jordan
90
Table 3.7: Primary screening of actinomycetes isolates for pancreatic lipase
inhibition:
S.No Culture number Percent of pancreatic
lipase inhibition at 75 µg
of crude extract
1 B2 Nil 2 B4 12.1 3 B8 Nil 4 B9 18.25 5 B10 12.34 6 B11 Nil 7 B13 17.5 8 B14 18.53 9 B17 16.12 10 B18 25.7 11 B20 10.5 12 B22 Nil 13 B24 Nil 14 B25 5.8 15 B28 Nil 16 B29 19.5 17 B32 Nil 18 B35 Nil 19 B37 7.8 20 B80 52.6 21 C2 18.4 22 C3 Nil 23 C4 35.7 24 C6 Nil 25 C7 10.7 26 C8 16.0 27 C11 Nil 28 C12 19.4 29 C15 8.7
Continued….
91
30 C18 25.8 31 C19 15.7 32 C22 17.8 33 C24 Nil 34 C26 10.4 35 C27 13.6 36 C28 15.8 37 C31 Nil 38 C32 Nil 39 V2 Nil 40 V4 10.46 41 V7 13.93 42 V8 2.83 43 V11 3.36 44 V12 12.55 45 V15 12.39 46 V16 12.35 47 V18 13.93 48 V19 Nil 49 V20 Nil 50 V22 66.6 51 V24 Nil 52 V27 Nil 53 V28 Nil 54 V41 35.9 55 S4 Nil 56 S6 Nil 57 E2 15.8 58 E6 15.2 59 E7 13.6 60 E9 Nil 61 E11 7.9 62 E12 1.5 63 E14 Nil 64 E15 Nil
Continued….
92
65 E16 Nil 66 E18 18.9 67 E21 Nil 68 E22 9.7 69 E24 Nil 70 E27 11.77
71 M9 Nil 72 M11 4.7 73 M23 Nil 74 M30 5.85 75 T3 8.56 76 T7 Nil 77 T14 Nil 78 F3 12.5 79 F5 15.8 80 F6 Nil 81 F7 11.6 82 F9 Nil 83 F11 6.8 84 F12 13.2 85 F15 12.2 86 F16 13.7 87 F19 Nil 88 F21 19.2 89 F23 13.4 90 F24 Nil 91 F26 Nil 92 F29 7.9 93 F30 Nil 94 F35 3.4 95 N1 14.32 96 N2 91.8 97 N8 4.5 98 N7 Nil
Continued….
93
99 N10 Nil 100 N12 Nil 101 N18 26.11 102 N20 14.2 103 N21 26.15 104 N24 Nil 105 N26 Nil 106 N27 Nil 107 N32 30.2 108 N35 25.1 109 N38 10.2 110 N39 7.0 111 N41 15.1 112 N46 25.38 113 N55 10.5 114 N59 Nil 115 N60 6.4 116 N62 61.4 117 N67 Nil 118 N69 9.5 119 N71 13.4 120 N73 Nil 121 N75 17.5 122 N76 7.5 123 N79 18.5 124 N82 33.7 125 K5 9.4 126 K4 Nil 127 K8 Nil 128 K10 33.08 129 K12 72.8 130 K16 Nil 131 K18 11.5 132 K22 10.1
Continued….
94
133 K25 13.07 134 K26 8.8 135 K28 8.88 136 K32 6.2 137 K34 Nil 138 K35 14.62 139 K36 4 140 K39 19.61 141 K40 Nil 142 K41 Nil 143 K44 58.2 144 K45 4.5 145 K46 15.96 146 K47 19.23 147 K49 12.31 148 K50 15.54 149 K51 6.66 150 K53 16.81 151 K55 12.41 152 K56 17.89 153 K57 13.84 154 K59 14.12 155 K60 20.0 156 K61 9.33 157 K64 19.33 158 K67 15.6 159 K72 Nil 160 K77 Nil 161 K81 13.08 162 K82 15.38 163 K83 16.53 164 G21 Nil 165 G36 8.9 166 G38 Nil 167 A2 16.15
Continued….
95
168 A4 11.16 169 A5 13.5 170 A6 13.4 171 A7 Nil 172 A9 Nil 173 A11 16.77 174 A12 18.53 175 A13 15.67 176 A15 5.8 177 A17 Nil 178 A18 14.6 179 A19 13.7 180 A21 26.7 181 A24 26.7 182 A26 Nil 183 A27 9.4 184 A29 Nil 185 A30 11.6 186 A32 Nil 187 A33 18.6 188 A34 25.7 189 A36 Nil 190 A37 80.2 191 A56 45.9 192 P2 12.7 193 P7 Nil 194 P8 Nil 195 P9 17.1 196 P10 11.7 197 P15 Nil 198 P24 Nil 199 D3 7.4 200 D4 25.67 201 D6 Nil 202 D7 31.6
Continued….
96
203 D9 Nil 204 D11 18.2 205 D14 Nil
206 D15 15.9 207 D16 Nil 208 D17 9.4 209 D21 Nil 210 D23 12.7 211 D24 Nil 212 D25 17.5 213 D28 Nil 214 D29 Nil 215 D30 Nil 216 H4 Nil 217 H5 26.53 218 H8 Nil 219 H9 13.7 220 H11 Nil 221 H12 Nil 222 H13 12.5
223 H18 14.9 224 H19 Nil 225 H21 13.6 226 H23 Nil 227 H26 18.7 228 H28 Nil 229 H29 14.8 230 H33 11.7
97
Table 3.8: Classification of actinomycete isolates based on inhibition (%) of
pancreatic lipase:
Inhibition (%) Sample code
Nil 1-10 11-20 21-50 >50
B 8 2 8 1 1
C 6 1 9 2 0
V 6 2 6 1 1
S 2 0 0 0 0
E 6 3 5 0 0
M 2 2 0 1 0
T 2 1 0 0 0
F 6 3 8 0 0
N 9 5 8 5 2
K 8 8 20 1 2
G 2 1 0 0 0
A 7 2 13 2 1
P 4 0 3 0 0
D 9 2 4 2 0
H 7 0 8 0 0
Total 84 32 92 15 7
98
Table 3.9: Distribution of actinomycetes isolates showing inhibition greater than
20% on soil sample type:
Sample code Sample type Positive cultures
B Forest 2
C Lake 2
V Tea plantation 2
M Coconut field 1
N Cow barn yard 7
K Lake 3
A Hill station 3
D Desert 2
Total: 22
Table 3.10: Distribution of actinomycetes isolates showing inhibition greater than
20% on soil pretreatment basis:
Sample code Pretreatment Positive cultures
B Calcium carbonate 2
C Calcium carbonate 2
V Calcium carbonate 2
M Calcium carbonate 1
N Calcium carbonate 5
N Phenol 2
K Phenol 1
K Calcium carbonate 2
A Calcium carbonate 2
A Heat 1
D Calcium carbonate 2
99
Table 3.11: Distribution of active actinomycetes isolates on the basis of colour:
Gray White Red Blue Green Yellow Cream Polymorhica
(pink or orange) No aerial mycelium
Total
Number of isolates 106 69 11 9 2 14 4 6 9 230
Number of active isolates 12 7 0 2 0 1 0 0 0 22
Percentage of active isolate 11.30 10.10 0 22.20 0 7.10 0 0 0 9.50
Results and Discussion
100
soils, found that the higher percentage of active isolates was found in red and gray series and
the lower percentage ones in green and white series. Arai (1976), however reported that most
active species of Streptomyces were found in the gray and yellow series of non chromogenic
type and no antibiotic producing species were described in the white and green series of
chromogenic type.
The comparison of the enzymatic inhibition between all colour classes against the
targeted enzymes showed that the isolate N2 and A37 in the gray series displayed the highest
enzyme inhibition against pancreatic lipase enzyme and no activity was noted in the green, red,
yellow, cream, polymorphic and no aerial mycelium series. The highest percentage of isolates
was found in blue series followed by gray and white. Most of the actinomycetes from gray
series inhibited lipase enzyme followed by white. Isolates in gray series were found to be the
most active against target enzymes. These differences in percentage of enzyme inhibition may
imply that the investigated actinomycetes isolates may produce different bioactive compounds.
In fact, it was observed that in the same colour class, most of the isolates showed a range
enzyme inhibition.
From the preliminary screening, seven actinomycete isolates were selected for
secondary screening, which showed inhibition greater than 50 % and were designated as K12,
V22, B80, N2, N80, A37 and K44 (Table 3.7). Production of pancreatic lipase inhibitor by selected
isolates and their consistency and reproducibility was checked on two different media,
production medium I and production medium II. This approach was useful to detect the effect
of various media components, genetic stability of the microbial culture and the yield.
The cultures were grown in four replicates in submerged fermentation. The inhibition
shown by the four replicates of each culture is represented in Table 3.12. Results indicated that
the isolate N2 not only produced the maximum amount of inhibitor but also showed greater
reproducibility. The remaining cultures K12, V22, B80, N62, A37 and K44 were not as potent as N2.
Production media (PM-II) was found to be the best (Table 3.12). As the inhibition shown by
actinomycete N2 in submerged fermentation were reproducible and more potent than the other
cultures, further studies were carried out using this culture.
Results and Discussion
101
Table 3.12: Secondary screening of actinomycetes cultures for reproducibility and
consistent production of lipase inhibitor:
Inhibition concentration (µg) to obtain 50% inhibition of pancreatic lipase
Run-I Run-II Run-III Run-IV
Culture
Number
PM-I PM-II PM-I PM-II PM-I PM-II PM-I PM-II
B80 77.01 71.29 76.42 73.03 78.21 72.80 78.84 72.38
V22 61.20 56.30 61.36 55.89 62.40 54.90 62.60 56.89
N2 39.42 33.00 40.01 33.80 40.26 34.20 41.23 32.40
N62 67.12 61.07 67.81 62.12 69.20 61.78 68.60 62.86
K12 58.89 51.51 57.62 53.36 57.98 52.90 59.20 53.42
K44 69.89 64.43 71.02 65.20 71.62 65.60 70.89 66.10
A37 51.28 46.75 53.21 48.20 54.62 47.45 55.16 47.98
Results and Discussion
102
.
3.B.1. Morphological characters of isolate N2:
The isolate N2 was a gram-positive, non-acid fast, non-motile actinomycete with
extensively branched substrate hyphae (0.3-0.5 µm in diameter). Aerial mycelia form complete
spiral chains of spores with more than 20 spores per chain. The colour of the spores was gray.
The size of the spores ranges from 1.3-1.0 x 1.5 µm, spore surface was warty (Figure 3.4) and
the spores were non motile. No synnemata, sclerotia or sporangia were observed. There were
no distinctive substrate mycelial pigments, diffusible pigments and no sensitivity of substrate
pigment and diffusible pigment to pH was observed. There were no spores on substrate
mycelium (Table 3.13).
3.B.2. Cultural characteristics of isolate N2 on different media:
The growth characteristics of isolate N2 in twelve different media are described in
Table 3.14. The growth was good on all the tested media except on peptone yeast extract iron
agar, nutrient agar and Bennets agar. Gray aerial mycelium was observed on oat meal, starch
casein agar and czapek dox agar, white coloured substrate on starch casein and czapek dox
agar, and dark brown on oat meal agar. White aerial mycelium and colourless substrate
mycelium was observed on nutrient agar. The aerial mycelium was grayish black on peptone
yeast extract iron, tyrosine and glycerol arginine agar, gray substrate mycelium on peptone
yeast extract iron and tyrosine, but colourless on glycerol arginine agar. The growth of isolate
N2 on glycerol aspargine agar was shown in Figure 3.5. Only on Bennet’s agar colourless aerial
and substrate mycelium was observed. The aerial mycelium was grayish white on inorganic
salts and starch, glycerol aspargine agar and potato dextrose agar, but reddish brown substrate
mycelium was observed on inorganic salts and starch, blackish brown substrate on glycerol
aspargine agar and dirty
Results and Discussion
103
Figure 3.4: Scanning electron micrograph showing N2 warty spores and spiral spore chains.
Results and Discussion
104
Table 3.13: Morphological characters of isolate N2:
S.No Morphological character
and pigmentation
Observation
1 Spore chain morphology Spirales
2 Spore chain ornamentation Warty
3 Colour of aerial spore mass Gray
4 No distinctive substrate
mycelial pigments
Nil
5 Pigmentation of substrate
mycelium
Nil
6 Production of diffusible
pigments
Nil
7 Sensitivity of substrate
pigment to pH
Nil
8 Sensitivity of diffusible
pigment to pH
Nil
9 Melanin production on
peptone yeast extract iron
agar
Nil
10 Melanin production on
tyrosine agar
Nil
11 Fragmentation of mycelium Nil
12 Sclerotia formation Nil
13 Sporulation on substrate
mycelium
Nil
Results and Discussion
105
Table 3.14: Cultural characteristics of isolate N2 on different media:
S.No Media Growth Aerial
mycelium
Substrate
mycelium
1 Yeast extract Malt
extract agar
Good Blackish gray Black
2 Oat meal agar Good Gray Dark brown
3 Inorganic salt and
starch agar
Good Grayish white Reddish brown
4 Glycerol aspargine
agar
Good Grayish white Blackish brown
5 Peptone yeast extract
iron agar
Moderate Grayish black Gray
6 Tyrosine agar Good Grayish black Gray
7 Starch casein agar Good Gray White
8 Potato dextrose agar Good Grayish white Dirty green
9 Nutrient agar Moderate White Colorless
10 Glycerol-arginine agar Good Grayish black Colorless
11 Bennets agar Moderate Colorless Colorless
12 Czapek dox agar Good Gray White
Results and Discussion
106
Figure 3.5: Culture growth on glycerol aspargine agar.
Results and Discussion
107
green colour on potato dextrose agar. Blackish gray aerial mycelium and black substrate
mycelium was observed on yeast extract malt extract. Soluble pigment was not produced
on any of the tested media. Melanin was also not produced.
3.B.3. Antimicrobial activity of isolate N2:
The culture and its aqueous extract was tested against various microorganisms
(Table 3.15). The test organisms were gram positive and gram negative bacteria,
Streptomyces and fungi. However, the culture did not show any antimicrobial activity
against the tested microorganisms (Table 3.15).
Table 3.15: Antimicrobial activity of isolate N2:
S.No Antimicrobial activity Observation
1 Bacillus subtilis NCIB 3610 Negative
2 Pseudomonas fluorescens
NCIB 9046
Negative
3 Escherichia coli NCIB 9132 Negative
4 Micrococcus luteus NCIB 196 Negative
5 Candida albicans CBS 562 Negative
6 Saccharomyces cerevisiaae
CBS 1171
Negative
7 Streptomyces murinus ISP
5091
Negative
8 Aspergillus niger LIV 131 Negative
Results and Discussion
108
3.B.4. Enzyme activity tests for isolate N2: Lecithinase, proteolysis, pectin, chitinolysis, hippurate hydrolysis and lipolysis tests:
Chitinolytic activity, proteolysis, lecithinase was detected by the appearance of
zones of clearing around the growth (Table 3.16). There was no zone of hydrolysis for
pectinolytic, hippurate and lipolysis.
Table 3.16: Enzyme activity of isolate N2:
S.No Enzyme activity Observation
1. Lecithinase Positive 2. Pectin Negative 3. Lipolysis Negative 4. Proteolysis Positive 5. Chitin Positive 6. Nitrate reduction Positive 7. Hydrogen sulphide
production Negative
8. Hippurate hydrolysis Negative 9. Milk coagulation Doubtful 10. Milk peptonization Negative
H2S production test:
No characteristic greenish brown, brown, blackish brown, bluish black or black
color of the substrate was observed indicating the absence of H2S production.
Nitrate reduction test:
Pink, red or orange colour was not observed, indicating the test to be negative.
Further, the presence of nitrate was confirmed by adding a pinch of zinc dust after
addition of reagents, wherein pink colour was observed, indicating the presence of nitrate
in the broth.
Results and Discussion
109
Results and Discussion
110
3.B.5. Degradation tests of isolate N2:
Clearance of the insoluble compounds around the growth area was scored as
positive for the degradation of adenine, hypoxanthine, xanthine and guanine. There was
no clearance of zone for aesculin, elastin and xylan indicating a negative test (Table
3.17). Blackening of the media was not observed, which indicated a negative test for
arbutin. Tween 20, 40, 60 and 80 plates were observed for opacity along with control.
There was no change in opacity of the plates and the test was negative. Absence of
orange yellow to pink or purple indicated negative result for allantoin. Bluish black or
black diffusible pigment was not observed and the test was negative for tyrosine.
Hydrolyzed zone was observed for both gelatin and starch which indicates the test to be
positive. The organism hydrolyzed urea producing a characteristic red- violet colour on
the plate. As incubation proceeded, the colour extended towards the bottom of the tube
indicating complete hydrolysis.
3.B.6. Antibiotics resistance of isolate N2:
The organism was found to be susceptible to ampicillin (10µg), chloramphenicol
(30µg), erythromycin (15µg), neomycin (30µg), oxytetracycline (30µg), penicillin G (10
IU), rifampicin (10µg), gentamycin (10µg) and streptomycin (10µg), but not susceptible
to kanamycin (30µg) (Table 3.18).
3.B.7. Effect of temperature and pH on growth of isolate N2:
Good growth was observed between 10 to 370C, no growth was observed at 40C
and poor growth was observed at 450C (Table 3.19). There was no growth at pH 2.0 and
the growth was good between pH 5 to 11 (Table 3.20).
3.B.8. Growth of isolate N2 in the presence of inhibitory compounds:
The growth was good in the presence of sodium chloride upto a concentration of
5% (w/v) after which there was no growth, on further increase in sodium chloride
Results and Discussion
111
concentration upto 13% (w/v). The growth was good on sodium azide, poor on phenol
and there was no growth in the presence of potassium tellurite, thallous acetate and
crystal violet (Table 3.21).
The chromogenic Streptomycetes (melanin producers) tended to be less tolerant to
sodium chloride than the non chromogenic species. Similarly, smooth spored species
were less tolerant than spiny spored forms. Gray coloured species of Streptomycetes were
more tolerable when compared with other colour series (Tresner et al, 1968). 50% of all
Streptomycetes fall in to low or high tolerance group, which is of considerable taxonomic
significance.
3.B.9. Test for carbon source utilization by isolate N2:
The ability of N2 to grow on the test media varied considerably. The growth in
control tube showed very little or no growth and those tubes in which isolates could
effectively utilize the particular carbon source had very profuse growth. Very slight
growth was observed with some carbon sources, indicating that the particular compound
was not adequate source of carbon in that concentration or that the materials used
contained traces of other compounds. The utilization of carbon sources by the isolate N2
is shown in Table 3.22.
The results recorded as follows:
Utilization positive: when growth on tested carbon source was significantly better than
on the basal medium without carbon source but some what less than on glucose.
Utilization doubtful: when growth on tested carbon source was only slightly better than
on the basal medium without carbon source and significantly less than with glucose.
Utilization negative: when growth was similar or less than growth on basal medium
without carbon source.
The isolate utilizes L-arabinose, starch, sorbitol, D-glucose, D-fructose, D-xylose,
meso-inositol, D-mannitol, L-rhamnose, maltose, D-mannose, D-lactose, trehalose, D-
melibiose, dextran, D-sorbitol, adonitol, D-galactose, cellobiose for growth, but not
sucrose, inulin, sodium malonate, sodium benzoate and sodium tartarate. Little or poor
growth was
Results and Discussion
112
Table 3.17: Degradation of various compounds by isolate N2:
S.No Degradation of
Observation
1 Hypoxanthine Degraded
2 Guanine Degraded
3 L- Tyrosine No degradation
4 Elastin No degradation
5 Adenine Degraded
6 Xanthine Degraded
7 Tween 20 No degradation
8 Tween 40 No degradation
9 Tween 60 No degradation
10 Tween 80 No degradation
11 Starch Degraded
12 Xylan Degraded
14 Urea Degraded
15 Allantoin No degradation
16 Gelatin Degraded
17 Aesculin No degradation
18 Arbutin No degradation
Results and Discussion
113
Table 3.18: Resistance of isolate N2 to antibiotics:
S.No Antibiotic Observation
1. Ampicillin (10µg) Not resistant
2. Chloramphenicol (30µg) Not resistant
3. Erythromycin (15µg) Not resistant
4. Neomycin (30µg) Not resistant
5. Oxytetracycline (30µg) Not resistant
6. Penicillin G (10 IU) Not resistant
7. Rifampicin (10µg) Not resistant
8. Gentamycin (10µg) Not resistant
9. Streptomycin (10µg) Not resistant
10. Kanamycin (30µg) Resistant
Results and Discussion
114
Table 3.19: Growth of isolate N2 at different temperatures:
S.No Temperature (0C) Observation
1 4 No growth
2 10 Good growth
3 20 Good growth
4 28 Good growth
5 37 Good growth
6 45 Doubtful growth
Table 3.20: Growth of isolate N2 at different pH
S.No PH Observation
1 2 No growth
2 5 Good growth
3 7 Good growth
4 9 Good growth
5 11 Good growth
Results and Discussion
115
Table 3.21: Growth of isolate N2 in the presence of inhibitory compounds:
S.No Inhibitory compound
(% w/v)
Observation
1 Sodium chloride (1) Good growth
2 Sodium chloride (3) Good growth
3 Sodium chloride (5) Good growth
4 Sodium chloride (7) No growth
5 Sodium chloride (9) No growth
6 Sodium chloride (11) No growth
7 Sodium chloride (13) No growth
8 Sodium azide (0.01) Good growth
9 Sodium azide (0.02) Good growth
10 Phenol (0.1) Doubtful growth
11 Potassium tellurite (0.001) No growth
12 Potassium tellurite (0.01) No growth
13 Thallous acetate (0.001) No growth
14 Thallous acetate (0.01) No growth
15 Crystal violet (0.0001) No growth
Results and Discussion
116
Table 3.22: Growth of isolate N2 on different carbon sources:
S.No Carbon source (1.0% w/v) Observation
1 L-Arabinose Positive
2 Cellulose Negative
3 Sucrose Negative
4 Starch Positive
5 Sorbitol Positive
6 D-Xylose Positive
7 Meso-inositol Positive
8 Mannitol Positive
9 D-Fructose Positive
10 D-Glucose Positive
11 L-Rhamnose Positive
12 Raffinose Negative
13 Maltose Positive
14 D-Mannose Positive
15 D-Lactose Positive
16 Inulin Negative
17 Trehalose Positive
18 D-Melibiose Positive
19 Dextran Positive
20 D-Galactose Positive
21 D-sorbitol Positive
22 Adonitol Positive
23 Cellobiose Positive
24 Xylitol Doubtful
25 Sodium acetate (0.1% w/v) Doubtful
26 Sodium citrate (0.1% w/v) Doubtful
27 Sodium malonate (0/1% w/v) Negative
28 Sodium propionate (0.1% w/v) Doubtful
29 Sodium pyruvate (0.1% w/v) Doubtful
30 Sodium benzoate (0.1% w/v) Negative
31 Sodium tartarate (0.1% w/v) Negative
Results and Discussion
117
observed with cellulose, xylitol, sodium acetate, sodium citrate, sodium propionate and
sodium pyruvate.
3.B.10. Test for nitrogen utilization by isolate N2:
Only slight growth was observed with some compounds, indicating that the
particular compound was not adequate source of nitrogen in that concentration or that the
compounds used contained traces of other compounds and results are shown in Table
3.23. The isolate was found to utilize alanine, L-dopa, L-leucine, glycine, ornithine
mono HCl, tyrosine, glutamic acid, tryptophan, L-hydroxy proline, L-histidine, L-
methionine, L-lysine, L-serine, L-threonine, L-cysteine, potassium nitrate and DL-
amino-n-butyric acid for growth, but not aspartic acid. Little or poor growth was
observed with L-valine, L-arginine and L-phenylalanine.
3.B.11. Test for production of acid and gas by isolate N2:
Production of acid was observed on starch, melibiose, inositol, arabinose, lactose,
rhamnose, fructose, glucose, galactose, maltose, inulin and xylitol. Production of acid
was not observed on mannose, xylose, cellulose, sucrose, sodium acetate and sodium
citrate. However, on none of the carbohydrates gas was produced (Table 3.24).
3.B.12. Chemotaxonomic characteristics of isolate N2:
3.B.12.1.Test for sugars and amino acids:
The extract of isolate N2 contains levo- diaminopimelic acid, which was
confirmed by comparison with the sigma standard samples of meso-diaminopimelic acid
and levo- diaminopimelic acid on the TLC plate.
The carbohydrates migrated in the following sequence from the origin (slowest to
fastest) galactose, glucose, arabinose, mannose, xylose, ribose and rhamnose. Yellowish,
Results and Discussion
118
brown and maroon coloured spots were not observed indicating the absence of hexoses
and pentoses in the cell wall composition of isolate N2. It was concluded that strain N2
has levo- diaminopimelic acid, no characteristic sugar was detectable in the cell wall
(wall chemotype I) (Lechevalier and Lechevalier, 1970) which is a characteristic feature
of Streptomyces.
3.B.12.2. Test for menaquinones:
Peaks corresponding to molecular ions (790, 792 and 788) were found in the mass
spectra of menaquinones isolated from strain (Figure 3.6). From the mass spectra, major
menaquinones detected were MK-9(H8), MK-9(H6) and MK-9(H4) which is a
characteristic feature of Streptomyces.
3.B.12.3. Test for mycolic acid:
The sample was compared with the standard sigma sample of mycolic acid. The
results indicated it to be absent, which is a characteristic feature of Streptomyces.
3.B.12.4. Test for fatty acids:
The resultant peaks were identified with a mixture of standard methyl esters with
gas chromatography. The cellular fatty acids consisted of 12-methyltetradeconic acid (ai-
15:0), hexadecanoic acid (16:0), 14-methylhexadecanoic acid (ai-17:0) and 14-
methylpentadecanoic acid (I-16:0) (Figure 3.7), which is a characteristic feature of
Streptomyces (Kroppenstedt, 1985).
3.B.12.5. Test for phospholipids:
All polar lipids appeared as dark spots on a light green ground
molybdophosphoric acid. Lipids containing amino groups appeared as pink-red spots
with Ninhydrin. Phosphorus containing lipids appeared as blue spots on a white back
ground with Zinzadze reagent.
Results and Discussion
119
Table 3.23: Growth of isolate N2 on sole nitrogen source:
S.No Nitrogen source
(0.1% w/v)
Observation
1 DL-α-Amino-n-butyric acid Good growth
2 Potassium nitrate Good growth
3 L-Cysteine Good growth
4 L-Valine Doubtful growth
5 L-Threonine Good growth
6 L-Serine Good growth
7 L-Phenylalanine Doubtful growth
8 L-Lysine Good growth
9 L-Methiionine Good growth
10 L-Histidine Good growth
11 L-Arginine Doubtful growth
12 L-Hydroxy proline Good growth
13 Tryptophan Good growth
14 Glutamic acid Good growth
15 Tyrosine Good growth
16 Ornithine mono HCl Good growth
17 Glycine Good growth
18 L-Leucine Good growth
19 Aspartic acid Good growth
20 Dopa Good growth
21 Alanine Good growth
Results and Discussion
120
Table 3.24: Production of acid and gas by isolate N2:
S.No Carbon source
(1% w/v)
Acid Gas
1 Starch Positive Negative
2 Melibiose Positive Negative
3 Meso-inositol Positive Negative
4 Arabinose Positive Negative
5 Mannose Negative Negative
6 Xylose Negative Negative
7 Mannitol Negative Negative
8 Lactose Positive Negative
9 Tyrosine Positive Negative
10 Rhamnose Positive Negative
11 Fructose Positive Negative
12 Glucose Positive Negative
13 Galactose Positive Negative
14 Maltose Positive Negative
15 Inulin Positive Negative
16 Xylitol Positive Negative
17 Sodium acetate Negative Negative
18 Sucrose Negative Negative
19 Sodium citrate Negative Negative
20 Cellulose Negative Negative
Results and Discussion
121
Glycolipids appeared as brown spots with α-naphthol. By comparing with the standard it
was observed that the sample contains the type II phospholipids, namely
phosphatidylinositol and phosphatidylmethy-lethanolamine (Lechevalier et al, 1977),
which is a characteristic feature of Streptomyces.
3.B.13. Phylogenetic analysis of isolate N2:
The assignment of N2 to the genus Streptomyces was also supported by the 16S
rRNA gene sequence analysis of N2. The almost complete sequence of 16S rRNA gene
(1464 nt) of N2 following BLAST analysis indicated that N2 is related to species of
Streptomyces ranging from a minimum of 95% [as in S. venezuelae (AB045890), S.
subrutilus (X80825), S. bikiniensis (X79851) and S. bottropensis (D63868)] to a
maximum of 99% [as in S. violceusniger (AJ391822)] (Table 3.25). There are many
other species to which N2 has a similarity ranging between 97 to 98% at the rRNA gene
level (Table 3.25), To further analyse the phylogenetic relationship of N2 a phylogenetic
tree was constructed using NEIGHBOR JOINING and DNAPARS and the results clearly
indicated that N2 falls within the genus Streptomyces (Figure 3.8). It is obvious from
Figure 3.8 that N2 forms a sub clade along with S. violaceusniger (AJ391822), with
which it is closely related (99%) and four other species namely S. malayensis
(AFF117304), S. yatensis (AFF336800), S. melanosporofaciens (AJ391837) and S.
rutgersensis (AY508511). These four species are related to N2 by 98% at the 16S rRNA
gene level and as anticipated form one clade with high bootstrap values (64 to 96%). Five
other species namely S. hygroscopicus (AB045864), S. kasugaensis (AB02442), S.
cebimarensis (AJ560629), S. erumpens (AJ621603) and S. rimosus (AB045883) despite
having 98% similarity with N2 were distributed in different clades and appeared to be
distanced away from the N2 clade and their grouping was not supported by high bootstrap
values. N2 could be differentiated from all the species having more than 97% similarity at
rRNA gene level based on a number of phenotypic and chemotaxonomic characteristics
as listed in Table 3.26. Table 3.27 lists the phenotypic differences between N2 and S.
violaceusniger with which it exhibits 99% similarity. Thus based on the overwhelming
differences in morphological and chemotaxonomic features and
Results and Discussion
122
Figure 3.6: Mass spectra of menaquinones isolated from N2
Results and Discussion
123
Figure 3.7: Cellular fatty acids of the isolate N2
Results and Discussion
124
16S ribosomal RNA gene partial sequence Working GenBank flatfiles:
LOCUS AY950450 1464 bp DNA linear BCT 21-MAR-2005
DEFINITION Streptomyces vayuensis 16S ribosomal RNA gene, partial sequence.
ACCESSION AY950450 VERSION AY950450
KEYWORDS .
SOURCE Streptomyces vayuensis
ORGANISM Streptomyces vayuensis
Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales; Streptomycineae;
Streptomycetaceae; Streptomyces.
REFERENCE 1 (bases 1 to 1464)
AUTHORS Shivaji,S., Prabagaran,S.R., Naveen Babu,K. and Sattur,A.P.
TITLE Streptomyces vayuensis sp. nov.: a new species of the genus Streptomyces
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1464)
AUTHORS Shivaji,S., Prabagaran,S.R., Naveen Babu,K. and Sattur,A.P.
TITLE Direct Submission
JOURNAL Submitted (02-MAR-2005) Microbiology, Centre for Cellular and
Molecular Biology, Uppal Road, Hyderabad, AP 500007, India
FEATURES Location/Qualifiers
Source 1..1464
/organism="Streptomyces vayuensis"
/mol_type="genomic DNA"
/db_xref="taxon:319468"
/note="taxonomic information"
rRNA <1..>1464
/product="16S ribosomal RNA"
Results and Discussion
125
1 tttgagtttt gatcctggct caggacgaac gctggcggcg tgcttaacac atgcaagtcg
61 aacgatgaac cggtttcggc cggggattag tggcgaacgg gtgagtaaca cgtgggcaat
121 ctgccctgca ctctgggaca agccctggaa acggggtcta ataccggata cgactgccga
181 ccgcatggtc tggtggtgga aagctccggc ggtgcaggat gagcccgcgg cctatcagct
241 tgttggtggg gtgatggcct accaaggcga cgacgggtag ccggcctgag agggcgaccg
301 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg
361 cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg gatgacggcc ttcgggttgt
421 aaacctcttt cagcagggaa gaagcgcaag tgacggtacc tgcagaagaa gcgccggcta
481 actacgtgcc agcagccgcg gtaatacgta gggcgcaagc gttgtccgga attattgggc
541 gtaaagagct cgtaggcggc ttgtcgcgtc ggatgtgaaa gcccggggct taactcccgg
601 gtctgcattc gatacgggca ggctagagtt cggtagggga gatcggaatt cctggtgtag
661 cggtgaaatg cgcagatatc aggaggaaca ccggtggcga aggcggatct ctgggccgat
721 actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac
781 gccgtaaacg ttgggaacta ggtgtgggcg acattccacg ttgtccgtgc cgcagctaac
841 gcattaagtt ccccgcctgg ggagtacggc cgcaaggcta aaactcaaag gaattgacgg
901 gggcccgcac aagcggcgga gcatgtggct taattcgacg caacgcgaag aaccttacca
961 aggcttgaca tacaccggaa acatccagag atgggtgccc ccttgtggtc ggtgtacagg
1021 tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg
1081 caacccttgt tctgtgttgc cagcatgcct ttcggggtga tggggactca caggagactg
1141 ccggggtcaa ctcggaggaa ggtggggacg acgtcaagtc atcatgcccc ttatgtcttg
1201 ggctgcacac gtgctacaat ggccggtaca atgagctgcg aagccgtgag gtggagcgaa
1261 tctcaaaaag ccggtctcag ttcggattgg ggtctgcaac tcgaccccat gaagtcggag
1321 tcgctagtaa tcgcagatca gcattgctgc ggtgaatacg ttcccgggcc ttgtacacac
1381 cgccccgtca cgtcacgaaa gtcggtaaca cccgaagccg gtggcccaac ccttgtggag
1441 ggagccgtcg aatgtgggac tggc
Results and Discussion
126
Table 3.25: rRNA gene sequence similarity of Streptomyces vayuensis sp. nov.
(strain
N2) with species of Streptomyces exhibiting > 95% similarity at the
rRNA gene level as determined by BLAST
Species RNA gene similarity homology (%)
Strain Accession no.
S. violaceusniger 99 NRRL B-1476 AJ391822 S. hygroscopicus 98 IFO 13598 AB045864 S. yatensis 98 DSM 41771 AF336800 S. kasugaensis 98 MB273-C4 AB024442 S. rutgersensis 98 DSM 40830 AY508511 S. cebimarensis 98 DSM 41798 AJ560629 S. erumpens 98 DSM40941 AJ621603 S. rimosus 98 JCM 4667 AB045883 S. malaysiensis 98 ATB-11 Af117304 S. melanosporofaciens 97.8 NRRL B-12234 AJ391837 S. platensis 97.7 SAFN-030 AY167807 S. sclerotialus 97.6 DSM 43032 AJ621608 S. mashuensis 97.5 DSM40221 X79323 S. griseocarneus 97.5 DSM40004 X99943 S. catenulae 97.5 DSM 40258 AJ621613 S. tubercidicus 97.5 DSM40261 AJ621612
S. niger 97.4 DSM 43049 AJ621607 S. olivaceiscleroticus 97.4 DSM 40595 AJ621606 S. sparsogenes 97.3 NRRL 2940 AJ391817 S. peucetius 97.1 JCM 9920 AB045887 S. thermodiastaticus 97.0 DSM40573 Z68101 S. yunnanensis 97.0 YIM 41004 AF346818 S. cattleya 97.0 JCM 4925 AB045870 S. thermocoprophilus 97.0 B19 AJ007402 S. lydicus 96 ATCC 25470 Y15507 S .sampsonii 96 DSM 40394 Z76680 S. thermoviolaceus 96 DSM 41392 Z68095 S. griseus 96 IFO 13550 AB045867 S. yeochonensis 96 CN 732 AF101415 S. somaliensis 96 DSM 40738 AJ007403 S. venezuelae 95 NRRL2277 AB045890 S. subrutilus 95 ATCC27467 X80825 S. bikiniensis 95 ATCC11062 X79851 S. bottropensis 95 ATCC25435 D63868
Results and Discussion
127
S t r a i n N 2
Figure 3.8: Neighbour-joining tree based on 16S rRNA gene sequences showing the
phylogenetic relationship between Streptomyces vayuensis sp. nov. and other species of
the genus Streptomyces and related reference microorganisms. Bootstrap values
(expressed as percentages of 1000 replications) greater than 50% are given at the nodes.
Results and Discussion
128
the phylogenetic analysis it is proposed to assign N2 the status of a new species for which
the name Streptomyces vayuensis sp. nov. is proposed.
3.B.14. Comparative studies of isolate N2:
Isolate N2 was compared with 19 similar strains and the comparative studies were
shown in Table 3.26. Although the N2 isolate was closely related to Streptomyces
violaceusniger at species level, however, it differed with the isolate on counts of, the
spore surface of N2 isolate was warty, where it was rugose in case S. violaceusniger. The
isolate N2 cannot utilize both raffinose and sucrose, but S. violaceusniger utilizes. S.
violaceusniger degrades both arbutin and tyrosine, whereas N2 isolate did not degrade. S.
violaceusniger hydrolyzes esculin, whereas N2 isolate did not hydrolyze. S.
violaceusniger shows antifungal activity against Aspergillus niger whereas N2 isolate did
not show the antifungal activity. Growth of the isolate N2 was good at 10 and 450C and
there was no growth in case of S. violaceusniger at both these temperatures (Table 3.27).
It is evident from the chemical, molecular, systematic and phenotypic data that the isolate
N2 should be given species status in the genus Streptomyces (Waksman and Henrici,
1943). Therefore, the name Streptomyces vayuensis is proposed for the isolate N2.
3.B.15. Final and brief description of isolate N2 as Streptomyces vayuensis sp.nov:
Streptomyces vayuensis sp. nov, gram-positive, non-acid fast, non motile
actinomycete with extensively branched substrate hyphae (0.3-0.5 µm in diameter).
Aerial mycelia form complete spiral chains of spores with more than 20 spores per chain.
The size of the spores ranges from 1.3-1.0 x 1.5 µm, spore surface was warty and the
spores are non motile. No symnemata, sclerotia or sporangia are observed. The reverse
sides of colonies are colorless/gray/reddish brown. The colour of the aerial and substrate
mycelia varied depending on the growth medium.
Melanin and soluble pigments were not produced on any media tested. Grows
between 10-37°C and pH 5-13. Tolerates upto 5% NaCl. No hydrolysis of starch, no
Results and Discussion
129
production of hydrogen sulphide, liquefies gelatin, coagulates milk and is positive for
urease but negative for catalase and is not reduced to nitrite. Degrades casein, xanthine,
adenine, hypoxanthine and guanine but not tyrosine, tryptophan, Tween 20, 40, 60, 80,
xylan, allantoin, aesculin and arbutin. Produces acid from L-arabinose, galactose,
cellulose, starch, D-melibiose, meso-inositol, D-lactose, tyrosine, L-rhamnose, D-
fructose, D-glucose, maltose, inulin, Xylitol and no acid production from mannose,
xylose, mannitol and sucrose. Cells are susceptible to ampicillin, chloramphenicol,
erythromycin, neomycin, oxytetracycline, penicillin G, rifamycin, gentamycin and
streptomycin; but negative to kanamycin. Utilizes L-arabinose, starch, sorbitol, D-
glucose, D-fructose, D-xylose, meso-inositol, D-mannitol, L-rhamnose, maltose, D-
mannose, D-lactose, trehalose, D-melibiose, dextran, D-galactose, cellobiose, alanine, L-
dopa, L-leucine, glycine, ornithine mono HCl, tyrosine, glutamic acid, tryptophan, L-
hydroxy proline, L-histidine, L-methionine, L-lysine, L-serine, L-threonine, L-cysteine,
potassium nitrate and DL- amino-n-butyric acid for growth, but not sucrose, inulin,
sodium malonate, aspartic acid and adonitol. Little or poor growth is observed with
cellulose, xylitol, sodium acetate, sodium citrate, sodium propionate, sodium pyruvate,
raffinose, L-valine, L-arginine and L-phenylalanine. Mycolic acids were absent. Major
menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). Cellular fatty acids consisted
of 12-methyltetradeconic acid, hexadecanoic acid, 14-methylhexadecanoic acid, 14-
methylpentadecanoic acid. The strain Streptomyces vayuensis has been deposited in
Microbial Type Culture Collection MTCC at IMTECH, Chandigarh with accession
number MTCC 5219.
Results and Discussion
130
Table 3.26: Phenotypic and chemotaxonomic characteristics that differentiate Streptomyces vayuensis sp. nov. (strain N2) from the closely related species of the genus Streptomyces
Test 1 2* 3* 4* 5* 6* 7* 8* 9* 10* 11* 12* 13* 14* 15 16* 17 18 19 20 Carbon utilization L-arabinose + + + + + - - + + + d - d + d d - + - d D-fructose + + + + + + d + + + d + d + d d + + d d D-galactose + + + + + + + + + + d - d + d d d + d d Meso-inositol + + + + + + + + + + - + + + - + + + - D-mannitol + + + + + - + + + + + + + + + + + - + + Raffinose + + + - + + + + - + d + d + - + + + - - L-Rhamnose + + + + + - + + + + + + - - - + - - - - Sucrose - + - + + - - - - + d + + - + d + - - +
D-xylose - + + + + - + + + + d + d - d d + - d d Enzymatic activity Hippurate - - + - - - - - - - d - - + d d d - d d Esculin + + + + + + + + - - - - d + d d d - d d Allantoin - - + - - - - - - - - - d + d d d - d d Hydrogen sulphide production
- - + - - - - - - - + - - - + + d - d +
Nitrate reduction - - - + - + + - - - - - - + - - d - d - Urea utilization + - - + + + - + - d - d + d d d - d d Starch hydrolysis + - + - + - + + + d + + d d d + d d Degradation Adenine + + - + + + + + + - d - d + d d d d d d Arbutin + + + + + - + - - - + - + + + + d d d + Elastin + + - - + + - - - - + + + + + + + + Xylan + + + + - - - - - - d - d + d d d d d d
Results and Discussion
131
Tween-80 + + + + - - - - - - d - d + d d d d d d Xanthine - - - + - - - - + - + - - + + + + + + L-tyrosine - + + + + + + + - - - + d d d d d d d Cellulose hydrolysis - - - - - - - - - - d - - d d d d d d d Gelatin liquefaction + + + + + + + + + - d - + d d d d d d d Milk coagulation - - - - - + - - - - - - - d d d d d d Milk peptonization + - - + - + - - - - - - - d d d d d d Melanin - - - - - - - + - - - - - - - - - - - - Melanin - - - - - - - + - - - - - - - - - - - - Antimicrobial activity
Bacillus subtilis - - - - - - - + - - - - + + - - d - - - Candida albicans - - + - - - - + - - - - - + + - d d - + Aspergillus niger + + + + + + + + - - - + - + + - d d - + Growth at (°C) 10 - - - - - - - - + + - + - + + d d d d d 45 - - - - - - - - + + - + - + - + - - - Sodium chloride 7% w/v
- - - - - - - - + + + + + + + + d d d d
+: good growth, -: no growth, ± : doubtful, d: data not available * Present study The numbers in the top row of the table represent characteristics of the following species: 1, S. yunnanensis (Qi Zhang et al., 2003); 2, S.hygroscopicus; 3, S. melanosporofaciens; 4, S. sparsogenes; 5, S. violaceusniger; 6, S. kasugaensis; 7, S. yatensis; 8, S. malaysiensis; 9, Strain N2; 10, S. erumpens; 11, S. thermodiastaticus; 12, S. olivaceiscleroticus; 13, S. platensis; 14, S. rimosus; 15, S. niger (Goodfellow et al, 1986c); 16, S. rutgersensis; 17, S. peucetius (Grein et al, 1963); 18, S. tubercidicus (Nakamura, 1961); 19, S. catenulae (Williams et al, 1989); 20, S. selerotialus (Williams et al, 1989).
Results and Discussion
132
Table 3.27: Differences in the phenotypic and chemotaxonomic features of
isolate N2 and S. violaceusniger, the phylogenetically nearest neighbour.
Characteristics Isolate N2 S. violaceusniger Spore surface Warty Rugose Utilization of raffinose Negative Positive Utilization of sucrose Negative Positive Esculin hydrolysis Negative Positive Degradation of arbutin Negative Positive Degradation of L-tyrosine Negative Positive Antimicrobial activity on Aspergillus niger Negative Positive Growth at 10°C Positive Negative Growth at 45°C Positive Negative
Results and Discussion
133
3.C.1. Isolation and purification of pancreatic lipase inhibitor:
The isolation of streptolipin was carried out by using of porcine pancreatic lipase assay
(Figure 3.9). At the end of fermentation, the biomass was separated from 6.5 litres cultured
broth by centrifugation and dried at 300C. The biomass (40 g) was then ground to small
granules to which ethyl acetate (1.6 litres) was added and kept on rotary shaker for two hours.
The organic layer was separated by centrifugation and concentrated under reduced pressure to
obtain crude extract (6.02 g). The crude extract was loaded on to silica gel column
chromatography and eluted successively with hexane, chloroform and methanol and their
combinations. The active fraction in hexane: chloroform (1:1) eluted as an yellow eluent
which upon concentration under reduced pressure (4.3 g) and was further chromatographed on
silica gel column chromatography with hexane and ethyl acetate at different ratios of
increasing polarity. The active fraction eluted as colourless fraction (2.6 g) and was further
chromatographed on Sephadex LH-20 with methanol. The active fractions were pooled (1.68
g) and further purified by preparative TLC using benzene: ethyl acetate (7:3) and benzene:
methanol (9:1) and benzene. The inhibitor was extracted from silica with diethyl ether to obtain
an off white solid (26.2 mg). The purified compound moved as a single spot on silica gel F254
with an Rf 0.34 in benzene: methanol (9:1). The structure was accomplished with this
compound.
3.C.2. Adaptation of the PNPB spectrophotometric assay to TLC system:
During the purification of the compound the spectrophotometric assay was adapted to
the TLC system. The adaptation to the TLC method was as follows: The principle of the assay
is that the substrate, p-nitrophenyl butyrate is hydrolyzed by lipase to give a p-nitrophenol
(yellow colour). The presence of the inhibitor is indicated by the non action of the enzyme on
the substrate and thereby appearing as a colourless spot. The inhibitor was run on a TLC plate
with a mobile phase Benzene: Methanol (9:1). The plate was then air dried to remove the traces
of solvent. After complete evaporation of the solvent, pancreatic lipase enzyme was sprayed
on the TLC plate at a concentration of 20 mg / ml with sprayer and incubated at 300C for 3
minutes, after which the substrate para-
Results and Discussion
134
Figure 3.9: Purification protocol of lipase inhibitor
Fermented broth (6.5 L) Centrifugation
Mycelium (40 g)
Ethyl acetate extraction
Crude extract (6.0 g)
Silica gel(#60-120) (4.3 g) Hexane: CHCl3 (1:1)
Silica gel(#60-120) (2.6 g)
Hexane: ethyl acetate (9:1)
Sephadex LH-20 (1.689 g) Methanol 40 fractions
12 active fractions checked on TLC Preparative TLC (1.2 g) Benzene
Benzene: methanol (9:1) Benzene: ethyl acetate (7:3)
Pure compound (26.2 mg)
Results and Discussion
135
nitro phenyl butyrate (100 mM) was sprayed and incubated again at 300C for 3 minutes. The
background of the plate was yellow and the inhibitor appears as a white spot. The procedure in
the form of a flow sheet is given in Figure 3.10.
Lipase Spectrophotometer Assay:
Lipase +
PNPB PNP Butyric acid
3.C.3. Physico-chemical properties of streptolipin
Streptolipin is an off white solid. It is freely soluble in chloroform, dimethyl sulphoxide
and acetone (Table 3.29). It is sparingly soluble in methanol, acetonitrile, diethyl ether and
ethyl acetate and insoluble in hexane, various buffers [phosphate buffer (pH 7.4 to 8.0), acetate
buffer (pH 5.0 to 6.0), trisbuffer (pH 8.0 to 9.0)], aqueous solutions [5% NaOH, 5% acetic
acid, 5% sodium bicarbonate] and water. The inhibitor decomposes at 184.4oC. The λ max nm
(ξ) in methanol 210 (21,752), 260 (11,400). HPLC analysis shows retention of 19 minutes on a
RP-C18 column with a gradient mobile phase of acetonitrile and water at 210 nm. Elemental
analysis: theoretical C, 67.86; H, 9.67; N, 3.68; P, 4.07; S, 4.21 found C, 67.02; H, 9.86; N,
3.94; P, 3.89; S, 4.36. IR (ν values in cm-1): 1434, 1312, 1046, 954, 761 for quinoxaline, 3436,
2913, 1771,1714, 1659, 1236, 667 (Figure 3.11). The molecular formula of the compound was
calculated as C43H73N2O5SP, based on the mass spectra and 1H and 13C NMR spectra. 1H and 13C NMR data (Figure 3.12,3.13, 3.14 and Table 3.26A). EI-MS m/z: 761 (M+),
Figure 3.10: Adaptation of the PNPB (p-nitrophenylbutyrate) spectrophotometric assay
to TLC system
O
NO2
C
O
(CH2)2 CH3
NO2
OH
CH3 (CH2)2 C
O
OH
Results and Discussion
136
TLC Plate with Inhibitor
Benzene: Methanol (9:1)
Sprayed with pancreatic lipase enzyme (20 mg/ ml)
Incubated for 3 min at 300C
Sprayed with Substrate (PNPB, 100 mM)
Incubated for 3 min at 300C
Inhibitor and Background
(white) (yellow)
Results and Discussion
137
Figure 3.11 IR spectrum of inhibitor, isolated from Streptomyces vayuensis.
Results and Discussion
138
Figure 3.12: 1H NMR spectrum of inhibitor from Streptomyces vayuensis
Results and Discussion
139
Figure 3.13: 13C NMR spectrum of inhibitor from Streptomyces vayuensis
Results and Discussion
140
Figure 3.14: LCMS of inhibitor from Streptomyces vayuensis
Results and Discussion
141
763 [M+2]+, [M-C5H11]+, 579, 295, 337, 225, 128, 113, 112,111 (Figure 3.14 and Table
3.28B). The lassaigne’s sodium test indicated the presence of nitrogen, phosphorous and
sulphur.
3.C.4. Structure elucidation of the inhibitor
The structure of the inhibitor was established with the help of extensive spectroscopic
studies such as UV, IR, NMR (1H and 13C; 1 and 2-D) and LC-MS along with the support from
elemental analysis and colour reactions. The elemental analysis showed the presence of P, N
and S and indicated the molecular formula as C43H73N2O5SP. The UV absorption spectra
showed the maxima at 210 and 260 nm, these corresponds to the aromatic -C=N (π- π*
transition) group in a quinoxaline nucleus. IR spectra of the inhibitor revealed the absorption
bands attributable to quinoxaline (1434, 1312, 1046, 954, 761, imine (2249 and 2123 cm-1),
OH or NH (3,452 cm-1) groups and a characteristic P–O and P–O–C stretching (1046 cm-1). 1H
NMR spectrum showed signals in the range of δ 7.05-7.15 as multiplet for the aromatic
protons. The 13C NMR spectrum showed six signals between δ 130-140 along with two more
signals at δ 155.3 and 171.1, which are characteristic of quinoxaline nucleus. The IR
absorption at 954 cm-1 and the doublet of doublet signal at δ 5.86 (16 Hz) for two protons in
proton NMR spectrum indicated the presence of a trans olefinic double bond in alkyl chain,
which is confirmed by the presence of two carbon signals at δ 122.4 and 147.8 ppm in Carbon
NMR spectrum. This is also supported by the presence of two multiplets δ 1.6-1.7 and 1.92-
1.96 in proton NMR spectrum and two carbon signals at δ 30.1 and 39.6 indicated the protons
and carbons adjacent to double bond. The signal at δ 179.7 shows the presence of carbonyl
carbon of an ester group. Mass spectral analysis disclosed the structure of the inhibitor further
(Figure 3.14). LC-EI-MS showed the M+ ion at m/z 761 along with a low abundance ion at
m/z 763 (M+2)+, which are in agreement with the molecular formula proposed from elemental
analysis. The ion at m/z 295 suggested the presence of nonadecenoyl moiety, while the ion at
m/z 579 with loss of 182 units (C13H26) moiety is due to the presence of double bond on C-6,7
in nonadecenoyl moiety. The presence of double bond in nonadeca-6-enoyl moiety was also
confirmed by the NMR signals as described earlier. The LC/MS data also substantiated the
presence of quinoxaline moiety [at m/z 128], which was further confirmed by the chemical
Results and Discussion
142
tests for the presence of both nitrogen and phosphorous. The ions at m/z 111, 112 and 113
confirmed the presence of thiophosphoryl group, that is suggested from the IR spectrum. The
triplet at δ 2.36-2.39 in proton NMR spectrum and signal at δ 24.3 indicated the OCH2 group
of hexadecyl moiety attached to thiophosphoryl group.
HMBC spectrum showed the connectivities between signals δ 147.8 and 122.4 ppm (C-
6’ and 7’ respectively) and the proton signals at δ 1.92-1.96 ppm (H-5’) related to the double
bond. The connectivities between the carbon signals δ 14.5 ppm (C-19’ and 16’’) and the
proton signals at δ 1.25-1.35 ppm (H-17’, 18’ and H-14’’, 15’’) were also observed. The
δ 171.1 ppm (C-2) signal showed weak connectivities with the proton signals at δ 7.05-7.15
ppm (H-8) and 2.04-2.12 ppm (H-2’) indicating the link between nonadeca-6-enoicacid and
quinoxaline nucleus. The carbon signal at δ 27.8 ppm (C-1’’) showed peak connectivity with
the proton signal at δ 5.37 ppm (hydroxyl proton of thiophoshphoryl group). The quaternary
carbon signals at δ 138.8 and 140.2 ppm (C-9 and 10 respectively) were not observed in the
HMBC spectrum due to the low concentration of the compound.
The ions at m/z 337 and 225 suggested the second alkyl chain as –O-P=S (OH) -O-
(CH2)15-CH3. Further conformation of the structure was obtained from 2DHMQCT and1H-1H
COSY. 2DHMQCT spectra also gave corresponding carbon signals wherever protons were
attached. The molecular formula C43H73N2O5SP was confirmed on the basis of high resolution
LC/MS [M+ 761, M+2 763] in combination with proton and carbon NMR data and finally
confirmed by its elemental analysis. Two dimensional NMR analysis including COSY, HMQC
and HMBC spectra led to the assignment of this compound as nonadeca-6-enoicacid-3-
(hexadecyloxy- hydroxy thiophosphoryloxy)-quinoxalin-2-yl ester.
Results and Discussion
143
Based on the above data, the most probable structure of the compound was found to be
as follows:
N
N O
OP
OS
OH
O
1'
2'3'
4'5'
6' 7'8'
9'10'
11'12'
13'14'
15'
12''
13''
14''
16'
16''
18'17'
15''
1''2''
3''
4''
5''
6''
7''
8''
9''
10''
11''
19'
1
2
3
45
6
7
89
10
Figure 3.15: Chemical structure of streptolipin
Based on this data, proposed structure of the inhibitor was chemically named as nonadeca-6-
enoic acid-3-(hexadecyloxy-hydroxy-thiophosphoryloxy)-quinoxalin-2-yl ester and is given as
Figure 3.15. A literature search revealed that this compound did not match with any reported
lipase inhibitors or of any Streptomyces metabolites. The inhibitor is henceforth designated as
streptolipin [Streptomyces, lipase inhibitor]. The isolated inhibitor exhibited a dose dependent
inhibition against pancreatic lipase inhibition at an IC50 of 349 nM (Figure 3.16).
3.C.5. Kinetic studies on the inhibition of streptolipin against pancreatic lipase: 3.C.5.1. Lineweaver-Burk (LB) plot of streptolipin inhibition by streptolipin:
In order to determine the nature of inhibition, pancreatic lipase was exposed at different
concentrations of streptolipin. The results illustrated in Figure 3.18 shows that increasing the
concentration of streptolipin resulted in a family of lines with a common intercept in the
second quadrant resulting in increase in Vmax with different slope values. However, as
streptolipin concentration was increased from 0.125 to 0.625 µM, Km value was same which
denoted that it is non competitive inhibition. The equilibrium B plot versus the inhibitor
concentration, which is linear as shown in the Figure 3.19 and the Ki value was found to be
0.714 µM. These results indicate that streptolipin is a non-competitive inhibitor.
Table 3.28.A: 1H and 13C NMR chemical shifts of compound
Results and Discussion
144
H/C No δ (Η) Multiplicity J (Hz) δ ©
2 171.1
3 155.3
5 130.4
6 128.4
7 128.2
8
7.05-7.15
m
7.0
130.1
9 138.8
10 140.2
1′ 179.7
2′ 2.04-2.12 t 8.0 25.1
5′ 1.92-1.96 m 39.5
6′
(CH=CH)
5.86-5.89 m 147.8
7′(CH=C
H)
5.86-5.89 m 16 122.4
8′ 1.6-1.7 m 10 30.3
19′ (CH3) 0.88 t 6.5 14.5
1″
(OCH2)
2.36-2.39 t 7.5 27.8
16″(CH3) 0.89 t 6.0 14.5
OH 5.37 s
-(CH2)n- 1.25-1.35 m 29.5-30.1
* :500 MHz ** :125MHz
Results and Discussion
145
Table 3.28.B: Mass spectrometry; fragmentation of streptolipin in EI-MS (m/z)
(m/z) Fragment
761.1 (M+)
763.4 [M+2]+
468.4 [M- X]+
427.4 [M- Y]+
579.4 [M-Z]+
514.4 [M-C18H33]
337 . PO3S-C16H34[Y]
295.2 O2C19H35 [X]
181 C13H26 [Z]
255.2 C15H15O2N2
112 . PO3HS
225.2 . CH2-(CH2)14-CH3
128 C8H4N2
Results and Discussion
146
Table 3.29: Physico-chemical properties of the inhibitor
Characteristics Compound data
Appearance Off white solid
Solubility Chloroform, dimethyl sulphoxide, acetone
U.V 210, 260
HPLC (Rt) (minute) 19
Melting point 184.40C
Theoretical value C: 67.86; H: 9.67; N: 3.68
Found C: 67.02; H: 9.86; N: 3.94.
Molecular formula C43H73N2O5S
Molecular weight 761.09
Exact mass 760.5
IR (cm-1) 1434,1312, 1046, 954, 761 for quinoxaline,
3436,2913, 1771,1714, 1659, 1236, 667
NMR See Table 3.28.A
LC/MS (EI-MS m/z)
Fragmentation
See Table 3.28.B
IUPAC name nonadeca-6,10-dienoic acid-3-
(hexadecylloxy-hydroxy-
thiophosphoryloxy)-quinoxalin-2-yl ester.
Designated as Streptolipin
Results and Discussion
147
3.C.5.2. Determination of irreversibility of the pancreatic lipase enzyme inhibition:
Streptolipin was checked for reversibility/irreversibility of specific activity of the
enzyme exposure to the inhibitor. Enzyme fraction eluting in the void volume was used for
reversibility/irreversibility studies. The inhibitor was not bound to Sephadex G-25 column
chromatography indicating irreversible nature.
3.C.6. Other biological activities of streptolipin:
Streptolipin did not show any inhibition against soybean 15-lipoxygenase, rat lens
aldose reductase, rat brain acetyl cholinesterase and other pancreatic enzymes such as
phospholipase A2, amylase, trypsin and chymotrypsin upto 200 µM. Streptolipin did not
show any activity against lipases like Mucor javanicus, Rhizopus oryzae, Candida rugosa,
Penicillium roqueforti, Pseudomonas sp, Candida rugosa, Candida antarctica and Humicola
sp upto 200 µM. Streptolipin revealed no antimicrobial activity upto a concentration of 200 µg
by disc plate method against Bacillus subtilis, B. pumilis, E. coli, Pseudomonas aeruginosa,
Penicillium notatum, Aspergillus niger, Saccharomyces cerevisiae and Candida utilis.
Results and Discussion
148
Figure 3.16: Concentration dependent inhibition of streptolipin on pancreatic lipase
0
20
40
60
80
100
120
0 100 200 300 400 500 600 700
Inhibitor concentration (nM)
Rem
aini
ng a
ctiv
ity (%
)
Results and Discussion
149
Figure 3.17:[V] versus [S] plot in the presence of different fixed concentrations of streptolipin
0
0.2
0.4
0.6
0.8
1
1.2
0 0.05 0.1 0.15 0.2 0.25 0.3
[S]
[V]
I=0(control) I=0.125 µM I=0.250µM I=0.375µMI=0.5 µM I=0.625 µM
Results and Discussion
150
Figure 3.18: L-B plot of 1/[V] vs 1/[S] in the presence of different fixed concentraions of streptolipin
-10
-5
0
5
10
15
20
25
30
35
40
-20 -10 0 10 20 30 40 50 60
1/[S]
1/[V
]
I=0(control) I=0.125 µM I=0.250µM I=0.375µM I=0.5 µM I=0.625 µM
Results and Discussion
151
Figure 3.19: The slope (km/Vmax) of the lines described from the double reciprocal
plot are plotted against the streptolipin concentration in order to derive
the Ki value for the inhibitor:
y = 0.1341x + 0.0408R2 = 0.8017
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
-2 0 2 4 6
Inhibitor Concentration
Slop
e of
rec
ipro
cal p
lot
Results and Discussion
152
3.D. Studies on media optimization for streptolipin production:
Large-scale fermentation processes are frequently developed with complex media that
contain a large variety of nutrients and growth factors. These media are generally economical
and able to support high product yields. However, many of these processes experience
unacceptable variability in the productivity and product profiles (especially trace
contaminants). This variability is usually attributed to changes in the composition of one or
more of the complex ingredients. One approach at addressing this problem is to develop
chemically defined media that use pure compounds in known proportions and have
traditionally been employed only in the laboratory to support biosynthetic studies. Defined
medium can offer other advantages such as low foaming, translucency and the relative ease of
product recovery and purification.
3.D.1. Standard HPLC curve for streptolipin: Different concentrations of pure streptolipin ranging from 10 to 50 µg, were injected
into HPLC and the peak area was determined. A standard curve was constructed by plotting the
peak area versus the streptolipin concentration (Figure 3.20). Based on this standard curve
streptolipin concentration in crude samples was determined.
3.D.2. Time course fermentation for streptolipin production:
A typical time course of fermentation is shown in Figure 3.21. The production medium
as used in screening studies (ISP medium I) was used for the experiment. It was seen that
biomass increased steadily till it reached the maximum of 8.35 g/L at 168 h after which it
reached stationary phase. The death phase was not reached till 240 h of fermentation time. The
initial pH of the medium was 7.2 and gradually increased to 8.8 and maintained till the end of
the fermentation. The course of extracellular and intracellular streptolipin production was quite
characteristic. The production of the inhibitor both extracellularly and intracellularly started
after 48 h and increased till it reached a maximum at 168 h. The maximum amount of
Results and Discussion
153
streptolipin produced extracellularly was 15 mg/L and intracellularly 42 mg/L after which it
decreased.
Figure 3.20: Standard curve for streptolipin
0100000002000000030000000400000005000000060000000700000008000000090000000
0 10 20 30 40 50 60
Streptolipin (µg)
Peak
are
a
As the intracellular inhibitor concentration was higher than extracellular, further studies
on this organism were carried out only on intracellular inhibitor. The optimum time of 168 h
for the production of streptolipin was taken for the termination of fermentation.
3.D.3. Optimization of physical parameters for production of streptolipin:
3.D.3.1. Selection of inoculum media for production of streptolipin:
Two inoculum media were tried in this study. The difference in the medium was the
presence of glycerol in the inoculum medium II and corn steep liquor and glucose in inoculum
medium I(Section 2.2.A.7). The inoculum medium I showed highest production of streptolipin,
than inoculum medium II (Table 3.30). The maximum biomass was 8.38 and 6.98 g/L and the
productivity of streptolipin was found to be 42.6 and 34.98 mg/L in the inoculum media I and
II, respectively. Inoculum medium I was used for further studies.
Results and Discussion
154
Figure 3.21: Time course fermentation for streptolipin production
0
5
10
15
20
25
30
35
40
45
0 24 48 72 96 120 144 168 192 216 240
Time (hrs)
Stre
ptol
ipin
(mg/
L)
0
1
2
3
4
5
6
7
8
9
10
pH, D
ry b
iom
ass
(g/L
)
Intracellular (mg/L) Extracellular (mg/L) pH Dry biomass(g/L)
Table 3.30: Selection of inoculum media for production of streptolipin:
Results and Discussion
155
Inoculum media Dry biomass (g/L) Streptolipin (mg/L)
I 8.38 42.60 II 6.98 34.98
3.D.3.2. Effect of temperature on production of streptolipin:
The effect of different temperature on streptolipin production was tested using the ISP
medium I and the results are shown in Figure 3.22. There was marked increase in the
production of streptolipin from 10 to 300C reach the highest 48.81 mg/L and biomass to 12.52
g/L. The productivity of Streptolipin increased from 0.05 to 0.29 mg/L/h from 10 to 300C and
further increase of the temperature decreased the productivity. Further increase of temperature
till 450C, decreased the production of streptolipin as well as biomass. The optimal cultivation
temperature on cell growth and streptolipin production was 300C and used for further studies.
3.D.3.3. Effect of initial pH of the medium on production of streptolipin:
The effect of initial pH from pH 2.0 to 9.0 was studied on the ISP medium I. The
results indicate that the production of streptolipin to be strongly dependent on the pH of culture
broth. The cell growth and streptolipin production increased from pH 2.0 to 7.0. Further
increase in pH from 7.0 to 9.0 decreased the dry biomass and streptolipin production (Figure
3.23). The streptolipin productivity increased from pH 2.0 to 7.0 (0.05 to 0.29 mg/L/h) and
further decreased. The highest level of biomass (12.29 g/L), streptolipin (48.99 mg/L) and the
maximum productivity (0.29 mg/L/h) was observed at pH 7.0.
Results and Discussion
156
Figure 3.22: Effect of temperature on production of streptolipin
0
10
20
30
40
50
60
10 20 25 30 35 40 45
Temperature 0C
Stre
ptol
ipin
(mg/
L),
Dry
bio
mas
s (g/
L)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Stre
ptol
ipin
pro
duct
ivity
(mg/
L/h
)
Streptolipin (mg/L) Dry biomass (g/L) Streptolipin Productivity (mg/L/h)
Results and Discussion
157
Figure 3.23: Effect of initial pH on production of streptolipin
0
10
20
30
40
50
60
2 5 7 8 9 10 11
pH
Stre
ptol
ipin
(mg/
L)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Dry
bio
mas
s (g/
L),
Stre
ptol
ipin
Pro
duct
ivity
(mg/
L/h
)
Streptolipin (mg/L) Dry biomass (g/L) Streptolipin Productivity mg/L/h
3.D.3.4. Effect of aeration on production of streptolipin:
Results and Discussion
158
The oxygen demand of a fermentation process is generally met by aeration. One
important aspect of aeration in fermentation is the resistance to the transfer of dissolved
oxygen through the medium to the microbial cell, which could be overcome by increased
agitation. The transfer of oxygen from air to solutions forms the second important factor in
aeration. It is usually was solved in shake flasks by modeling working volume (medium to
flask volume) ratios. The effect of aeration on streptolipin production was carried out with a
flask volume: flask ratio (medium to flask volume) from 0.05 to 0.4.
Biomass production increased gradually as the volume: flask ratio increased (Figure
3.24). The highest biomass was reached at 17.66 g/L at a volume: flask ratio of 0.4. On the
other hand, streptolipin production increased dramatically from 0.05 to reach the highest of
48.93 mg/L at 0.2. At ratios above this, the inhibitor yield fell to a low of 9.01 mg/L at a ratio
of 0.4.
3.D.4. Optimization of nutritional parameters for the production of streptolipin: 3.D.4.1. Screening of standard media for the production of streptolipin
The composition of the growth medium has an important influence on metabolite
production (Porter, 1975). There is no generalized medium applicable to all organisms,
standard media reported in literature for production of various secondary metabolites (Table
3.31) were screened for the production of streptolipin and the results shown in Table 3.32. The
poor growth of the strain on Czapek-Dox medium was evidently due to the inability of the
organism to utilize sucrose. Among the International Streptomyces Project (ISP) production
media, accumulation of biomass and streptolipin production was highest on ISP media I
(biomass 12.766 g/L, streptolipin 48.81 mg/L) followed by ISP V (biomass 12.57 g/L,
streptolipin 45.78 mg/L) and ISP VIII (biomass 11.96 g/L, streptolipin 44.92 mg/L) and
among other media tried, accumulation of biomass and streptolipin production was highest on
carbohydrate carcode medium (biomass 15.61 g/L,
Results and Discussion
159
Figure 3.24: Effect of aeration on production of streptolipin
0
10
20
30
40
50
60
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Volume: Flask ratio
Stre
ptol
ipin
(mg/
L),
Dry
bio
mas
s (g
/L)
Dry biomass weight (g/L) Streptolipin (mg/L)
streptolipin 47.79 mg/L) followed by Baron’s media (biomass 10.04 g/L, streptolipin 25.20
mg/L) and Numerof’s medium (biomass 10.60 g/L, streptolipin 20.32 mg/L). However ISP
Results and Discussion
160
medium I showed almost the same highest productivity of streptolipin as carbohydrate carcode
medium (0.29 mg/L/h and 0.28 mg/L/h respectively). This could be due to the presence of Zinc
a micronutrient in the ISP production medium I that could have played role in the maximum
production of the compound. The organism when grown on complex organic medium
containing higher amounts of carbon sources and the micronutrients Ca++, Na+, K+ did not
show the production of the compound although it accumulated maximum amount of biomass.
Highest yield 35.21mg/L of streptolipin and a low biomass of 2.925 g/L on Lindenberg
synthetic medium suggested a role of Mg++ and Fe++ in the production of compound. However
reduced biomass in this medium may be due to the glycerol, which is not a good carbon source
for this culture. Out of 20 media, ISP production media I showed the highest productivity of
streptolipin and was taken as the basal medium for further experiments since the organism
produced the highest amount of inhibitor on this medium.
3.D.4.2. Effect of different carbon sources on production of streptolipin:
Different carbon sources such as polysaccharides, oligosaccharides (starch, dextrin,
cellulose), trisaccharides (raffinose and rhamnose), disaccharides (melibiose, sucrose, maltose,
xylose and lactose), monosaccharides (glucose, galactose, arabinose, fructose, mannose and
inositol), sugar alcohols (mannitol, xylitol), sodium salts of organic acids (sodium acetate,
citrate and pyruvate) and a control (without carbon source) were screened for optimizing media
components for the production of streptolipin. Each carbon source was incorporated at 15 g/L
level in to the basal medium in place of glucose.
Removal of glucose from the ISP medium I was taken as control. The biomass and
streptolipin production was very low (Table 3.33). These results showed poor production of
streptolipin on the polysaccharides > trisaccharides > disaccharides > sugar
Results and Discussion
161
Table 3.31: A list of standard media for the production of antibiotics:
Media Metabolite Reference
Czapek – Dox medium Inulinase Gill, 2003 O – Brien synthetic medium Streptomycin O’Brien et al, 1956 Complex organic medium Streptomycin Shirato and Nagatsu, 1965
Dulaney medium Streptomycin Dulaney, 1948 Thornberry medium Streptomycin Shirato and Motoyama, 1966 Baron medium Streptomycin Shirato and Motoyama, 1966 Numerof medium Streptomycin Numerof et al, 1954 Lumb medium Streptomycin Shirato and Motoyama, 1966 Cornmeal salt medium Streptomycin Shunzo et al, 1982 Carbohydrate carcode medium
Nystatin Jonsbu et al, 2002
Hobb medium Streptomycin Hobb et al, 1989 Lindenberg synthetic medium
Actinorhodin Elibol et al, 1995
Results and Discussion
162
Table 3.32: Screening of standard media for the production of streptolipin
Media Dry Biomass (g/L)
Streptolipin (mg/L) Streptolipin productivity (mg/L/h)
Czapek – Dox medium 1.92 8.00 0.04 O – Brien synthetic medium 3.27 8.96 0.05 Complex organic medium 0.32 8.10 0.04
Dulaney medium 1.80 12.80 0.07 Thornberry medium 2.20 6.63 0.03 Baron medium 4.05 25.20 0.15 Numerof medium 4.61 20.32 0.12 Lumb medium 2.72 4.82 0.02 Cornmeal salt medium 0.91 11.17 0.06 Carbohydrate carcode medium
15.62 47.79 0.28
Hobb medium 3.64 9.54 0.05 Lindenberg synthetic medium
2.93 35.21 0.20
ISP Production Medium I 12.77 48.82 0.29 ISP Production Medium II 10.37 37.75 0.22 ISP Production Medium III 9.53 30.20 0.17 ISP Production Medium IV 4.29 15.45 0.09 ISP Production Medium V 12.57 45.78 0.27 ISP Production Medium VI 10.58 26.95 0.16 ISP Production Medium VII 11.65 39.99 0.23 ISP Production Medium VIII 11.97 44.92 0.26
Results and Discussion
163
Table 3.33: Effect of different carbon sources on production of streptolipin:
Supplement (15 g/L) Dry biomass (g/L) Streptolipin (mg/L) None (control) 0.82 1.20 Carbon sources Monosaccharides
D-glucose 12.70 48.05 D-Galactose 6.87 47.76 Arabinose 6.48 44.74 Fructose 9.05 43.23 Mannose 6.98 38.23 Inositol 8.57 13.39 Disaccharides
Melibiose 6.49 13.33 Maltose 7.79 23.26 Sucrose 7.80 15.90 Lactose 6.90 29.22 Xylose 5.90 31.82 Trisaccharides
Raffinose 6.13 10.70 Rhamnose 4.81 15.89 Polysaccharides
Starch 10.56 19.29 Cellulose 18.51 12.90 Dextrin 9.08 19.69 Sugar alcohols
Mannitol 8.46 25.72 Xylitol 7.95 11.16 Sodium salts
Sodium acetate 5.73 29.27 Sodium citrate 4.43 12.48 Sodium pyruvate 5.79 46.05
Results and Discussion
164
alcohols and higher productivity on monosaccharides and sodium salts of acids. Poor
production with polysaccharides may be due to poor metabolic turn over of the compound.
Among monosaccharides tried, the maximum biomass was accumulated from glucose
(12.70 g/L) followed by inositol and fructose and the maximum streptolipin yield (48.05 mg/L)
was from glucose followed by galactose and arabinose. Among disaccharides studied, the
maximum biomass was accumulated from sucrose (12.70 g/L) followed by maltose and lactose
and the maximum streptolipin yield (48.05 mg/L) was from xylose followed by lactose and
maltose. The trisaccharides tried were rhamnose and raffinose. The maximum biomass was
accumulated from raffinose (6.13 g/L), but the maximum streptolipin yield (15.89 mg/L) was
from rhamnose. Among polysaccharides tried, the maximum biomass was accumulated from
cellulose (15.51 g/L) followed by starch and dextrin and the maximum streptolipin yield (19.69
mg/L) was from dextrin followed by starch and cellulose. Sugar alcohols tried were mannitol
and Xylitol. The maximum biomass and streptolipin was from mannitol (8.46 g/L of biomass
and 25.72 mg/L streptolipin). Among sodium salts tried, the maximum biomass was
accumulated from sodium pyruvate (5.79 g/L) followed by sodium acetate and sodium citrate
and the maximum streptolipin yield (46.05 mg/L) was from sodium pyruvate followed by
sodium acetate and sodium citrate.
Among all carbon sources sodium pyruvate and galactose showed highest yield (46.046
and 47.7 mg/L respectively) followed by arabinose and fructose. Significant rise on the yield of
the compound on sodium pyruvate suggests that pyruvate could be one of the intermediate
compounds in the biosynthetic pathway of the inhibitor. However significant reduction in the
biomass on sodium pyruvate and galactose suggests higher metabolic turnover of the
compound by the organism on the carbon sources. But on glucose, not only highest production
of the metabolite but also higher accumulation of biomass was observed. Hence it was a good
carbon source for the production of streptolipin and glucose was used as a carbon source for
screening of nitrogen sources.
Results and Discussion
165
3.D.4.3. Effect of inorganic nitrogen sources on production of streptolipin:
Seven nitrogenous compounds i.e., (ammonium acetate, urea, sodium nitrate,
ammonium nitrate, ammonium hypophosphate, ammonium sulphate and potassium nitrate)
were used as single sources in the basal medium at a level of 15 g/L in place of soyabean meal.
In addition calcium carbonate was added at a level of 3.5 g/L, to keep the pH relatively neutral
and allow the organism to grow and produce the inhibitor.
It was generally observed that ammonium salts gave low biomass and relatively low
streptolipin values (Table 3.34). Sodium nitrate and potassium nitrate, however was readily
utilized. The results indicate that a medium containing sodium nitrate as the sole nitrogen
source was more suitable for streptolipin production than in a medium with ammonium
nitrogen. When sodium nitrate served as the sole nitrogen source, the pH remained relatively
neutral and yields of approximately 41.92 mg per liter were obtained. However, when the
sodium nitrate was tried at a higher level (20 g/L), it resulted in little or no inhibitor formation,
indicating the critical nature of sodium nitrate in the production of streptolipin. Such critical
nutritional studies on inorganic salts was generally poor nitrogen sources (Martin and Daniel,
1977) have been reported for polyene production.
3.D.4.4. Effect of organic nitrogen sources on production of streptolipin:
A number of complex organic nitrogen sources were tested at 15 g/L for their effect on
the production of streptolipin. The results are shown in Table 3.35. It was observed that the
organic nitrogen sources had a marked effect on the fermentation. Growth occurred more
readily and significantly higher streptolipin levels were reached. The most remarkable increase
in the streptolipin yield was obtained by the addition of yeast extract (54.248 mg/L). A number
of authors have suggested that stimulation of higher yields of secondary metabolites might
been possible due to free amino acids and short peptide (two to three amino acids long) and
also more growth factors than other protein hydrolysates in yeast extract and soyabean meal,
Results and Discussion
166
perhaps a single amino acid or a combination of amino acids (Aasen et al, 2000; De Vuyst,
1995; De Vuyst and combinat-
Table 3.34: Effect of inorganic nitrogen sources on production of streptolipin:
Inorganic nitrogen sources (15 g/L)
Dry biomass (g/L) Streptolipin (mg/L)
Potassium nitrate 11.65 36.94 Sodium nitrate 12.01 41.20 Ammonium nitrate 9.12 26.60 Ammonium acetate 7.62 17.82 Ammonium sulphate 6.92 13.38 Ammonium hypophosphate 8.92 24.42 Urea 5.74 18.80
Table 3.35: Effect of organic nitrogen sources on production of streptolipin:
Complex nitrogen sources (15 g/L)
Dry biomass (g/L) Streptolipin (mg/L)
Peptone 9.12 24.13 Casein 11.86 41.85 Soyabean meal 11.94 48.79 Yeast extract 12.62 54.25 Malt extract 8.92 23.27 Corn meal 7.89 18.00 Corn steep liquor 5.92 16.22 Skim milk 11.75 40.87
Table 3.36:Effect of different amino acids on production of streptolipin: Amino acids ( 15 g/L) Dry biomass (g/L) Streptolipin (mg/L) Glycine 1.25 8.64 Histidine 5.56 18.31 Isoleucine 6.00 27.10 Aspargine 5.23 32.94 Tryptophan 6.07 16.37
Results and Discussion
167
ion of amino acids (Aasen et al, 2000; De Vuyst, 1995; De Vuyst and Vandamme 1992;1993;
Egorov et al, 1971; Kozlova et al, 1972).
This possibility was investigated by adding 5 amino acids, glycine, tryptophan,
histidine, isoleucine and aspargine separately at a level of 15 g/L instead of soya bean meal.
The results are given in the Table 3.36. Asparagine showed the highest yield of streptolipin.
This could be due to the presence of two amino groups in aspargine. Higher yield of inhibitor
on aspargine may be due to the long carbon chain (6 carbons) of the amino acid, which would
have contributed to the biomass accumulation. Growth was little on medium supplemented
with glycine.
3.D.4.5.Effect of trace elements on production of streptolipin: ISP medium I, used as basal medium contains 5 trace elements. The effect of
individual trace element from the medium was studied by removing one trace element at a
time.
Results showed that removing the trace elements of potassium and zinc reduced the
production of streptolipin from 48.75 to 38.24 and 41.91 respectively (Table 3.37). The results
indicate both dipotassium hydrogen phosphate and zinc sulphate play a role in
Table 3.37: Effect of trace elements on production of streptolipin:
Basal medium without Dry biomass weight (g/L) Streptolipin (mg/L)
NaNO3 12.06 47.81 K2HPO4 11.92 38.24
NaCl 12.77 47.02 ZnSO4 12.35 41.91 CaCO3 12.66 46.99 Control 12.82 48.75
Results and Discussion
168
Figure 3.25:Effect of dipotassium hydrogen sulphate on production of streptolipin
12.2
12.4
12.6
12.8
13
13.2
13.4
13.6
13.8
0.01 0.025 0.05 0.1 0.5 1
Dipotassium hydrogen sulphate (g/L)
Dry
bio
mas
s (g/
L)
44
46
48
50
52
54
56
58
Stre
ptol
ipin
(mg/
L)
Dry biomass weight (g/L) Streptolipin(mg/L)
Results and Discussion
169
Figure 3.26: Effect of zinc sulphate on production of streptolipin
11.8
12
12.2
12.4
12.6
12.8
13
13.2
13.4
0.01 0.025 0.05 0.1 0.5 1 Zinc sulphate (g/L)
Dry
bio
mas
s (g
/L)
46
47
48
49
50
51
52
53
54
55
56
Stre
ptol
ipin
(mg/
L)
Dry biomass (g/L) Streptolipin (mg/L)
the production of streptolipin. So, the next experiments were carried out on different
concentrations (0.01g/L to 1g/L) of dipotassium hydrogen phosphate and zinc sulphate. The
Results and Discussion
170
optimum concentration of dipotassium hydrogen phosphate and zinc sulphate wasfound to be
0.5 g/L and 0.1 g/L respectively (Figure 3.25 and 3.26). These gave the highest Streptolipin
values 56.76 mg/L and 54.80 mg/L of dipotassium hydrogen phosphate and zinc sulphate
respectively.
3.D.4.6. Effect of lipids on production of streptolipin:
Preliminary experiments revealed that this organism grew well on a medium
containing soya bean meal and glucose, the soya bean meal was replaced by lipids including
cottonseed oil, coconut oil, groundnut oil, olive oil, stearic acid and lauric acid. The biomass
was very less when compared with the control. The growth was sparse in groundnut oil (Table
3.38). This suggests the interference of lipids with primary metabolism of the organism which
in turn would have lead to reduction in biomass. The maximum biomass was observed in the
medium supplemented with olive oil (5.02 g/L) and the highest streptolipin was observed in
coconut oil (18.95 mg/L). However, total production of the biomass and is very less compared
to control indicating that lipids cannot be used as inhibitory source of raw material for
production of Streptolipin.
Table 3.38: Effect of lipids on production of streptolipin:
Lipids (10 g/L) Dry biomass weight (g/L) Streptolipin (mg/L) Olive oil 5.02 12.75 Cotton seed oil 2.54 4.63 Ground nut oil 0.37 3.03 Coconut oil 4.02 18.95 Stearic acid 1.15 9.36 Lauric acid 0.32 9.06 Control 12.36 48.99
Results and Discussion
171
Figure 3.27: Effect of molasses on production of streptolipin
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
10 20 40 60 80 100
Molasses (g/L)
Dry
bio
mas
s (g
/L)
0
2
4
6
8
10
12
14
16
18
20
Stre
ptol
ipin
(mg/
L)
Dry biomass weight (g/L) Streptolipin (mg/L)
Results and Discussion
172
3.D.4.7. Effect of molasses on production of streptolipin: Earlier reports (Lan et al, 2002) suggested increased productivity of antibiotics on
molasses when used as a commercial source. Different concentrations of molasses, varying
from 1 to 10% were used for the production of streptolipin. There was gradual increase in the
compound with increase in molasses concentration to reach a highest of
17.81 mg/L at 8% after which that inhibitor concentration declined (Figure 3.27). As,
production of Streptolipin was significantly low when compared with the basal medium,
further optimization on molasses was not done.
3.D.4.8. Formulation of medium for the production of streptolipin:
From results shown in Table 3.33, it was observed that glucose, galactose and sodium
pyruvate were the best carbon sources for the production of streptolipin and from the Table
3.34 and 3.35, casein, soyabean meal, yeast extract and sodium nitrate to be the best nitrogen
sources.
Effect of different nitrogen sources on the production of streptolipin in the presence of
either galactose or sodium pyruvate was carried out, since there was highest productivity of the
compound on galactose and sodium pyruvate. Results (Table 3.39 and Table 3.40) showed the
yields on sodium nitrate in the presence of either galactose or sodium pyruvate to be high.
However, the streptolipin production was highest with glucose as the carbon source and yeast
extract as nitrogen source (Table 3.33 and Table 3.34).
Table 3.39: Production of streptolipin on different nitrogen sources in presence of 15 g/L Galactose:
Nitrogen source, (15 g/L )
Dry biomass weight (g/L) Streptolipin (mg/L)
NaNO3 5.54 14.55 Casein 8.30 20.54 Yeast extract 9.45 32.71 Soya bean meal 7.06 31.35 Table 3.40: Production of streptolipin on different nitrogen sources in presence of
15 g/L sodium pyruvate:
Results and Discussion
173
Nitrogen source, (15 g/L) Dry biomass weight (g/L) Streptolipin (mg/L) NaNO3 4.79 16.53 Casein 7.40 23.73 Yeast extract 7.39 28.80 Soya bean meal 8.36 31.35
3.D.4.9. Effect of different glucose concentrations on the kinetics of streptolipin
production:
The results of nutritional studies on carbon sources indicated glucose to be the best for
achieving the highest streptolipin production (Section 3.D.4.2.). Hence, various concentrations
of glucose from 10 to 80 g/L were used to study the kinetics of streptolipin production. In these
experiments, the yeast extract concentration was fixed at 15 g/L along with the other salts of
basal medium.
Kinetics of Biomass formation:
Figure 3.28 shows the time course of biomass growth with initial glucose concentration
(So) as the parameter. The biomass increased with increasing glucose concentrations. Table
3.41 shows that the maximum biomass accumulation increased with increasing glucose
concentration up to 50 g/L, above which there was a decrease in the biomass accumulation. It
was also noted that the time taken to reach the stationary phase increased with increasing
glucose concentration (Table 3.41). Beyond a concentration of So=40 g/L, however, stationary
phase was not reached and biomass continued to increase, albeit, slowly.
In case of So = 10 g/L, the stationary phase was reached very early during the
fermentation (96 h) to reach a total biomass of about 6.04 g/L. Further increase in the initial
glucose concentration increased the biomass, till 168 h to 23.22 g/L (Table 3.41) and stationary
phase.
The biomass growth rate is described by the equation
dx/dt=µX---- (1)
Results and Discussion
174
Where µ is the specific growth rate.
This is the most frequently applied model for biomass growth. The specific growth rate related
to the substrate concentration as given by Monods equation
µ=µmax.So/(Ks + So)----- (2)
Where µmax was the maximum specific growth rate
When specific growth rates are calculated, it is important to take only the initial specific
growth rates (i.e µi) as in the early hours of fermentation, the cells are in active (logarithmic)
growth phase.
Table 3.41: Effect of initial glucose concentration on biomass synthesis:
Glucose g/L 10 20 30 40 50 80
Maximum
biomass g/L
6.04 12.02 15.83 18.48 21.85 22.92
Time to reach
stationary phase
96 132 144 168 Not
reached
Not
reached
For our experiments, through the plots of ln X vs time (Figure 3.29) at different initial
glucose concentrations, corresponding specific growth rates were obtained from the slopes of
these plots.
Specific growth rates thus calculated were plotted against the initial glucose
concentration (So) in Figure 3.30. The effect of initial glucose concentration on the specific
growth rate can be seen by a double reciprocal plot of initial specific growth rate against the
initial substrate concentration (Figure 3.31). This gives a straight line with an intercept 1/Vmax
on Y-axis. The Monods constants obtained from Figure 3.31 are: Ks value was 3.85 and the
µmax was 1.1 h-1.
Results and Discussion
175
Kinetics of Streptolipin production:
Figure 3.32 shows the effect of initial glucose concentration on the streptolipin
accumulation pattern. For all glucose concentrations tested, streptolipin accumulation started
after 24 hours of growth except So=20g/L. The accumulation of streptolipin increased with
increase of initial glucose concentration till 50 g/L after which it decreases (Figure 3.32). At
the lower glucose concentration (So=10 g/L) the accumulation of streptolipin started after 44h.
The time taken to reach the maximum streptolipin concentration increased with initial glucose
concentration.
The maximum streptolipin concentration was observed for So=50 g/L at 168 h and
further increase was not observed (Table 3.42). It may be noted that the maximum streptolipin
concentration reached at different initial glucose concentrations occurred during the secondary
metabolism at 132 h for So=10 and 20 g/L, and 168 h above So=20 g/L.
A plot of the rate of streptolipin synthesis (dp/dt) against time indicated that, generally
in all glucose tested, the rate of product synthesized increased drastically in the early hours of
fermentation after which it slowed and then dropped drastically (Figure 3.33). The rate of
streptolipin production increased with increase of glucose concentration till So=40 g/L and
further increase decreased. The overall rate of streptolipin increased from So=10 to 20 g/L, but
on further increase above So=20 g/L the rate decreased. Here, the production of streptolipin
was dependent on the biomass, which might be the reason for the decrease of time of
streptolipin production, when the initial glucose concentration increased.
Kinetics of sugar utilization:
Results and Discussion
176
Figure 3.34 shows the substrate utilization pattern at various initial glucose
concentrations. It was observed that, as the glucose concentration increased, the
Figure 3.28: Variation of total biomass with initial glucose concentration
0
5
10
15
20
25
0 50 100 150 200
Time (h)
Bio
mas
s g/L
Glucose (g/L)10 20 30 40 50 80
Results and Discussion
177
Figure 3.29: Semilogarithmic plot of biomass with time
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
0 50 100 150 200
Time (h)
Inx,
g/L
Glucose (g/L) 10 20 30 40 50 80
Results and Discussion
178
Figure 3.30: Monod's plot of initial specific growth rates vs substrate concentration
0
0.5
1
1.5
2
2.5
3
3.5
0 10 20 30 40 50 60 70 80 90
Glucose, So, g/L
ul, h
Results and Discussion
179
Figure 3.31: Double reciprocal plot of specific growth rate against the initial glucose concentration
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
-1 -0.5 0 0.5 1 1.5
1/So, lit/mol
1/u1
, h-1
Results and Discussion
180
Figure 3.32: Variation of total streptolipin with initial glucose concentration
-20
0
20
40
60
80
100
120
0 50 100 150 200
Time (h)
Stre
ptol
ipin
(mg/
L)
Glucose (g/L)10 20 30 40 50 80
Results and Discussion
181
Figure 3.33: Rates of streptolipin formation at different initial glucose concentration
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 20 40 60 80 100 120 140 160 180
Time (h)
dp/d
t mg/
L/h
Glucose (g/L) 10 20 30 40 50 80
Results and Discussion
182
Table 3.42: Effect of initial glucose concentrations on streptolipin synthesis:
Glucose g/L 10 20 30 40 50 80
Maximum streptolipin
concentration mg/L
23.32 58.42 72.96 80.12 92.42 90.27
Time of starting
streptolipin production
(h)
60 48 36 36 36 36
Time to reach
maximum streptolipin
concentration (h)
132 132 168 168 168 168
Overall rate of
streptolipin
synthesized (mg/L/h)
0.28 0.35 0.29 0.24 0.22 0.14
Table 3.43: Effect of initial glucose concentration on glucose utilization pattern:
Glucose g/L 10 20 30 40 50 80
Percent of glucose
utilization( g/L)
100 100 100 100 100 75.4
Glucose exhausted (h) 144 144 144 144 168 >168
Overall rate of glucose
utilization (g/L/h)
0.0069 0.013 0.02 0.027 0.029 0.028
Results and Discussion
183
Figure 3.34: Time course of initial glucose consumption during fermentation
-10
0
10
20
30
40
50
60
70
80
90
0 50 100 150 200
Time (h)
Glu
cose
g/L
Glucose (g/L) 10 20 30 40 50 80
Results and Discussion
184
Figure 3.35: Rates of glucose utilization at different initial glucose concentration
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0 50 100 150 200
Time (h)
ds/d
t g/L
/h
Glucose (g/L) 10 20 30 40 50 80
Results and Discussion
185
time taken to exhaust the glucose also increased. Since all the fermentation runs were
terminated at 168 h for all the experiments, glucose utilization was incomplete for So=80 g/L
(Table 3.43). The Table also reveals that overall rate of glucose utilization increased with
increasing glucose concentrations till So= 50 g/l, then stayed just about constant.
From the Figure 3.35, the rate of glucose utilization (ds/dt) increased steeply to reach
its maximum value of 0.012 g/l/h to 0.069 g/L/h from 10 to 80 g/L. The overall rate of
streptolipin increased from 10 to 80 g/L of initial glucose concentration. The results of the
glucose kinetics indicates that the initial sugar, glucose, or 50g/L to be optimum for the best
production of streptolipin.
3.D.4.10. Effect of different concentrations of yeast extract on streptolipin production:
Studies on the optimization of nitrogen sources (both organic and inorganic
nitrogen sources) showed that yeast extract at a concentration of 15 g/L resulted in the highest
concentration of streptolipin (54.25 mg/L) (section 3.D.4.4). Since maximum streptolipin
production corresponded to the maximum concentration of cell mass, achieving a high biomass
indicated as a prerequisite for a successful fermentation. In order to improve the streptolipin
yields by further generating higher cell densities, cell growth and streptolipin production were
determined over a period of time as a function of yeast extract concentration in the range of 10
to 80 g/L. The glucose concentration was fixed at 15 g/L along with the other salts of basal
medium.
Kinetics of Biomass formation:
The time course of biomass growth with respect to initial yeast extract concentration as
the parameter was followed. The maximum dry biomass accumulation increased from 5.08 to
19.05 g/L with increase of initial yeast extract (YEo) concentration (YEo= 10 to 80 g/L)
(Figure 3.36). It was also observed that the time taken to reach the stationary phase increased
with increasing YEo=10 to 80 g/L (Table 3.44).
Results and Discussion
186
Kinetics of Streptolipin production:
When the YEo concentrations increased from 10 to 50 g/L, the maximum streptolipin
production increased concomitantly. Streptolipin production started after 44 h of fermentation
(Figure 3.37). Interestingly, at all the yeast extract concentrations tried, the inhibitor was
detected when the biomass was around 35% of the final biomass (Xmax) had reached.
Table 3.44: Effect of initial yeast concentrations on biomass formation:
Yeast extract concentration (g/L)
Dry biomass (g/L) Xmax
Time taken to reach stationary phase (h)
10 5.08 108 20 10.05 120 30 12.23 120 40 15.84 120 50 18.44 144 80 19.05 144
Table 3.45: Effect of initial yeast extract concentrations on streptolipin synthesis:
Yeast extract concentration (g/L)
Maximum streptolipin concentration reached (mg/L)
Time taken to reach highest streptolipin concentration (h)
Rate of streptolipin production (mg/L/h)
10 23.40 108 0.14 20 55.00 132 0.33 30 66.90 132 0.40 40 80.64 132 0.48 50 92.71 144 0.55 80 90.01 144 0.54
Results and Discussion
187
The streptolipin production increased with increase YEo, the streptolipin yield was 23.4
mg/L where YEo=10 g/L and it increased to 92.62 mg/L when YEo=50 g/L, but on further
increase (YEo =50 g/L) streptolipin production declined. Table 3.45. The time taken to reach
stationary phase, increased from 108 h for YEo=10 g/L to 144 h for YEo= 80 g/L for
streptolipin production. Rate for streptolipin production increased with increase of YEo, from
YEo=10 g/L (0.13 mg/L/h) to YEo=50g/L (0.55 mg/L/h) and further increase of initial yeast
extract decreased the rate of streptolipin production to 0.53 mg/L/h (Table 3.45).
Increasing YEo concentration not only led to an increase in the biomass and streptolipin
production, but also led to an increase in the rate of streptolipin production from 0.139 mg/L/h,
for YEo=10g/L to 0.55 mg/L/h for a YEo=50g/L. Further increase in the YEo led to a decline
in the rate of streptolipin production and the time taken to reach highest streptolipin
concentration increased from 108 h to 144 h with increase of initial yeast extract concentration
(Table 3.45).
Achieving maximum cell density at the end of the fermentation seems to be a very
important parameter as the inhibitor is intracellular and biomass concentration at 144 h is the
highest. In the current studies above 50 g/L of yeast extract, streptolipin production was
decreased. Figures 3.36 and 3.37 indicate that, dry cell weight and streptolipin yield increased
with increasing concentration of yeast extract reaching an optimum at 50.0 g/L.
3.D.4.11. Production of streptolipin with optimum conditions on shake flask:
Based on the above observations, fermentation studies were carried out by using
optimized production medium, which contained glucose as carbon source and yeast extract as
nitrogen source. The trace elements were added as indicated in the optimization studies.
Results and Discussion
188
Figure 3.36: Influence of initial yeast extract concentrations on cell growth
0
5
10
15
20
25
0 50 100 150 200
Time (h)
Dry
bio
mas
s (g/
L)
Yeast extract (g/L) 10 10 g 20 g 30 g 50 g 80 g
Results and Discussion
189
Figure 3.37: Influence of initial yeast extract concentrations on streptolipin production
-20
0
20
40
60
80
100
0 50 100 150 200
Time (h)
Stre
ptol
ipin
(mg/
L)
10 g 20 g 30 g 40 g 50 g 80 g
Composition of the final optimized production media (g/L):
Results and Discussion
190
Glucose 50.0
Yeast extract 50.0
NaNO3 4.0
K2HPO4 0.5
NaCl 2.5
ZnSO4 0.1
CaCO3 0.4
Table 3.46. Comparison of medium studies before and after optimization in shake
flask:
Shake flask fermentation
(before media optimization)
Shake flask fermentation
(after media optimization)
Maximum biomass (g/L)
(Xmax)
8.35 28.01
Time taken to reach
highest biomass (h)
168 84
Maximum streptolipin
concentration (mg/L)
42 212.60
Time taken to reach
highest streptolipin (h)
168 84
Spore suspension was added from a well-sporulated glycerol aspargine agar slant to the above
production media and incubated at 300C at an initial pH 7.0, on a rotary shaker (220 rpm).
Medium flask volume ratio was maintained at 0.2 level and fermentation was carried out for
168 h. The maximum streptolipin and biomass was reached at 72 h. The pH decreased from 7.0
to 6.4 till 84 h, where glucose was exhausted, at this point, the pH increased to pH 8.0 till 168h
(Figure 3.38). The specific growth rate (µ) during early exponential growth was phase 0.42,
maximum biomass (Xmax) 28.01 (g/L) and final
Results and Discussion
191
Figure 3.38: Production of streptolipin with optimum conditions on shake flask
0
5
10
15
20
25
30
0 12 24 36 48 60 72 84 96 108 120 144 168
Time (h)
Bio
mas
s (g/
L),
pH
0
50
100
150
200
250
Stre
ptol
ipin
(mg/
L)
pH Biomass weight (mg/L) Streptolipin (mg/L)
Results and Discussion
192
streptolipin concentration 212.6 mg/L. The maximum biomass increased from 8.35 to 28.01
g/L from initial unoptimized studies to the optimum shake flask conditions and the time taken
to reach the highest biomass was decreased from 168 to 84h. The maximum streptolipin
concentration increased from 42 to 212.6 mg/L and the time taken to reach the streptolipin
decreased from 168 to 84 h (Table 3.46). Therefore, there was significant improvement in the
production of streptolipin was observed with the modified medium with decrease of
fermentation time from 168 to 84 hours (Table 3.46).
3.D.4.12. 10 L Fermentor study:
Batch culture for production of streptolipin was carried with all the optimum conditions
of the shake flask optimization studies. pH was controlled at 7.0 and the initial DO was set at
100% in a stirred tank fermentor (Figure 3.39.A). The maximum streptolipin (199.1 mg/L) and
biomass (28.4 g/L) was found at 84 hrs, although there was some linear growth till 108 hours,
where the glucose was completely exhausted. When the biomass finally reached 30.47g/L
(Figure 3.39.B).
Table 3.47: Comparison between shake flask and fermentor studies after
optimization of media:
Shake flask fermentation Fermentor studies
Maximum biomass (g/L)
(Xmax)
28.01 30.47
Time taken to reach
highest biomass (h)
84 84
Specific growth rate (µ) 0.42 0.39
Maximum streptolipin
concentration (mg/L)
212.60 199.10
Time taken to reach
highest streptolipin (h)
84 84
Results and Discussion
193
Figure 3.39.A. Production of streptolipin in 10 L laboratory fermentor
Results and Discussion
194
Figure 3.39.B: 10 L Fermentor study
0
50
100
150
200
250
0 12 24 36 48 60 72 84 96 108 120 144 168
Time in Hours
Stre
ptol
ipin
(mg/
L)/G
luco
se p
erce
nt
0
5
10
15
20
25
30
35
Bio
mas
s wei
ght (
g /L
)
Streptolipin (mg/L) Percent of glucose in the broth
Biomass weight (g/L)
Results and Discussion
195
The specific growth rate during early exponential growth phase (µmax) was 0.39. The
DO% had decreased from 100 to 63.5% along with increase of biomass. Therefore, significant
improvement in the production of streptolipin was observed with the modified medium.
The maximum biomass obtained from fermentor studies and shake flask fermentation
and the time taken to reach highest biomass was same in the both shake flask and fermentor
studies (84 h) (Table 3.47). The specific growth rate (µ) was 0.42 g/L in the shake flask, where
as in fermentor studies it was 0.39 g/L. The maximum streptolipin concentration decreased
212.6 to 199.1 g/L from shake flask to the scale up studies, whereas, the time taken to reach the
highest streptolipin production was 84 h for both shake flask and scale-up studies (Table 3.47).
The result clearly demonstrated that streptolipin production was predominantly growth
associated. The optimized medium developed was fairly simple containing the basic nutrients
and succeeded in quickly developing a reliable and very productive defined medium process,
which yielded to rapid large-scale cultivation study for streptolipin production.
3.D.5. Optimization of down stream processing conditions for streptolipin extraction:
3.D.5.1. Choice of the extraction solvent:
The effect of organic solvents was studied on the extraction of streptolipin from the dry
biomass. Both polar and non polar Organic solvents, were tried for efficient extraction of
streptolipin. Among the different organic solvents tried, ethyl acetate extraction gave
maximum recovery of streptolipin from the biomass, followed by chloroform and acetone
(Table 3.48).
Results and Discussion
196
Table 3.48: Choice of the extraction solvent:
Solvent Streptolipin (mg/10 g of dry biomass weight)
Ethyl acetate 50.26
Chloroform 46.94
Hexane 8.6
Methanol 4.3
Ether 13.26
Ethanol 12.76
Acetone 46.35
3.D.5.2. Biomass to solvent ratio:
The effect of biomass to solvent ratio on extraction of streptolipin was followed by
varying the amount of ethyl acetate to the 10 g (Figure 3.40). When the biomass to solvent
ratio increased from 1:5 to 1: 40, extraction of streptolipin, increased from 9.48-to 50.6-mg/10
g of dry biomass weight. Biomass to solvent ratio above 1:40 did not further increase
streptolipin yields. These results indicate that biomass to ethyl acetate ratio 1:40 to be ideal for
efficient Streptolipin extraction.
3.D.5.3. Effect of static and agitated conditions during extraction:
The extent of recovery was studied using the optimized conditions of biomass to ethyl
acetate ratio of 1:40 (w/v) in conical flasks under static and agitated conditions at 300C.
Agitation of the fermented biomass with the solvent was carried out by incubating the flasks on
a rotary shaker at 220 rpm. Under static conditions, a maximum of 9.12mg/ 10 g of dry
biomass could be extracted after 60 min of extraction. Beyond this time, streptolipin
concentration in the extract remained constant (Figure 3.41). Further it was observed that under
agitation conditions, the rate of extraction of streptolipin increased gradually till 60 min
(49.91mg). As compared to static extraction, the agitated mode of extraction gave much higher
yields. This was particularly true with extraction times of less than 60 minutes. For both modes
of extraction, the optimum time of extraction was 60 minutes.
Results and Discussion
197
3.D.5.4. Repeated extraction:
Repeated extractions were carried out by incubating the biomass with ethyl acetate
(1:40 w/v ratio) on a rotary shaker at 300C for 30 minutes each. At the end of incubation, the
solvent was removed and fresh solvent was added to the same biomass. Six such cycles were
carried out to study the efficiency of repeated extraction, which was a function of the number
of cycles needed to achieve maximum streptolipin yield from the biomass. It was observed
that, the first extract had maximum streptolipin concentration was 49.89 mg/10 g dry biomass,
while the second extract had 15.6 mg/g dry biomass, while the third extract 8.4 mg/g dry
biomass, while the fourth 4.6 mg/g dry biomass. There was no streptolipin extracted in the next
subsequent two extractions. This indicated that the biomass be extracted for four times to
extract all the streptolipin.
3.D.5.5. Sequential extraction:
This mode of extraction was studied in three flasks in which the solvent containing
streptolipin extracted from biomass was added to the fresh biomass, taking are to maintain the
solvent to biomass ratio (1:40). This was done for three flasks (Figure 3.42). The extract
obtained from the third flask was thus in contact with biomass for a total of 180 minutes. The
concentration of streptolipin was checked at the end of each step in the process. The
concentration of streptolipin in the extract increased from 48.23 mg in the first extraction to
63.84 mg/g of dry biomass in the final extraction. This would mean that there was substantial
increase in the overall yield of streptolipin extraction from the biomass. One advantage, using
this method was ethyl acetate could be reused for at least three cycles of extraction. This would
mean significant savings in terms of overall downstream processing costs.
Results and Discussion
198
Figure 3.40: Effect of biomass to ethyl acetate ratio on extraction of streptolipin
0
1
2
3
4
5
6
0 10 20 30 40 50 60
Ratio of biomass : ethyl acetate
Stre
ptol
ipin
(mg/
g dr
y bi
omas
s)
3.E. In vivo efficacy of streptolipin on experimental animal models:
Results and Discussion
199
Obesity and hyperlipidemia are to a relevant degree related to a high dietary fat intake.
The major ingredient (Over 95%) of dietary fat is triglyceride. The key enzyme that hydrolyses
dietary triglyceride during digestive process is lipase secreted by pancreas. Pancreatic lipase
(poured through pancreatic juice) exerts its action in the intestinal lumen at the water-lipid
interphase, in conjunction with bile juice and co-lipase (also present in pancreatic juice). A
clear demonstration that such an inhibition of dietary fat absorption results from inhibition of
the lipase activity has been made in the case of Orlistat isolated from Streptomyces toxytricini.
Orlistat has been observed to be a very potent, selective and irreversible inhibitor of pancreatic
lipase.
Investigations were made on laboratory mice to examine the health beneficial anti-
obesity effects of streptolipin by inhibition of lipase and hence interference with digestion and
absorption of dietary fat. This was done in both single dose as well as multiple dose
streptolipin administered animals.
3.E.1. Influence on lipase activity and dietary triglyceride absorption In this investigation, streptolipin has been administered at 3 different doses by gavage
(as 0.2 ml suspension in 5% gum arabic in 5% defatted milk) namely 5, 10 and 20 mg/kg body
weight. The middle dose (Weibel et al, 1987) i.e. 10 mg/Kg body weight corresponds to the
dose of reference drug Orlistat.
In the single dose study, the fat intake and faecal excretion of triglycerides were
monitored for 48 hours following the drug administration. In the multiple dose study, the
animals were administered with the compound on three alternate days (Day-1, Day-3 and Day-
5). Fat intake and faecal excretion of dietary triglycerides were followed until 48 hours after
the last dose. In single dose study, the animals were sacrificed on the third day and in multiple
dose, on the seventh day.
Faecal excretion of triglycerides in streptolipin administered animals is presented in
Table 3.49 and 3.50. Excretion of triglyceride in faeces was 164, 297 and 334% higher in three
Streptolipin groups compared to the control animals during first 24 hour of the drug dosage.
Thus, there was a dose dependent higher excretion of triglyceride as a result of streptolipin
Results and Discussion
200
administration. Further, the effect of Streptolipin was higher than that of Orlistat given at the
same dose level. Higher faecal excretion of triglyceride continued on the second day, but to a
much lesser degree compared to day 1. Faecal excretion of triglycerides on day 3 after
streptolipin administration was compared to controls.
Significantly, higher excretion faecal triglyceride was evidenced in the multiple dose
study throughout the duration of the experiment. As expected, the extent of higher excretion of
triglyceride was much higher on days of drug administration Day-1, Day-3 and Day-5).
Compared to the second day after drug administration.
Figure 3.43 and 3.44 present amounts of dietary triglyceride ingestion, faecal
triglyceride excretion and net triglyceride absorbed in mice administered streptolipin. It is very
clear that triglyceride absorption was dose -dependently lower in streptolipin-treated mice, and
was concomitant with higher faecal excretion of triglycerides.
Activity of lipase was examined in the mucosal scrapings of small intestine in
streptolipin administered animals (Figure 3.45). Lipase activity resident in intestinal mucosa
was significantly lowered (29 to 42%) in animals administered streptolipin, at the end of two
days following single oral administration. The activity was concurrable to controls, in Orlistat
administered mice. Similarly, lower activity of lipase in intestinal mucosa was seen in the
multiple dose two days after the third dose of administration of streptolipin. The decrease in
lipase activity was 19 to 41 %. Multiple dose of Orlistat does not have any inhibitory effect on
lipase resident in intestinal mucosa.
Results and Discussion
201
Table 3.49: Effect of streptolipin on faecal excretion of triglycerides (Single dose study)
Mouse Group Day 1 Day 2
Day 3
Control 20.9 + 4.85 19.2 + 3.25 18.2 + 3.25
Orlistat (10 mg/kg b.w.) 77.5 + 6.2 42.7 + 7.35 19.2 + 3.5
Streptolipin (5 mg/Kg b.w.) 54.5 + 5.25 36.4 + 7.9 18.3 + 2.6
Streptolipin (10 mg/Kg b.w.) 82.7 + 6.35 45.5 + 6.65 19.3 + 3.2
Streptolipin (20 mg/Kg b.w.) 90.4 + 6.75 45.6 + 4.25 19.1 + 2.35
Values expressed as mg triglyceride/ mice are mean ± SEM of 10 number of animals per group.
Results and Discussion
202
Table 3.50: Effect of streptolipin on faecal excretion of triglycerides (Multiple dose study) Mouse Group Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Control 17.2 + 3.6 18.3 + 2.9 19.2 + 4.5 38.3 + 9.0 19.9 + 2.8 20.1 + 4.05
Orlistat (10 mg/Kg b.w.) 66.6 + 4.7 37.5 + 5.8 77.6 + 8.4 83.5 + 17.6 76.3 + 6.25 39.3 + 7.95
Streptolipin (5mg/Kg b.w.) 58.7 + 4.65 36.0 + 9.3 53.5 + 9.05 61.5 + 8.9 54.2 + 7.3 33.2 + 6.2
Streptolipin (10 mg/Kg b.w.) 74.7 + 5.25 42.0 + 4.8 81.9 + 8.05 80.3 + 9.6 78.9 + 5.95 44.4 + 2.75
Streptolipin (20 mg/Kg b.w.) 69.6 + 4.5 42.7 + 6.15 78.8 + 6.0 45.3 + 5.65 82.3 + 6.05 46.6 + 4.7
Values expressed as mg triglyceride/ mice are mean ± SEM of 10 number of animals per group.
Results and Discussion
203
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Figure 3.43: Effect of streptolipin on absorption of dietary triglyceride (Single dose)
0
50
100
150
200
250
Control Orlistat 10mg/kg b.w
Streptolipin 5mg/kg b.w
Streptolipin 10mg/kg b.w
Streptolipin 20mg/kg b.w
�������� Total triglyceride intake Faecal triglyceride
���������� Absorbed triglyceride
Results and Discussion
204
��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������
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Figure 3.44: Effect of streptolipin on absorption of dietary triglyceride (Multiple dose)
0
100
200
300
400
500
600
700
800
Control Orlistat 10mg/kg b.w
Streptolipin 5mg/kg b.w
Streptolipin 10mg/kg b.w
Streptolipin 20mg/kg b.w
���������� Total triglyceride intakes Faecal triglyceride
�������� Absorbed triglyceride
Results and Discussion
205
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Figure 3.45: Effect of streptolipin on activity of intestinal lipase
66.2 67
46.8
39.4 38.1
54.6 53.8
44.5
32.3
41.1
0
10
20
30
40
50
60
70
80
Control Orlistat (10mg/kg b.w)
Streptolipin (5mg/kg b.w)
Streptolipin(10 mg/kg b.w)
Streptolipin(20 mg/kg b.w)
Spec
ific
activ
ity (U
nits
/mg
prot
ien)
�������� 3 days 7 days
Values expressed as TBARS represent mean ± SEM of 10 animals in each group.
Results and Discussion
206
Thus, the higher excretion of fecal triglycerides is attributal to the strong lipase
inhibitory potential of streptolipin. It is probable that streptolipin has similar inhibitory
potential on pancreatic lipase which in the key enzyme in the digestive hydrolysis of
dietary triglycerides. In the absence of inhibitory effect of Orlistat on lipase activity of
intestinal mucosa, the higher excretion of fecal triglycerides, as a result of Orlistat
administration should be attributable to previously reported inhibitory influence of
Orlistat on pancreatic lipase.
3.E.2. Influence on blood and liver lipid profile: Serum lipid profile of streptolipin administered mice are presented in Table 3.51
and 3.52. Concomitant with decreased dietary triglyceride absorption. Serum triglyceride
levels were significantly lower in streptolipin treated mice. The decrease in serum
triglyceride was in the range 17–29% as a result of single dose (Table 3.51) and in the
range of 19-36% as a result of multiple dose (Table 3.52). Although, total cholesterol
concentration in serum remained unchanged, as a result of single of administration of
streptolipin, the proportion of cholesterol associated with LDL fraction was somewhat
lower in these test groups. Such a decrease in LDL cholesterol was accompanied by a
proportionate increase in HDL cholesterol (Table 3.51). Similar trend serum cholesterol
was also evidenced in streptolipin administered animals in the multiple dose study (Table
3.52).
Liver lipid profile in streptolipin administered mice, as a result of single as well as
multiple dose is prevented in Table 3.53 and 3.54. Triglyceride content of liver was
significantly lower, as a result of multiple dose streptolipin administration (Table 3.54).
As a result, the total lipid content of the liver was also proportionally lower.
Perirenal fat (depot fat) was significantly lowering in experimental mice, as a
result of streptolipin administration (Table 3.55 and 3.56). The decrease in perirenal fat
content and liver triglyceride content are in agreement to interferance of streptolipin with
dietary triglyceride absorption. These effects of streptolipin appear to exceed the effect of
Orlistat (Reference drug) administered with an equal dose. In otherwise, streptolipin is
Results and Discussion
207
Table 3.51: Effect of streptolipin on serum lipid profile (Single dose study)
Cholesterol Groups/parameters Total triglycerides Total HDL LDL
Total phospholipids
Control 178.2 ± 18.6 228.3±8.32 51.8±1.46 176.5±7.98 160.5±2.13
Orlistat (10 mg/kg b.w.) 207.5 ± 11.2 220.5±4.26 64.8±1.05 155.8±5.09 129.6±10.0
Streptolipin (5 mg/Kg b.w.) 134.2 ± 9.37 204.7±3.67 64.3±0.57 140.4±4.10 171.0±10.2
Streptolipin (10 mg/Kg b.w.) 146.9 ± 17.6 212.1±4.35 79.2±0.60 133.0±4.42 129.0±2.09
Streptolipin (20 mg/Kg b.w.) 125.8 ± 7.11 213.5±3.32 82.7±1.21 130.8±4.13 123.3±1.23
Values expressed as mg/dl are mean ± SEM of 10 number of animals in each group
Results and Discussion
208
Table 3.52: Effect of streptolipin on serum lipid profile (Multiple dose study)
Cholesterol Groups/parameters Total triglycerides Total HDL LDL
Total phospholipids
Control 168.2 ± 7.73 205.2 ± 10.9 48.5 ± 0.44 156.7 ± 10.4 95.3 ± 1.38
Orlistat (10 mg/kg b.w.) 136.8 ± 3.37 181.9 ± 7.81 65.3 ± 0.62 116.7 ± 7.80 125.6 ± 9.95
Streptolipin (5 mg/Kg b.w.) 137.0 ± 2.74 188.9 ± 7.94 66.9 ± 0.88 122.0 ± 7.92 128.3 ± 9.30
Streptolipin (10 mg/Kg b.w.) 113.2 ± 3.49 199.4 ± 3.88 79.2 ± 0.58 120.3 ± 3.58 147.8 ± 7.04
Streptolipin (20 mg/Kg b.w.) 107.6 ± 4.87 168.0 ± 10.8 80.6 ± 1.35 107.4 ± 14.7 149.4 ± 5.85
Values expressed as mg/dl are mean ± SEM of 10 number of animals in each group
Results and Discussion
209
Table 3.53: Effect of streptolipin on liver lipid profile (Single dose study)
Groups/parameters Total lipids Triglycerides Cholesterol Phospholipids Control 75.2 ± 2.88 38.9 ± 1.37 8.11 ± 0.12 20.6 ± 0.95
Orlistat (10 mg/kg b.w.) 71.9 ± 1.87 41.4±2.44 7.34±0.22 24.5±0.88
Streptolipin (5 mg/Kg b.w.) 96.6 ± 3.04 46.9±1.28 7.72±0.23 24.5±0.88
Streptolipin (10 mg/Kg b.w.) 94.2 ± 6.33 45.0±1.93
9.23±0.33 32.4±1.02
Streptolipin (20 mg/Kg b.w.) 79.7 ± 2.19 40.5±1.08 7.43±0.26 26.4±0.99
Values expressed as mg/gm are mean ± SEM of 10 number of animals in each group.
Results and Discussion
210
Table 3.54: Effect of streptolipin on liver lipid profile (Multiple dose study)
Groups/parameters Total lipid Triglyceride Cholesterol Phospholipids
Control 98.7 ± 3.98 31.9 ± 0.67 8.97 ± 0.25 22.9 ± 1.29
Orlistat (10 mg/kg b.w.) 67.2 ± 3.88 18.4 ± 1.67 6.28 ± 1.67 21.0 ± 1.57
Streptolipin (5 mg/Kg b.w.) 85.8 ± 4.12 25.5 ± 0.52 7.49 ± 0.49 18.3 ± 0.32
Streptolipin (10 mg/Kg b.w.) 77.6 ± 3.33 22.1 ± 0.78 7.05 ± 0.49 20.8 ± 0.94
Streptolipin (20 mg/Kg b.w.) 83.6 ± 6.16 23.8 ± 1.55 8.56 ± 0.63 19.1 ± 1.52
Values expressed as mg/gm are mean ± SEM of 10 number of animals in each group.
Results and Discussion
211
Table 3.55: Effect of streptolipin on liver and adipose tissue weight (Single dose study)
Perirenal fat Liver Groups/parameters Perirenal fat
(mg) mg% Perirenal
fat Total liver (g) % Liver weight
Control 130.0 ± 1.20 479.7 ± 4.48 1.11 ± 0.031 4.09 ± 0.114
Orlistat (10 mg/kg b.w.) 103.1 ± 3.00 389.1 ± 11.32 1.05 ± 0.05 3.96 ± 0.188
Streptolipin (5 mg/Kg b.w.) 110.0 ± 10.0 404.4 ± 36.7 1.04 ± 0.026 3.82 ± 0.095
Streptolipin (10 mg/Kg b.w.) 99.4 ± 2.00 357.6 ± 7.19 1.05 ± 0.016 3.77 ± 0.057
Streptolipin (20 mg/Kg b.w.) 96.0 ± 2.82 349.1 ± 10.2 1.07 ± 0.015 3.89 ± 0.054
Results and Discussion
212
Table 3.56: Effect of streptolipin on liver and adipose tissue weight (Multiple dose study)
Perirenal fat Liver Groups/parameters Perirenal fat (g) mg% Perirenal fat Total liver (g) % Liver weight
Control 167.0 ± 6.02 592.2 ± 21.3 1.24 ± 0.04 4.39 ± 0.131
Orlistat (10 mg/kg b.w.) 122.6 ± 9.06 433.2 ± 32.0 1.26 ± 0.04 4.45 ± 0.141
Streptolipin (5 mg/Kg b.w.) 118.4 ± 10.1 409.7 ± 34.9 1.33 ± 0.04 4.60 ± 0.138
Streptolipin (10 mg/Kg b.w.) 94.7 ± 12.1 341.9 ± 43.7 1.31 ± 0.05 4.72 ± 0.173
Streptolipin (20 mg/Kg b.w.) 99.6 ± 9.79 353.2 ± 34.7 1.29 ± 0.03 4.57 ± 0.109
Values represent mean ± SEM of 10 animals in each group.
Results and Discussion
213
evidenced in this animal study to be a more potent inhibitor of lipase activity and hence
of dietary triglyceride absorption, when compared to Orlistat.
3.E.3. Influence on antioxidant status: Lipid peroxide values in serum and liver streptolipin administered mice is
presented in Figure 3.46. Significantly decreased serum (or) circulatory lipid peroxide
content was evidenced in streptolipin administered mice. This decrease was as much as
50% in the highest dose. Similarly, hepatic lipid peroxide content was lower as a result
of streptolipin administration. The extent of decrease in lipid peroxide content of both
circulation and hepatic tissue was higher in streptolipin administration as compared to the
effect produced by an equal dose of Orlistat.
Activities of three antioxidant enzymes in the catalase, GSH peroxidase, superoxide
dismutase in the liver of streptolipin administered mice are presented in Table 3.57.
Streptolipin administration produced a significant increase in the activities of hepatic
catalase, both as a result of single dose as well as multiple dose. There was no much
difference in activities of plasma non-specific enzymes in streptolipin administered mice
in multiple dose study (Table 3.58).
Similarly, the activities of hepatic glutathione peroxidase and of superoxide
dismutase were significantly higher in streptolipin administered mice especially as a
result of multiple dose. This effect was higher compared to the effect produced by an
equal dose of Orlistat. Thus, concomitant with lowered lipid peroxides, the endogenous
antioxidant enzymes were beneficially modulated in hepatic tissue of streptolipin
administered mice.
Thus, in addition to its lipase inhibitory activity, streptolipin is evidenced in this
animal study to exert beneficial influence on the antioxidant status of the animal.
Results and Discussion
214
Figure 3.46: Effect of streptolipin on lipid peroxides in serum (Circulatory)
and liver (Multiple dose study)
Se
rum
µM/dl
0
10
20
30
40
Liver
nM/m
g pro
tein
0
1
2
3
4
5
Serum µM/dl Liver nM/mg protein
Values expressed as TBARS represent mean ± SEM of 10 animals in each group.
Control Lipstatin Streptolipin Streptolipin Streptolipin (10 mg/kg b.w) (5 mg/kg b.w) (10 mg/kg b.w) (10 mg/kg b.w)
Results and Discussion
215
Results and Discussion
216
Table 3.57: Effect of streptolipin on activities of hepatic antioxidant enzymes (Multiple
dose study) :
Mouse Group Streptolipin
Liver
antioxidant Enzymes
Control
Orlistat (10 mg/kg b. w) 5 mg/ Kg
b.w 10 mg/ Kg b.w
20 mg/ kg b. w
Catalase 3 days 214.4 ± 4.5 290.6 ± 8.7 243.3 ± 5.6 304.3 ± 3.7 326.9 ± 5.6 7 days 155.0 ± 11.9 297.5 ± 26.7 286.2 ±
19.2 389.0 ± 27.9
428.1 ± 15.6
Peroxidase 3 days 27.1 + 2.9 27.8+ 2.8 26.2 + 2.2 30.6+ 4.2 31.9 + 1.8 7 days 16.8 + 2.0 23.5 + 0.6 20.0 + 3.2 27.5 + 7.7 29.1 + 5.5 Super Oxide Dismutase 3 days 7.0+2.9 7.7 + 0.1 7.5 + 0.2 10.0 + 1.2 14.7 + 1.2 7 days 6.4 + 1.3 8.1 + 0.9 8.1 + 1.2 10.1+2.4 15.4+1.1
Results and Discussion
217
Table 3.58: Effect of streptolipin on the activities of plasma non-specific enzymes (Multiple dose study).
Mouse Group Streptolipin
Serum Enzyme
Control Orlistat
10 mg/Kg b.w. 5 mg/ Kg b.w. 10 mg/ Kg b.w. 20 mg/ Kg b.w. Alanine
aminotransferase
137.7 + 2.90 132.7 + 3.33 127.4 + 1.19 124.6 + 1.51
129.0 + 2.31
Aspartate
aminotransferase
293.2 + 1.59 271.2 + 5.40 269.5 + 1.98 268.6 + 4.13
279.0 + 3.89
Results and Discussion
218
Figure 3.42: Effect of sequential extraction on streptolipin recovery from the biomass
0
10
20
30
40
50
60
70
80
1 2 3
Sequential extraction number
Stre
ptol
ipin
(mg/
10 g
dry
bio
mas
s wei
ght)
Results and Discussion
219
Summary and highlights of the investigation:
15 fungal cultures were screened for lipase inhibition from CFTRI culture collection
center and none of the fungal cultures have shown potent inhibition against pancreatic lipase.
Several soil samples were collected from randomly chosen locations, taking sufficient care to
see that the points of collection had, as widely varying characteristics as possible with regard to
physical and physiological characters. Several methods for pretreatments of soil sample and
media were tried for isolation of actinomycetes, out of which calcium carbonate treatment was
found to be the best for isolation of actinomycetes and starch casein media was the best among
the media tried.
230 isolates were isolated from terrestrial samples. According to color of the mature
sporulated aerial mycelium, isolates of the gray series were found to represent 46 percent of the
total number of isolates and the lowest occurrence was noted for the green series (0.86%).
22 isolates were found to produce pancreatic lipase inhibitors. Among these, 7 were
recovered from the soil sediment collected from cow barnyard, 5 from lakes, 2 from desert, 2
from tea plantations, 3 from hill station, 2 from forest and 1 from coconut field, the greatest
number of active strains were isolated from samples, which were pretreated with calcium
carbonate followed by phenol.
From these 22 isolates, 7 actinomycete isolates which showed inhibition greater than 50
% were selected for secondary screening. Results indicated that only one isolate N2 not only
produced the maximum amount of inhibitor but also showed greater reproducibility. Two
different types of media were employed for the production of lipase inhibitor for secondary
screening. This approach was useful to detect the effect of various media components, genetic
stability of the microbial culture and the yield. As the inhibition shown by actinomycete N2 in
submerged fermentation were reproducible and more potent than the other cultures, further
studies were carried out using this culture.
Results and Discussion
220
Identification of the actinomycete N2 was carried out. It is gram-positive, non-acid fast,
non motile with extensively branched substrate hyphae was assigned to Streptomyces genus.
The assignment of N2 to the genus Streptomyces was also supported by the 16S rRNA
gene sequence analysis of N2. The almost complete sequence of 16S rRNA gene (1464 nt) of
N2 following BLAST analysis indicated that N2 is related to species of Streptomyces and
further analyse the phylogenetic relationship of N2 a phylogenetic tree was constructed using
NEIGHBOR JOINING and DNAPARS. A detailed study on morphological and
chemotaxonomic features complemented by phylogenetic analysis showed overwhelming
differences with other closely related Streptomyces species, which led to a proposal to assign
N2 the status of a new species for which the name Streptomyces vayuensis sp. nov. was given.
The extraction and purification of the lipase inhibitor carried out. Techniques such as 1H, 13C NMR, 2DHMQCT, 1H-1H COSY, HMQC and HMBC spectra along with LC/MS and
elemental analysis were used which led to the identification of a novel metabolite as nonadeca-
6-enoicacid-3-(hexadecyloxy- hydroxy thiophosphoryloxy)-quinoxalin-2-yl ester with a
molecular formula C43H73N2O5PS with a molecular weight of 761.
A literature search revealed that this compound did not match with any reported lipase
inhibitors or of any Streptomyces metabolites. The inhibitor is henceforth designated as
STREPTOLIPIN [Streptomyces lipase inhibitor]. This has an IC50 of 349 nM and did not
show inhibition against plant and fungal lipases. Streptolipin revealed no antimicrobial activity
upto a concentration of 200 µg by disc plate method. Studies on the mode of inhibition of
streptolipin against pancreatic lipase revealed it to be irreversible with a non competitive type
of inhibition with a Ki value of 0.714 µM.
The optimisation of the fermentation and nutritional parameters and downstream
processing of production of streptolipin through submerged fermentation was studied. An
HPLC method was developed to determine the streptolipin concentration in crude samples
from the standard graph. Streptolipin production was influenced by carbon, nitrogen and trace
salt supplements. Streptolipin production was highest when glucose and yeast extract were
used at 50 g/L and incubation at 300C for 84 h. The maximum streptolipin obtained at
optimized physico-chemical parameters was 212 mg/L.
Results and Discussion
221
Extraction of the biomass using ethyl acetate ratio 1:10 at pH under agitated conditions
resulted in maximum inhibitory recovery. Sequential extraction showed substantial increase in
the overall yield of inhibitor extraction. At optimum levels of physico-chemical and down
stream process parameters streptolipin yield was found to be 75.9 mg/10 g of dry biomass.
There was a dose dependent higher excretion of triglyceride as a result of streptolipin
administration. It is very clear that triglyceride absorption was dose -dependently lower in
streptolipin-treated mice, and was concomitant with higher faecal excretion of triglycerides.
The higher excretion of fecal triglycerides is attributal to the strong lipase inhibitory potential
of streptolipin. Total cholesterol concentration in serum remained unchanged, the proportion of
cholesterol associated with LDL fraction was somewhat lower in these test groups. The
decrease in perirenal fat content and liver triglyceride content are in agreement to interferance
of streptolipin with dietary triglyceride absorption.
The activities of hepatic glutathione peroxidase, catalase and superoxide dismutase
were significantly higher in streptolipin administered mice especially as a result of multiple
doses. Thus concomitant with lowered lipid peroxides, the endogenous antioxidant enzymes
were beneficially modulated in hepatic tissue of streptolipin administered mice. Streptolipin is
evidenced in this animal study to exert beneficial influence on the antioxidant status of the
animal along with a more potent inhibition of lipase activity and hence of dietary triglyceride
absorption, when compared to Orlistat.
Recommendations for further work:
• Studies on strain improvement for further increase of streptolipin production is also
another area which is needed for the commercialization of the process.
• Spectroscopic studies such as circular dichroism and fluorescence studies are to be
carried out to get a detailed insight into the mode of action of the inhibitor at the
molecular level.
Results and Discussion
222
• Biochemical and physiological parameters such as interactions with serum proteins to
determine bioavailability and dosage development would be valuable in determining
the efficiency of the inhibitor in vivo.
• Fermentation techniques such as fed-batch fermentation to enhance the streptolipin
production are also recommended.
Results and Discussion
223
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List of publications and patents:
Papers communicated:
1. Naveen Babu, K., Shivaji, S. and Sattur, A.P. Streptomyces Vayuensis sp. nov: A new
species of Genus Streptomyces. International Journal of Systematic and Evolutionary
Microbiology (Communicated).
2. Naveen Babu, K., Jagan Mohan Rao, L. and Sattur, A.P. Streptolipin, a novel
pancreatic lipase inhibitor. Journal of Antibiotics (Communicated).
3. K.Naveen Babu and A.P.Sattur. Streptolipin production by Streptomyces vayuensis:
kinetics and the influence of nutrients. Prcocess Biochemistry (Communicated).
US product patent:
Sattur, Avinash Prahalad; Babu, Kilaru Naveen; Rao Lingamallu Jagan Mohan; Karanth,
Naikanakatte Ganesh; An inhibitor compound and its isolation and method of treatment
against pancreatic lipase for use as an anti- obesity agent (Filed).