Chapter 3

MATERIALS AND METHODS

3.1 Layout of the Research

Sampling

Isolation

Purification by Three-Point Inoculation

Screening for Pectinase on two different media

Identification of Potential Isolates by Colony Morphology and Microscopy

Selection of single Isolate using AHP for Optimization

Optimization by three parameters: Temperature, pH, and Substrate Level

Assessment of Enzyme Activity by DNS Method

3.2 Materials

Glass Petri Plates

Glass Test Tubes
Eppendorf’s
3,5 Dinitrosalicyclic Acid
Malt Extract
Citrus Pectin
Agar
D-Galacturonic acid
3.3 Sampling

Rotten samples of four different fruits namely Grapes (Vitis vinifera), Mangoes
(Mangifera indica), Apples (Malus domestica) and Bananas (Musa acuminata) were
collected from Government Sabzi Mandi (Vegetable and Fruit Market) located south of
Lahore City. The grounds of sabzi mandi are mostly not covered in concrete and most of
the vendors just dump their non-desirable fruits on the ground (Appendix I) and with no
proper cleaning methods implemented, the ground forms a feeding heaven for microbes
such as bacteria and fungi in which case it was highly probable that most of the microbes
that would infect fruits in such places would be enzyme producers. The samples were put
in polythene bags with a little opening for crossing of air and were carefully transported
Forman Christian College University’s mycology lab.

3.4 Isolation

Isolation was done by adopting the method described by (Thiyam and Sharma 2013).
Rotten fruits were washed with distilled water to remove any soil present on the surface
of the fruits and then their surface was sterilized by dipping them into 1% NaClO
Solution and then afterwards three subsequent rinsing in distilled water were done
(Appendix II). Rotten parts of every fruit were cut into small pieces measuring roughly 2-
5mm sized squares. These pieces were placed onto petri plates containing freshly
prepared MEA media containing (g/L): 20.0g-Malt Extract, 20.0g-Agar with 0.1%
ampicillin added to restrict bacterial growth and were left for 3 days for colony
development. After 3 days, distinct colonies were picked by hyphal tips technique and
placed onto new MEA media plates for purification.

3.5 Purification by three-point inoculation method and preparation of

suspension culture

3.5.1 Purification by three-point inoculation

Purification was done by repeated sub-culturing onto MEA media plates by adopting
three-point inoculation method as described by (Waing et al. 2015). The benefit of using
three-point inoculation (Fig 3.1) is that is allows one to clearly see the patterns of growth
by allowing three spaces which are sizable enough for complete growth of the colony and
can be compared with each other for any distinct features. It makes it much easier to
distinguish between similar looking colonies of fungi so colony morphology can be
studied with more accuracy.

Fig 3.1: Three-point inoculation method

3.5.2 Preparation of suspension cultures:

Suspension cultures of the pure isolates were prepared by placing plates of pure cultures
in dark with no access to sunlight for 72 hours. Fungi produce spores when subjected to
stress conditions. After 72 hours, discs were cut from pure plates and placed in test tubes
containing 15ml distilled water under aseptic conditions. Test tubes were covered and
stored in refrigerator at 4oC for further use

3.6 Screening for Pectinase on two different media

3.6.1 Pectinase Screening by Czepak-Dox Agar modified with Citrus Pectin

Screening for pectinase assay were done by method described by (Priya and Sashi 2014).
Czepak-Dox Agar modified with commercial pectin was prepared containing (g/L): 1.0g-
K2HPO4, 0.50g-MgSO4.H2O, 0.01g-FeSO4, 0.50g-KCl, 3.0g-NaNO3, 30g-Sucrose, 15.0g-
Agar with 1.5% commercial pectin as sole carbon source. 0.1% ampicillin was added to
restrict bacterial growth. Furthermore, pH was adjusted to 5.5 and then subsequently the
media was autoclaved at 121oC for 15 minutes. Afterwards the media was poured onto
petri plates and was allowed to cool down and solidify. For cultivation of isolates, 100µl
of spore suspension was centrally inoculated and then afterwards incubated at 28 oC±2 for
5 days. Pectinase activity was studied by flooding the plates with freshly prepared Iodine-
Potassium iodide solution containing (g/330ml): 1.0g-Iodine, 5.0g-Potassium iodide).
Staining with this solution gives dark color to media containing pectin and shows clear
zones in areas where pectin has been hydrolyzed indicating pectinase activity.

3.6.2 Pectinase Screening by Minimal Salt Media modified with Citrus Pectin

Screening for pectinase assay were done by method described by (Tariq and Latif 2012).
Minimal Salt media modified with commercial pectin was prepared containing (g/L):
3.0g-K2HPO4, 0.1g-MgSO4.H2O, 20g-NH4Cl, 5g-NaCl, 6.0g-Na2HPO4, 15.0g-Agar with
0.2% commercial pectin as sole carbon source. 0.1% ampicillin was added to restrict
bacterial growth. Furthermore, pH was adjusted to 6.0 and then subsequently the media
was autoclaved at 121oC for 15 minutes. Afterwards the media was poured onto petri
plates and was allowed to cool down and solidify. For cultivation of isolates, 100µl of
spore suspension was centrally inoculated and then afterwards incubated at 28 oC±2 for 10
days. Pectinase activity was studied by flooding the plates with freshly prepared Iodine-
Potassium iodide solution containing (g/330ml): 1.0g-Iodine, 5.0g-Potassium iodide).
Staining with this solution gives dark color to media containing pectin and shows clear
zones in areas where pectin has been hydrolyzed indicating pectinase activity.

3.7 Identification of Potential Isolates

Isolates which showed highest pectinolytic activity measured by diameter of hydrolysis

zone and zone ratios as described by (Bansal et al. 2011) on both media were further
selected for micromorphological identification. In addition to macromorphological
characters such as colony size, color, texture, and growth pattern, micromorphological
characters such as size and shape of conidia and spores, structure of hyphae etc. The
isolates were identified till genus level.

3.8 Selection of Single Isolate for Optimization

Final selection of single isolate was made after identification by combining the data of
identification and screening and comparing the selected isolates. It means that if there is
no to little difference between these isolates then the isolate belonging to a particular
genus which has been proved by past literature to be the best producer of pectinase and
has been frequently used for optimization would be selected as the final choice for
optimization.

3.9 Optimization of culture conditions for enhanced production of

pectinase in Submerged Fermentation (SmF) assessed by DNS Method

Single isolate which was selected was further subjected to different cultural conditions.
For optimization of pectinase production, submerged fermentation (SmF) was adopted,
and the method described by (Madika et al. 2020) was followed. Hankins Broth
containing (g/L): 1.4g-NaNO3, 0.35g-KCl, 0.35g-MgSO4.7H2O, 0.7g-K2HPO4, 0.007g
FeSO4.7H2O with 1.5% commercial pectin added was used as standard media which was
modified with different cultural parameters i.e., temperature, pH, and substrate
concentration. To observe Polygalacturonase (PG) activity, DNS Method by (Miller
1959) was adopted to quantify the amount of reducing sugars released as an indicator of
enzyme activity.

3.9.1 Standard Curve of D-Galacturonic Acid and Quantification of Enzyme

Activity using DNS Method

Standard curve of D-galacturonic acid was prepared by following the method described
by (Khan 2018). 0.1g of D-galacturonic acid was added in 50ml distilled water and the
volume was raised to 100ml to prepare 0.1% stock solution of D-galacturonic acid. Ten
dilutions of D-galacturonic acid measuring from 0.0mg/ml to 1.0mg/ml were prepared in
0.1M sodium acetate buffer with a pH of 5.5. Afterwards, 3ml of DNS reagent was mixed
with 1ml sample from each tube and was put in boiling water for 10 minutes for color
development. The test tubes were allowed to cool and were diluted with distilled water of
7ml in each flask and absorbance of each tube was measured spectrophotometrically at
540nm after with blank in parallel to set zero. Slope of curve was noted and was used to
estimate pectinase activity of test samples.
Quantification of enzyme activity was done by taking 2ml of broth from each sample and
then centrifuging it at 3000rpm for 10 minutes. The supernatant obtained was used as
crude enzyme. Afterwards, 500µl of crude enzyme was mixed with 500µl of 1% pectin
solution in equal amount of 0.1M sodium acetate buffer with pH 5.5 and was put in water
bath at 50℃ for ten minutes. Then 3ml DNS reagent was added to stop the hydrolysis
process and the solution was put in boiling water bath for 10 minutes for color
development. Then 5ml distilled water was added to dilute the solution and absorbance of
the solution was read spectrophotometrically at 540nm. One unit of pectinase activity is
defined as the amount of enzyme that releases 1µ mol of galacturonic acid per min under
standard assay conditions.

3.9.2 Optimization of temperature

Six 100ml Erlenmeyer flasks each containing 50ml of Hankins’s Broth with 1.5%
commercial pectin as sole carbon source was prepared and pH was adjusted to 5.5 before
being put in autoclave at 121oC for 15 minutes. Afterwards, 1ml fungal spore suspension
was inoculated in each of the flasks and the flasks were put on rotatory shaker for 5 days
at 150rpm at different temperatures namely 25℃, 30℃, 35℃, 40℃, 45℃, 50℃. After
5 days, 2ml of media was picked from each flask and was centrifuged at 3000rpm for 10
minutes. The supernatant obtained was used as crude enzyme used for quantification of
enzyme activity by using DNS Method (Miller 1959).

3.9.3 Optimization of pH

Six 100ml Erlenmeyer flasks each containing 50ml of Hankins’s Broth with 1.5%
commercial pectin as sole carbon source was prepared and pH was adjusted in different
values namely 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 before being put in autoclave at 121 oC for 15
minutes. Afterwards, 1ml fungal spore suspension was inoculated in each of the flasks
and the flasks were put on rotatory shaker for 5 days at 150rpm. After 5 days, 2ml of
media was picked from each flask and was centrifuged at 3000rpm for 10 minutes. The
supernatant obtained was used as crude enzyme used for quantification of enzyme activity
using DNS Method (Miller 1959).

3.9.4 Optimization of substrate concentration

Six 100ml Erlenmeyer flasks each containing 50ml of Hankins’s Broth with different
substrate concentrations of commercial pectin such as 1.5%, 2.0%, 2.5%, 3.0%, 3.5%,
4.0%, 4.5% and the pH was adjusted to 5.5 before being put in autoclave at 121oC for 15
minutes. Afterwards, 1ml fungal spore suspension was inoculated in each of the flasks
and the flasks were put on rotatory shaker for 5 days at 150rpm. After 5 days, 2ml of
media was picked from each flask and was centrifuged at 3000rpm for 10 minutes. The
supernatant obtained was used as crude enzyme used for quantification of enzyme activity
using DNS Method (Miller 1959).
 APPENDICES
Annexure I

Figure I.1: Ground of local sabzi mandi, fruits can be seen as dumped on the ground

Figure I.2: Banana market in sabzi mandi, sludge can be seen everywhere.
 Appendix II

Figure II.1: Rotten samples of Apple, Mango, Grape, and Banana

Figure II.2: Surface sterilization and inoculation on plates

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