WVSU Research Journal Vol. 11 No. 1 pp.

36-50 (June 2022)

Isolation, Screening, and Characterization of Fungal Isolates with

Pectinolytic Activity from Decaying Fruits

Rey G. Tantiado
College of Arts and Sciences
West Visayas State University
Luna St., La Paz Iloilo City, Philippines
Corresponding author: rtantiado@wvsu.edu.ph

Abstract

Filamentous fungi are potential source of pectinolytic enzyme production

used in fruit processing industry can be isolated from decaying fruits as
substrates. Thus, this study was conducted to isolate, screen, characterize
pectinase-producing fungi from decaying fruits and assess their pectinolytic
activity. Decaying fruits samples were collected from fruit stands of Iloilo City
Terminal Market. Pectinolytic fungi were grown and screened for pectinolytic
activity using pectinolytic activity plate assay based on the decolorization
coefficient value. Results showed three genera of fungi isolated from various
decaying fruits which include Aspergillus (9 isolates), Rhizopus (1 isolate),
and Mucor (1 isolate). Aspergillus spp. was the most dominant fungal
isolate obtained from decaying fruits of ampalaya (Momordica charantia),
pear (Pyrus pyrifolia), pineapple (Ananas comosus), tomato (Lycopersicon
esculentum), lemon (Citrus limon), melon (Cucumis melo), calamansi (Citrus
microcarpa), and mango (Mangifera indica). The Aspergillus spp. showed
significant pectinolytic activity among the fungi effectively screened based
on the decolorization coefficient value obtained. Thus, common fungi with
pectinolytic activities are mitosporic genera isolated from decaying fruits that
can be applied in agro-biotechnological industry.

Keywords: decaying fruits, fungal isolates, pectin, pectinolytic activity

© WVSU Research Journal

ISSN 2244 - 4335 (print)
ISSN 2651 - 6659 (online) https://doi.org/10.59460/wvsurjvol11iss1pp36-50

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 Isolation, Screening, and Characterization of Fungal Isolates... 37

Introduction

Awareness on health issues of what we are putting in our body

especially liquid leads to increase in consumption of natural fruit juices.
Drinking fruit juices makes an alternative nutritious drink over caffeinated
beverages like coffee or soft drinks (Tapre & Jain, 2014). Due to the increase
in the consumption of fruit juices, it goes along with the increase in the demand
for fruits to make into juices in the market specifically tropical fruit juices
(Blanco et al., 1999). However, tropical fruits are pulpy or fibrous to yield
juice by mechanical pressing which is quite laborious and strenuous. Most
of the time, it results to a meagre juice yield. Currently, as an alternative to
yield a clear juice, pectinases are utilized in the chemical processing of fruits
for the clarification of juices. These enzymes are utilized in processing plant
material as the main raw source in a juice manufacturing industry (Cheirsilp
& Umsakul, 2008). Through the enzymatic activity of these enzymes, fruit
juices become clear by breaking down the pectin, eliminating color changes
and maintaining the stability of the juice (Tapre & Jain, 2014). Pectinases
are also helpful in fruit puree production and wine clarification (Carneiro et
al., 2002). Microbial production of pectinase from filamentous fungi allows
the use of agricultural wastes as substrates for enzyme production which are
renewable and abundant in fruits as wastes which represent a potent low-cost
raw material for enzyme production. However, expensive pectinase production
is a limiting factor for wide scale production and utilization of the enzyme.
According to Gragasin et al. (2012), the cost analysis for the production cost
for United States Pharmacopeia (USP) grade pectin in the laboratory scale
set-up was estimated to be 5,667.51 Php/kg while the imported pectin was
at 27,122.56 Php/kg. Thus, the production of pectin out of decaying fruits
locally is technically and economically feasible. Filamentous fungi are the
common source of commercial pectinase productions (Merín et al., 2015).
This research study utilizes and recycles organic wastes from decaying
fruits in one of the fruit stands in Iloilo Terminal Market. Fruits and vegetable
wastes from the area are not only collected and sent to a dumping site but
rather before they reach their end point, they are fully explored for their
possible discovery of novel fungi with promising pectinolytic activity. It is also
relevant to agro-industrial sectors which involves the degradation of pectin
for a particular process. It seeks to discover novel pectin enzyme-producing
microorganisms from a diverse microbial world with a very interesting
application in the fruit industry. In addition, the genes of fungi which encode
pectinase enzymes can be obtained producing strains of interest (Sieiro et

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al., 2012). It is an alternative way of producing ecofriendly biotechnological

process in the production of microbial enzymes as an efficient tool taken
from indigenous fungi from a local commercial site in the vicinity (Ossola &
Galante 2004; Sharma et al. 2017). Thus, this research is conducted to isolate,
screen and characterize pectinolytic fungi from decaying fruits in Iloilo City
Terminal Market garbage wastes.

Methods

Description of the sampling site and fruits sample collection. The

sampling site is in Iloilo City Terminal Market at Fuentes-De Leon streets
Iloilo City, Region 6, Philippines with its geographical coordinates are
10.7202° N, 122.5621°E. Decaying fruits samples were collected in sterile
heat resistant (polypropylene, PP) plastic containers under sterile conditions
from fruit stands of Iloilo City Terminal Market. Samples were transferred
to sterile plastic bags, maintained in aseptic conditions and immediately
transported to the Central Science Laboratory (CSL) of West Visayas State
University (WVSU) for further processing.
Acquisition of Aspergillus niger BIOTECH 3080 as positive control.
The test fungus was purchased from the Philippine National Collection of
Microorganisms (PNCM) University of the Philippines Los Baños (UPLB)
Biotech in Los Baños, Laguna isolated from fruits. Fungal isolate from
decaying fruits was chosen for its known pectinolytic activity (Antier et al.,
1993; Mill, 1966; Sieiro et al., 2009; 2012). Culture of fungus in agar slants
was scraped with the aid of an inoculating loop and aseptically transferred to
the prepared nutrient broth solution.
Preparation and serial dilution of samples. About 10 g of mixed
decaying fruit samples was homogenized in 90 ml normal saline solution
(NSS). One, 1mL of the homogenized sample was transferred into the into the
10-2 bottle containing 9ml NSS. The solution was homogenously mixed with
the aid of a vortex mixer. Serial dilution was repeated until the 10-6 dilution.
Preparation of potato dextrose agar (PDA) plates. Approximately
39 grams of dehydrated PDA was dissolved in 1 liter of distilled water and
mixed. The culture medium solution was autoclaved for 15 minutes at 121°C
and 15psi. After autoclaving, two (2) ml per liter of ciprofloxacin antibiotic
solution was added to the media to prevent bacterial contamination of the
medium. The medium was then dispensed in several sterile petri dishes
aseptically, solidified and stored for further use.

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Plating of Samples. A 100μL micropipetter was used in taking 0.1ml

of aliquot from tubes 10-2, 10-4, and 10-6 tubes of serially diluted samples.
After introducing 0.1 ml of aliquot sample at the center of agar surface, a
sterile L-shaped spreader was used to spread the inoculum evenly around
the plate. All plates were inverted after 3 minutes or until all the liquid has
absorbed into the surface of the agar. All plates were incubated for one week
at room temperature.
Selection for pectinolytic activity (plate assays). A procedure by
Raju and Divakar (2013) was followed with modification. For plate assay,
molds were spot inoculated in the solid medium with 2% apple pectin and
incubated for 72 h at 280C. After incubation, 50 mM iodine-potassium iodide
solution or 6 M HCI was flooded. Plates were left undisturbed for 5-10
minutes. Then iodine or HCl solution was decanted, and clear zones were
observed to detect clearance halos around the colonies. Positive strains were
further isolated and evaluated.
Isolation and purification of fungal cultures. Plates were monitored
regularly to verify the growth of pectinolytic fungi. Individual distinct
colonies that grow in PDA plates was cut in a small block and aseptically
transferred in a new PDA plate. Purification was repeated once for three weeks
until one colony grow in a new PDA plate. Pure fungal isolates were then
photographed, morphologically described based on colonial characteristics,
and microscopically analyzed for cell characteristics. Macroscopic
characteristics of the colonies both top and reverse were described based on
appearance in culture, colony pigmentation, agar pigmentation, colony shape,
and texture. Fungal isolates were then preserved in microbiological tubes
containing agar slants, maintained at 2 to 4 °C, periodically subcultures every
three months.
Identification of fungal isolates. A sheet of sterile filter paper was
placed in a Petri dish with sterile filter paper with a U-shaped glass rod
was prepared aseptically. It was moistened with 4ml sterile water. A 5 mm
square block of potato dextrose agar was cut with the aid of gently flamed
sterilized scalpel. The agar block was picked transferred aseptically to the
center of the slide. The sides of the agar square were inoculated with spores
or mycelial fragments of the fungal sample. A sterile cover slip was placed
on the upper surface of the agar cube. After inoculation, the Petri dish was
covered then, incubated at room temperature for 48 hours. After 48 hours,
the slide was examined under low power to high power objectives. A single
drop of lactophenol cotton blue stain was placed on a clean microscope slide.

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The cover slip from the slide culture was removed, added with 95% ethanol
and evaporated. The cover slip was placed, mold side-down, on the drop of
lactophenol cotton blue stain on the slide. The agar block was discarded.
The fungus on the slide was examined under microscope. Microscopic
characteristics were noted which include type of hypha, the mycelia (stipe,
phialide, vesicle, and conidia length and width was measured) and spore
characteristics (size, shape, number of cells, attachment, wall thickness,
appendages, wall characteristics) and their color were noted for fungal
identification by Ainsworth et al. (1973) and Crous et al. (2009).
Pectinolytic Assay Proper. A method by Aneja (1996) was followed
with modification. Pure pectinolytic fungal isolate was cut in a small block
with a size of 10 mm2. It was placed at the center of the agar surface media
with 2% pectin. It was incubated for 72 h at 280C. After incubation, 50 mM
iodine-potassium iodide solution was flooded. Plates were left undisturbed for
5-10 minutes. Then iodine or HCl solution was decanted and clear zones were
observed to detect clearance halos around the colonies. Zone of pectinolytic
activity was measured with the aid of Vernier caliper, recorded, evaluated and
analyzed.
Data Collection Procedure and Analysis
Determination of Decolorization Coefficient. The decolorization
disc diameter and growth disc diameter (cm), of each fungal sample
were measured. The numerical values obtained were used to calculate the
decolorization coefficient (A) by Antier et al. (1993) as shown below:

( H1 Href )
A= C1 Cref
Href Cref
where H - decolorization disc diameter; C - growth disc diameter.
ref - reference strain Aspergillus niger BIOTECH 3080
Descriptive and Inferential Statistics were employed in the study.
For the descriptive data analysis, mean decolorization coefficient value was
used to determine the absence or presence of pectinolytic activity of fungi.
A scale was used to evaluate the degree of pectinolytic activity based on the
decolorized surface of the agar plate (Tan Gana et al., 2014). This is shown
in Table 1.

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Table 1.
Degree of Pectinolytic Activity

Figure Descriptive Scale

- negative = opaque; no enzymatic activity

+ positive = slightly clear (<5 mm); weak

enzymatic activity

++ positive = moderately clear (5-10 mm);

moderate enzymatic activity

+++ positive = very clear; (> 10mm); high

enzymatic activity

For inferential data analysis, One-Way Analysis of Variance

(ANOVA) was used to determine any significant difference on the zone of
pectinolytic activity among the fungi isolates in decaying fruits from Iloilo
City. Furthermore, Least Significant Difference (LSD) was used for pairwise
comparison between treatments. The level of significance was set at α = 0.05.

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Results
Pectinolytic Fungal Isolates and their Characteristics. Figures 1 shows
the characterized and identified genera of the fungal isolates obtained from
decaying fruits from Iloilo Terminal Market. The eleven isolates belong to the
genera of Rhizopus, Aspergillus, and Mucor.

Figure 1. Fungal isolates from decaying fruits showing cultural characteristics

(L) and microscopic characteristics (R). Note: A= Rhizopus sp., B-J=
Aspergillus spp., K= Mucor sp.

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 Isolation, Screening, and Characterization of Fungal Isolates... 43

Table 2 shows the description of the fungal isolates obtained from

decaying fruits from Iloilo Terminal Market.
Table 2.
Genera and their description and sources of decaying fruits

Figure Descriptive Scale

Rhizopus sp. Ampalaya
Cultural Characteristics (Momordica
Colony obverse – white, reverse – white. Circular, flat, entire, dull charantia), mango
and opaque on PDA. Colony diam. 40 x 32 mm after 7 days of (Mangifera
incubation at room temperature. indica), tomato
Microscopic Characteristics (Lycopersicon
Hypha: coenocytic; smooth stipe, brown, 20um X 3 um, biseriate. esculentum)
Vesicle: spatulate. Spores: 2um, clusters, thin walled, smooth and
colorless, 1-celled produced inside sporangia. Sporangiophores:
with rhizoids (branched “roots”) at base
Aspergillus sp. 1 Ampalaya
Cultural Characteristics (Momordica
Colony obverse - Dark olive green, reverse - yellow with green charantia), squash
margin. Lobate, dry texture with spores dull and opaque on PDA. (Cucurbita maxima)
Colony diam. 40 x 50 mm after 7 days of incubation at room
temperature.
Microscopic Characteristics
Hypha: Coenocytic, smooth stipe, > 30 um X 5um, biseriate,
spatulate, brown or black. Spores: 2um, thin walled, smooth and
colorless, 1-celled. Conidiophores: swollen head.
Aspergillus sp. 2 Ampalaya
Cultural Characteristics (Momordica
Colony obverse – white, reverse -white. Translucent, entire, dry charantia), pear
texture with spores on PDA. Colony diam. 20 x 50 mm after 7 (Pyrus pyrifolia)
days of incubation at room temperature.
Microscopic characteristics
Hypha: coenocytic, smooth, stipe >30 um X 5um, biseriate and
spatulate, brown or black. Spores: 2um, thin walled, smooth, and
colorless, 1-celled, clusters. Conidiophores: swollen head.
Aspergillus sp. 3 Lemon (Citrus
Cultural Characteristics limon)
Colony obverse - Dark olive green, reverse – creamy white.
Lobate, dry texture with spores dull and opaque on PDA. Colony
diam. 24 x 50 mm after 7 days of incubation at room temperature.
Microscopic characteristics
Hypha: Coenocytic, smooth stipe, > 30 um X 5um, biseriate and
uniseriate, globose, brown or black. Spores: 2um, thin walled,
smooth and colorless, 1-celled. Conidiophores: with vesicle
bearing bottle-shaped phialides.

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Table 2 continued...
Figure Descriptive Scale
Aspergillus sp. 4 Ampalaya (Momordica
Cultural Characteristics charantia), pineapple
Colony obverse: black, reverse: white. Entire and dry texture (Ananas comosus)
with spores on PDA. Colony diameter 15 X 45 mm after 7 days of
incubation at room temperature.
Microscopic characteristics
Hypha: coenocytic, smooth stipe, 40 um X 5um, biseriate and
globose, brown or black. Spores: 2um, thin walled, smooth and
colorless, 1-celled. Conidiophores: vesicle bearing bottle-shaped
phialides.
Aspergillus sp. 5
Cultural Characteristics Ampalaya (Momordica
Colony obverse: Dark green, reverse: light green. Translucent, charantia), tomato
(Lycopersicon
lobate and furry texture with spores on PDA. Colony diameter 35
esculentum), pineapple
X 45 mm after 7 days of incubation at room temperature. (Ananas comosus),
Microscopic characteristics dalandan (Citrus
Hypha: Coenocytic, smooth stipe, 50 um X 5um, biseriate and aurantium), calamansi
spatulate, brightly colored. Spores: 2um, thin walled, smooth, and (Citrus microcarpa)
colorless, 1-celled, spores produced in chains. Conidiophores:
with a swollen head.
Aspergillus sp. 6 Ampalaya (Momordica
Cultural Characteristics charantia), Squash
Colony obverse: Dark olive green, reverse: yellow with green (Cucurbita maxima),
margin. Translucent, undulate and velvety texture with spores on tomato (Lycopersicon
esculentum), pear
PDA. Colony diameter 25 X 55 mm after 7 days of incubation at
(Pyrus pyrifolia),
room temperature. pineapple (Ananas
Microscopic characteristics comosus), eggplant
Hypha: coenocytic, smooth stipe, 30 um X 5um, biseriate and (Solanum melongena),
spatulate, brown or black. Spores: 2um, thin walled, smooth and melon (Cucumis melo),
colorless, 1-celled. Conidiophores: vesicle bearing bottle-shaped avocado (Persea
phialides. americana)
Aspergillus sp. 7
Cultural Characteristics: Squash (Cucurbita
maxima), lemon
Colony obverse: Dark olive green, reverse: creamy white.
(Citrus limon)
Translucent, lobate dry texture with spores on PDA. Colony
diameter 15 X 45 mm after 7 days of incubation at room
temperature.
Microscopic Characteristics
Hypha: coenocytic, smooth stipe, 45um X 5um, biseriate and
spatulate, brown or black. Spores: 2um, thin walled, smooth and
colorless, 1-celled and in clusters. Conidiophores: with vesicle
bearing bottle-shaped phialides.

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 Isolation, Screening, and Characterization of Fungal Isolates... 45

Table 2 continued...
Figure Descriptive Scale
Aspergillus sp. 8 Mango (Mangifera
Colony obverse: white, reverse: white. Translucent, undulate dry indica)
texture with spores on PDA. Colony diameter 25 X 35 mm after 7
days of incubation at room temperature.
Microscopic Characteristics
Conidia: white with interspersed grey-green patches of conidia,
conidial heads are short, columnar and uniseriate. Conidiophore:
stipes smooth-walled and constricted at the neck. Vesicles:
subglobose. Conidia: globose 2-3.2 μm, smooth.
Aspergillus sp. 9 Rambutan
Cultural Characteristics (Nephelium
Colony obverse: grayish white, reverse: grayish white. lappaceum)
Translucent, undulate dry texture with spores on PDA. Colony
diameter 15 X 45 mm after 7 days of incubation at room
temperature.
Microscopic Characteristics
Sporangiophores: abundant rhizoids, hyaline, 2 um X 20 μm,
branched all ending in a sporangium, 3-11 × 2-7 μm. Sporangia:
spherical,100 μm, columella cylindrical, 40 μm.
Mucor sp.
Cultural Characteristics
Colony obverse: Dark olive green, reverse: white. Translucent, Fruits- Tomato
undulate, dry texture with spores on PDA. Colony diameter 15 X (Lycopersicon
45 mm after 7 days of incubation at room temperature. esculentum), pear
Microscopic Characteristics (Pyrus pyrifolia),
Hypha: Coenocytic, rough stipe 45um X 5um, biseriate and lemon (Citrus
globose; brown or black. Spores: 2um, thin walled, smooth and limon), melon
colorless, 1-celled, spores produced in chains. Conidiophores: (Cucumis melo),
with vesicle bearing bottle-shaped phialides. calamansi (Citrus
microcarpa)

Pectinolytic Activity and Decolorization Coefficient of Fungal Isolates.

Table 3 shows the decolorization coefficient values of pectinolytic fungal
isolates. There were only four fungal isolates (Aspergillus sp. 2, Aspergillus
sp. 4, Aspergillus sp. 7, and Aspergillus sp. 9) considered as effective
degraders of pectin based on obtained decolorization coefficient value. There
is a significant difference in the decolorization coefficient among the isolates
obtained, F (11,96) =4.17, p (0.000) < 0.05. It is supported by large effect
size (eta squared =0.323). It shows a significant variation in terms of positive
decolorization results indicating degradation of the pectic components of the
decaying fruits and the capability of the isolates to degrade pectin.

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Table 3
Decolorization Coefficient Values of Pectinolytic Fungal Isolates

Isolate Decolorization Coefficient Remarks/ Interpretation

1. Rhizopus spp. 0.985* No decolorization; (-)
2. Aspergillus sp. 1 0.991* No decolorization; +++
3. Aspergillus sp. 2 1.039*a (1,2,4,6,7,9,+) Positive decolorization; +++
4. Aspergillus sp. 3 0.981* No decolorization; (-)
5. Aspergillus sp. 4 1.006* a (10,9) Positive decolorization; +++
6. Aspergillus sp. 5 0.995* No decolorization; (-)
7. Aspergillus sp. 6 0.992* No decolorization; (-)
8. Aspergillus sp. 7 1.048* a (1,11,2,4,6,7,9,+) Positive decolorization; ++
9. Aspergillus sp.8 0.980* No decolorization; (-)
10. Aspergillus sp. 9 1.059* a (1,11,2,4,5,6,7,9,+) Positive decolorization; +++
11. Mucor spp. 0.999* No decolorization; (-)
Positive (+) - 1.000* a (10,8) Positive decolorization; ++
Aspergillus niger
BIOTECH 3080

Note: Score +1=positive decolorization; 0=no decolorization; *P < 0.05 is

significant. (-) negative, opaque, no enzymatic activity; (+) slightly clear,
weak enzymatic activity; (++) positive, moderately clear, moderate enzymatic
activity; (+++) positive, very clear, high enzymatic activity. aSignificant
difference using LSD; Eta squared values 0.01=small, 0.06=medium,
0.14=large effect size.
Discussion
In the present study, Aspergillus spp. Was the most dominant fungal
isolate obtained from decaying fruits from Iloilo Terminal market. The
following fruits contain the Aspergillus isolate which known to have very
good pectinolytic activity: ampalaya (Momordica charantia), pear (Pyrus
pyrifolia), pineapple (Ananas comosus), tomato (Lycopersicon esculentum),
lemon (Citrus limon), melon (Cucumis melo), calamansi (Citrus microcarpa),
and mango (Mangifera indica).
The present study parallels to the result of Damásio et al. (2011)
showing that agro-industrial wastes from fruit industry isolated a filamentous
fungus from lemon peel. The agro-industrial residues used for the enzyme
production obtained from fruits represent an effective carbon source for the
fungus, and significantly contributes to the reduction of waste accumulation
in the environment (Gainvors et al., 1994). These carbon sources for fungi
might reduce the enzyme production costs in the global market too.

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Pectinases are used in fruit juice industry and in wine production

(Tressler & Joslyn, 1971). Rhamnogalacturonase, a pectinase found in
Aspergillus aculeatus is used in apple juice preparation was described by
Schols et al. (1990). The result of this study showed that Aspergillus spp. Was
isolated from the various fruits collected from Iloilo Terminal market. With
similar results reported to Aspergillus (Mill, 1966) and Rhizopus (Trescott
& Tampion, 1974). However, only the genus Aspergillus was proven to be
effective in pectinase degradation.
Based on the result of the study, the genus Aspergillus is associated
with fruits (Morais et al., 1995; Fleet, 2003) or fruit pulp (Trindade et al., 2002)
in the enzymatic production of pectinase. Pectinolytic activity is a mechanism
for survival and growth which is responsible for the decomposition of fruits
(Gainvors et al., 1994). Pectinase secretion from filamentous fungi is a way of
consuming fruits as carbon sources to increase survival (Trindade et al., 2002;
Blanco et al., 1994; 1999). The isolated pectinase-producing fungi in tropical
fruits degrade pectin to modify the sugars at the side chains of pectin. In
addition, pectinase cannot be only used in the fruit juice extraction but also in
textile industry, caffeinated beverages fermentation like coffee and tea and in
poultry feed additive production. Pectinase production in the laboratory is of
low cost and pectic substances are readily available in the locality (Gragasin
et al., 2012). Hence, local production of pectinase from fungi isolated from
decaying fruits at a commercial scale has great potential. It can help in
creating job opportunities to agro-industrial sectors and saves the country’s
dollar reserves. Moreover, the utilization of huge solid wastes from decaying
fruits from various fruit stands collected is environment friendly. Therefore,
it pays in saving the earth from diminution which is a major global concern.

Conclusions
The genus Aspergillus isolated from various decaying fruits was the
dominant filamentous fungus with pectinolytic activity screened based on the
decolorization coefficient value obtained. This fungus needs to be studied
more at the species level for proper identification. There is also a need to
isolate and purify the pectinase produced from by fungi to determines its
stability and for upgraded commercial use in juice preparations.

Acknowledgement
This research would like to acknowledge the University Research
Development Center for the support and approval of this study.

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