Geetha et al.
SCREENING OF PECTINASE PRODUCING BACTERIA AND
FUNGI FOR ITS PECTINOLYTIC ACTIVITY USING FRUIT
WASTES
M. Geetha, P.Saranraj*, S. Mahalakshmi and D. Reetha
Science Instinct Publications
Abstract
Fruit peel wastes, one of the polluting solid wastes has been utilized for the production of
pectinase enzymes by solid state and submerged fermentation employing pectinolytic
microorganism isolated from fruit peel wastes. Pectinolytic bacterial and fungal cultures were
isolated from fruit peel wastes. The bacterial cultures were identified as Bacillus sp. and
Pseudomonas sp. and the fungal cultures were identified as Aspergillus niger, Aspergillus
flavus and Penicillium chrysogenum. All the bacterial and fungal cultures were found to
produce appreciable level of pectinolytic enzyme viz., pectin esterase and pectate lyase.
Among the different levels of pectin tested, all the cultures recorded maximum pectinase
activity at one percent pectin concentration. Maximum pectate lyase and pectin esterase
activity was expressed by Bacillus sp. grown in medium with one percent pectin
concentration. All the fungal isolates showed maximum activity of pectin esterase and
pectate lyase activity. Among the fungal isolates, Aspergillus niger recorded maximum
pectin esterase and pectate lyase activity. Under submerged fermentation, the fungal isolate
Aspergillus flavus recorded higher pectin esterase and pectate lyase. The Pectinase
production from fruit peel was higher in solid state fermentation than in submerged
fermentation and Aspergillus performed better than other organisms.
Keywords: Fruit wastes, Pectinase, Bacteria, Fungi, Pectin esterase, Pectate lyase and
Fermentation.
*
P.Saranraj
Department of Microbiology,
Annamalai University,
Annamalai Nagar,
Chidambaram – 608 002.
E.mail: microsaranraj@gmail.com
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Geetha et al.
Introduction
ectinases are group of enzymes that attack pectin and depolymerize it by hydrolysis and
trans-elimination as well as by de-esterification reactions, which hydrolyses the ester
bond between carboxyl and methyl groups of pectin [1]. These enzymes act on pectin, a class
of complex polysaccharides found in the cell wall of higher plants and cementing material for
the cellulose network. Pectinases accounts for 10% of global industrial enzymes produced
and their market is increasing day by day [2].
Pectinases are the groups of enzymes, which cause degradation of pectin that, are chain
molecules with a rhamnogalacturonan backbone, associated with other polymers and
carbohydrates. These pectinases have wide applications in fruit juice industry and wine
industry. In fruit juice industry, it is used for clarification, where reduction in viscosity is
caused which ultimately leads to formation of clear juice. They increase the yield of juices by
enzymatic liquefaction of pulps; these pectinases also helps in formation of pulpy products by
macerating the organized tissue into suspension of intact cells. In wine industry, pectinases
are mainly used for decreasing astringency by solubilizing anthocyanins without leaching out
procyadin polyphenols, and pectinases also increase pigmentation by extracting more
anthocyanins [3].
Pectinases can be produced by both submerged and solid state fermentation (SSF).
Submerged fermentation is cultivation of microorganisms on liquid broth. It requires high
volumes of water, continuous agitation and generates lot of effluents. SSF incorporates
microbial growth and product formation on or within particles of a solid substrate under
aerobic conditions, in the absence or near absence of free water, and does not generally
require aseptic conditions for enzyme production [4]. Many filamentous fungi like
Aspergillus niger, Aspergillus. awamori, Penicillium restrictum, Trichoderma viride, Mucor
piriformis and Yarrowia lipolytica are used in both submerged as well as solid state
fermentation for production of various industrially important products such as citric acid,
ethanol etc. Fungi like Aspergillus niger, Aspergillus oryzae, Penicillium expansum, which
are generally regarded as safe (GRAS) by United States Food and Drugs Administration
(USFDA) are employed in food industry. Some bacteria (Bacillus licheniformis, Aeromonas
cavi, Lactobacillus, etc), yeasts like Saccharomyces, Candida and Actinomycetes like
Streptomycetes are also used. Amongst these, the filamentous fungi are most commonly
employed [5].
Fungi can produce both intracellular as well as extracellular enzymes. All fungi are
heterotrophic, and rely on carbon compounds synthesized by other living organisms. Small
molecules like mono disaccharides fatty acids and amino acids can easily pass through but for
breaking down of larger complex compounds like pectin, fungi secrete extracellular enzymes.
It is well known that as compared to intracellular enzymes, the extra cellular enzymes are
easier to be extracted. Intracellular enzymes require more time and costly chemicals for
extraction. Till date, substrates used for solid-state fermentation are materials of plant origin
like grains such as rice, corn, root, tubers, and legumes. Apart from these, pomace, mango
peels, orange waste like peels and other fruit and vegetable industry waste are also being in
much use [6].
Pectinases are frequently used in fruit and vegetable industry, and pectin is also employed
widely in food industry. Peel oil finds many useful applications in both food and
pharmaceutical industry. It is good for the skin citrus solvent is a biodegradable solvent
occurring in nature as the main component of citrus peel oil. Citrus Solvents have pleasant
aroma and FDAGRAS rating ("generally recognized as safe") makes it suitable to be used as
P
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Geetha et al.
solvent, citrus solvent can replace a wide variety of products, including mineral spirits,
methyl ethyl ketone, acetone, toluene, glycol ethers, and of course fluorinated and chlorinated
organic solvents. Dietary fibers are the most recent value added product and are used as
means of roughage. The present study was carried out to evaluate the pectinase activity of the
pectinolytic bacteria and fungi using fruit peel waste as a feed substrate. Pectinase production
by different organisms in submerged state fermentation has received more attention and is
found to be cost-prohibitive because of high cost of process engineering.
Materials and methods
Collection and preparation of fruit peels
The disposed fruit peels like Orange peel, Citrus peel and Potato peel were collected from the
Annamalai University canteen, Annamalai Nagar. The collected peels were sorted out
manually based on their fine texture and rigidity. The collected peels were minced to pieces
and were hot air oven dried at 55°C until constant weight was achieved. The dried peel was
diminuted in a Ball mill and they were clarified in a sieve shaker. The peel fractions from the
mesh size 12 were used for the extraction in the Soxhelet extractor.
Isolation of pectinolytic bacteria and fungi
Pectin degrading fungi and bacteria were isolated from the fruit peel wastes. For isolation
using sterile pestle and mortar, fruit peels were ground and made into slurry using sterile
distilled water. An aliquot of this sample was serially diluted with dilution blanks and plated
on Hankin's medium containing one percent pectin for bacteria and modified Czapek's
medium with 1% pectin and 1 ml of 1000 ppm streptomycin sulphate for fungi [7]. After
incubation, the isolated bacteria were purified by streak plate technique and the fungal
cultures were purified by single hyphal tip method (Riker and Riker) and maintained in pectin
Czapek's agar slopes under refrigerated conditions.
Characterization of bacterial and fungal isolates
The isolated bacterial cultures were characterized based on morphological tests such as shape
and Gram reaction. In addition to morphological characters, the isolated bacterial cultures
were cultivated in selective media and certain biochemical tests were also performed.
Purified fungal cultures were characterized by their morphology, hyphal characteristics,
presence or absence of asexual spores, arrangement of conidia and reproductive structures.
Assay of pectinolytic enzymes
Pectin esterase
Pectin esterase activity can be measured either by measuring the amount of methanol released
or increase in free carboxyl group by titration using pH meter [8]. For assaying pectin
esterase activity, 20 ml of one percent pectin dissolved in 0.15 M NaCl (pH 7.0) and four ml
of crude enzyme extract were taken in a beaker and incubated for one hour. After incubation,
the solution was titrated against 0.02 N NaOH to reach the pH 7.0 using phenolphthalein as
indicator. The heated crude enzyme extract was used as control. Pectin esterase activity was
calculated by using the following formula:
Pectin esterase activity = Vs – Vb (Normality of NaOH)  100/Vt
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Where , Vs - Volume of NaOH used to titrate sample (ml), Vb - Volume of NaOH used to
titrate blank (ml), V - Volume of incubation mixture (ml), t - Reaction time (hours). Pectin
esterase activity is expressed as milli equivalents of NaOH consumed min-1 ml-1 of crude
enzyme extract under the assay conditions.
Pectate lyase
Pectate lyase activity of the isolated cultures was assayed by measuring the increase in
absorbance at 232 nm due to the production of unsaturated bonds during the
depolymerization of galacturonic acid [9]. The reaction mixture was prepared with 2.5ml of
0.5 percent sodium pectate dissolved in 50 mM Tris HCl (Sigma) buffer and 1mM calcium
chloride and the activity was measured at 232 nm. The crude enzyme extract heated at high
temperature was kept as control. One unit of pectate lyase activity was defined as the amount
of enzyme that caused absorbance at 232 nm to increase at a rate of 0.1 OD min-1 ml-1 of
crude enzyme extract under the assay conditions.
Effect of different levels of pectin on pectinolytic enzymes
To find out the optimum level of pectin in the medium for the induction of pectinolytic
enzymes, the 24 hour old bacterial cultures were inoculated into medium containing 0.5, 1.0
and 1.5 percent pectin and incubated in a rotary shaker (100 rpm) for 48 hours at room
temperature. After the growth the culture broth was centrifuged at 10,000 rpm for 15 minutes.
The supernatant was collected and used for assaying different pectinolytic enzyme activities.
For fungi, the cultures were grown in medium containing pectin each at 0.5, 1.0 and 1.5
percent. After 5 days, the fungal mats were removed by filtering through Whattman No.1
Filter paper and then the filtrate was centrifuged at 10,000 rpm for 15 minutes and used for
pectinolytic enzymes assay.
Production of pectinolytic enzymes by submerged fermentation employing bacterial and
fungal cultures
To find out the efficiency of fungal cultures in producing pectinolytic enzymes from fruit
peel wastes waste by submerged fermentation, 50g of fruit peel waste was taken and
powdered. To this, 100ml of water was added and pH was adjusted to 5.0. It was sterilized
at 120°C for 15 minutes and then inoculated with fungal cultures and incubated at room
temperature. On 8th day of fermentation, it was filtered through Whatman No. 1 filter paper
and then centrifuged at 10,000 rpm for 15 minutes. This crude enzyme extract was used for
measuring pectinase activity. To find out the effect of ammonium nitrate on pectinase
production, ammonium nitrate was sprayed on the waste at 0.2 percent as inducer. Samples
were withdrawn and extracted with suitable ratio of water (1:2) and pectinolytic enzymes
were assayed.
Effect of different ammonium nitrate concentrations on pectinase activity.
To find out the effect of different concentrations of Ammonium nitrate on pectinase enzymes
activity, media with the best level of pectin (1%) were prepared for bacteria and fungi along
with Ammonium nitrate each at four different levels (0.1, 0.2, 0.3 and 0.4 percent) and
incubated at room temperature in a rotary shaker at 100 rpm and the extracts were used for
enzymes assay.
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Production of pectinolytic enzymes by solid-state fermentation employing bacterial and
fungal cultures
To extract pectinase enzyme complex from fungal cultures grown on fruit peel waste was
taken in metal trays and moistened with water to maintain the moisture at 50 percent. The
trays were covered with muslin cloth and sterilized. After sterilization the fungal cultures
grown on the modified Czapeck's broth were sprayed over the substrates and incubated at
room temperature. The moisture content of the tray was monitored periodically and water
spray was given to avoid drying of the substrate. Samples were withdrawn after eight days of
fermentation and enzyme activities were measured. To find out the effect of ammonium
nitrate on pectinase production under solid state fermentation, ammonium nitrate was sprayed
on the waste at 0.2 percent as inducer. Samples were withdrawn and extracted with suitable
ratio of water (1:2) and pectinolytic enzymes were assayed.
RESULTS AND DISCUSSION
Microbes associated with fruit peelings waste
Two pectinolytic bacteria and three fungal cultures were isolated from fruit wastes. The
bacterial isolates were characterized based on Gram staining, plating on selective media and
several biochemical reactions. Purified fungal cultures were characterized by their
morphology, hyphal characteristics, presence or absence of asexual spores, arrangement of
conidia and reproductive structures. Bacterial isolates were identified as Bacillus and
Pseudomonas, while the fungal isolates were characterized as Aspergillus niger, Aspergillus
flavus and Penicillium chrysogenum. The results of the isolates confirmed the findings of
Rolz et al. and Roussos et al., who reported that pectinolytic bacteria were the important
fraction of the microbial population on whole fruit wastes and pulped fruits [10 & 11].
Pectinolytic organisms isolated from fruit peel wastes were found to produce an array of
pectinolytic enzymes collectively called as pectinases. All the bacterial cultures recorded
significant quantities of pectinolytic enzymes such as pectin esterase and pectate lyase.
Higher levels of pectinase activities were expressed by the bacterial isolate Pseudomonas sp.
Chatterjee et al. also reported that several bacteria like Erwinia sp., Pseudomonas sp.,
Bacillus sp., were able to produce pectinases [12]. The result of the present study is also in
confirmation with the findings of Liao et al. [13]. Many species of fungi are capable of
degrading pectin by producing pectic enzymes. The fungal isolates of fruit pulp wastes were
also found to produce pectinases. Production of pectin enzymes by fungi such as Alternaria,
Cladosporium, Colletotrichum, Mucor, Penicillium, and Trichoderma was confirmed by
Isshiki et al. and Kapat et al. [14 & 15].
The isolated cultures were screened for further studies based on the activity of pectinolytic
enzymes such as pectin esterase and pectate lyase (PL). Among the bacterial isolates,
Pseudomonas sp., and Bacillus sp. recorded maximum Pectinase activities (Figure 1 and 2).
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Fig. 1. Pectin esterase activity of the cultures from fruit peeling waste
Fig. 2. Pectate lyase activity of the cultures from fruit peeling waste
The fungal cultures viz., Aspergillus niger, Aspergillus flavus and Penicillium chrysogenum
exhibited highest pectinolytic enzyme activities and hence these isolates were selected for
further experiments.
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Effect of pectin on pectic enzymes
Many organisms are stimulated to increase enzymes synthesis by adding an inducer to the
culture media. This inducer may be the substrate of the enzyme or the modification of the
substrate. Any method which can supply the inducer to the growing organism will reduce
catabolite repression and result in enhanced enzyme yields. Most of the pectinase are
inducible enzymes and hence substrate rich in pectin content must therefore be added to the
medium to stimulate enzyme production. In addition to the inducing properties, pectin also
enhances the release of pectic enzymes into the fermentation medium. Pandey also observed
the inducement of enzymes by substrates [16].
The effect of pectin on the production of pectinolytic enzymes was studied by inoculating
pectinolytic cultures into the medium containing different levels of pectin and the results
were showed in Figure-3 and 4.
Fig. 3. Effect of pectin on bacterial pectin esterase activity
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Fig. 4. Effect of pectin on bacterial pectate lyase activity.
The present investigation revealed that at one percent pectin concentration, maximum pectin
esterase activity was observed in all the bacterial isolates Bacillus sp. and Pseudomonas sp.
Pectin esterase activities of the isolate Bacillus sp. was significantly different from
Pseudomonas sp. Nagel and Vaugh [17] observed the pectin induced pectin esterase activity
in Bacillus polymyxa. The fungi isolates also showed significantly higher pectin esterase
activity at one percent concentration of pectin. Among the isolates tested, Aspergillus niger
recorded maximum activity at one percent concentration. It was followed by Aspergillus
flavus and Penicillium chrysogenum (Figure-5 and 6).
Fig. 5. Effect of pectin on fungal pectin esterase activity
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Fig. 6. Effect of pectin on fungal pectate lyase activity
The result was in confirmation with the findings of McMillan and Permbelon [18] who
observed a high pectin esterase activity at one percent pectin concentration in Botrytis
cineria.
Pectate lyase performs a non hydrolytic breakdown of pectate and characterized by a
transeliminative split of pectic polymer. In the present study, Bacillus sp. produced maximum
activity of 2.74 Unit ml-1 at one percent pectin concentration. This higher activity might be
due to the role of pectin as inducer of enzyme. Zucker and Hankin [19] also observed the
inducible synthesis of extracellular pectate lyase in Erwinia carotovora and Pseudomonas
fluorescens. The same inducible trend was also observed by Ganibaldi and Bateman [20] in
Xanthomonas compestris strain grown in polygalacturonic acid. Most of the fungi required
one percent pectin concentration for higher pectate lyase activity. In the present investigation,
maximum pectate lyase was exhibited by fungus, Penicillium chrysogenum at one percent
pectin. Similar trend in pectate lyase activity of Fusarium oxysporum was reported by Pietro
and Roncero [21] who stated that one percent (w/v) of polygalacturonic acid enhanced the
pectate lyase activity.
Effect of different concentrations of ammonium nitrate on Pectin enzymes
Different concentrations of ammonium nitrate were reported to enhance the pectinase
production. In the present investigation the effect of ammonium nitrate on pectinase
production by the microbial cultures was studied. Pectin esterase activities of the bacterial
isolates were maximum in medium supplemented with 0.4% ammonium nitrate. Same trend
was noted by Papdiwal and Deshpande [22] in Xanthomonas malvacearum in which yeast
extract at 0.4 to 2.0 percent level stimulated the production of pectin esterase. The present
study on fungal cultures recorded the higher production of pectin esterase by Penicillium
chrysogenum at 0.4 percent ammonium acetate. Ikotun [23] obtained maximum production of
pectin esterase with Penicillium chrysogenum at 0.1 percent citrus pectin and 0.2 percent of
yeast extract. Ammonium nitrate in addition to serving as nitrogen source can also be
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considered as growth stimulant. This might be the main reason for the increased enzyme
production in the medium with ammonium nitrate (Figure-7).
Fig. 7. Effect of ammonium nitrate on pectin esterase activity
Pectate lyase activities of the most of the bacterial isolates were high in medium prepared
with yeast extract and one percent of pectin. The bacterial isolate Pseudomonas sp. at 0.4
percent ammonium nitrate exhibited maximum Pectate lyase activity. In the case of fungi,
Aspergillus niger recorded maximum enzyme activity at 0.4 percent ammonium nitrate. Ward
and Fogarty [24] reported that Bacillus subtilis and Flavobacterium pectinovorum preferred
glutamine or yeast extract as both nitrogen source and an inducer (Figure-8).
Fig. 8. Effect of ammonium nitrate on pectate lyase activity
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Production of pectinase enzymes from fruit peel waste by solid state fermentation and
submerged fermentation
Utilization fruit peel waste in one or other form is immediate necessity from economical and
environmental protection point of view. Production of pectinases is one of the alternative
options due to the availability of the substrate almost at throw away price. Agro-industrial
residues are generally considered to be the best substrate for the solid state fermentation
process and hence the fruit peel wastes were found to be an ideal substrate for producing
pectinase [25].
In the present study, only fungi are utilized for the production of pectinases from fruit peel
wastes by solid state fermentation. Filamentous fungi alone can grow in the absence of free
water. In general, the hydrolytic enzymes like pectinases are produced by fungal cultures
since such enzymes are used in nature by fungi for their growth. In the present study, species
of fungi such as Aspergillus niger, Aspergillus flavus and Penicillium chrysogenum were
used for producing pectinase from fruit peel wastes (Figure-9 and 10).
Fig. 9. Production of pectinase by submerged fermentation
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Fig. 10. Production of pectinase by solid state fermentation
David et al. were also of same opinion that for producing pectinases commercially, moulds
can be used and not bacteria [26].
Solid state fermentation was followed to produce pectinases from fruit peel wastes. In the
present investigation also the pectinase activity was lower in submerged fermentation than in
solid state fermentation. It is in conformity with the reports of Archana and Satyanarayana
who stated that enzyme production was 22 fold higher in solid state fermentation system than
in submerged system [27]. The use of higher water contents in submerged process for
production of enzyme by microbial strain also results in the dilution of the product in the
fermenting medium as compared to the solid state fermentation process [28].
Conclusion
Most of the microorganisms can capable to produce various extracellular and intracellular
enzymes using various cheap sources. The effect of enzyme activity is higher than the plant
derived enzymes. Pectinase is an extracellular enzyme, which is produced from various
organisms including bacteria, fungi, and also some actinomycetes. Research on pectinase has
progressed very rapidly over the last five decades and potential industrial applications of the
enzyme especially in solid waste management have been identified. Major impediments to
exploit the commercial potential of pectinases are the yield, stability and cost of enzyme
production. Although, terrestrial strains of microbes have been extensively studied by many
researchers. The bacteria Pseudomonas sp. and Bacillus sp. and the fungi Aspergillus niger,
Aspergillus flavus and Penicillium chrysogenum were used for the production of pectinase
and fruit peel wastes were used as a substrate.
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