Culture Optimization for Enhanced Production of Microbial Pectin Methylesterase under Submerged Conditions
The present investigations were undertaken with the objective to isolate PME producing microbial strain and to standardize conditions to have cost effective production of the enzyme. Based on the ability of pectinolysis, out of 20 microbial strains isolated, one fungal strain was selected and got identified from Institute of Microbial Technology (IMTECH), Chandigarh (India), as Aspergillus heteromorphus. The strain was added to its collection with identification number MTCC 9262. Various process variables viz., type and pH of the medium, temperature of growth, time of incubation, inoculum concentration and type and concentration of carbon and nitrogen sources were optimized as per the conventional method of one variable at a time approach which involves varying a single independent variable and maintaining others at a constant level. Modified pectin medium-A, in which both Fe++ and Cu++ were omitted, gave the maximum growth of A. heteromorphus. Maximum PME production was obtained when an inoculum size of 4x106 spores mL-1 was incubated in modified pectin medium-A for 144 h at pH 4.5 and at a temperature of 30°C under static conditions in submerged fermentation. Out of the various nitrogen and carbon sources tested, dibasic ammonium phosphate and pectin were found to be the best N and C source, respectively. After standaradizing various process variables, a three fold increase in PME production by A. heteromorphus was achieved. Orange peel, a waste byproduct of fruit industry could effectively replace pectin as C-source thus, making the process cost effective. The PME produced by A. heteromorphus had the pH optimum in acidic range-a characteristic which could be exploited for its use in fruit processing industry.
Pectins, the complex high molecular weight heterogeneous and acidic structural
polysaccharides are the major constituents of primary cell wall of cereals,
vegetables, fruits and fibers. The main component of pectin backbone is galacturonic
acid with neutral sugars such as arabinose, galactose and xylose present in
the side chains, whereas rhamnose constitutes a minor component of pectin. Particularly,
in fruits and vegetables, pectins are present in large amounts contributing
to the strength of these tissues. They are one of the important functional food
ingredients in jams, jellies, fruit juices, confectionery products, bakery fillings
and are used for stabilization of acidified milk drinks and yoghurts. Pectinolytic
enzymes, that profoundly affect the fine structure of pectin, find applications
in the various industrial processes. The most commonly used industrial pectinases
which account for about 10% of the global industrial enzymes (Stutzenberger,
1992), include polygalacturonases, pectin lyases and pectin methylesterases
(PMEs). Acidic PME is used in extraction, clarification and liquification of
fruit juices (Kaur et al., 2004), while alkaline
PME finds potential applications in paper and textile industry (Ahlawat
et al., 2009). In addition, these enzymes also have the potential
of producing low degree methylated pectins. Biotechnological potential of microorganisms
producing PME in textile, paper and fruit processing industry (Reid
and Ricard, 2000) has drawn a great deal of attention worldwide. Though
numbers of PME producing organisms such as Aspergillus niger (Bhat,
2000; Kashyap et al., 2001). Aspergillus
awamori (Blandino et al., 2002), Aspergillus
foetidus (Sebastian et al., 1996; Hours
et al., 1988), Aspergillus japonicum (Semenova
et al., 2003), Aspergillus carneus (El-Sheekh
et al., 2009), Aspergillus flavus (Yadav
et al., 2008) and Bacillus subtilis (Ahlawat
et al., 2009) have been reported, selection of potential isolates
remains a tedious task, especially, when physiologically potential strains are
to be obtained to achieve maximum yield (Pandey et al.,
1999). Several researchers have studied the optimization of culture conditions
for enhanced PME production from various microorganisms (Maldonado
et al., 1986; Maldonado and de-Saad, 1998;
Joshi et al., 2006; Ahlawat et al., 2009;
El-Sheekh et al., 2009). We report here, the
standardization of process variables for the maximum production of PME in submerged
fermentation of a physiologically potential strain of Aspergillus isolated
from the soil debris of fruit and vegetable markets.
MATERIALS AND METHODS
Micro-Organism and Inoculum
The present investigations were carried out in the Department of Biochemistry,
CCS Haryana Agricultural University, Hisar (Haryana), India from Nov. 2008 to
April, 2009. Microorganisms were isolated from soil samples collected from different
fruit and vegetable markets of the city. Based on the ability of pectin hydrolysis
and potential of PME production, a total of twenty isolates were screened. Finally,
one fungal isolate was selected as the potential producer of PME which was got
identified from the Institute of Microbial Technology (IMTECH), Chandigarh,
India as Aspergillus heteromorphus. It had been included to their collection
at IMTECH with identification mark MTCC 9262. The inoculum was prepared in Potato
Dextrose Medium (PDM) containing pectin as the sole C-source, by harvesting
spores from 120 h old cultures grown at 30°C.
Submerged fermentation was carried out in 250 mL Erlenmeyer flasks by taking
50 mL pectin medium containing (%, w/v). (NH4)2SO4-0.3;
KH2PO4-1.0; MgSO4-0.2; Na2B4O7-0.07;
CuSO4-0.0003; MnSO4-0.0001; ZnSO4-0.007; Pectin-0.3
(SRL). Flasks were inoculated with inoculum size of 1x105 spores
mL-1 and incubated at 30°C for 144 h under static conditions
in a BOD incubator (Remi, India).
For maximum production of PME, various culture conditions viz., type and concentration
of carbon and nitrogen sources, type of medium, inoculum size, pH, temperature
and incubation period were optimized by conventional method of standardization
by one variable at a time approach which involves varying a single independent
variable at a time while maintaining the others at a constant level. Fungal
isolate was grown in different media, containing 0.3% pectin, at pH 4.2 for
120 h. Medium giving maximum growth was further used to standardize the inoculum
size by inoculating different concentrations (1-5x106 spores mL-1)
of inoculum. Similarly, the temperature and time of incubation were optimized
by growing the fungal isolate at different temperatures (25, 30, 35, 40, 45
and 50°C) for different time periods (72-192 h), respectively. Different
organic and inorganic nitrogen sources viz., yeast extract, peptone, ammonium
chloride, ammonium hydrogen phosphate, ammonium sulfate and sodium nitrate were
supplemented separately to a final concentration of 0.3% (w/v) to study the
microbial growth and PME activity. Glucose, fructose, sorbitol, sucrose, maltose,
pectin and citrus peel at a final concentration of 1% were used as the carbon
Effect of carbon and nitrogen sources and their concentrations, type of
medium, inoculum size, pH, incubation temperature and time period, was examined
in submerged condition. Statistical analysis of standard deviations was done
to determine the effect of variables on production of PME using software Statistical
Package for Agricultural Scientists OPSTAT.
Enzyme Isolation and Assay
After 144 h of growth, the fungal broth was filtered through 4 layers of
muslin cloth and centrifuged at 8000 rpm for 15 min at 4°C in a refrigerated
centrifuge. The supernatant taken as enzyme extract containing extracellular
PME, was used to assay the enzyme activity. Ten milliliter of 1% pectin in 0.15
M NaCl (pH 7.0) was incubated with 2 mL of enzyme extract in 100 mL flask at
35°C for 1 h. The reaction was stopped by keeping the flask in boiling water
bath for 10 min. The contents were cooled and the free carboxyl groups released
were determined by titrating against 0.02 N NaOH using phenolphthalein as indicator
(Kertesz, 1955). One unit of PME was defined as the amount
of enzymes required to release one micro equivalent of carboxyl group/mL/h.
Identification of PME Producing Microorganism
Out of twenty wild cultures of fungal isolates, only five isolates showing
clear zone (Fig. 1a, b) on agar plates with pectin medium
were selected as PME producers. These isolates were further screened for the
maximum production of PME on two media viz., potato dextrose medium and pectin
medium (Fig. 2) at pH 4.2 with 0.3% pectin as sole carbon
zone formation showing PME activity on pectin agar medium (a) after 3
days of incubation and (b) after 5 days of incubation|
of PME by five fungal isolates on potato dextrose (PD) and pectin medium|
of different media on PME production by Aspergillus heteromorphus
As is clear from the results, isolate SK-2, exhibited highest PME production
in both the media (160 and 213 units in potato dextrose medium and pectin medium,
respectively) and was used for further studies for optimizing culture conditions
to get maximum production of PME. This fungal isolate was got identified from
IMTECH, Chandigarh as Aspergillus heteromorphus and was included to their
collection at the centre with identification mark MTCC 9262.
Optimization of Culture Medium
The Aspergillus heteromorphus MTCC 9262 was initially grown on four
different media (pH 4.2) viz., Potato Dextrose Medium (PDM), Pectin Medium (PM),
Pectin Minimum Medium (PMM; it contained no micronutrients) and Czapeck Dox
Medium (CDM). As is clear from the results presented in Fig. 3,
the enzyme activity was maximum (213 units) in PM containing all the micronutrients
and minimum in CDM (136 units). Since, pectin minimal medium which contained
no micronutrients also showed 76% of the maximum activity, it was thought worthwhile
to omit the micronutrients one by one and see the effect on growth and enzyme
activity. When either Zn or Mn or B or Mo was omitted individually or in combination
from pectin medium, the activity was same as observed in pectin minimal medium,
indicating that these micronutrients were the absolute requirements for the
growth of the A. heteromorphus and could not be omitted from the medium.
However, when either Cu or Fe was omitted, there was rather improvement in PME
activity over that of pectin medium. Keeping this in view, A. heteromorphus
was grown in three modified pectin media viz., A (where, Fe and Cu both were
omitted), B (where Cu alone was omitted) and C (where Fe alone was omitted).
Results presented demonstrate that when grown in modified pectin medium-A, in
which both Cu and Fe both were omitted, Aspergillus heteromorphus showed
maximum PME producing activity (305 units) and the activity was even higher
than that observed with pectin medium (213 units).
Optimization of Culture Conditions
The size of inoculum seems to have a profound effect on growth of the microorganisms
and enzyme production. PME production activity was studied by varying the inoculum
size from 1mL (1x106spores) to 5 mL (5x106 spores). The
maximum enzyme production (361 units) was observed with 4 mL inoculum i.e.,
4x106 spores. When inoculum size was further increased to a size
more than 4 mL, enzyme production declined (Fig. 4a-d).
The production of PME by A. heteromorphus MTCC 9262 was studied by
growing the fungus in PM-A medium with pH ranging from 4.0 to 6.5 in submerged
fermentation. Results (Fig. 4) revealed that pH 4.5 was optimum
for the maximum production of PME. On either side of the optimum pH (4.5), the
enzyme production declined.
||Effect of different cultural conditions on the PME production
by Aspergillus heteromorphus MTCC 9262, (a) inoculum size (mL), (b)
pH, (c) days of incubation and (d) incubation temperature
The PME production was detected after three days i.e., after 72 h of incubation.
After 72 h, the activity was measured at 24 h interval till 192 h. The results
in Fig. 4 demonstrate maximum PME production to occur after
144 h of incubation with the yield of 377 units. After 144 h, the activity declined.
To find out the optimum temperature of growth and enzyme production, the
organism was grown at different temperatures ranging from 25 to 45°C. Optimum
temperature observed in this study was 30°C (Fig. 4) for
the maximum production of PME by A. heteromorphus in submerged conditions.
When different inorganic and organic nitrogen sources were supplemented
in the medium to achieve maximum PME production by A. heteromorphus,
peptone and dihydrogen ammonium phosphate were observed to be better as they
enhanced the PME production to 480 (28% of control) and 520 (39% of control)
units, respectively (Fig. 5). Among the various nitrogen sources
tested, ammonium sulphate was at number three.
The carbon source in the medium appears to exert a profound effect on the
enzyme production behaviour of the fungus. Pectin at 1% concentration gave the
maximum PME production (613 units). Citrus peel at a concentration of 1% showed
almost identical PME production (543 units) which was next to the activity observed
with 1% pectin (Fig. 5).
of different nitrogen and carbon sources on PME production by Aspergillus
heteromorphus MTCC 9262|
of PME production by Aspergillus heteromorphus MTCC 9262 under
initial and optimized culture conditions|
Based on these observations, citrus peel being a readily available waste by
product of citrus fruit industry, could be suggested as an efficient carbon
source for PME production by Aspergillus heteromorphus. No PME activity
could be observed when pectin was replaced by sucrose, maltose, dextrose and
fructose as the carbon sources suggesting that PME in A. heteromorphus
is substrate inducible.
Finally, the fungal strain of Aspergillus heteromorphus MTCC 9262 was grown in 250 mL Erlenmeyer flask containing 50 mL of modified pectin medium-A with 0.3% citrus peel as the C-source and 0.2% dihydrogen ammonium phosphate as the N-source at pH 4.5 and 30°C for 144 h inoculated with 4x106 spores mL-1. Results presented in Table 1 reveal that optimization of process variables resulted in 3-fold increase in PME production.
In recent years, considerable attention has been paid to the use of micro-organisms
in industrial fermentation processes, especially enzyme production There has
been a surge of interest in the production of fungi having the ability to produce
acidic pectinases which can be used in the fruit processing industry where they
play a crucial role in the extraction and clarification of fruit juices. In
order to obtain concentrates of high quality, it is essential to degrade pectin
and clarify the juices with the help of pectolytic enzymes. It is very well
reported that pectolytic enzymes produced by certain sprouting strains like
Aspergillus niger, Aspergillus oryzae, Rhizopus oryzae
etc., have long been used in fruit juice clarification because they have been
observed to have desirable characteristics to be utilized for that purpose.
However, these fungal strains have not been exploited to scale up the production
of these enzymes for the purpose of their purification and immobilization. The
search for specific enzymatic activities is still increasing due to their demand
for clarification of new types of fruit juices. For this reason, the present
investigations were undertaken with the objective to harness the valuable product
namely fungal/microbial isolate and to examine its potential to produce pectin
methylesterase. During the present investigations, twenty isolates were screened
on the basis of their ability of pectin hydrolysis on agar plates containing
pectin and the dye ruthidium red. The appearance of clear zone was taken as
the growth of fungus. Similar strategy of screening of fungal cultures was adapted
by Boccas et al. (1994). Out of twenty, five
isolates giving appreciably good amount of PME were grown on potato dextrose
and pectin media. The isolate-2 gave maximum activity in both the media and
was selected for further studies. Microbial cultures, regardless of the nature
of their end product and the type of bioprocess have certain specific requirements
for their growth which have to be optimized for their maximum production. The
growth conditions optimized for the fungal strain selected in the present investigations
included type of medium, its temperature, pH, inoculum concentration, incubation
time and type and concentration of C- and N-source. When grown on different
media, it was observed that the modified pectin medium A in which both Fe and
Cu were omitted gave the maximum PME production. This may be due to inhibitory
effect of Cu and Fe on PME activity (Celestino et al.,
2006). Cu has been reported to inhibit the growth of the microorganisms
while the Fe has been found to interfere with the color of ruthidium red while
counting the microbial colonies. Maximum production of PME when inoculated with
4x106 spores of A. heterolorphus and decrease in its production
on increasing the inoculum size (Fig. 4) could be due to competition
between microorganism colonies for nutrients and probably the non-availability
of nutrients for the large population limits the fungal growth. Similar observations
have been made by Maldonado and Callieri (1989) where
inoculum size of 4x106 spores has been shown to give maximum growth.
However, there are several reports showing the need of either less (Maldonado
and de-Saad, 1998; Bai et al., 2004) or more
(Cavalitto et al., 1996) size of inoculum for
maximum production of PME from different sources. This might be due to differences
in the nutrient requirements by different microorganisms.
When grown in medium with different pH values, maximum production of enzyme
was observed at pH 4.5. On either side of this optimum pH, the PME producing
ability declined. The variation in PME production due to change in pH may be
because of maximum availability of nutrients at that particular pH, as has been
reported by Joshi et al. (2006). Semenova
et al. (2003) also reported that the production of pectinase by A.
japonicum was maximum in acidic medium. The influence of wide range of pH
from 2.3 (Sebastian et al., 1996) to 7.2 (Parley
and Page, 1971) on the production of microbial pectinase from different
substrates has been reported. Wide range of pH of the medium during upstream
bioprocesses makes the end product either acidic or alkaline, which tends to
have varied applications (Hoondol et al., 2002).
Acidic pectinases are useful in extraction, clarification and liquifaction of
fruit juices (Kaur et al., 2004) and wines (Favela
et al., 2005), while alkaline pectinases are widely used in fabric industries
for setting of plant fibers such as flax, hemp and jute, in biopreparation of
cotton fabrics, in enzymatic polishing of jute/cotton blended fabrics in the
paper industry to solve the retention problems in mechanical pulp bleaching,
in the treatment of pulp and paper effluents and for improving the flavour of
black tea (Sharma and Satyanarayana, 2004; Favela
et al., 2005). Aspergillus heteromorphus, studied during the
present investigations, is suggested to be the producer of acidic PME and may
find its applications in fruit juice industry.
Maximum production of PME at 144 h of incubation as has been observed in the
present investigations has also been reported by Sunnotel
and Nigam (2002). However, in their case, prolonged fermentation up to 288
h resulted in comparatively very small decline in enzyme activity, whereas in
the present case, the activity declined significantly after 144 h of incubation
suggesting that enzyme produced by A. heteromorphus was more unstable
than the one described by Sunnotel and Nigam (2002).
Temperature is another very important factor which is known to influence the
metabolic rate of the organism involved in the process, which in turn determines
the amount of the end product. Temperature has been observed to be one of the
major process variables affecting the production of pectinases in submerged
conditions. Optimum temperature of 30°C for maximum production observed
in present case falls within the range of 21°C (Joshi
et al., 2006) to 35°C (Soni and Bhatia, 1981)
When different inorganic and organic nitrogen sources were supplemented in
the medium to achieve maximum PME production by A. heteromorphus, peptone
and dihydrogen ammonium phosphate were observed to be better as they enhanced
the PME production to 480 (28% of control) and 520 (39% of control) units, respectively
(Fig. 5). Ammonium phosphate (dibasic) has been reported to
be the best nitrogen source for the growth of A. niger (Joshi
et al., 2006). Ammonium chloride, ammonium nitrate and yeast extract
inhibited PME production. Kashyap and Soni (2003) have
also reported ammonium nitrate to be inhibitory for pectinase production by
Bacillus. To find out its optimum concentration, the fungal isolate was
grown in different concentrations (ranging from 0.1-0.4%) of dibasic ammonium
phosphate and it was observed that 0.2% gave the maximum PME production. The
present results are in accordance with those reported by Joshi
et al. (2006) where A. niger has been observed to produce
maximum PME with 0.2% dibasic ammonium phosphate.
In the present investigations, the PME production activity observed with citrus
peel at a concentration of 1% was almost identical to the activity observed
with 1% pectin thus, making the process cost effective. Similarly, very recently
(El-Sheekh et al., 2009), orange peel and pulp
scrap excluded as waste in orange juice and canning industry, have been successfully
used for the production of pectinase by Aspergillus carneus NRC 1. No
PME activity could be observed when pectin was replaced by sucrose, maltose,
dextrose and fructose as the carbon sources suggesting that PME in A. heteromorphus
is substrate inducible. Many researchers have used pectin as a sole carbon source
for PME production and found the enzyme to be inducible one (Oretga,
1994; Maldonado and de-Saad, 1998; Jaffar
and Oommen, 1993). Kashyap and Soni (2003) however,
reported that carbon sources such as polygalacturonic acid, pectin and lactose
enhanced pectinase production by 11-44% while glucose, galactose, sucrose, maltose
and xylose inhibited pectinase production by 25-60% in Bacillus.
After optimization of process variables, a 3-fold increase in PME production
was observed. A similar increase of 2.5 fold has been obtained in the activity
of tyrosine phenol lyase by Citrobacter freundii MTCC 2424 after optimizing
the culture conditions (Chandel and Azmi, 2008). Sharma
and Satyanarayana (2006) reported about 34-41 fold increase in pectinases
production after optimization of process variables by statistical method of
Response Surface Methodology (RSM).
Isolation and multi-step screening method employed in the study led to the isolation of one efficient strain of Aspergillus heteromorphus for production of PME. Results obtained on optimization of process variables under submerged fermentation revealed that citrus peel could be effectively used for PME production. About 3 fold enhancement in acidic PME production by A. heteromorphus MTCC 9262 was achieved when an inoculum size of 4x106 spores mL-1 was incubated in modified pectin medium-A at pH 4.5 and 30°C for 144 h. The low cost of its production and acidic nature of enzyme may further broaden the scopes for its use in fruit juice industries.
Researchers are thankful to Haryana State Council of Science and Technology, Chandigarh, India for the financial assistance in the form of the research project entitled Production and immobilization of microbial enzymes for fruit processing industry.
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