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Journal of the Science of Food and Agriculture
J Sci Food Agric 79:1054±1059 (1999)
Pectinolytic and cellulolytic activities of heat
resistant fungi and their macerating effects on
mango and African mango
J Obeta Ugwuanyi* and Jason AN Obeta
Department of Microbiology, University of Nigeria, Nsukka, Nigeria
Abstract: Five heat resistant fungi (HRF), Neosartorya ®scheri, N ®scheri var spinosa, N quadricincta, Paecilomyces varioti and Byssochlamys nivea, were studied for production of pectinolytic and
cellulolytic activities. All isolates produced considerable hydrolase, lyase and pectinesterase activities.
Hydrolase activities were signi®cantly higher in fruit tissue (mango and African mango) media than in
pectin medium (P < 0.01) when assayed by both cup plate and viscometric methods. Activities
produced in both fruit media were comparable in N ®scheri, N ®scheri var spinosa and P varioti but
not in N quadricincta and B nivea. All isolates produced greater lyase activities in pectin medium than
in fruit tissue media except for N quadricincta, while the converse was the case for pectinesterase. P
varioti did not utilise carboxymethyl cellulose or produce cellulase activity. Other isolates produced
cellulase with B nivea producing the greatest activity. Each isolate caused considerable maceration of
arti®cially inoculated mango and African mango fruits, which is not directly related to measurable
pectinase or cellulase. The possibility of co-operation between pectinase and cellulase activities in the
disintegration of fruit tissues is discussed.
# 1999 Society of Chemical Industry
Keywords: heat resistant fungi; Neosartorya spp; Paecilomyces varioti; Byssochlamys nivea; cellulase; pectinases;
macerating activity; mango; African mango
INTRODUCTION
Spoilage of heat processed fruits and fruit products
due to heat resistant fungi has been frequently
reported.1±3 Earlier studies suggested that moulds of
the genus Byssochlamys were associated with spoilage
of packaged heat processed fruits and so have been
extensively studied.4±7 More recent investigations have
implicated Neosartorya spp, Talaromyces spp, as well as
Eupenicillium spp in the spoilage of processed
fruits.8±11 These organisms are soil saprophytes and
have been isolated from garden and farm soils, from
where they contaminate fruits.1,12±16 They are able to
cause spoilage because they produce ascospores which
survive heat treatment and subsequently germinate in
processed packaged fruits and fruit products, thereby
causing disintegration of such fruits.
It is believed that members of the genus Byssochlamys are able to cause fruit disintegration by their
ability to produce various pectinolytic and disintegrative enzymes.17±20 Textural disintegration of fruit
tissues by Neosartorya spp has been documented,21,22
and is the basis of the speculations that these
organisms may also produce pectinolytic and disintegrating enzymes.2 To our knowledge, however, there
is no report on tissue macerating enzymes in these
organisms whose role in fruit spoilage is becoming
increasingly important. In this paper we report on the
pectinolytic and cellulolytic activities of some Neosartorya spp and other heat resistant fungi isolated from
Nigerian soils as well as their disintegrative effects on
some arti®cially inoculated tropical fruit tissues.
EXPERIMENTAL
Test isolates of fungi
The isolates used for this study include Neosartorya
®scheri (Wehmer) IMI 333695, N ®scheri var spinosa
(Raper and Fennell) IMI 333692, and Paecilomyces
varioti (Bainier) IMI 333694 all isolated from Nsukka
Southern Nigeria.23 Neosartorya quadricincta (Yuill)
IMI 312830 and Byssochlamys nivea were kindly
supplied by Dr RN Okagbue. All isolates were
maintained on slants of freshly prepared potato
dextrose agar (PDA) and stored at 4 °C.
Production of pectinolytic enzymes in pectin
medium
The pectin medium employed contained, in distilled
water (g lÿ1): citrus pectin (30% DE (Sigma)), 5.0;
yeast extract powder (Oxoid), 3.0; Na2HPO42H2O,
* Correspondence to: Jeremiah Obeta Ugwuanyi, Department of Bioscience and Biotechnology, University of Strathclyde, Royal College
Building, 204 George Street, Glasgow G1 1XW, U.K
E-mail: jeremiah.ugwuanyi@strath.ac.uk
(Received 8 August 1997; revised version 2 November 1998; accepted 17 December 1998)
# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50
1054
Cellulolytic and pectinolytic activities of heat resistant fungi
7.2; KH2PO4, 3.5; pH (before sterilisation) 6.8.24 The
medium (50 ml) in 250 ml Erlenmeyer ¯asks sterilised
at 121 °C for 15 min were inoculated in duplicate with
three agar plugs (6 mm diameter) of 7-day-old
mycelial growth of test isolate grown on PDA. Two
control ¯asks were inoculated with similar but sterile
PDA plugs. All ¯asks were incubated in an orbital
shaker (Gallenkamp) at 28 °C and 150 rpm for 7 days.
The broth culture from each ¯ask was separated from
mycelial growth by ®ltration through a GF/C ®lter
(Whatman). The ®ltrate was dialysed twice against 50
volumes of double distilled water at 2±4 °C for 24 h to
remove salts and small molecules. Cellular growth of
isolates was determined by drying the ®ltered mycelia.25
Production of pectinolytic enzymes in fruit tissue
medium
The basal medium was modi®ed from Obi and
Moneke24 and contained (litreÿ1): (NH4)2SO4, 2.0 g;
FeSO4.7H2O, 1.0 mg; MgSO4, 0.2 g; CaCl2, 1.0 mg;
H3BO4, 1.0 mg; ZnSO4, 70 mg; CuSO4, 50 mg;
(NH4)6Mo7O24.4H2O, 10 mg; Na2HPO4 0.2 g;
KH2PO4, 1.2 g; pH (before sterilisation) 6.2. Mineral
medium was dispensed in 50 ml quantities in 250 ml
Erlenmeyer ¯asks and sterilised at 121 °C for 15 min.
Twenty mesocarp cores (0.6 2.0 cm) obtained aseptically using a sterile cork borer from healthy, freshly
plucked mango (Mangifera indica) at the early stages of
ripening (`colour break') and freshly picked African
mango (Irvingia gabonensis) were suspended in mineral
medium. Triplicate ¯asks of each fruit were inoculated
with test isolate as previously described. Controls were
set up as before. All the inoculated ¯asks were
incubated in an orbital shaker (Gallenkamp) at 28 °C
and 150 rpm for 7 days. Time of complete disintegration of the fruit tissues was recorded. Flasks in which
there was incomplete disintegration of tissues after 7
days were mechanically shaken with sterile glass beads
to achieve complete disintegration. Crude enzyme was
obtained from culture broth by centrifuging at
5000 g and 4 °C for 15 min. Crude enzyme was
prepared for assay as described above.
Production of cellulolytic enzyme
The ability of isolates to produce cellulolytic activity
was determined in the medium of Bravery26 containing in distilled water (g lÿ1): carboxymethyl cellulose sodium salt (BDH), 10; KH2PO4, 1.0; KCl, 0.5;
MgSO4.7H2O, 0.2; CaCl2, 0.1; (NH4)2SO4, 0.54;
thiamine.HCl, 0.001; pH (before sterilisation) 6.2.
Medium (50 ml) sterilised in 250 ml Erlenmeyer ¯asks
were inoculated in duplicate with test isolate and
incubated as described for pectin medium. Cellular
growth was determined as described before.
Assay of pectinase activities by cup plate assay
(CPA) method
Hydrolase, pectinesterase and lyase activities were
assayed by a modi®cation of the cup plate method of
J Sci Food Agric 79:1054±1059 (1999)
Dingle et al 27 as described by Obi and Moneke.24
Details of the experimental protocols have been
recently described.28 Distinction between activities
was based on the nature of the zones of clearing.
Hydrolase activity was detected as a transparent
clearing around the wells, pectinesterase as a cloudy
precipitation around the wells and lyase activity as ring
halo of precipitate around well. Four determinations
were made on at least two plates and the means
determined. The assay was repeated twice. Activity
measured in fruit tissue medium blanks were not
statistically signi®cant in comparison with test activity
and so were considered as baseline (zero) control and
subtracted from test activity.
Assay of
methods
pectinase
activities
by
viscometric
Pectinolytic activity resulting in decrease in viscosity of
pectin solution at 40 °C was determined for hydrolase
and lyase activities using an Ostwald-type viscometer
with distilled water ¯ow time of 5 s as described
previously.28 Enzyme activity was calculated as percentage decrease in viscosity of pectin solution using
the formula of Capellini29:
T0 ÿ Tx
100
T0 ÿ Tw
where T0, Tx and Tw are the ¯ow times for substrate
control, test and water, respectively. The assays were
carried out in triplicate and repeated twice on different
days. As in CPA, activity measured in fruit tissue
medium blanks were not statistically signi®cant in
comparison with test activity and so were considered
as baseline control and subtracted from test activity.
Assay for cellulolytic enzyme
Crude enzyme extract was examined for carboxymethyl cellulase activity by the dinitrosalicylic acid
method30 as previously reported.28 One unit of
CMCase was de®ned as the enzyme that liberates
1 mM reducing sugar as glucose minÿ1 from the assay
mixture under the de®ned assay conditions. The
enzyme assay was set up in triplicate and repeated
twice.
Determination of tissue maceration ability of isolates
Tissue macerating ability of isolates was determined
on healthy freshly plucked mango fruits (M indica) at
early stages of ripening and freshly picked African
mango (I gabonensis) by the cork borer method of
Ogundana et al.31 For each isolate, agar plugs of
mycelial growth from 7-day-old PDA plate culture
were inoculated into wells (0.6 2.0 cm) made in the
fruit mesocarp. Eight replicates were set up for each
fruit type and each isolate. Wells made in control fruits
were inoculated with sterile PDA plugs. All treated
and control fruits were stored insect-proof at room
temperature (28 ° 2 °C) in an airy room for up to 10
days. After 7 days, a transverse section was cut through
1055
JO Ugwuanyi, JAN Obeta
Table 1. Cup plate assay of pectinolytic activities produced in pectin and fruit tissue media as diameter of clear zone (cm)
Pectin medium activity
Isolate
N ®scheri
N ®scheri var spinosa
N quadricincta
P varioti
B nivea
Mango tissue medium activity
Af
Growth
(g 100 mlÿ1)
Hydrolase
Lyase
Pectinesterase
Time (days)
Hydrolase
Lyase
Pectinesterase
0.40
0.35
0.33
0.29
0.30
0.32
0.27
0.25
0.45
0.44
0.85
0.82
0.42
1.05
0.94
0.90
0.66
0.79
0.94
0.26
5±6
5
5±6
4±5
5
0.98
1.02
0.84
1.08
0.90
0.57
0.41
0.63
0.44
0.44
1.24
0.91
0.82
0.69
1.0
a
African mango tissue medium activity
Isolate
N ®scheri
N ®scheri var spinosa
N quadricincta
P varioti
B nivea
a
Time a (days) Hydrolase Lyase
4±5
5
5±6
5±6
5
1.20
0.95
0.60
1.15
0.75
Pectinesterase
0.69
0.53
0.46
0.68
0.52
1.15
1.14
0.82
1.19
1.15
Time (days) taken for fruit cores to disintegrate in culture ¯asks.
each of at least 4 test and 3 control fruits and examined
for the nature of tissue damage in colour, odour and
texture or strength. An isolate was considered disintegrative of fruit tissue if it caused damage of at least
1.0 cm diameter greater than the damage recorded in
control fruit and was recovered in pure culture from
advancing zone of disintegrating fruit tissue.
Analysis of results
Results of enzyme assays were subjected to analysis of
variance (one-way classi®cation) using the InstatR
statistical package. Differences between treatment
means were tested using the Tukey±Kramer multiple
comparison test.
RESULTS
Assay of enzymes
The results of cup plate assay (CPA) of pectinolytic
activities produced by the HRF isolates are shown in
Table 1 for activity produced in pectin medium and
fruit tissue media. The isolates produced varying levels
of hydrolytic, lyase and pectinesterase activities in both
media. P varioti produced the greatest mean pectinase
activities in pectin medium while producing the least
biomass. Statistical analyses of hydrolytic activities
obtained in pectin medium indicate that there was no
difference in activities between the Neosartorya spp and
between P varioti and B nivea. P varioti and B nivea,
however, had signi®cantly higher activities than the
Neosartorya spp (P < 0.01). N quadricincta produced
signi®cantly less (P < 0.01) lyase activity than other
isolates whose activities were similar. Pectinesterase
activities varied in a slightly different way with B nivea
producing signi®cantly lower activity than the other
isolates whose activities did not differ. Cup plate assay
of hydrolytic activities produced in mango tissue
1056
medium (MTM) indicated comparable activities for
all ®ve isolates (P > 0.05). N quadricincta produced
greater lyase activity in MTM than the other isolates
(P < 0.05) whose activities were similar. N ®scheri
produced the greatest pectinesterase activity of all the
isolates in MTM (P < 0.05) while P varioti produced
the lowest activity. N ®scheri produced the greatest
hydrolytic activity in African mango tissue medium
(AMTM), which was comparable to P varioti and N
®scheri var spinosa activities but greater than the
activities of other isolates. Comparable lyase activities
were produced by N ®scheri and P varioti and these
were signi®cantly greater (P < 0.05) than those of the
other isolates, which were comparable. Analysis of
pectinesterase activity produced in AMTM by cup
plate assay indicated that high and comparable
activities were produced by all the isolates except for
N quadricincta which produced signi®cantly lower
activity (P < 0.01).
Viscometric analysis of hydrolytic activities produced in pectin medium showed that P varioti and B
nivea produced signi®cantly greater activities than N
®scheri (Table 2). No activity could be detected by this
method in either N ®scheri var spinosa or N quadricincta. B nivea and P varioti produced signi®cantly
(P < 0.01) greater lyase activities than the Neosartorya
spp whose activities were similar. P varioti produced
the greatest hydrolase (P < 0.01) activity in MTM
when assayed by this method while N quadricincta
activity was the lowest. B nivea, P varioti and N ®scheri
had comparable lyase activities in this medium while N
®scheri var spinosa activity was signi®cantly less
(P < 0.05) than the others. Analysis of activities
produced in AMTM showed that there was no
signi®cant difference in hydrolytic activities produced
by N ®scheri, B nivea and P varioti and these were
greater than activities produced by N quadricincta and
J Sci Food Agric 79:1054±1059 (1999)
Time a (days)
4±5
5
5±6
5±6
5
Cellulolytic and pectinolytic activities of heat resistant fungi
Pectin medium
Isolate
a
Lyase
Hydrolase
Lyase
Hydrolase
Lyase
18
ND a
ND
27
30
30
34
30
63
44
72
76
51
82
71
64
57
63
69
72
78
70
42
84
85
62
40
30
61
94
ND, not detectable.
Table 3. Cellulolytic activity produced by isolates in carboxymethyl cellulose
medium
Isolate
Cellular growth
(g 100 mlÿ1)
CMCase activity
(U mlÿ1)
0.57
0.52
0.60
NG a
0.45
160
120
120
0.0
180
N ®scheri
N ®cheri var spinosa
N quadricincta
P varioti
B nivea
a
African mango medium
Hydrolase
N ®scheri
N ®cheri var spinosa
N quadricincta
P varioti
B nivea
Table 2. Viscometric assay of
pectinolytic activities produced in
pectin medium and fruit tissue media
Mango medium
NG, no growth.
N ®scheri var spinosa (P < 0.05). B nivea produced
greater lyase activity in this medium than other isolates
(P < 0.01).
All ®ve isolates produced signi®cantly (P < 0.01)
more hydrolase activity in fruit tissue media than in
pectin medium in both assay procedures. Activities
produced in both fruit media were comparable in N
®scheri, N ®scheri var spinosa and P varioti. N
quadricincta produced greater activity in MTM than
in AMTM (P < 0.05) and so did B nivea (P < 0.01)
(CPA). In the case of lyase enzyme, greater activities
were recorded in pectin medium than in fruit tissue
media for all isolates except N quadricincta which
produced comparable activities in all three media
(CPA). In the viscometric assay, greater lyase activity
was produced in fruit tissue media by N ®scheri and B
nivea. N quadricincta also produced comparable
pectinesterase activities in all three media. P varioti
produced lowest pectinesterase activity in MTM
(P < 0.05) while the other isolates produced slightly
greater activities in the fruit tissue media.
Four of the ®ve isolates, the Neosartorya spp as well
as B nivea grew in CMC medium and produced
considerable carboxy methyl cellulase activities (Table
3). B nivea produced the highest activity (180 Umlÿ1)
while the lowest activity was produced by N quadricincta (120 Umlÿ1) and N ®scheri var spinosa. P varioti
did not utilise CMC as sole carbon source and did not
grow in CMC medium.
During cultivation in fruit tissue media, all ®ve
isolates caused complete (by visual observation)
disintegration of fruit cores in 6 days or less (Table
1). All the fruit cores used in the control, however,
remained at the end of 7 days shaken cultivation.
Fruit tissue maceration
The result of the tissue maceration experiment is
shown in Table 4. N ®scheri and B nivea produced the
most extensive mean tissue damage in mango tissue. A
similar pattern of spoilage occurred in inoculated
African mango. The nature of the smell, however,
appeared to depend more on the type of fruit than on
the spoilage fungus and was only considered as a guide
to actual spoilage.
Table 4. Maceration of fruit tissues caused by isolates
Mango fruit
Isolate
Spoilage
diameter a (cm)
N ®scheri
3.35
N ®cheri var
spinosa
N quadricincta
2.80
3.10
P varioti
2.60
B nivea
3.20
a
Nature of spoilage
slight browning; extensive softening;
mouldy smell
extensive maceration of fruit tissue;
mouldy smell
luxuriant growth; moderate softening/
browning; strong mouldy odour
moderate softening of tissue; light
penetrating smell
luxuriant growth; extensive softening
of tissue, strong penetrating odour
African mango fruit
Spoilage
diameter a (cm)
2.40
1.80
1.65
1.15
2.00
Nature of spoilage
slight tissue softening; strong rotten
smell
slight browning; extensive maceration;
faint rotten smell
strong browning; moderate softening of
tissue; faint rotten smell
strong browning; moderate softening;
slightly mouldy odour
luxuriant growth; slight browning;
extensive softening of tissue; strong
mouldy smell
Diameter of spoilt tissue (cm) caused after incubation of fruits for 7 days.
J Sci Food Agric 79:1054±1059 (1999)
1057
JO Ugwuanyi, JAN Obeta
DISCUSSION
Cell separation due to pectinase activities has been
previously reported.32 Pectinolytic activity is widely
distributed among fungi particularly within the genus
Aspergillus33 where it is widely associated with fungal
invasiveness.34,35 In this work, pectinase activity is
reported in heat resistant Neosartorya spp for the ®rst
time. This capacity follows the trend in members of
the genus Aspergillus which are well documented to be
extensive producers of pectinase activities.36 Among
members of the genus Byssochlamys, pectinase activities have been widely reported and believed to be
responsible, at least in part, for the ability of these
organisms to cause maceration and spoilage of
processed fruit tissues.1,2 In this study Byssochlamys
nivea and Paecilomyses varioti, both members of the
Paecilomyces group, are also shown to be capable of
producing various pectinase activities.
All the isolates caused disintegration of arti®cially
inoculated mango and African mango fruit tissues.
The level of maceration caused by these organisms,
however, did not seem to be dependent on the
measurable overall activity of pectinolytic enzymes.
This supports the position of Chesson32 that all groups
of pectic enzymes are not able by themselves to cause
maceration. This ability is restricted to those endoacting pectinases that are able to cause extensive
solubilisation of pectic materials by cleavage of
relatively few linkages.37,38 It is dif®cult, however,
based on data obtained from this experiment (softening of inoculated fruits, Table 4, and disintegration
time of fruit cores in fruit tissue media, Table 1) to
suggest outstanding endo-activity for enzymes produced by any particular isolate in the media used. N
quadricincta, which produced lowest hydrolase activity
(undetectable in pectin medium by viscometry), was
able to cause considerable maceration of fruit tissues
and solubilised fruit cores in about the same time as
more hydrolytically active isolates. This organism,
however, produced considerable lyase activity which,
alone, has been shown to be capable of tissue
maceration in potatoes.39 On the other hand, P varioti,
which produced very high hydrolytic activity, failed to
produce corresponding maceration of fruit tissues.
Although comparable activities were produced in
media made with these fruits, greater tissue destruction was recorded in mango than in African mango
fruits, suggesting that these fruits differ in their
susceptibility to spoilage by these organisms. There
is probably a complex type of interaction between
intrinsic parameters of fruits and fungal enzymes, such
as cell wall structure and amenability of enzymes to
catabolite repression, in the maceration of infected
plant materials. Such complex interaction has been
alluded to in the pectinase activities of Aspergillus
spp.35 Ampe and Brauman40 reported interaction
between bacterial and endogenous pectinases in the
retting of cassava tubers. Tani41 also suggested
interaction between endogenous and microbial pectinases during maceration of Diospyros kaki following
1058
infection with Gleosporium kaki. Such interactions
could have occurred in the disintegration of these test
fruits even though no signi®cant fruit activities were
detected in the fruit media blanks.
Several workers have reported cellulase activities in
rot-causing organisms and have sought to associate
tissue softening ability with cellulolytic activities.24,28,32 A possible role for CMCase activity in
fruit tissue maceration in this work is still open for
debate by the ability of P varioti, which did not
produce cellulase activity, to cause considerable tissue
damage. Whether this lack of cellulase activity led to
comparative weakness of P varioti in tissue maceration
when compared to other isolates with less hydrolytic
activity but greater tissue maceration, however, is not
clear. El-Refai et al 42 reported that polygalacturonase
was responsible for the macerating effect of a
commercial pectinase and that neither cellulase nor
hemicellulase was required. Chesson32 suggested that
the role of cellulases may be limited to degradation of
wall polymers to provide invading organisms with
readily available sugars as a carbon source. Obviously,
this is unlikely to be a problem for fruit macerating
organisms since fruits often have abundant assimilable
sugars. Ampe and Brauman40 could not demonstrate
cellulase and xylanase activities during bacterial retting
of cassava tubers. It can therefore be concluded that,
while cellulases may aid or enhance tissue maceration
in some instances, it is not absolutely necessary for the
organism to be able to cause (fruit) tissue destruction.
The inability to detect hydrolytic activity in N
quadricincta and N ®scheri var spinosa pectin medium
broth by viscometry points to the dif®culty associated
with measurement of pectinase activity by this
method. This dif®culty may be understandable from
the following: viscometric assay is not sensitive for
measuring exo-acting enzymes; with endo-acting enzymes, for which this method is sensitive and dependable, it may become subjective when activity is very
low. Variations exist between different microbial
species activities which may make comparison dif®cult. Maiorano et al 43 reported that viscometric
activity in microbial pectinases changes with the
secreting species and thus cannot be depended upon
for an accurate comparison of pectinase activities.
Wherever possible, activities should be measured by
the more precise spectrophotometric methods.
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