food and bioproducts processing 9 4 ( 2 0 1 5 ) 727735

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Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Biodegradation of coffee pulp tannin by Penicillium

verrucosum for production of tannase, statistical
optimization and its application
Roopali N. Bhoite, Pushpa S. Murthy
Plantation Products Spices & Flavour Technology Dept, CSIR-Central Food Technological Research Institute, India

a b s t r a c t
Coffee pulp is a primary by-product produced during coffee processing and represents 30% of the coffee fruit on a
dry-weight basis. A novel potential tannin degrading fungi was isolated from coffee by-products. Among the various
fungi isolated, Penicillium sp. CFR303 was found to be potent with 66.5 0.9% tannin degradation. The potent tannin
degrader was identied as Penicillium verrucosum using internal transcribed spacers (ITS) 5.8S rDNA analysis. Solid
state fermentation was carried out on coffee pulp as a sole carbon source and yielded 28.173 1.4 U/gds of tannase.
Further, 3.93 fold increase in tannase production (115.995 U/gds) was achieved using central composite rotatable
design, a statistical approach. Model validations showed excellent agreement between the experimental results
and the predicted responses with a condence level of 95%. Coffee pulp accounts to 810% tannin content and the
present study demonstrates coffee pulp as an excellent substrate for production of value added products. Aonla
and pomegranate juice were treated with partially puried tannase and the degradation of tannins was evident by
changes in the physicochemical parameters of the juice. Thus, the present investigation signies utilization of coffee
pulp for production of tannase as value addition and its potential application in the food industry.
2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Penicillium verrucosum; Internal transcribed spacer (ITS); Solid-state fermentation (SSF); Response surface
methodology (RSM); Tannase; Coffee pulp

1.

Introduction

Coffee is one of the international agricultural products and is

the second largest commodity traded in the world next to oil.
Annually, large amount of coffee by-products are generated
during coffee processing. The disposal of these by-products is
an environmental concern (Bhat et al., 1998). With the advent
of biotechnology, attempts have been made globally to make
potential use of agro-industrial residues for value addition
by production of enzymes, organic acids, bioactive secondary
metabolites and single cell protein etc. (Chatterjee et al., 1996).
Tannins are toxic to fungi, bacteria and viruses. However, many microorganisms have developed the mechanisms
to overcome the effects of tannins. These mechanisms
include tannin modication, degradation and dissociation of

tannin-substrate complexes, tannin inactivation by high

afnity binders, membrane modication and metal ion
sequestration (Belur et al., 2010). Tannase is implicated for
biodegradation of tannins and is an ecologically important
biocatalyst. Tannase (tannin acyl hydrolase EC 3.1.1.20) is an
inducible enzyme which catalyzes the hydrolysis of ester and
depside bonds in hydrolysable tannins such as tannic acid that
releases glucose and gallic acid (Lekha and Lonsane, 1997).
Also, tannase is used as a clarifying agent in some wines, fruit
juices and in refreshing drinks with a coffee avor (Rout and
Banerjee, 2006).
Solid state fermentation (SSF) involves the use of agro byproducts, which are inexpensive, easily available, afford high
volumetric yield and are reproducible (Krishna, 2005). Among
microbes, some of the fungi as enzyme producers have many

Corresponding author. Tel.: +91 821 2512352; fax: +91 821 2517233.
E-mail addresses: pushpa@cftri.res.in, pushpamurthys@yahoo.com (P.S. Murthy).
Received 30 May 2013; Received in revised form 22 July 2014; Accepted 13 October 2014
Available online 22 October 2014
http://dx.doi.org/10.1016/j.fbp.2014.10.007
0960-3085/ 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

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food and bioproducts processing 9 4 ( 2 0 1 5 ) 727735

advantages as they are normally GRAS (generally regarded as

safe) strains and produce extracellular enzymes which makes
its easy recuperation from fermented broth. Filamentous fungi
of Aspergillus and Penicillium genus have been widely used for
tannase production and have better thermal and pH stability
(Lekha and Lonsane, 1994).
The optimization of the fermentation process by conventional one-factor-at-a-time leads to confusion in understanding of process parameters. Also, this method neglects the
interaction among variables and thus is unable to reach the
true optimum. Therefore, statistical method of optimization
such central composite rotatable design (CCRD) based on
response surface methodology (RSM) improve product yield,
reduce time and overall process costs (Wee et al., 2009). In this
investigation, we aim to carry out SSF using coffee pulp for
production of tannase and optimize the process parameters
by CCRD. Further, the partially puried tannase was treated
as clariers on aonla, pomegranate juice.

2.

Materials and methods

2.1.

Media and analytical chemicals

All chemicals used in the study were of analytical grade and

were procured from Hi-Media, India, Sigma-Aldrich, USA.

2.2.

Substrate and preparation of inoculum

Coffee pulp was obtained from coffee plantation, Coorg, India.

Organic components of coffee pulp was proximally analyzed
under standard experimental conditions for determination
of tannins, total pectic substances, reducing sugars, nonreducing sugars, caffeine, chlorogenic acid and total caffeic
acid. Prior to storage, coffee pulp was washed with water and
sun dried. The fungal strains were isolated from coffee byproducts and were grown on potato dextrose agar (PDA) slants
for 6 days at 28 C in an incubator (Technico Ltd., India) and
stored at 4 C. Spore suspension was prepared using sterile
water solution containing 0.4% (v/v) tween 80 and the viable
spores were enumerated using the plate count technique and
volume of 1 mL containing 106 spores was used as inoculum.

2.3.

Screening of fungal organisms

Screening of isolates for tannase production was carried out

on tannic acid agar plate (Couri and Farias, 1995), containing
(w/v): tannic acid 1%, NaNO3 0.3%, KH2 PO4 0.1%, MgSO4 7H2 O
0.05%, KCl 0.05%, FeSO4 7H2 O 0.001%. Point inoculation was
carried on agar plates with pure cultures and incubated at
30 C for 96 h. The growth diameter of the colony formed was
measured to screen the best potential tannin degrader. Further, secondary screening was done by inoculating 0.5 mL of
fungal spore directly on coffee pulp (5 g) and incubated at
30 C for 96 h. The extracellular enzyme was recovered using
citrate buffer (50 mM, pH 5.0) and the percentage degradation of tannic acid (20 mM) by recovered tannase enzyme was
determined by the method of Mondal et al. (2001).

2.4.
Fungal DNA isolation and molecular identication
of fungi
The fungal strain was grown on potato dextrose broth for
7 days and the genomic DNA was extracted using fungal
DNA isolation kit procured from Chromous Biotech Pvt Ltd.,

Bangalore, India. The isolated DNA was evaluated using

agarose (1%) gel electrophoresis and quantied spectrophotometrically. The fungal DNA amplication was carried using the
Primers ITS5 (5 -GGAAGTAAAAGTCGTAACAAGG-3 ) and ITS4
(5 -TCCTCCGCTTATTGATATGC-3 ). The PCR reaction was performed in a 25 L nal volume containing 1 L (0.510 ng) of
total DNA, 2.5 L of 10X PCR buffer (without Mg2+ ), 0.5 mol l1
of each primer, 200 mol l1 of each dNTP, 2.5 mmol l1 of
MgCl2 and 1.5 U of Taq DNA polymerase. The reaction conditions were as follows: initial denaturation at 94 C for 4 min
followed by 34 cycles of denaturation at 94 C for 30 s, annealing at 58 C for 45 s and primer extension at 72 C for 1 min;
followed by a nal extension at 72 C for 7 min (White et al.,
1990). Further, the PCR product was puried with a PCR purication kit (Sigma-Gen Elute PCR clean-up Kit) and Internal
Transcribed Spacerss (ITS)-5.8s rDNA was sequenced by Chromous Biotech Pvt Ltd, Bangalore, India. In order to analyze
the Penicillium sp. CFR303s DNA sequence, related sequence
were obtained from GenBank using BLASTN program and were
aligned using MEGA 5 software. Distances were calculated and
phylogenetic tree was constructed using the neighbor-joining
method. The robustness of tree topology was tested using
bootstrap analysis with 1000 replicates (Tamura et al., 2011).

2.5.

Solid state fermentation and tannase activity

Coffee pulp was pre-treated with steam explosion at 121 C

for 15 min before using as a substrate in culture medium.
The pH and moisture modulation was done using salt solution (NH4 NO3 0.5%, NaCl 0.1%, MgSO4 7H2 O 0.1% at pH 5.5)
(Paranthaman et al., 2009a,b). The contents were sterilized at
121 C for 20 min. The cooled sterilized solid substrate (10 g) in
100 mL conical ask was inoculated with 1 mL of the Penicillium
sp. CFR303 spore inoculum, mixed uniformly and incubated at
30 C for 96 h. Tannase was extracted by suspending fermented
substrate in 5 times citrate buffer (50 mM, pH 5.0) using orbital
shaker at 30 C, 150 rpm for 60 min. The extracts were pooled
by ltration through whatman No. 1 lter paper, centrifuged
at 5000 g at room temperature for 10 min and supernatant
obtained was used as the crude tannase. The tannin content
in the coffee pulp was determined before and after fermentation according to Leifa et al. (2001). The tannase activity
was carried out as per Sharma et al. (2000) using gallic acid
as standard. The reaction mixture containing 0.5 mL of 10 mM
tannic acid in citrate buffer (0.05 M, pH 5.0) and 0.5 mL of extracellular enzyme was incubated at 30 C for 10 min followed
by the addition of 0.3 mL of methanolic rhodanine (0.667%
w/v). After 5 min of incubation, the mixture was treated with
0.2 mL of 0.5 M KOH. The pink color developed was read at
520 nm using a spectrophotometer (Shimadzu UV-1800). One
unit of tannase activity was dened as the amount of enzyme
required to liberate one micromole of gallic acid per minute
under dened reaction conditions and expressed as units/g
coffee pulp {A520 = (Atest Ablank ) (Acontrol Ablank )}.

2.6.
Optimization of bioprocess parameters and
analysis of experimental data
The CCRD, a method of RSM, was used to maximize tannase production. Generally, the independent parameters that
predominantly affect the tannase production in SSF are pH
(A), moisture (B; %) and fermentation period (C; h) (Aguilar
et al., 2001). The values of the variables used were A (46), B
(4060%), and C (72120 h) each at ve coded levels (2, 1,

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food and bioproducts processing 9 4 ( 2 0 1 5 ) 727735

0, +1, +2). CCRD with 3 factors in one block encompassing 20

runs were employed for the study. Experiments were randomized in order to maximize the effects of unexplained variability
in the observed responses due to extraneous factors. For the
analysis, a statistical software package (Design Expert 8.0.6.1,
Stat-Ease, Inc., Minneapolis, USA) was used.
The response (tannase activity) obtained was subjected to
analysis of variance (ANOVA) and R2 to evaluate lack of t
(LOF). A second-order polynomial equation was tted to the
data using a multiple regression procedure. All tests were performed in duplicates, and the represented data are a mean of
the two. The model was tested for validity and adequacy by
randomizing the value of variables within the dened range
and also at optimal value of variables determined by the
model.

2.7.
Studies on growth characteristics and tannase
production
The fungal population was determined using standard microbiological method. The growth count were enumerated at a
regular interval of 24 h for 6 days by spread plate method
and expressed in terms of colony forming units per mL. The
Protein and sugar constituents of the fermented broth were
estimated by Bradfords method (Bradford, 1976) and Miller
method (Miller, 1959) respectively. Experiments were conducted in duplicates and the results described are mean of
two replicates.

2.8.
juices

3.

Results and discussion

3.1.

Solid substrate

The analysis of coffee pulp on dry weight basis resulted to

contain 810% tannins, 6.5% pectic substances, 12.5% of fermentable sugars, 1.3% caffeine, 2.6% chlorogenic acid and
1.6% caffeic acid. Coffee pulp could be appropriate substrates
for production of microbial enzymes (Pushpa Murthy and
Madhava Naidu, 2010). Generally, agro industrial residues
possess unique buffering action and have advantages for
enzyme production. In a SSF process, the solid substrate not
only supplies the nutrients to the microbial culture growing in
it but also serves as an anchorage for the cells. The selection
of a substrate for enzyme production in a SSF process depends
upon several factors, mainly related with cost and availability
of the substrate. India being a fth largest coffee producing
country has a high potential of utilizing these by-products for
enhanced economic returns.

Application of tannase for clarication of fruit

The crude tannase was treated with activated charcoal (9%)

and allowed to stand with intermittent shaking for 60 min,
centrifuged at 5000 g for 10 min at room temperature and the
supernatant obtained was further puried by 80% ammonium
sulphate precipitation and utilized for food applications. Fresh
pomegranate (Punica granatum) and aonla (Phyllanthus emblica)
were purchased from a local market in Mysore. The fruits were
washed, peeled and juice was extracted using distilled water
(1:10) using the house hold blender. The resulting pulp was
squeezed using cheese cloth. 200 mL of fruit juice was treated
with 10 mL partially puried tannase (303.86 U/mg) and incubated at 37 C for 2 h. The untreated juice was used as control
and 10 mL water was used instead of tannase. The enzyme
was inactivated by heating the suspension at 90 C for 5 min
in a water bath, ltered and centrifuged at 3000 g for 10 min
and nally, the supernatant was used to determine physicochemical parameters of treated and untreated juice.

2.9.

measured using digital pH meter (Ranganna, 1978). The brix

value was recorded at room temperature (2022 C) using a
portable refractometer (Lecia Abbe Mark II, Model 10480, Buffalo, NY, USA). The ratio of brix value to percentage acid
which is a sugar acid ratio was also determined. The tannin
content and total phenolics in the fruit juice were measured
by Hagerman and Butler (1978) and Singleton et al. (1999)
respectively. The ascorbic acid content was determined using
2,6-dichlorophenolindophenol (2,6-DCIP) (Ranganna, 1978).

3.2.
Isolation, screening and identication of potential
tannase producer
Selection of potent strain for the fermentation process is a crucial factor and is one of the major factors that affect microbial
synthesis of enzymes in a SSF system. Direct measurement
of the colony diameter through tannic acid agar plate method
was a good indicator of tannic acid utilization as the diameter
of growth zone could be linearly correlated with efciency of
fungal organism to secrete tannase. Compared to the reported
literature on degradation of tannins, our organism potentially degrades tannins with the largest growth zone of 37 mm
(Paranthaman et al., 2009a,b). Furthermore, SSF was carried
out with coffee pulp as substrate to screen positive fungal
organism for production of tannase. Penicillium sp. CFR303 was
evident to be potential tannase producer with 66.5 0.9% tannin degradation (Table 1).
The identication of potent tannase producing fungus
was carried out based on morphological and molecular

Physicochemical parameters of the juice

Impact of enzymatic treatment on aonla and pomegranate

juice was determined by measuring and comparing physicochemical parameters of treated and untreated juice. The
color of the juice was measured using Hunter Lab Colorimeter Ultra scan, model SN 7877 Hunter Associates Lab. Inc.
Virginia (Busch-Kschiewan et al., 2004). The juice clarity was
determined by measuring the absorbance at 660 nm using a
UVvis spectrophotometer (Model UV-1800, Shimadzu corporation, Japan) (Sin et al., 2006). The titratable acidity of the
claried juice was measured by titrating the juice against 0.1 N
NaOH using phenolphthalein as an indicator and expressed
as % titratable acidity. The pH values of juice samples were

Table 1 Screening of tannase producing fungi from

coffee by-products.
Organism
Penicillium sp. CFR303
Penicillium sp. CFR304
Aspergillus sp. CFR305
Aspergillus sp. CFR306
Neurospora sp. CFR307
Rhizopus sp. CFR308
Pleurotus orida CFR309

Growth
diameter (mm)
37
31
33
31
27
33
26

4.0
3.0
5.0
7.0
3.0
7.0
6.0

Percentage
degradation (%)
66.5
52.5
47.0
56.1
33.2
58.4
38.9

0.9
0.5
0.2
0.5
0.7
1.3
0.4

Data shown is mean and standard deviation of triplicates.

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food and bioproducts processing 9 4 ( 2 0 1 5 ) 727735

Penicillium simplicissium strain KUC5153(HM469430.1)

88
86

Penicillium oxalicum strain KUC1674(HM469410.1)

Penicillium steckii strain KUC1681-1(HM469415.1)

80

Penicillium verrucosum(AF003357.1)

Penicillium KM213866 (ISOLATE)

96

Eupenicillium javanicum isolate AFTOL-ID 429(EF413621.1)

84

Penicillium cyclopium(AF003356.1)
84

Penicillium aurantioogriseum(AF003355.1)
Eupenicillium limosum AFTOL-ID 2014(EF411064 1)

Fig. 1 Phylogenetic tree based on the ITS-5.8S rDNA sequence of the Penicillium sp. CFR303 and related strains.
characterization using 5.8S rDNA internal transcribed spacer
(ITS). The color of the hyphae and mycelia were greenish
along with septate hyphae. Morphological characters revealed
that organisms belonged to Penicillium spp. The identication
by molecular characterization targeting ITS regions of fungal
specicity grouped Penicillium sp.CFR303 with Penicillium verrucosum (96 supports in ML values) and hence the isolated fungus
was identied as P. verrucosum (Fig. 1). The gene sequences
(878 bp) are deposited in the NCBI, GenBank database, under
the accession number KM213866 for the P. verrucosum.

3.3.

Steam pre-treatment of coffee pulp

Steam explosion is an attractive pre-treatment process

due to its low use of energy and chemicals. Tannase
production by steam pre-treated coffee pulp was fairly high
(28.173 U/gds) compared to tannase production without steam
pre-treatment (18.548 U/gds). This could be due to an increase
in surface area or pore size of coffee pulp which breaks up
polyphenolic complex resulting in easy accessibility for microbial attack to tannins present in coffee pulp. Steam treatment
was found to be effective on coffee substrates (Pushpa Murthy
and Madhava Naidu, 2010) and therefore was considered

during the optimization process. Generally, steam pretreatment of solid wastes has been found useful to improve its
digestibility and increase the efciency of hydrolysis through
reduction of the lignin content and cellulose crystallinity
(Kristensen et al., 2008).

3.4.
Optimization of process parameters for tannase
production by P. verrucosum
CCRD was used to develop a correlation between pH, moisture
and fermentation duration to improve the tannase production
by P. verrucosum. By multiple regression analysis, the polynomial equation with the coefcient of the full regression model
equation and their statistical signicance was determined.
ANOVA is a statistical technique that subdivides the total
variation of a set of data into component associated to specic sources of variation and is important in determining
the adequacy and signicance of the quadratic model. The
analysis of variance (ANOVA) for the CCRD is presented in
Table 3. To test the good t of the model, regression coefcient R2 was calculated. The model represented a high R2 value
(R2 = 0.9787) indicating about 97.87% variability in responses.
The rest (2.13%) of the total variation was not explained by

Table 2 Experimental design indicating coded values of variables including experimental and predicted response for
tannase productiona .
Run #

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15(C)
16(C)
17(C)
18(C)
19(C)
20(C)
a

A: pH

4 ()
4 ()
4 ()
4 ()
6 (+)
6 (+)
6 (+)
6 (+)
3.31 ()
6.68(++)
5 (0)
5 (0)
5 (0)
5 (0)
5 (0)
5 (0)
5 (0)
5 (0)
5 (0)
5 (0)

B: Moisture (%)

40 ()
40 ()
60 (+)
60 (+)
40 ()
40 ()
60 (+)
60 (+)
50 (0)
50 (0)
33.18 ()
66.81 (++)
50 (0)
50 (0)
50 (0)
50 (0)
50 (0)
50 (0)
50 (0)
50 (0)

Experimental tannase activity was average of duplicates.

C: Fermentation period (h)

72 ()
120 (+)
72 ()
120 (+)
72 ()
120 (+)
72 ()
120 (+)
96 (0)
96 (0)
96 (0)
96 (0)
55.63 ()
136.36 (++)
96 (0)
96 (0)
96 (0)
96 (0)
96 (0)
96 (0)

Tannase activity, Y (U/gds)

Yexp

Ypred

19.665
58.331
54.331
93.662
49.331
58.331
63.664
68.997
83.33
74.663
40.998
81.663
22.332
54.331
105.329
115.995
114.995
109.662
106.329
112.995

20.945
59.014
56.873
93.441
48.075
54.312
61.504
66.24
80.063
80.004
42.254
82.495
21.379
57.373
110.824
110.824
110.824
110.824
110.824
110.824

food and bioproducts processing 9 4 ( 2 0 1 5 ) 727735

731

Fig. 2 Response surface graphs of (A) Tannase activity (U/gds) as a function of moisture (%, w/v) and pH; (B) Tannase
activity (U/gds) as a function of fermentation period (h) and moisture (%, w/v) (C) Tannase activity (U/gds) as a function of
fermentation period (h) and pH at 30 C.
the model. The Predicted R2 of 0.9433 is in reasonable agreement with the adjusted R2 of 0.9596 which indicate that the
model is good. For a good statistical model, the R2 should
be in the range of 01.0 and nearer to 1.0 the value is, the
more t the model is deemed to be. The adequate precision value of the model is 20.173. The lower value of the
coefcient of variation (CV % = 9.41) showed that the experiments were precise and reliable. The Model F-value of 100.81
implies the model is signicant. The P value of the model was
<0.0001 (P < 0.05) and insignicant lack of t suggested good
t. According to the P value, (the value, in case of below 0.05,

Table 3 ANOVA for the quadratic polynomial model for

tannase production.
Source
Model
A - pH
B - Moisture
C - Fermentation time
AB
AC
BC
A2
B2
C2
Lack of t

Mean
square

F value

p-value
Prob > F

1924.15
4.29E03
1954.7
1563.94
253.1
506.64
1.13
1707.06
4228.55
9196
18.06

100.81
2.25E04
102.41
81.94
13.26
26.54
0.059
89.44
221.54
481.79
0.9

<0.0001
0.9883
<0.0001
<0.0001
0.0045
0.0004
0.813
<0.0001
<0.0001
<0.0001
0.5457

R2 : 0.9787; Adj R2 : 0.9596; Pred R2 : 0.9433; Adeq Precision: 20.173;

Std. Dev: 0.15; C.V. %: 9.41.

indicated signicance level) B, C, AB, AC, A2 , B2 , C2

were signicant whereas, pH and interaction term (moisture fermentation period) were found to be insignicant. In
general, fungal tannase is an acidic protein. The crude tannase activity from Penicillium verrucosum was unaffected with
the change in pH and could be due to presence of inhibitors
at active site of an enzyme, which could possibly prevent ionization of amino acids, making tannase activity unaffected by
salt concentration. Minimum moisture content is sufcient to
maintain water activity and subsequent tannase production
till 96 h, after which there is a drastic decline in tannase production irrespective of moisture and other conditions. This
could be the reason for insignicance of interaction term
(moisture vs fermentation period). The nal model for tannase
response after eliminating insignicant terms can be written
as follows
Y = 2.62 + 0.25(B) + 0.21(C) 0.13(AB) 0.16(AC)
0.34(A2 ) 0.47(B2 ) 0.64(C2 )

(1)

where Y is tannase yield and A, B and C were the coded

variables for the pH, moisture (%) and fermentation duration (h), respectively. Based on the above equation, the
coefcients with one factor represented the linear effect, while
the coefcients with two factors and those with secondorder terms represented the interaction effect and quadratic
effect, respectively. The positive sign in front of the terms
indicated synergistic effect, whereas the negative sign indicated antagonistic effect (Joshi et al., 2008). The obtained

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food and bioproducts processing 9 4 ( 2 0 1 5 ) 727735

Fig. 3 Desirability prole showing optimum values of independent process parameters for tannase production (pH 5;
Moisture 50%; Fermentation period 96 h).
equation was used to predict the tannase yield presented in
Table 2.
The response surface plots illustrate the inuence of
the linear and interaction effects on the response variable
(tannase yield). Fig. 2A depicts the interaction of pH and moisture. Tannase activity was found to increase with the increase
in pH and moisture to optimum values of pH 5 and moisture
50%, respectively. The moisture content is a critical factor in
solid-state fermentation. Thus, it is crucial to provide optimized water content, and control the water activity (aw ) of the
fermenting substrate. With the increase in moisture beyond
50%, surface curvature is declining indicating negative effect
on tannase production. Higher moisture levels can cause a
reduction in enzyme yield due to steric hindrance of the
growth of the producer strain due reduction in porosity (inter
particle spaces) of the solid matrix, thus interfering oxygen
transfer (Mrudula and Murugammal, 2011). Fig. 2B reveals the
interaction effects of fermentation period and pH. Tannase
production remained almost unaltered with an increase in pH
after 5 but was found to decrease after 96 h. Extended period
of incubation lead to the decomposition of enzyme because
of interaction with other components in the media (Ramesh
and Lonsane, 1987). Fig. 2C exhibits the effect of interaction
of fermentation period and moisture. Maximum tannase production was observed at 96 h of fermentation period and the
tannase yield decreased after 96 h which could be due to the
accumulation of gallic acid or appearance of toxic metabolites during fermentation. Similar results have been reported
by Rakesh et al. (2007), with maximum production of tannase
at 96 h in case of Aspergillus spp.

3.5.

Numerical optimization and process validation

The optimized levels of variables (A, B and C) were determined using desirability proles (Fig. 3) for tannase activity
by assigning 0 to least desired level of response and 1 to
most desired response. The aim was to achieve maximum
tannase activity using P. verrucosum by SSF. Solutions with
higher desirability gave optimum pH 5, 50% moisture and 96 h
of fermentation at which highest tannase activity could be
achieved. The optimum values of process variables obtained in
the present investigation was in agreement with the previous
reports of Lekha and Lonsane (1997). Under optimized conditions, tannase activity of 115.995 U/gds was obtained. The
yield obtained in the present study is higher as compared to
the other reported maximum tannase yield under SSF with

fungal organisms such as Aspergillus ruber, 69 U/gds (Rakesh

et al., 2007); Aspergillus acuelatus, 2.93 U/gds (Banerjee et al.,
2007); Aspergillus niger, 67.5 U/gds (Pinto et al., 2001).
Model validation is used to determine the compatibility
of accurate representation of the model to the real system.
Verication of randomized variables and optimized process
variables, predicted by the model was carried out in shake
asks in triplicates. Under optimal conditions predicted by the
model such as pH 5, 50% moisture and 96 h of fermentation,
experimental value (107.92 1.4 U/gds) was found to be close
to the predicted value (110.824 U/gds) and hence, the model
was successfully validated. Similar results with the model prediction indicated that our model was accurate and reliable for
predicting the production of tannase by SSF from Penicillium
verrucosum.

3.6.

Time course analysis of tannase production

The kinetics of tannase production using P. verrucosum was

studied at 30 C, initial pH of 5.0. The effect of tannase activity
with growth, glucose and protein concentration is presented
in Fig. 4. Samples (whole asks) were withdrawn at regular intervals of 24 h for 6 days and analyzed. The tannase
production was found to increase gradually from 46 h of the
fermentation period when the growth of the microorganism
reaches the early exponential phase which reveals that tannase production is growth associated. The maximum tannase
activity was found in the mid-exponential phase of P. verrucosum. Maximum tannase production was found at 96 h of
incubation, beyond which it started to decline since growth of
the organisms would have reached a stage that indirectly stimulates the production of secondary metabolites (Febe et al.,
2002) due to insufcient availability of nutrients such as protein and glucose. Also, lower rate of hydrolysis (after 96 h of
fermentation) and inhibition effects of secondary metabolites would have had negative impact on Penicillium growth
and metabolism. Protein and glucose concentration increased
with fermentation time of 120 h beyond which it reduced.
Tannase production was found to increase till 96 h with an
increase in glucose level till 170.4 mg/mL beyond which it
reduced. Same trend was followed for glucose, tannase production increased linearly until glucose concentration reached
205.6 mg/mL beyond which it reduced as shown in Fig. 4. The
presence of glucose and protein in the fermentation broth was
apparently used to promote initial growth of biomass (Van

733

food and bioproducts processing 9 4 ( 2 0 1 5 ) 727735

(B)
160

300
Growth (CFU/g)

Tannase activity (U/g)

(A)
140
120
100
80
60
20

40

60

80

100

120

250
200
150
100
50

140

20

Fermentation period (h)

(C)

80

100

120

140

280

200

260

180

Glucose (mg/ml)

Protein (mg/ml)

60

(D)

220

160
140
120
100
80
60
40

40

Fermentation period (h)

240
220
200
180
160
140
120
100

20

40

60

80

100

120

140

Fermentation period (h)

20

40

60

80

100

120

140

Fermentation period (h)

Fig. 4 Time course analysis of tannase production; (A) Tannase activity (U/g) vs Fermentation period (h); (B) Growth (CFU/g)
vs Fermentation period (h); (C) Protein (mg/ml) vs Fermentation period (h); (D) Glucose (mg/ml) vs Fermentation period (h).
de Lagemaat and Pyle, 2001) which enhanced better tannase
productivity.

3.7.
Clarication of fruit juice by partially puried
tannase
Fruit juices such as pomegranate and aonla have recently been
acclaimed for health benets due to their high nutritional
value and antioxidant potential. However, presence of high
tannin content in those fruits is responsible for haze, sediment
formation, undesirable color, bitterness, and astringency of
the juice upon storage. In general, the fruit juices are prone
to damage by conventional method and also are not effective in de-bittering process therefore, enzymatic treatment is
explored in the present study. Enzymatic treatment of fruit
juices to reduce bitterness has got benets in executing higher
quality juice with better taste and low haze (Aguilar et al.,
2007). The crude extracellular tannase was partially puried
using activated charcoal (9%) and 80% ammonium sulphate.
The specic activity was improved 3.5 fold times (108.41 U/mg)
on decolourisation and 9.81 folds on precipitation with ammonium sulphate (303.86 U/mg).
Physicochemical parameters of the treated and untreated
juice are depicted in Table 4. The claried juice quality is
directly proportional to its visual appearance, which is an
important sensory attribute. The Commision Internationale
de LEclairage (CIE) system reference measures the lightness (L* value) on a numerical scale where, white = 100 and
black = 0 (Busch-Kschiewan et al., 2004). The L* value improved
from 73.17 0.9 and 56.85 0.3 to 72.65 0.7 and 52.32 0.7,
respectively for aonla and pomegranate juice after tannase
treatment. The Clarity is an important index of claried
juice (Sin et al., 2006) and is desirable to reach international

standard. After tannase treatment, decrease in absorbance

value from 0.10 to 0.04 for aonla juice and 2.59 to 1.69 for
pomegranate juice indicated signicant clarication. The turbidity of the fruit juice is mainly caused by the presence of
polysaccharides. Brix is an estimate of total soluble solids in
the juice. These soluble solids are primarily sugars; sucrose,
fructose, and glucose. Sugar concentration increased after
enzyme treatment for both aonla and pomegranate juice and
could be due to action of tannase on the complex sugar moieties present in the juice resulting in release of free sugars.
Alternatively, it also could be due to hydrolytic reaction of tannase resulting in the release of gallic acid and glucose which
increase the sugar levels in fruit juices (Aguilar et al., 2001).
Sugar/acid ratio contributes toward giving many fruits their
characteristic. The sugar/acid ratio increased after tannase
treatment for both aonla and pomegranate juice as a result
of hydrolytic activity of tannase, sugars are released inducing
characteristic avor. Titrable acidity is a measure of the total
acid present in a juice. The amount of acid present in juice is
reported as percent citric acid. Higher acid levels in fruit are
often associated with lower pH value and vice versa. Thus, the
acids of the fruit have a signicant bearing on pH. In addition,
they also play a signicant role in taste, color, and microbial
stability of the juice. % Citric acid reduced in case of aonla
juice after tannase treatment, although for pomegranate juice
it remained unchanged.
Aonla is one of the richest sources of vitamin C, minerals, phenolics and are known for its therapeutic value. But
due to astringent taste, the consumer appeal to the product
is not alleviated; hence enzymatic de-bittering is very essential. When aonla juice was treated with tannase, 25.71 0.06%
reduction of ascorbic acid was observed. Tannin content was
reduced by 27% and 37% respectively and total polyphenol was

734

food and bioproducts processing 9 4 ( 2 0 1 5 ) 727735

Table 4 Physico-chemical composition of untreated and treated Aonla and Pomegranate juice.
Parameters

Aonla juice
Un-treated juice

pH
Color (L* value)
Clarity (abs)

Brix
The sugar acid ratio
Protein (mg/100 mL)
Titrable acidity (% Citric acid)
Ascorbic acid (mg/100 mL)

3.02
73.17
0.10
1.0
6.25
12.06
0.16
99.99

0.00
0.9
0.00
0.03
0.7
0.3
0.03
1.6

Pomegranate juice
Treated juice
3.69
72.65
0.04
2.0
15.62
10.71
0.12
74.29

0.3
0.7
0.01
0.05
1.3
0.5
0.01
1.5

Un-treated juice
4.20
56.85
2.59
13.8
86.25
56.68
0.16
71.42

0.2
0.3
0.00
0.3
0.9
0.2
0.05
1.1

Treated juice
4.54
52.32
1.69
15.0
93.75
47.18
0.16
62.28

0.7
0.7
0.01
0.7
1.2
0.6
0.04
0.9

Data shown is mean and standard deviation of triplicates.

reduced by 7.8% and 13% respectively in the case of aonla and

pomegranate juice. Enzymatic hydrolysis of tannins to gallic acid in aonla juice is advantageous and also effective in
reducing astringency with minimum loss of vitamin C. The
reduction in tannins and polyphenols could be attributed to
hydrolysis reaction of the ester bonds present in hydrolysable
tannins and gallic acid esters. Haze component in fruit juice
is a result of protein-carbohydrate complex. The surface of
the protein molecules as well as carbohydrates is negatively
charged. Underneath the negative charged coat, there are positively charged proteins. Partial hydrolysis of the negatively
charged coat leads to the exposure of positively charged surface and attraction between unlike charged molecules leads
to ock formation (Sandeep and Reena, 2004). In the present
study, partial hydrolysis of tannase resulted in formation of
ocks which could be separated by ltration resulting in significant clarication of aonla and pomegranate juice. Therefore
enzyme treatment and ltration of juice successfully claried
and removed astringency of fruit juice. Thus, use of partially
puried tannase for fruit juice clarication is economically
promising for industrial application.

4.

Conclusion

A new prospective tannin degrader, P. verrucosum was isolated.

This is the rst report on cost effective production of tannase using coffee pulp by SSF. The RSM based on CCRD helped
in accomplishing the optimal solutions and in critically analyzing the interactive effects of most inuential bioprocess
variables on tannase production. The current study suggests
that partially puried tannase could be used as a clarifying aid
for fruit juices rich in tannins. Since, coffee pulp is available in
enormous quantity; it could be used for production of valueadded metabolites to meet the increasing demand of food and
pharmaceutical industry.

Acknowledgements
We thank Director, CFTRI, Mysore and Dr. P. Srinivas, Head,
PPSFT for constant encouragement. The nancial help from
CSIR, New Delhi, is gratefully acknowledged.

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