Application of Tannase in Wine Clarification Produced Under Submerged Fermentation…

IJTA © Serials Publications

Application of Tannase in Wine Clarification Produced Under Submerged

Fermentation Using Aspergillus ruber
Rakesh Kumar*,**, Jitender Sharma*, Anju Kumari**, Sonia Ahlawat*, Vikas Beniwal** & Sunita Dalal*#

Abstract: Tannase producing fungal strains were isolated from different locations including garbages, forests and orchards etc.
The strain giving higher enzyme yield was identified to be Aspergillus ruber. Enzyme production was studied under submerged
fermentation using tannin rich substrates ber leaves (Zyzyphus mauritiana) and amla leaves (Phyllanthus emblica). Tannase
production was optimized under submerged cultivation conditions. Aspergillus ruber produced maximum enzyme after 72 h of
incubation at 30oC having pH 5.5, with mannitol as carbon source and sodium nitrate as nitrogen source. End product (Gallic
acid) in the growth media completely inhibited the enzyme production at 0.3% concentration. Supplementation of tannic acid
(0.6%) medium with Ber leaves and Amla leaves at a concentration of 0.6% (w/v) resulted in higher tannase production
compared to tannic acid (2%) medium. Under optimized conditions here the enzyme production by Aspergillus ruber was 18
Uml-1. Tannase produced was used in clarification of jamun wine and grape wine (red wine). For red wine (grape) and jamun
wine 20 Uml-1 tannase was found enough to degrade a significant amount of tannins.
Keywords: Submerged Fermentation, Tannase, Aspergillus ruber, Ber leaves, Amla leaves, wine clarification.

INTRODUCTION Raw material: Leaves of ber and amla were

Tannase (tannin acyl hydorlase, E.C.3.1.1.20)
specifically breaks the galloyl ester bond of tannins
and produce gallic acid and glucose. Because of this
property, the enzyme has been used extensively in
foods, beverages, pharmaceuticals and chemical
industries. Microorganisms have been reported to
produce tannase capable of hydrolyzing tannins to
gallic acid during fermentation (Lekha and Lonsane,
1997; Mukharjee and Banerjee, 2003). Gallic acid, the
product of tannin hydrolysis finds application in many
fields like dye making, pharmaceuticals, leather and
chemical industries (Hadi et al., 1994). Agrowastes like
jamun leaves, ber leaves and amla leaves were
reported as substrate for tannase production under
solid state fermentation (Kumar et al., 2007).
MATERIALS AND METHODS
Microorganisms and Growth: Isolated fungal strain was
identified as Aspergillus ruber and was maintained on
Czapek-dox agar slants at 4oC.
collected from local orchard. Leaves were properly
dried at 60oC and powdered in a grinder.
Inoculum preparation: Sporulated culture lawn of
the Aspergillus ruber was picked up and mixed well in
sterilized distilled water with 0.1% tween 80.
Tannase assay: Tannase activity was determined
colorimetrically using method of Mondel et al. (2001).
The reaction mixture contained 0.3 ml of tannic acid
(0.5% in 0.2M sodium acetate buffer, pH 5.5), 0.1 ml
of enzyme and was incubated at 30 o C for one
hour. The enzymatic reaction was stopped by addition
of 3 ml of BSA solution, which precipitates the
remaining tannic acid. The tubes were centrifuged
(5000 g X 10 min) and the resultant precipitate
was dissolved in 3 ml SDS-triethanolamine solution.
One ml of FeCl3 reagent was added to each tube
and was kept for 15 min at room temperature
for stabilization of the color. The absorbance was
read at 530 nm against the blank (i.e. without
tannic acid). One unit enzyme activity is defined as

* Department of Biotechnology, Kurukshetra University, Kurukshetra-136 119, Haryana, India, (# Corresponding author: E-mail:
sunitadalal69@gmail.com)
** Department of Biotechnology, MMEC, Maharishi Markandeshwar University, Mullana-Ambala- 133203

Vol. 28, No. 3-4, July-December 2010 529

 Rakesh Kumar, Jitender Sharma, Anju Kumari, Sonia Ahlawat, Vikas Beniwal & Sunita Dalal
the amount of enzyme required to hydrolyze 1 µmol
of tannic acid in 1 min.
Production of tannase under submerged fermentation:
Tannic acid medium (Yamada et al.,1968) having
NaNO3 (0.2 %), KH2PO4 (0.1 %), MgSO4.7H2O (0.05%),
KCl (0.05%) and tannic acid (1.0%) in tap water ( Cl-
0.08%, Ca++, Mg++ 0.5%, HCO3- 0.4%) having pH 6.0 was
inoculated with 0.1 ml of spore suspension (109- 1010
spores/ml) and incubated on shaker (200 rpm) at 30oC.
Enzyme extraction: After 72 h of incubation, the
extract was passed through muslin cloth and
centrifuged at 10000 g for 5 min. Supernatant was
stored at 4oC for enzyme assay.
Effect of incubation temperature: The SmF was
carried out at different temperatures such as 28, 30,
33 and 35oC for 72 h and the enzyme was assayed.
Effect of incubation time: After inoculation, the flasks
were incubated at 30oC for different time periods
ranging from 48 h to 120 h.
Effect of pH: SmF was carried out using tap water
with different pH ranging from 5.0 to 7.5. The flasks
were incubated at 30 oC for 72 h and the enzyme
production was measured as described earlier.
Effect of tannin rich agrowastes: Agrowastes
including ber leaves and amla leaves were added in
tannic acid medium for extra cellular production of
tannase. Ber leaves and amla leaves were added in
individual and combined form, enzyme was harvested
as described above.
Effect of Tannic acid: Tannic acid was supplemented
to the production media at different concentration to
see its effect on enzyme production.
Effect of carbon source supplementation: The
production medium was supplemented with different
carbon sources like Glucose, sucrose, mannitol,
maltose and starch at a concentration of 2 % (w/v).
The production was carried out at the conditions
optimized earlier and enzyme was assayed as
described above.
Effect of nitrogen source supplementation: Different
nitrogen sources were added externally to the
production media and all other conditions were kept
same. Different nitrogen sources added were urea,
ammonium chloride, ammonium nitrate and sodium
nitrate.
Effect of metal ions: Metal ions including Mg, Mn,
K, Cd, Ca, Zn and Na were added to the medium
while other conditions were kept the same as
optimized earlier to see their effect on tannase
production.
Application of tannase for wine clarification: Partially
purified tannase was used to clarify the jamun and
530 International Journal of Tropical Agriculture © Serials Publications, ISSN: 0254-8755
grape wine. Tannase was added to wine at different
concentration (5 Uml-1 to 30 Uml-1) and tannin content
was estimated after every half an hour.
RESULTS AND DISCUSSION
Tannic acid medium (Yamada et al.,1968) was used
for the production tannase under submerged
fermentation. Optimum temperature was found to be
at 30oC with a pH of 5.5 (Figure 1, 3). Temperature
above and below reduces its growth and may be
the reason for low tannase production, also its
favourable temperature for fungal growth. These
results are in agreement with the previos reports
of tannases from Aspergillus niger Aa 20 (Anguilar
et al., 2001); Aspergillus ruber (Kumar et al.,
2007); Aspergillus awamori (Chhokar et al., 2010)
and Aspergillus heteromorphus (Chhokar et al.,
2010).
The maximum tannase production was observed
after 72 h of incubation (Figure 2). Thereafter a
decrease in tannase activity was observed that might
be due to the end product inhibition as reported earlier
by Bradoo et al. (1997).
Tannase was reported as inducible enzyme, thus
tannic acid (substrate) concentration plays a major role
in higher production of tannase. Higher tannase
production was observed at 2% of tannic acid
concentration (Figure 4). Thereafter a decrease in
tannase production was observed, that may be due to
antimicrobial activity of tannic acid. The concentration
of tannic acid adequate for tannase production ranged
from 0.1 – 10% (Aoki et al., 1976; Lekha and Lonsane,
1997).
Ber leaves and amla leaves contains tannins
(6.7mg/g dry leaves and 45.5mg/g dry leaves
respectively) (Kumar et al., 2007). These were exploited
as substrate for tannase production in different
combinations and showed maximum tannase
production in combined form having ber leaves, amla
leaves and tannic acid (Figure 5). Many other
substrates were also reported such as palm kernel cake
(PKC), tamarind seed powder (TSP) (Sabu et al., 2005)
and wheat bran (Sabu et al., 2005; Gustavo et al., 2001).
In this part of world ber leaves and amla leaves are
easily available in abundant. Different carbon sources
and nitrogen sources were also added to study their
effect on enzyme production (Figure 6, 7). Mannitol
as carbon source and sodium nitrate as nitrogen source
were found to be the best for extracellular production
of tannase under submerged fermentation, however,
mannitol as carbon source did not show significant
increase over control.
 Application of Tannase in Wine Clarification Produced Under Submerged Fermentation…
Figure 1: Extracellular Tannase Production by Aspergillus
ochraceus at Diffrent Temperature
Figure 2: Effect of Incubation Time on Extracellular Tannase
Production
Figure 3: Effect of pH on Extracellular Tannase Production
Figure 4: Effect of Tannic Acid Concenration on Tannase
Production
Vol. 28, No. 3-4, July-December 2010
Figure 5: Effect of Ber and Amla Leaves as Tannin Rich
Substrate on Extracellular Tannase Production
Figure 6: Effect of Carbon Sources on Tannase Production
Figure 7: Effect of Different Nitrogen Sources on Tannase
Production
Metal ions independently affect the production of
tannase. Na and Mg ions increased tannase
production over the control (Figure 8), wherever all
other showed a decrease in tannase production at a
concentration of 0.2%. End product inhibition was also
studied and enzyme was completely inhibited at 0.3%
concentration of end product (Gallic acid). Gallic acid
at concentration of 0.05 and 0.1 showed no inhibition
and thereafter a steep inhibition was observed and
completely inhibited at 0.3% (Figure 9). Bradoo et al.,
(1997) also reported end product gallic acid as
531
 Rakesh Kumar, Jitender Sharma, Anju Kumari, Sonia Ahlawat, Vikas Beniwal & Sunita Dalal
inhibitor as it strongly inhibited both when produced
constitutively (glucose as carbon) and in induced
conditions (tannic acid as carbon). It has been also
reported that pyrogallols, gallic acid and
gallaldehydes competitively inhibited the tannase of
Aspegillus niger (Mahadevan and Sivaswamy, 1985).
Filtered suspension of jamun and red wine was
treated with tannases at different enzyme doses and
different interval of time. Tannases at a concentration
of 20 IU/ml was found suitable to reduce tannin from
both the wins when incubated for two and half an
hour (Figure 10). After 2.5 hour of incubation, the
Figure 8: Effect of Different Metal ions on Tannase Production
Figure 9: Inhibition of Tannase Production by End Product
(Gallic Acid)
Figure 10: Optimization of Tannase Concentration for Wine
Clarification
532 International Journal of Tropical Agriculture © Serials Publications, ISSN: 0254-8755
colloidal suspension of wines becomes clear. The
original tannin content in red and jamun wine was 90
µg/ml and 122 µg/ml respectively. The tannin content
was reduced to 69 and 95 µg/ml respectively after 2.5
hour of incubation (Figure 11). Chae et al., (1983)
explored the tannases treatment in manufacturing of
acron wine. The enzyme hydrolysed chlorogenic acid
to caffeic and gallic acid, which favorably influenced
the taste.
Figure 11: Tannin Reduction in Jamun and Grape Wine (Red)
CONCLUSION
We have isolated Aspergillus ruber capable of
producing tannase under SmF conditions. This is the
first time that ber leaves and amla leaves have been
used for tannase production under submerged
fermentation. The yield of about 18 U/ml was
achieved under optimum growth conditions. Ber
leaves and amla leaves attemped as substrates were
rich in tannins and are easily available in abundance
as agro-wastes. Cost is one of the reasons that tannases
are not being used widely. By using cheaper
substrates, we have tried to lower down the input cost
for enzyme production, thereby, making it possible
to be used at a larger scale in industries. Tannases
application was tried here in clarification of red and
jamun wines and found efficient to degrade tannins
in just two and half an hour.
REFERENCES
Anguilar, C.N.,C. Augur, E. Favela-Torre and G. Viniega-
Gonzalez (2001), ”Production of Tannase by Aspergillus
niger Aa-20 in Submerged and Solid State Fermentation:
Influence of Glucose and Tannic Acid”. J Ind Microbiol
Biotechnol, 26(5): 296-302.
Aoki K., R,Shinke and H. Nishira (1976), ”Purification and
some Properties of Yeast Tannase”. Agi Biol Chem.,
40:79-85.
Bradoo, S., R. Gupta and R. K. Saxena (1997), ”Parametric
Optimization and Biochemical Regulation of
 Application of Tannase in Wine Clarification Produced Under Submerged Fermentation…
Extracellular Tannase from Aspergillus japonicus.”
Process Biochem, 32(2): 135-39.
Chhokar, V., V. Seema Beniwal, V. K. Salar., K. S., Nehra,
A. Kumarand and J. S. Rana (2010), ”Purification and
Characterization of Extracellular Tannin Acyl
Hydrolase from Aspergillus heteromorphus MTCC 8818.”
Biotechnology and Bioprocess Engineering, 15: 793-799.
Chhokar, V., M. Sangwan., V. Beniwal., K. Nehra and K. S.
Nehra (2010), “Effect of Additives on the Activity of
Tannase from Aspergillus awamori MTCC9299”. Applied
Biochemistry and Biotechnology, 160: 2256-64.
Gustavo, A.S., G.F. Selma Leite, C. T. Selma and S. Couri
(2001), ”Selection of Tannase-producing Aspergillus
Niger Strains”, Brazilian J. Microbiol, 32: 24-26.
Hadi, T.A., R. Banerjee, B.C. Bhatacharya (1994), ”
Optimization of Tannase Biosynthesis by a Newly
Isolated Rhizopus Oryzae.” Bioprocess Engg., 11: 239-243.
Kumar, R., J. Sharma and R. Singh (2007), “Production of
Tannase from Aspergillus ruber Under Solid State
Fermentation Using Jamun (Syzygium cumini) Leaves.”
Microbiol Res., 162(4): 384-90.
Lekha, P.K. and B.K. Lonsane (1997), ”Production and
Application of Tannin Acyl Hydrolase: State of the
Art.”, Adv. Appl. Microbiol, 44: 215-260.
Vol. 28, No. 3-4, July-December 2010
Mahadevan, A. and S. N. Sivaswamy (1985), ”Tannins and
Microorganisms”. In Frontiers of Applied
Microbiology, eds. K.G. Mukerji, N.C. Pathak and
V.P. Singh. Rastogi and Company, Meerut, India.
pp. 327-47.
Mondal, K.C., D. Banerjee., M. Jana, B.R. Pati (2001), ”
Colorimetric Assay for Determination of Tannin Acyl
Hydrolase (E.C. 3.1.1.20) Activity”, Analytical
Biochemistry 295:168-171.
Mukharjee, G., R. Banerjee (2003), ”Production of Gallic
Acid”: Biotechnological Route (Part I). Chimica Oggi
Chem. Today, 21(1/2):59-62.
Sabu A., G.S. Kiran and A. Pandey (2005), ”Purification and
Characterization of Tannin Acyl Hydrolase from
A. niger ATCC 16620". Food Technol Biotechnol, 43(2):
133-138.
Sabu, A., A. Pandey, M.J. Daud and G. Szakacs (2005),
”Tamarind Seed Powder and Palm Kernel Cake: Two
Novel Agro Residues for the Production of Tannase
Under Solid State Fermentation by Aspergillus niger
ATCC 16620”, Bioresour Technol, 96(11): 1223-1228.
Yamada, H., O. Adachi, M., Watanabe and N. Sato (1968),
”Studies on Fungal Tannase”, Agi. Biol. Chem., 32(9):
1070-78.
533