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Application of Tannase in Wine Clarification Produced Under Submerged Fermentation…

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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) collected from local orchard. Leaves were properly
specifically breaks the galloyl ester bond of tannins dried at 60oC and powdered in a grinder.
and produce gallic acid and glucose. Because of this Inoculum preparation: Sporulated culture lawn of
property, the enzyme has been used extensively in the Aspergillus ruber was picked up and mixed well in
foods, beverages, pharmaceuticals and chemical sterilized distilled water with 0.1% tween 80.
industries. Microorganisms have been reported to Tannase assay: Tannase activity was determined
produce tannase capable of hydrolyzing tannins to colorimetrically using method of Mondel et al. (2001).
gallic acid during fermentation (Lekha and Lonsane, The reaction mixture contained 0.3 ml of tannic acid
1997; Mukharjee and Banerjee, 2003). Gallic acid, the (0.5% in 0.2M sodium acetate buffer, pH 5.5), 0.1 ml
product of tannin hydrolysis finds application in many of enzyme and was incubated at 30 o C for one
fields like dye making, pharmaceuticals, leather and hour. The enzymatic reaction was stopped by addition
chemical industries (Hadi et al., 1994). Agrowastes like of 3 ml of BSA solution, which precipitates the
jamun leaves, ber leaves and amla leaves were remaining tannic acid. The tubes were centrifuged
reported as substrate for tannase production under (5000 g X 10 min) and the resultant precipitate
solid state fermentation (Kumar et al., 2007). was dissolved in 3 ml SDS-triethanolamine solution.
One ml of FeCl3 reagent was added to each tube
MATERIALS AND METHODS and was kept for 15 min at room temperature
Microorganisms and Growth: Isolated fungal strain was for stabilization of the color. The absorbance was
identified as Aspergillus ruber and was maintained on read at 530 nm against the blank (i.e. without
Czapek-dox agar slants at 4oC. 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 grape wine. Tannase was added to wine at different
of tannic acid in 1 min. concentration (5 Uml-1 to 30 Uml-1) and tannin content
Production of tannase under submerged fermentation: was estimated after every half an hour.
Tannic acid medium (Yamada et al.,1968) having
NaNO3 (0.2 %), KH2PO4 (0.1 %), MgSO4.7H2O (0.05%), RESULTS AND DISCUSSION
KCl (0.05%) and tannic acid (1.0%) in tap water ( Cl- Tannic acid medium (Yamada et al.,1968) was used
0.08%, Ca++, Mg++ 0.5%, HCO3- 0.4%) having pH 6.0 was for the production tannase under submerged
inoculated with 0.1 ml of spore suspension (109- 1010 fermentation. Optimum temperature was found to be
spores/ml) and incubated on shaker (200 rpm) at 30oC. at 30oC with a pH of 5.5 (Figure 1, 3). Temperature
Enzyme extraction: After 72 h of incubation, the above and below reduces its growth and may be
extract was passed through muslin cloth and the reason for low tannase production, also its
centrifuged at 10000 g for 5 min. Supernatant was favourable temperature for fungal growth. These
stored at 4oC for enzyme assay. results are in agreement with the previos reports
Effect of incubation temperature: The SmF was of tannases from Aspergillus niger Aa 20 (Anguilar
carried out at different temperatures such as 28, 30, et al., 2001); Aspergillus ruber (Kumar et al.,
33 and 35oC for 72 h and the enzyme was assayed. 2007); Aspergillus awamori (Chhokar et al., 2010)
Effect of incubation time: After inoculation, the flasks and Aspergillus heteromorphus (Chhokar et al.,
were incubated at 30oC for different time periods 2010).
ranging from 48 h to 120 h. The maximum tannase production was observed
Effect of pH: SmF was carried out using tap water after 72 h of incubation (Figure 2). Thereafter a
with different pH ranging from 5.0 to 7.5. The flasks decrease in tannase activity was observed that might
were incubated at 30 oC for 72 h and the enzyme be due to the end product inhibition as reported earlier
production was measured as described earlier. by Bradoo et al. (1997).
Effect of tannin rich agrowastes: Agrowastes Tannase was reported as inducible enzyme, thus
including ber leaves and amla leaves were added in tannic acid (substrate) concentration plays a major role
tannic acid medium for extra cellular production of in higher production of tannase. Higher tannase
tannase. Ber leaves and amla leaves were added in production was observed at 2% of tannic acid
individual and combined form, enzyme was harvested concentration (Figure 4). Thereafter a decrease in
as described above. tannase production was observed, that may be due to
Effect of Tannic acid: Tannic acid was supplemented antimicrobial activity of tannic acid. The concentration
to the production media at different concentration to of tannic acid adequate for tannase production ranged
see its effect on enzyme production. from 0.1 – 10% (Aoki et al., 1976; Lekha and Lonsane,
Effect of carbon source supplementation: The 1997).
production medium was supplemented with different Ber leaves and amla leaves contains tannins
carbon sources like Glucose, sucrose, mannitol, (6.7mg/g dry leaves and 45.5mg/g dry leaves
maltose and starch at a concentration of 2 % (w/v). respectively) (Kumar et al., 2007). These were exploited
The production was carried out at the conditions as substrate for tannase production in different
optimized earlier and enzyme was assayed as combinations and showed maximum tannase
described above. production in combined form having ber leaves, amla
Effect of nitrogen source supplementation: Different leaves and tannic acid (Figure 5). Many other
nitrogen sources were added externally to the substrates were also reported such as palm kernel cake
production media and all other conditions were kept (PKC), tamarind seed powder (TSP) (Sabu et al., 2005)
same. Different nitrogen sources added were urea, and wheat bran (Sabu et al., 2005; Gustavo et al., 2001).
ammonium chloride, ammonium nitrate and sodium In this part of world ber leaves and amla leaves are
nitrate. easily available in abundant. Different carbon sources
Effect of metal ions: Metal ions including Mg, Mn, and nitrogen sources were also added to study their
K, Cd, Ca, Zn and Na were added to the medium effect on enzyme production (Figure 6, 7). Mannitol
while other conditions were kept the same as as carbon source and sodium nitrate as nitrogen source
optimized earlier to see their effect on tannase were found to be the best for extracellular production
production. of tannase under submerged fermentation, however,
Application of tannase for wine clarification: Partially mannitol as carbon source did not show significant
purified tannase was used to clarify the jamun and increase over control.

530 International Journal of Tropical Agriculture © Serials Publications, ISSN: 0254-8755


Application of Tannase in Wine Clarification Produced Under Submerged Fermentation…

Figure 1: Extracellular Tannase Production by Aspergillus Figure 5: Effect of Ber and Amla Leaves as Tannin Rich
ochraceus at Diffrent Temperature Substrate on Extracellular Tannase Production

Figure 2: Effect of Incubation Time on Extracellular Tannase


Figure 6: Effect of Carbon Sources on Tannase Production
Production

Figure 3: Effect of pH on Extracellular 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
Figure 4: Effect of Tannic Acid Concenration on Tannase completely inhibited at 0.3% (Figure 9). Bradoo et al.,
Production (1997) also reported end product gallic acid as

Vol. 28, No. 3-4, July-December 2010 531


Rakesh Kumar, Jitender Sharma, Anju Kumari, Sonia Ahlawat, Vikas Beniwal & Sunita Dalal

inhibitor as it strongly inhibited both when produced colloidal suspension of wines becomes clear. The
constitutively (glucose as carbon) and in induced original tannin content in red and jamun wine was 90
conditions (tannic acid as carbon). It has been also µg/ml and 122 µg/ml respectively. The tannin content
reported that pyrogallols, gallic acid and was reduced to 69 and 95 µg/ml respectively after 2.5
gallaldehydes competitively inhibited the tannase of hour of incubation (Figure 11). Chae et al., (1983)
Aspegillus niger (Mahadevan and Sivaswamy, 1985). explored the tannases treatment in manufacturing of
Filtered suspension of jamun and red wine was acron wine. The enzyme hydrolysed chlorogenic acid
treated with tannases at different enzyme doses and to caffeic and gallic acid, which favorably influenced
different interval of time. Tannases at a concentration the taste.
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 11: Tannin Reduction in Jamun and Grape Wine (Red)

Figure 8: Effect of Different Metal ions on Tannase Production


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
Figure 9: Inhibition of Tannase Production by End Product for enzyme production, thereby, making it possible
(Gallic Acid) 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.
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532 International Journal of Tropical Agriculture © Serials Publications, ISSN: 0254-8755


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