Microbial production of tannase - Aseanbiotechnology.info

Microbial production of tannase: an enzyme with potential use in
foodindustry
Ruth Belmares a , Juan Carlos Contreras-Esquivel a ,Ra!ul Rodr!ıguez-Herrera a ,
Ascensi!on Ram!ırez Coronel b , Crist!obal Noe Aguilar a, *
a Food Research Department, School of Chemistry, Universidad Autonoma de Coahuila, Unidad Saltillo, Blvd. Venustiano Carranza, P.O. BOX 252,
ZIP 2500, Coahuila, Mexico
b Institut de Recherche pour le Développement, Laboratorio de Microbiologie, Université de Provence, ESIL, Case 925, Avenue de Luminy, 132888,
Marseille, Cedex 9, France
Abstract
Tannase catalyses the hydrolysis of gallic acid esters and hydrolysable tannins. This enzyme is produced by plants and
microorganisms and it is industrially used as catalysts in the manufacture of gallic acid. Also, it is potentially used in beverage and
food processing. Two critical factors, production costs and insufficient knowledge of the basic characteristics, physicochemical
properties, catalytic characteristics, regulation mechanisms andpotential uses, limit the use of tannase at the industrial level. This
work reviews the state of critical aspects relatedto the tannase, emphasizing aspects such as sources, substrates, metabolic regulation
mechanisms, physicochemical properties, inhibitors, production, applications and potential uses.
r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Tannase; Tannins; Regulation mechanism; Properties; Production; Applications
1. Introduction
Tannase or tannin acyl hydrolase (EC, 3.1.1.20)
catalyses the hydrolysis reaction of the ester bonds
present in the hydrolysable tannins and gallic acid esters.
Its production at industrial level is in a microbial way
using submergedculture (SmC), where the activity is
expressedmainly of intracellular form, implying additional
costs in its production (Lekha & Lonsane, 1994).
Tannase is recently commercializedby Biocon (India),
Kikkoman (Japan) ASA Specilaeznyme GmbH (Germany)
andJFC GmbH (Germany) with different
catalytic units depending of the product presentation.
However, several studies have reported interesting
advantages between the tannase produced by solid state
culture (SSC) in relation with that produced by SmC.
On this topic there are few reports, but they are clearly
*Corresponding author. Tel.: +52-844-416-9213; fax: +52-844-439-
0511.
E-mail address: cag13761@mail.uadec.mx (C.N. Aguilar).
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interesting (Barthomeuf, Regerat, & Pourrat (1994);
Garc!ıa-Pen˜ a (1996); Chaterjee, Dutta, Banerjee, &
Bhattacharyya, 1996; Lekha & Lonsane, 1997; Garc!ıa-
Pen˜ a et al., 1999; Ram!ırez-Coronel, Viniegra-Gonz!alez,
& Augur, 1999; Aguilar, Augur, Viniegra-Gonz!alez, &
Favela, 1999; Aguilar, Augur, Favela, & Viniegra-
Gonz!alez, 2001a, b; Aguilar, Favela-Torres, Viniegra-
Gonz!alez, & Augur, 2002; Van de Lagemaat & Pyle,
2001). In these, attractive advantages indicated, are the
high-production titles (up to 5.5 times more than in
SmC), the extracellular nature of the enzymes andthe
stability to wide pH and temperature ranges (Lekha &
Lonsane, 1994). Aguilar et al. (1999) reportedproductivities
of 6.667 UE/Lh and1.275 UE/Lh for SSC and
SmC, respectively, the tannase activity maximum
expressedintracellularly is also 18 times more in SSC
than in SmC, while the extracellular activity is 2.5 times
higher in SSC that in SmC.
At the moment, the biggest commercial applications
of the tannase are given in the elaboration of
instantaneous tea or of acorn liquor andin the
0023-6438/$30.00 r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2004.04.002

production of the gallic acid (Coggon, Graham, &
Sanderson, 1975; Chae & Yu, 1983; Pourrat, Regerat,
Pourrat, & Jean, 1985; Garc!ıa-N!ajera, 2002), which is an
important intermediary compound in the synthesis of
the antibacterial drug, trimetroprim, used in the
pharmaceutical industry (Sittig, 1988) andalso in the
foodindustry; gallic acidis a substrate for the chemical
or enzymatic synthesis of the propylgallate, a potent
antioxidant. Also, the tannase is used as clarifying agent
in some wines, juices of fruits andin refreshing drinks
with coffee flavour (Lekha, Ramakrishna, & Lonsane,
1993).
In the case of the wines, it is important to state that
the main tannins present are catechins andepi-catechins,
which can get a complex with galacto-catechins and
others galloyl-derivated. The amount of catechin in
white wines is aroundof 10 to 50 mg/l, while in other
wines it can reach 800 mg/l (Ribereau-Gayon, 1973).
Fifty percent of the colour of the wines is due to the
presence of the tannins, however, if these compounds
are oxidized to quinones by contact with the air it could
be formed an undesirable turbidity, presenting severe
quality problems. The use of the tannase can be a
solution to these problems.
In the manufacture of beer, the tannase couldbe used
since the tannins are present in low quantities. When the
proteins of the beer are in considerably high quantities
an undesirable turbidity is presented by the accomplishing
with these tannins. This problem couldbe resolved
with the employment of the tannase. The tannery
effluents contains high amounts of tannins, mainly
polyphenols, which are dangerous pollutants, for this
reason the use of the tannase represents a cheap
treatment andcash for the removal of these compounds.
2. Tannase substrates
Tannins are widely distributed in different parts
(bark, needles, heartwood, grasses, seeds and flowers)
of vascular plants. They are a group of complex
oligomeric chains substances characterizedby the
presence of polyphenolic compounds. They have molecular
weight higher than 500, reaching values above
20000 kDa. One of the major characteristic of tannins is
its ability to form strong complexes with protein and
other macromolecules such as starch, cellulose and
minerals. Tannins are classifiedin two major groups:
hydrolysable and condensed tannins (Lekha & Lonsane,
1997; Aguilar & Gutiérrez-S!anchez, 2001).
The hydrolysable tannins are constituted by several
molecules of organic acids, such as gallic, ellagic, digallic
andchebulic acids, esterifiedto a molecule of glucose.
Molecules with a core of quinic acidinsteadof glucose
have been also considered as hydrolysable tannins.
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Fig. 1 presents some examples of hydrolysable tannins
(Mueller-Harvey, 2001).
In order to maintain its binding capacity, gallotannins
must have more than two gallic acidconstituents
esterifiedto the glucose core. Hydrolysable tannins can
be easily hydrolysed under mild acid or alkaline
conditions; with hot water or enzymatically (L!opez-
R!ıos, 1984).
Condensed tannins or Proanthocyanidins (Fig. 2) are
complex compounds constituted by flavonoid groups
(from 2 to 50) which are considered not to be
hydrolysable. Their major constituents are cyanidin
and delphinidin which are responsible for the astringent
taste of fruit andwines (Sanchez, 2001).
The negative effect of tannins in animal nutrition is
due to their capacity to bind macromolecules rendering
them undigestible (Mendez, 1984). Tannins form stable
complex with enzymes andminerals requiredfor the
ruminal microorganisms. They are also responsible of a
bitter taste, which considerably reduces the feed intake.
However, low tannin concentrations in feedhave
demonstrated to increase nitrogen assimilation in
ruminants, rendering higher growth rates and milk
production (Nip & Burns, 1969).
3. Tannase
Tannase catalyses the breakdown of hydrolysable
tannins such as tannic acid, methygallate, ethyl gallate,
n-propylgallate andisoamylgallate. A typical reaction of
tannase is the hydrolytic cleavage of (-)epigallocatechin-
3-ol gallate (Fig. 3) (Bajpai & Patil, 1997; Lekha &
Lonsane, 1997).
Although tannase is present in plants, animals and
microorganisms, it is mainly produced by microorganisms
(Ayed& Hamdi, 2002; Nishitani & Osawa, 2003).
Table 1 presents some of the microorganisms studies for
tannase production.
Tannase production and application have been
extensively studied, studies related to strain isolation
andimprovement, process development andapplication
of tannases have conducted to a great number of
scientific publications andpatents. Table 2 presents
some of the publishedpatents regarding tannase
production and application.
4. Production of tannase
Filamentous fungi of the Aspergillus genus have been
widely used for tannase production. Although tannase
production by Aspergillus can occur in the absence of
tannic acid, this fungi tolerates tannic acid concentrations
as high as 20% without having a deleterious effect
on both growth and enzyme production. Studies on

HO
HO
HO
HO
(B)
OH
OH
(D)
HO
(A)
HO
HO
O
C
O
C
HO
HO
HO
HO
HO
HO
O
O
HO
HO
O
C=O
OH OH
OH
OH
O
C
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O
O
COOH
COOH
O
C=O
O
O
C
C
O
O
OH OH
O
OH
O
C
OH
O
Fig. 1. Hydrolysable tannins and some of their constituents. A. Gallotannin, B. Ellagitannin, C. Ellagic acid, D. Hexahydroxyphenic acid and E.
Gallic acid.
HO
HO
HO
O
OH
OH
HO OH
HO
HO
O
OH
OH
OH
OH
OH
Fig. 2. Condensed tannins or Proanthocyanidins.
O
O
OH
OH
OH
HO
O
OH
OH
O
C
OH
OH
HO
(C)
HO
O
OH
O
OH
(E)
O
HO
O
C
O
HO
OH
HO
HO
O
OH
O
OH
OH
OH
OH
OH
OH
COOH
O
OH
OH
O
O
C
OH
HOOC
Fig. 3. Typical reaction of tannase.
OH
OH
OH
OH
OH
OH
OH
OH

tannase production by Aspergillus have been carriedout
on submergedandsolidstate cultures. Depending on the
strain andthe culture conditions, the enzyme can be
constitutive or inducible, showing different production
patterns. Phenolic compounds such as gallic acid,
pyrogallol, methyl gallate andtannic acidinduces
tannase synthesis (Bajpai & Patil, 1997). However, the
induction mechanism has not been demonstrated and
there is some controversy about the role of some of the
hydrolysable tannins constituents on the synthesis of
tannase (Deschamps, Otuk, & Lebeault, 1983). For
instance, gallic acid, one of the structural constituents of
some hydrolysable tannins, such as tannic acid, has been
reported as an inducer of tannase synthesis under
submergedfermentation, whilst it represses tannase
Table 1
Microorganisms usedfor tannase production
Bacteria Bacillus pumilus
Bacillus polymyxa
Corynebacterium sp
Klebsiella pneumoniae
Streptococcus bovis
Selenomonas ruminantium
Yeast Candida sp.
Saccharomyces cerevisiae
Mycotorula japonica
Fungi Aspergillus niger
Aspergillus oryzae
Aspergillus japonicus
Aspergillus gallonyces
Aspergillus awamori
Penicillium chrysogenum
Rhizopus oryzae
Trichoderma viride
Fusarium solani
Mucor sp.
Table 2
Publishedpatents relatedto tannase production or application
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synthesis under solid state fermentation. Nevertheless,
independently of the involved mechanism, it has been
well acceptedthat due to the complex composition of
the hydrolysable tannins, some of their hydrolysis
products induces tannase synthesis (Aguilar et al., 2002).
Addition of carbon sources such as glucose, fructose,
sucrose, maltose, arabinose to the culture medium at
initial concentrations from 10 to 30 g/l improves tannase
production by Aspergillus niger. Nitrogen requirements
can be suppliedby different organic andinorganic
sources. Inorganic nitrogen can be supplementedas
ammonium salts (sulphate, carbonate, chloride, nitrate,
monohydrated phosphate) or nitrate salts (sodium,
potassium or ammonium). Other nutritional requirements
such as potassium, magnesium, zinc, phosphate
andsulphur are suppliedas salts. Although A. niger
does not require cofactor supplementation, folic and
pantothenic acidare eventually addedto the culture
medium. A typical medium for tannase production by
Aspergillus niger is presentedin Table 3.
Tannase production in submerged culture by Aspergillus
sp. is improvedat high aeration rates. It is
favouredat 30–33 C andinitial pH values from 3.5 to
Table 3
Typical culture medium for tannase production by Aspergillus niger
Constituent Initial concentration (g/l)
Tannins 50
Glucose 10
(NH4)2HPO4
5
K2HPO4 1
MgSO4 7H2O 1
ZnSO4 7H2O 0.1
NaCl 0.005
Year Title Patent No.
1974 Conversion of green tea andnatural tea leaves using tannase USP3812266
1975 Production of tannase by Aspergillus JP7225786
1975 Tea soluble in coldwater UKP1280135
1976 Extraction of tea in coldwater GP2610533
1976 Enzymatic solubilization of tea cream USP3959497
1985 Gallic acidester(s) preparation EP-137601
1985 Preparation of gallic acidesters e.g. propylgallate EP-137601
1985 Enzymatic treatment of black tea leak EP135222
1987 Preparation of tannase JP62272973
1987 Manufacturing of tannase with Aspergillus JP62272973
1988 Production of tannase by Aspergillus oryzae JP63304981
1988 Elaboration of tannase by fermentation JP63304981
1989 Preparation of spray-concrete coating in mining shaft SUP1514947
1989 Antioxidant catechin and gallic acid preparation JP01268683
1989 Tannase production by culture of Aspergillus tamarii EP-339011
1989 New Aspergillus niger B1 strain EP307071
1989 Tannase production process by Aspergillus andits application to obtain gallic acid EP339011
1992 Tannase preparation method JP4360684

6.5. The maximal enzymatic activity is attainedafter 1 to
3 days of cultivation.
Tannase production has been mostly studied in
submergedfermentation; however, few studies have
been also carriedout under solidstate fermentation
conditions. Wheat bran has been used as support and
sole source of nutrients for tannase production by
Aspergillus niger in SSC. Innert supports, such as sugar
canne pith or polyurethane foam, impregnatedwith a
defined culture medium have been also used (Aguilar
et al., 2001a, b). Two major differences are found when
submerged and solid state conditions are compared: (i)
tannase yield production and productivity are higher in
SSC than in SmC; (ii) tannase location under SSC
conditions is mostly extracellular, whilst it is bounded to
the mycelium under SmC conditions.
It is important to note that such higher tannase
activity levels in SSC than SmC have been clearly
associatedwith the concomitant production of proteolytic
activities in the last culture system (Aguilar et al.,
2002; Viniegra-Gonz!alez et al., 2003). Also, in SSC the
tannase produced exhibits a higher tolerance to wide
range of pH andtemperature (Lekha & Lonsane, 1994).
For optimization of tannase production, Pinto,
Bruno, Hamacher, Terzi, andCouri (2003) evaluated
the tannic acid/wheat bran ratio, different moisture
levels, addition of supplementary nitrogen sources,
addition of supplementary phosphate and the concentration
of supplementary nitrogen and phosphate added
to the medium. Their results showed that best medium
was with 15% of tannic acid, 37.5% of initial moisture,
1.7 ammonium sulphate and2.0% of sodium phosphate.
The presence of phosphate showeda great
importance for optimization, because promotedincrease
in the synthesis level anda very expressive decrease in
the maximum production time, from 72 to 24 h of
fermentation. The optimizedprocess promoteda
increase 861% in yieldand2783% in productivity.
5. Tannase extraction
Tannase extraction strongly depends on the fermentation
system used. Since tannase is mostly extracellular
when produced by SSC, it can be easily extracted with
water or a buffer. Two to three volumes of the agent
extraction is well mixedwith the fermentedmass and
pressedto obtain the enzymatic extract. Tannase
location during its production by SmC depends on the
cultivation time (Rajakumar & Nandy, 1983). It is
mainly intracellular at the beginning of the culture andit
is further secretedto the culture medium. However, up
to 80% of tannase remainedboundedto the mycelium
when the maximum overall tannase titter is attained.
Bounded tannase can be extracted after cell wall
hydrolysis with digestive enzymes such as chitinase.
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The cells can be also mechanically disrupted to recover
the bounded tannase. Recently, Ramirez-Coronel,
Viniegra-Gonzalez, Darvill, andAugur (2003) produced
an extracellular tannase by solid-state cultures of
Aspergillus niger. The enzyme was purifiedto homogeneity
from the cell-free culture broth by preparative
isoelectric focusing andby FPLC using anion-exchange
andgel-filtration chromatography. SDS-PAGE analysis
as well as gel localization studies of purified tannase
indicated the presence of two enzyme forms.
6. Tannase immobilization
Since a fraction of the produced tannase remains
bounded to the cell under submerged fermentation
conditions, the produced biomass may be recycled as a
biocatalyst. Several strategies can be usedfor tannase
concentration or purification andimmobilization after
extraction from the biomass (submergedfermentation)
or from the culture medium (submerged an solid state
fermentation). In order to increase the specific activity of
the enzymatic preparation, tannase shouldbe concentrated.
For that, classical methods such as salt or solvent
precipitation, ultrafiltration followedby ion exchange or
size exclusion chromatography, as well as solvent
extraction can be used(Lekha & Lonsane, 1994). Once
the tannase activity has been concentratedandeventually
purified, it can be immobilized to reuse the
enzymatic preparation. Agarose, chitosan, alginate and
different derivatized silicious materials can be used for
tannase immobilization (Sharma, Bhat, & Gupta, 2002).
Abdel-Naby, Sherif, El-Tanash, and Mankarios (1999)
immobilizedtannase from Aspergillus oryzae on various
carriers, however, that enzyme immobilizedon chitosanglutaraldeheyde
showed the highest activity. The bound
enzyme retained20.3% of original specific activity. On
the other hand, Sharma et al. (2002) immobilized
tannase from A. niger on concavalin A-Sepharose via
bioaffinity interaction. The immobilizedpreparation
was quite stable to reuse, there was no loss of enzyme
activity after three cycles andit retained81% activity
even after the sixth cycle. Ester hydrolysis using the
immobilizedenzyme ledto a 40% conversion into gallic
acidas comparedwith 30% obtainedwith the free
enzyme.
7. Properties of tannase
The tannase of some Aspergillus strains has a
molecular weight around150–350 kDa. Their activity
andstability pH are 5–6.0 and3.5–8.0, respectively,
whilst optima temperatures from 35 Cto40C have
been reported. Tannase is stable for several months at
30 C. Tannase produced by Penicillium strains present

similar characteristics in terms of pH andtemperature
activity andstability. When tannic acidis usedas
substrate Km values of 11.25 mM and0.048 mM were
obtainedwith Aspergillus and Penicillium, respectively.
(Yamada, Iibuchi, & Minoda, 1968; Adachi, Watanabe,
& Yamada, 1971; Libuchi, Minoda, & Yamada, 1972;
Aoki, Shinke, & Nishira, 1976; Chae & Yu, 1983).
Ramirez-Coronel, Viniegra-Gonzalez, Darvill, and
Augur (2003) produced two tannase forms by solid
state culture with molecular masses of 90 kDa and
180 kDa. The tannase hadan isoelectric point of 3 8, a
temperature optimum of 60–70 C anda pH optimum of
6 0. The substrate specificity of the tannase was
determined by HPLC analysis of tannin substrates and
products. The enzyme was able to remove gallic acid
from both condensed and hydrolysable tannins. Internal
sequences were obtainedfrom each of the gel-purified
and trypsin-digested tannase forms. The peptide sequences
obtainedfrom both forms were identical to
sequences within a b-glucosidase from Aspergillus
kawachii. The purifiedtannase was testedfor bglucosidase
activity and was shown to hydrolyse
cellobiose efficiently. However, no b-glucosidase activity
was detected when the enzyme was assayed in the
presence of tannic acid.
The effect of metal ions on tannase activity was
studied recently by Kar, Banerjee, andBhattacharyya
(2003). One mM Mg +2 or Hg + activatedtannase
activity. Ba +2 ,Ca +2 ,Zn +2 ,Hg +2 andAg + inhibited
tannase activity at 1.0 mM concentration andFe +3 and
Co +2 completely inhibitedtannase activity. Ag + ,Ba +2 ,
Zn +2 andHg +2 competitively inhibitedtannase activity.
Among the anions studied, 1 mM Br or S2O3 2
enhancedtannase activity. Tween 40 andTween 80
enhancedtannase activity whereas Tween 60 inhibited
tannase activity. Sodium lauryl sulfate and Triton X-100
inhibitedtannase activity. Urea stimulatedtannase
activity at a concentration of 1.5 M. Among the
chelators chosen for the present study, 1 mM EDTA
or 1,10-o-phenanthrolein inhibitedtannase activity.
Dimethyl sulphoxide and b-mercaptoethanol inhibited
tannase activity at 1 mM concentration whereas soybean
extract inhibitedtannase activity at concentrations
varying from 0.05% to 1.0% (w/v). Among the nitrogen
sources selectedammonium ferrous sulfate, ammonium
sulfate, ammonium nitrate andammonium chloride
enhancedtannase activity at 0.1% (w/v) concentration.
8. Tannase applications
One of the major applications of tannase is in the
manufacturing of instantaneous tea. Tannase applications
in food and beverage industrial products contribute
to remove the undesirable effects of tannins
(Boadi & Neufeld, 2001). Other important application
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of tannase is the production of gallic acid and
propylgallate (Kar, Banerjee, & Bhattacharyya, 2002).
The former one is usedin the pharmaceutical industry
for the synthesis of antibacterial drugs and in the food
industry as substrate for the chemical synthesis of food
preservatives such as pyrogallol andgallates. On the
other hand, propylgallate is a very important food
antioxidant (Sharma & Gupta, 2003). Tannase may
contribute to plant cell wall degradation by cleaving
some of the cross-links existing between cell wall
polymers (Garcia-Conesa, Ostergaard, Kauppinen, &
Williamson, 2001). Tannase can be potentially usedfor
the degradation of tannins present in the effluents of
tanneries, which represent serious environmental problems
(Van de Lagemaat & Pyle, 2001). Also, it can be
usedin the preparation of animal feeding using as
culture support the mycelial wastes from penicillin
manufacture (Nuero & Reyes, 2002).
9. Concluding remarks
The use of tannase from different microbial sources
may have benefits for different areas such as food,
beverage, cosmetic andpharmaceutical industries as
well as environmental depollution. For that, more effort
is needed in order to develop more productive process
for tannase production. In this sense, solid state
fermentation presents more advantages that the submergedtype
of culture. Improvements on tannase
immobilization are needed for the development of
cheaper process. For that, enzymatic preparations with
improved catalytic characteristics must be developed.
Acknowledgements
C.N. Aguilar thanks CONACYT-SEP (Project:
42244) andCOAH-CONACYT (COAH-2002-
CO1.2565 and4652) for financial support. The present
work was conducted within the framework of the ECOS
program (M02A02). R. Belmares andA. Ram!ırez-
Coronel are the recipients of a M.Sc. andPhD scholarships
from CONACYT-Me´ xico, respectively.
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