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228 Recent Patents on Biotechnology, 2013, 7, 228-233

Recent Advances in Industrial Application of Tannases: A Review


Vikas Beniwal1*, Anil Kumar2, Jitender Sharma3 and Vinod Chhokar2

1
Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Ambala- 133203 (Haryana), India;
2
Department of Bio and Nano Technology, Guru Jambheshwar University of Science & Technology, Hisar-125001
(Haryana), India; 3Department of Biotechnology, Kurukshetra University, Kurukshetra-136119 (Haryana), India

Received: February 01, 2013 Revised: April 08, 2013 Accepted: June 21, 2013

Abstract: Tannin acyl hydrolase (E.C. 3.1.1.20) commonly referred as tannase, is a hydrolytic enzyme that catalyses the
hydrolysis of ester bonds present in gallotannins, ellagitannins, complex tannins and gallic acid esters. Tannases are the
important group of botechnologically relevant enzymes distributed throughout the animal, plant and microbial kingdoms.
However, microbial tannases are currently receiving a great deal of attention. Tannases are extensively used in food, feed,
pharmaceutical, beverage, brewing and chemical industries. Owing to its diverse area of applications, a number of patents
have been appeared in the recent past. The present review pretends to present the advances and perspectives in the indus-
trial application of tannase with special emphasis on patents.
Keywords: Tannase, tea, tannic acid, tannin, gallic acid.

INTRODUCTION these have emphasis on the industrial application of the en-


zyme to its full potential. The current review updates us
Tannase (tannin acyl hydrolase, EC 3.1.1.20) is an induc- about the recent progress on industrial application of micro-
ible enzyme that catalyzes the hydrolysis of ester and dep- bial tannase with special reference to the patent published.
side bonds in hydrolyzable tannins by releasing glucose and
gallic acid [1]. Gallic acid is extensively used as an ingredi-
INDUSTRIAL APPLICATION OF TANNASE
ent of developer in photography and printing inks. It also
serves as a precursor for the commercial production of an Tannase is a versatile enzyme finding application in vari-
anti-microbial drug-trimethoprim, a food preservative- ous industries. A number of patents in tannase application
propylgallate and some dyestuffs. Besides this, gallic acid have been granted over the years. Table 1 presents some of the
possesses wide range of biological activities, such as anti- published patents on industrial application of tannase [8-31].
oxidant, antibacterial, antiviral, analgesic etc. As antioxidant
gallic acid acts as an antiapoptotic agent and helps to protect TEA
human cells against oxidative damage. Gallic acid is also
The main components of tea leaves are tea catechins,
found to show cytotoxic activity against cancer cells, without
namely (-)-epigallocatechin gallate (EGCG), (-)-
harming normal cells [2]. Besides gallic acid production, the
epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and
enzyme is extensively used in the manufacture of instant tea
(-)-epicatechin (EC); these compounds represent 20-30% of
and acorn wine. Tannase has potential application in the the dry weight and 80% of total polyphenol in fresh tea
clarification of beer and fruit juices, manufacture of coffee leaves. As catechins are not destroyed during manufacturing
flavored soft drinks, and improvement in flavor of grape of green tea, they are the major part of green tea extracts
wine, and as an analytical probe for determining the structure responsible of several health benefits such as scavenger of
of naturally occurring gallic acid esters [3]. Tannase has also reactive oxygen and nitrogen species, free metal chelation,
been applied for cleavage of poly phenolics such as dehy- inhibition of transcription factors and oxidative enzymes
drodimer crosslinks present in the cell wall of plants, which such as lipoxygenase and cycloxygenase [32].
is necessary for plant cell wall digestibility [4]. Tannase is
also used in the manufacture of sensitive analytical probes High cold water solubility is a large problem in the
for determining the structure of naturally occurring gallic manufacturing of instant tea, as tea cream is formed when
acid esters [5]. Moreover, it is incorporated into the manu- the tea is stored at or below temperatures of 4°C [1]. This
facture of high grade leather [6] and is used to clean up the haze formation is due to the coacervation of tea flavonoids.
hard and acidic industrial effluent containing tannin materi- Tea polyphenols also form hydrogen bonds with caffeine,
als [7]. A number of review have appeared in the recent past which leads to the cream formation. In conventional process
describing the production, purification and characterization for preparing instant tea, the hot water extract of tea is sub-
of tannase from various microbial sources, however none of jected to low temperatures with agitation, followed by cen-
trifugation of tea cream [33]. The tea cream is usually dis-
carded, which represents a considerable loss of the major
*Address correspondence to this author at the Department of Biotechnology,
Maharishi Markandeshwar University, Mullana, Ambala- 133203 (Haryana)
flavor components [34]. The chemical method for solubiliz-
India; Tel: +919416768062; Fax: +911731274375; ing tea cream involves treatment sulfite and molecular oxy-
E-mail: beniwalvikash@gmail.com gen together with an alkali [8]. Instant tea powder produced

2212-4012/13 $100.00+.00 © 2013 Bentham Science Publishers


Recent Advances in Industrial Application of Tannases: A Review Recent Patents on Biotechnology, 2013, Vol. 7, No. 3 229

by chemical methods, when reconstituted as a hot beverage, by mixing 1.1 to 2.0 wt% of enzymes (cellulase, pectinase,
reacts very badly on addition of milk by forming a dull- and tannase) with 98 to 98.9 wt% of red wine liquid concen-
blakish, unpleasant coloration [34]. Therefore, elimination of trate [31].
such poorly soluble compounds is probably important for
producing instant tea with good color, solubility and good FRUIT JUICE
yield [8]. Tannase treatment has been widely used in the
manufacture of instant tea to increase the extractability and Enzymatic treatment of fruit juices to reduce the bitter-
cold water solubility of tea compounds Catechins indirectly ness has got advantages such as the higher quality of juice
affects the formation of tea aroma, one of the critical aspects due to the lower haze and non-deterioration of juice quality.
of tea quality. A decrease in catechin content is associated New fruit juices (pomegranate, cranberry, raspberry, cold
with an increase in the content of total aroma compounds, tea, etc.) have recently been acclaimed for their health bene-
especially monoterpene alcohols, which are regarded as be- fits, in particular, for its disease-fighting antioxidant poten-
ing very important to black tea flavor [35]. Wang et al., tial. The presence of high tannin content in those fruits is
found that after tannase treatment, the content of EGCG and responsible for haze and sediment formation, as well as for
ECG decreased greatly in a green tea infusion, as a result, the color, bitterness, and astringency of the juice upon storage.
bitterness of the infusion was reduced [36]. Sanderson and Due to the inability of conventional fruit juice debittering
coggon [8] discloses a method that involves converting green processes to remove the bitterness effectively, enzymatic
tea to black using tannase and natural tea enzymes. The debittering should be preferred. Preliminary results reported
method includes tannase pre-treatment followed by oxidation by Rout and Banerjee, for pomegranate juice demonstrated
by natural tea enzymes to convert green tea into black, and that tannase treatment resulted in 25% degradation of tannin,
generate tea powders, which are both hot and cold water while a combination of tannase and gelatin (1:1) resulted in
soluble. Sanderson et al. [9] patented a method for making a 49% of tannin degradation [40]. Srivastava and kar reported
cold water soluble leaf tea extract. Tea leaves were pre- the enzymatic treatment of aonla/myrobalan (Phyllanthus em
treated with tannase anaerobically to generate cold-water blica) juice with 68.8 % removal of tannin content resulting
infusing tea showing good colour, yield and flavour. Tsai in considerable loss of astringency [41]. Oded et al., reported
discloses treating black tea with tannase along with other a enzyme from mutant species CMI CC 324, 626 of Asper-
cell-wall digesting enzymes to generate cold-water soluble gillus niger B1 showing activity of endo -glucosidase, tan-
instant tea powders [10]. Goodsall et al., reported a process nase and anthocyanase capable of enhancing taste and flavor
in which green tea leaves were macerated, treated with tan- of fruit juices and fermentation products [42]. A japenese
nase and fermented in the presence of hydrogen peroxide to patented described that tannase-treated black tea extract and
activate endogenous peroxidases. Gallic acid and other com- fruit juice in high concentrations can be preserved over a
pounds that are liberated by the tannase treatment were oxi- long period without causing clouding and precipitation, ex-
dized by endogenous peroxidases and dried to give black hibiting excellent taste, flavor and quality [15]. Motoichi et
leaf tea that infuses in hot or cold water to give good flavour al., claims that fruit and vegetable juice can be stored for
and colour [19]. Mullick et al., developed a process for pre- longer duration without showing any turbidity or precipita-
paring a theaflavin-enhanced tea product comprising enzy- tion when they are treated with carboxylic ester hydrolase, in
matic fermentation in the presence of added exogenous epi- particular tannase or chlorogenase [17].
catechin [30].
ANIMAL FEED
WINE AND BEER Tannins are present in large number of plant materials
Masschelein and Batum [37] reported that tannase from that are used as feed, e.g., tree leaves, agro-industrial by-
A. flavus dramatically reduce the haze formation in beer after products, agricultural wastes and are considered one of the
storage. This implicates tannase in the hydrolysis of wort most common antinutritional factors. Tannins present in
phenolics which complex with the other chemicals in the plants can, in general, adversely affect animal nutrition by
beer mixture and results in the haze formation [37]. Gio- reducing intake, protein digestibility, inhibiting digestive
vanelli showed that upon treatment of the stored beer with enzymes or by direct systemic toxicity [43]. Other deleteri-
tannase the potential of haze formation was dramatically ous effects of tannin include damage to the mucosal lining of
reduced [38]. In the case of wines, it is important to consider the gastrointestinal tract, alteration in excretion of certain
that the main tannins present are catequins and epicatequins, cations, and increased excretion of certain protein and essen-
which can create a complex with galactocatequins and others tial amino acids [1]. This leads to a reduction in their feed
galoyl-derivates. In the early days wine was treated chemi- intake, adversely affects rumen fermentation and signifi-
cally to remove the unfavoured phenolics. Now tannase is cantly depresses digestibility of almost all the nutrients.
being employed to hydrolyse chlorogenic acid to caffeic acid The use of enzymes in animal feeds is gaining impor-
and quinic acid, which influences the taste of the wine fa- tance nowdays, tannase in the pretreatment of tannin contain-
vourably [39]. Hennink et al., patented a process for produc- ing feed may prove beneficial in removal of the undesirable
ing beer having an improved flavour stability by adding an compounds and also improve digestibility. Xu patented a
oxygen scavenging enzyme (glucose-oxidase, laccase, tan- method of producing a cellulosic material reduced in tannin
nase, catechol oxidase, sulfhydryl-oxidase, superoxide dis- content by treating the cellulosic material with an effective
mutase etc) prior to and/or during the mashing stage of the amount of tannase leading to reduce the inhibitory effect of
brewing [21]. Jeong and kang have recently reported a high tannin on enzymatic saccharification of cellulosic material
polyphenol red wine, having antioxidant activity produced [44].
230 Recent Patents on Biotechnology, 2013, Vol. 7, No. 3 Beniwal et al.

Table 1. Patent Published on Industrial Application of Tannase.

Year Title Patent No Original Assignee Reference

Green tea conversion using tannase and natural tea enzymes:


1974 Comprising the steps of contacting green tea with tannase in the presence of US 3812266 Thomas J. Lipton Inc. [8]
water and within a temperature range in which the tannase is active

Cold water extractable tea leaf and process:


1977 Cold water extractable tea leaf is prepared by treatment of fresh green tea with US 4051264 Thomas J. Lipton, Inc. [9]
the enzyme tannase under anaerobic conditions

Enzymatic treatment of black tea leaf:


US4639375, The Procter & Gamble
1988 Black tea leaf is wetted with an enzyme solution comprising tannase and at [10]
EP0135222 B1 Company
least one cell-wall-digesting enzyme

Enzymatic synthesis of gallic acid esters:

1989 An improved method of synthesizing gallic acid esters consists in incubating EP0137601 B1 Corning Glass Works [11]
gallic acid with an alkyl alcohol or diol in the presence of an immobilized
tannase to yield the corresponding alkyl gallate ester

Process of preparing a tea product:


EP0391468 B1, Unilever N.V.,
1993 The present invention relates to a process of preparing a tea extract of im- [12]
EP0391468A1 Unilever Plc
proved colour without turbidity.

Enzyme extraction process for tea:


Enzymatic extraction of black tea leaf with water containing tannase and one Lipton, Division of
1999 US 5919500 [13]
or more cell wall digesting enzymes, such as cellulase, pectinase, hemicellu- Conoco, Inc.
lase or viscozyme

Method of enhancing color in a tea-based foodstuff:


A method of inducing the formation of color determining compounds in a tea-
Lipton, Division of
1999 based food product, characterized in that the tea leaves are treated with an US5879730A [14]
Conoco Inc.
exogenous laccase, polyphenol oxidase or peroxidase, in combination with a
pretreatment with a tannase.

Black tea drink containing fruit juice:


The black tea drink containing fruit juice is preferably prepared by mixing the Kikkoman Corp.,
2000 JP 2000037164 [15]
tannase-treated black tea extract with a fruit juice treated with chlorogenic acid Nippon Del Monte Corp
esterase

Tea concentrate prepared by enzymatic extraction and containing xan-


than gum which is stable at ambient temperature:
2000 A tea concentrate prepared by enzymatic extraction of the tea leaf with a com- US6024991A Thomas J. Lipton Co. [16]
bination of cell wall lysis enzymes and tannase is disclosed where the concen-
trate is stabilized where necessary by xanthan gum

Fruit or vegetable juice-containing protein beverage:


Kyowa Hakko Kogyo
2001 A method for producing the fruit and/or vegetable juice-containing protein JP 2001-340069 [17]
Co. Ltd
beverage using carboxylic ester hydrolase (tannase or chlorogenase)

Tooth whitening products and procedures:


2001 Discovered that the tannase enzyme as well as dioxiranes effectively can EP0010698 - [18]
whiten teeth

Cold water infusing leaf tea:


Green tea leaves are macerated, treated with tannase, fermented in the pres- EP1150575 B1,
Unilever N.V.,
2003 ence of hydrogen peroxide in an amount that is sufficient to activate endoge- EP1150575A1, [19]
Unilever Plc
nous peroxidases to oxidise gallic acid and other compounds that are liberated US6482450
by the tannase treatment, and then dried.
Recent Advances in Industrial Application of Tannases: A Review Recent Patents on Biotechnology, 2013, Vol. 7, No. 3 231

(Table 1) Contd….

Year Title Patent No Original Assignee Reference

Producing a tea extract:


2003 Aqueous extract of tea leaves, treated at a temperature of 20-65 °C. with tan- EP0777972A1 - [20]
nases whose glycoside part is covalently immobilized on an insoluble support

Process for the production of beer having improved flavour stability:


The invention relates to a process for producing beer having an improved
flavour stability by adding an oxygen scavenging enzyme prior to and/or dur- EP1252285,
2004 Quest International B.V. [21]
ing the mashing stage of the brewing. Wherein the oxygen scavenging enzyme US20030157217
is combined with another type of enzyme, (combination of laccase and tan-
nase)

Diagnostic and examination method for cancer of the colon using tannase
as indication:
2004 A diagnostic agent for colon cancer, which comprises a reagent for detecting a 20040137549A1 - [22]
tannase high-producing bacterium (Staphylococcus lugdunensis) or measuring
an amount of tannase contained in an intracolonic microflora sample.

Diagnostic agent and test method for colon cancer using tannase as index:
2006 A test method for colon cancer, which comprises the step of detecting US07090997B2 Eisai Co., Ltd. [23]
Staphylococcus lugdunensis contained in an intracolonic microflora sample

Process for producing purified green tea extract:


A process for producing a purified green tea extract including mixing a green
US20090081350,
2008 tea extract treated with tannase in a mixed solution containing an organic Kao Corporation [24]
EP2008525 A1
solvent and water at a mass ratio from 60/40 to 90/10 and separating a precipi-
tate thus formed.

Compositions comprising lactobacillus plantarum strains in combination


with tannin and new lactobacillus plantarum strains:
2009 The invention refers to a composition comprising one or more tannase- US07507572B2 - [25]
producing strains of Lactobacillus having the ability to adhere to the human
intestinal mucosa in combination with tannin.

Method for producing purified tea extract:


A process for producing a purified tea extract, comprising hydrolyzing (en-
zyme, a fungus or a culture having a tannase activity) a tea extract, allowing EP2189065 A1,
2010 Kao Corporation [26]
the tea extract to adsorb on an activated carbon, and then bringing a basic US20100184167
aqueous solution or an aqueous solution of an organic solvent into contact
with the activated carbon to elute non-polymer catechins.

Process for producing purified tea extract:


A first step comprises eliminating water-insoluble solids from a first tea ex- EP2225952 A1,
2010 tract and then obtaining a second tea extract. Kao Corporation [27]
US20100278972
Second step which comprises subjecting the second tea extract (non-polymer
catechins, 0.05 to 4.0 wt%) to tannase treatment.

Method for producing low-caffeine tea extract:


Takasago International
2010 A method for reducing a caffeine amount in a caffeine-containing tea extract US 20100055243 A1 [28]
Corporation
liquid without deteriorating the original flavor of tea.

Methods of reducing the inhibitory effect of a tannin of the enzymatic


hydrolysis of cellulosic material:
A method of producing a cellulosic material reduced in tannin content by EP2215242 A2,
2010 Novozymes Inc. [29]
treating the cellulosic material with an effective amount of tannase leading to US20090123979
reduce the inhibitory effect of tannin on enzymatic saccharification of cellu-
losic material
232 Recent Patents on Biotechnology, 2013, Vol. 7, No. 3 Beniwal et al.

(Table 1) Contd….

Year Title Patent No Original Assignee Reference

Process for the preparation of theaflavin-enhanced tea products:


EP2096937 A1, Unilever N.V.
2011 Process for preparing a theaflavin-enhanced tea product including a step of EP2096937 B1, [30]
subjecting a tea-source to enzymatic fermentation in the presence of added US20080131559 Unilever Plc
exogenous epicatechin.

Red wine liquid concentrate composition:

2011 High polyphenol red wine, having antioxidant activity produced by mixing 1.1 KR2010/008184 Hibio CO., Ltd [31]
to 2.0 wt% of enzymes (cellulase, pectinase, and tannase) with 98 to 98.9 wt%
of red wine liquid concentrate.

PRODUCTION OF GALLIC ACID CURRENT AND FUTURE DEVELOPMENTS


Tannase hydrolyzes tannic acid to glucose and gallic acid Tannase is a versatile enzyme finding application in a
[1]. Gallic acid is mainly used as a synthetic intermediate for number of industries. However, the major commercial appli-
the production of pyrogallol and gallic acid esters used in cation of the enzyme is limited to the manufacture of instant
foods, cosmetics, hair products, adhesives, and lubricant tea and production of gallic acid, which is used for the manu-
industry [45-47]. Gallic acid is extensively used as an ingre- facture of an antimalarial drug, trimethoprim and in the syn-
dient of developer in photography and printing inks. It also thesis of esters as propyl gallate used as antioxidants in the
serves as a precursor for the commercial production of an food industry. In spite of the publication of many studies that
anti-microbial drug-trimethoprim, a food preservative- deal with the technological potential of tannases, the real use
propylgallate and some dyestuffs [48,49]. Besides this, gallic of the enzyme remains limited. There are some studies indi-
acid possesses wide range of biological activities, such as cating the application of enzyme for the treatment of tannin-
antioxidant, antibacterial, antiviral, analgesic etc. As antioxi- rich effluents, such as wastewater from the leather industry.
dant gallic acid acts as an antiapoptotic agent and helps to However, efforts are required to overcome practical hurdles
protect human cells against oxidative damage. Gallic acid is such as inhibition of the enzyme or strains by various heavy
also found to show cytotoxic activity against cancer cells, metals present in the effluent. There is a possibility for the
without harming normal cells [50]. Antioxidants such as development of tannase based biosensor to determine the
gallic acid and prolygallate have been synthesized in fungal level of tannic acid/gallic acid in food industries as con-
SmC (submerged culture) [51,52] SSC (solid state culture) trolled level of tannic acid and gallic acid are known to be
[52] and in modified SSC [45] using microencapsulated tan- beneficial and higher level can develop some undesired
nase in chitosan and as free enzyme [46] or by mycelium- characteristics.
bound tannase in organic solvent like 1-propanol [47]. An
improved method of synthesizing gallic acid esters by incu- CONFLICT OF INTEREST
bating gallic acid with an alkyl alcohol or diol in the pres-
ence of an immobilized tannase was described [11]. The authors confirm that this article content has no con-
flicts of interest.
MISCELLANEOUS
ACKNOWLEDGEMENTS
Tannase may find use in cosmetology to eliminate the
turbidity of plant extracts and in leather industry to homoge- Declared none.
nize tannin preparation for high grade leather tannins [6].
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