Tannin acyl hydrolase acts on tannic acid and release its monomers namely gallic acid and glucose. Glucose reacts with dinitrosalicylic acid reagent to form colour component (3 amino 5 nitrosalicylic acid, red brown), which is measured spectrophotometrically at 575 nm and expressed as µM of glucose release per ml per h of rumen digesta.
Animals were supplemented with two types of diet containing 0 and 4 per cent taimin (tannic acid equivalent). Acacia nilotica pods was used as a source of tannin in the diet. After 21 days of preliminary feeding on experimental diets, rumen digesta (Strained Rumen Liquor) was collected at 0 and 4h post feeding to determine the tannin acyl hydrolase activity.
This enzyme is useful for the treatment of tea extract. It breaks down the turbidity complex in the tea extract to produce clearer and more delicious drinks with higher concentration of ingredients and to raise the high anti-oxidative activity. It has wide scope for the use in beverage and tannery industries also. The assay is easier, cheaper and less time consuming. Reagents
a) Citrate buffer (pH 5.5): Citric acid (obtained from SISCO Research Laboratories, AR extra pure, 99.7 %) 5.25 g was dissolved in 400 ml distilled water and pH was adjusted by adding 80 ml 1N NaOH (AR grade, obtained from S.D.Fine Chemicals, Ltd., Mumbai-400 025, India) solution.
b) Substrate (0.7 % Tannic acid): Tannic acid (T-0125) obtained from Sigma Company, USA was dissolved (0.7 g) in cool citrate buffer on the day of use and volume was made to 100ml with citrate buffer. Different substrate concentrations namely 0.3, 0.5, 0.6, 0.7 and 1.0 % tannic acid were tried and 0.7 % tannic acid was foimd optimum for tarmin acyl hydrolase activity.
c) DNS solution: Sodium hydroxide (5.0 g) was dissolved in 200 ml distilled water. Then 5.0 g DNS (Dinitro salicylic acid, LR grade, 97%, obtained from Qualikems Fine Chemicals Pvt. Ltd., New Delhi, India) and 1.0 g phenol crystal (obtained from BDH, a Division of Glaxo Laboratories Ltd, Dr. Annie Besant Road, Mumbai-18, India) was dissolved in the resultant solution and volume was made
to 500 ml with distilled water. Sodium sulphite (AR grade, obtained from Central Drug House Pvt. Ltd, Mumbai) of 0.05 per cent (0.25 g w/v) concentration was added just before use. Solution was kept in amber colour bottle to prevent the effect ©flight.
d) Na-K tartrate (40 %) solution: It was prepared by dissolving 40.0 g Na-K tartrate (obtained from E. Mereck Pvt. Ltd. Worli, Mumbai - 400018, India (AR grade, 99.0 % pure) in distilled water and then volume was made up to 100 ml.
e) Glucose solution (0.1 %): Glucose solution was prepared by dissolving 0.1g glucose (obtained from S.D.Fine Chemicals, Ltd., Mumbai-400 025, India.) in distilled water and volume was made up to 100 ml.
Collection of rumen digesta (strained rumen liquor) and sample preparation
Rumen digesta was collected at 0 and 4h post feeding using vacuum suction from rumen fistulated cattle fed on diet containing Acacia nilotica pods as a source of tannin. Immediately after filtration through muslin cloth, rumen digesta was collected in 100 ml plastic bottle and brought to laboratory in an ice chamber. Then 20 ml SRL (strained rumen liquor) was taken in a 50 ml capacity beaker and sonicated for 5 minutes. Then sample was centrifiiged at 8000 rpm at 4 °C for 15 min using K-24 centrifuge (Janetzki, Germany) and supernatant was kept in refrigerator at 4 °C until analysis. Analytical procedure
One ml cifrate buffer was taken in test tube and to it 0.5 ml sample (rumen digesta) and 0.5 ml substrate were added and incubated at 38°C for 40 minutes and then stopped reaction by adding 3.0 ml DNS solution. Similarly, control was prepared with exception that substrate was added after addition of DNS. Blank contained 2.0 ml distilled water and 3.0 ml DNS. Standard glucose was prepared by taking 0.25, 0.5, 0.75, 1.0,1.25 ml of 0.1 % glucose solution and volume was made to 2.0ml by adding distilled water and then 3.0 ml DNS solution. All the tubes were kept in boiling water for 15 minutes and then 1.0 ml Na-K tartrate solution was added to each tube. The content of the tube was cooled at room temperature and final volume was made to 20 ml with distilled water and OD was taken at 575 nm. OD of control was subtracted from test sample to get the actual reading.
Preparation of standard curve for Tannin acyl hydrolase
Two hundred milligram of lyophilize enzyme (500unit/g enzyme, obtained from
Kikkoman Corporation, Bio-chemicals Division, Edogawa Plant, 376-2, Kamihanawa,
Noda City, Japan) was dissolved in 10 ml cool citrate buffer to give a concentration of 10
unit/ml as a stock solution. From this 1, 2 , 3, 4, 5 and 6 unit/ml was prepared by taking
0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml mixture and diluted to 1.0 ml with citrate buffer. One ml
citrate buffer was taken in a test tube along with 0.5 ml standard tannin acyl hydrolase
from 1, 2, 3, 4, 5 and 6 unit/ml solution and then 0.5 ml substrate was added to each tube
and incubated in water bath at 38 °C for 40 minutes followed by processing of sample as
described above.
PROTOCOL
TEST: one ml citrate buffer + 0.50 ml sample (SRL) + 0.50 ml substrate and incubated
in water bath at 38°'C for 40 minutes + 3.0 ml DNS (to stop reaction) + Kept in
boiling (100°C) water bath for 15 min + 1.0 ml Na-K tartrate and cool at room
temperature. Volume was made to 20 ml and reading was taken at 575 nm.
CONTROL: one ml citrate buffer + 0.50 ml sample (SRL) + 3.0 ml DNS solution
(mixed by vortexed to deactivate enzyme action) + 0.50 ml substrate and
incubated in water bath at 38 °C for 40 minutes + Kept in boiling (100 °C) water
bath for 15 min + 1.0 ml Na-K tartrate and cool at room temperature. Volume
was made to 20 ml and reading was taken at 575 nm.
STANDARD: one ml citrate buffer + 0.50 ml tannase solution + 0.50 ml substrate and
incubated in water bath at 38 °C for 40 minutes + 3.0 ml DNS (to stop reaction)
+ Kept in boiling (100 °C) water bath for 15 min + 1.0 ml Na-K tartrate and
cool at room temperature. Volume was made to 20 ml and reading was taken at
575 nm.
BLANK: Two ml citrate buffer + 3.0 ml DNS solution + 1.0 ml Na-K tartrate solution.
Volume was made to 20 ml and reading was taken at 575 nm.
CALCULATION
Changed in OD = Test - Control
Enzyme activity (µM glucose/ml/h) = µg glucose/(T x S x 160)
Where, T = Time of incubation, S = Sample volume, 160 = Molecular weight of
glucose.

We claim that
1. The assay is easier, economic, less time and labour consuming and easily
reproducible, ii. Tannin acyl hydrolase breaks down the tannic acid into gallic acid and
glucose which gives a good colour (red brown) with assay solution
compared to that obtain by Rhodamine dye while estimating gallic acid. It is
difficult to get optimum colour with Rhodamine during gallic acid
estimation as experienced in present investigation, iii. Tannin acyl hydrolase activity is persisted in rumen digesta even without
pre-exposure to tannin containing diet, iv. Activity of the enzyme increased in the rumen digesta upon exposure to
taimin containing diet. V. Standard tannin acyl hydrolase was used to standardize the substrate
concentration (0.7% tarmic acid), vi. Tannin acyl hydrolase activity can be expressed in units/ml/h in presence of
standard tannin acyl hydrolase otherwise, expressed as µM glucose/ml/h in
absence of standard enzyme.