FIELD OF INVENTION
The invention relates to a process for the preparation of gallic
acid by co-culture.
BACKGROUND OF THE INVENTION
Enzymes are specific proteins of living tissues that act as
biocatalysts. Enzymes can accomplish those reactions at normal
temperatures and pressure which would otherwise require expensive,
energy demanding high temperature and/or pressures, or might not
be possible at all. That is why, use of enzymes in industries is
growing.
Many micro-organisms produce extra-cellular enzymes. They are
chiefly hydrolasesand are involved primarily in the degradation of
macromolecules to units capable of being taken into the living cell.
With the versatility of micro-organisms in producing enzymes, new
methods of making many industrially important chemicals are being
explored.
Current developments in biotechnology are yielding new
applications for microbial enzymes. In addition to the conventional
applications in food and fermentation industries, microbial enzymes
have attained significant role in biotransformations involving
organic solvent media specially bioactive compounds. Alongwith
the use of micro-organisms to produce biomass and microbial
metabolites, microbial cells may be used to catalyse the conversion
of a compound into a financially more valuable compound.
Microbial processes have the advantage of specificity over the use of
chemical reagents and of operating at relatively low pressures and
temperatures.
An important enzyme having various industrial applications is
tannase (tannin acyhydrolase), which is an extra-cellular enzyme
falling under the hydrolase group of enzymes.Tannins are water-
soluble, phenolic compounds with molecular weight (500-3000) that
have the property of combining with proteins, cellulose, gelatin and
pectin and can be classified into two distinct groups based on their
structural configuration, the hydrolysable tannins and the condensed
tannins.
Tannase is widely used in the food and chemical industries. Further,
tannase is used in wine making where it hydrolyses chlorogenic acid
and quinic acid, which favourably influences taste in the process of
wine making. Tannase also has potential in the manufacture of acom
wine. Tannase is also used alongwith lactase to treat grap juice and
grape musts
So as to remove phenolic substances for stabilization of the
beverage. Tannase significantly reduces chill haze formation in beer.
Disclouration and haze development during beer storage could be
prevented by the enzymatic hydrolysis of wort phenolics with
tannase.
Further, tannase is used in a preconversion treatment of fresh green
tea flush in the production of instant tea. Tannase is also used in the
determination of the structure of naturally occurring gallic acid
esters.
Tannin acyl hydrolase commonly called tannase catalyzes, the
hydrolysis of ester and depside bonds in such hydrolysable tannins
as tannic acid, thereby releasing glucose and gallic acid.
Gallic acid (3,4,5-Trihydroxybenzoic acid) finds various uses. In the
pharmaceutical industry, gallic acid is used in the manufacture of
trimethoprim. It is an antibacterial and is administered jointly with
sulphonamide and together provide a broad spectrum action for
medical treatment. TMP inhibits dihydrofolate reductase thereby
blocking transformation of dihydrofolate to tetrahydrofolate. The
consumption co-efficient of gallic acid in the manufacture of
trimethoprim is 4.8. In the tannery industry gallic acid is used for
homogenization of tannins, for the preparation of high grade leather
tannins. Gallic acid is also used in the manufacture of ordinary
writing inks and dyes, as a photographic developer. It also finds use
in the enzymatic synthesis of gallic acid esters like propyl gallate,
which is mainly used as an antioxidant in fats and oils, and also in
beverages. It is used in the testing of mineral acids, dihydroxy
acetone, and alkaloids, and as a synthetic intermediate for the
production of pyrogallol which is used for the production of
pyrogallol which is used for staining fur. leather, hair, etc.
Use of various fungal species to produce tannase is blown in the art.
A number of micro-organisms including bacteria like {Bacillus
pumilis, B. polymyxa, Corynebacterium sp., Klebsiella pneumoniae)
fungi (Ascochyta sp. Penicillium sp.) & yeasts (Candida sp.) have
been reported to produce tannase .
Production of tannase by a strain of Aspergillus niger was reported
by Tourrat et al. They found tannase activity was maximum when
fermentation was carried out in submerged culture at a constant air
flow. The enzyme activity was determined by gas chart method and
the maximum enzyme activity was reported to be 5.5 n Kat/ml.
R. Banerjee et.al, has reported the activity of tannase biosynthesis by
a newly isolated R. oryzae. The experimental conditions were
optimized in shake flask cultures. Maximum enzyme activity was
found to be 6.12 U/ml.
R. Banerjee et al have also reported the production of tannase by solid
state fermentation using R. oryzae.
Production of extracellular tannase by bacterial strains (B. polymyxa,
B. puminis, klebsiclla pnon & Corynebacterium) within few hours of
culture with simultaneous release of gallic acid & glucose has been
reported by Deschamp et al.
Raj Kumar et al. have reported the isolation, purification and some
properties of Penicillium chrysogcnwn tannase.
The enzyme is stable up to 30 C and within the pH range of 4 0 - 6.0
Km value was found to be 0.48 X 10 M with tannic acid as the
substrate. Metal salts at 20 Mm inhibited with enzyme.
Continuous production of gallic acid from tara tannin in a bioreactor
using Penicilium chrysogenum immobilized on sodium alginate &
CaCl2 is known from Yamada et al. They have also reported the use of
tannase in nine making industry.
Not much is known about the biotransformation of tannins to gallic acid.
The literature available mainly relates to chemical process wherein the yield
of gallic acid is was very low. Kan B. et al have reported the
biotransformation of tannins to gallic acid by SSF and SmF process using
gallo seed cover and Rhizopus oryzae as the raw material.
However, the processes of the art suffer from various drawbacks ie they
require a long time and tlie yield reported is also low.
OBJECTS OF THP; INVENTION
It is therefore an object of this invention to propose a process for the
preparation of gallic acid using mixed culture process which requires less
time compared to conventional techniques
It is a further object of this invention to propose a process for the preparation
of gallic acid using mixed culture technique which gives better yield and
employs a novel substrate.
DESCRIPTION OF THE INVENTION
Thus according to this invention is provided a process for the preparation of
gallic using co-culture, comprising providing a tannin-rich mixed substrate
and a culture medium in fluid communication with each other, adding an
induced innoculum comprising the fungi Rhizopus oryzae and Aspergillus
foetidus to the substrate to obtain a fermented mass and gallic acid,
extracting the gallic acid from the culture medium and the fermented mass
using an organic solvent followed by evaporation of the organic solvent to
obtain gallic acid.
In accordance with this invention, the raw materials used as mixed substrates
are myrobalan fruit powder (Terminalia chebula, seedless type, PUNJAB
variety) and gallo seed cover powder (Caesalpinia digyna) in a proportion of
1:3 to 3:1. These are substrates rich in tannin. Myrobalan seed contains 33-
45% tannin and gallo seed cover contains about 58% tannin. Two strains of
the filamentous fungi Rhizopus oryzae (RO IIT RB13,NRRL-21498) and
Aspergillus foetidus (GM.RB013), isolated from the soil of IIT. Kharagpur
Campus have been used. The organisms are maintained on 2% malt extract
agar slant. A subculture of the micro-organisms is done on slants. A malt
extract agar (MEA) medium is sterilized and a portion of it is transferred
into sterile test tubes and allowed to cool in slanting position.
After solidification, the sterile slants are inoculated with pure cultures
oWhizopus oryzae (RO III RBI3), NRRL-21498) and Aspergillus foetidus
(GMRR 013, MTCC 3557) and kept in an incubator. The slant cultures are
then used for further work or stored. As tannase is an adaptive enzyme, a
pre-induced innoculum for the culture is prepared wherein tannic acid in
modified Czapekdox medium is sterilized and inoculated with a spore
suspension prepared from the cultured slants They are then kept in a BOD
incubator under shaking, to produce the induced innoculum. For subsequent
studies of MSSF (Modified Solid State Fermentation), this induced
innoculum is used.
The two microorganisms were used together where the ratio of the
organisms varied from 0.5:2 to 1:1, where the optimum ratio was 1:1. It was
found in general, that upon carrying out the co-culture technique, the tannase
and gallic acid produced was higher than that produced under pure culture
conditions, and also the incubation period required was shorter.
Fermentation is carried out in batch process u nder modified solid state
fermentation in a modified tray type reactor. The raw materials used as
mixed substrates are myrobalan fruit powder and gallo seed cover powder
taken in a proportion of 1:3 to 3:1. This substrate mixture is placed on the
float of the tray reactor. The tray reactors ordinarily used are modified
byproviding a perforated float. This offers the unique advantages of greater
heat dissipation during fermentation which prevents the biomass from
getting denatured. Liquid modified Czapekdox medium in the ratio of 0.2:1
to 0.8:1 (solid-liquid ratio) is taken beneath the float in the tray.Czapekdox
medium is modified by using tannic acid as the carbohydrate source, instead
of glucose. Thus, the mixed substrate placed on the float comes in contact
with the liquid medium in the tray. The modified tray reactor is then
autoclaved. Fermentation of the substrates on the float is carried out by
adding an appropriate amount of induced innoculum of Rhizopus oryzae and
Aspergillus foetidus, ie by co-culture method about 1 X 10' to 2 X 10s
spores per ml, preferably 2 X 106 spores per ml is added to 20 gms of the
substrate. The micro-organizm added to the substrate converts the substrate
into the desired product, which leaches into the liquid medium. The
fermented material is removed, water is added and heated. It is then cooled
to room temperature and gallic acid is extracted using an organic solvent
such as diethyl ether and ethyl acetate.
The optimum conditions of pH, temperature, humidity and the effects of
quantity of innoculum, particle size and moisture on the production of gallic
acid have been evaluated.
The invention will now be explained in greater detail with the help of the
following non-limiting examples:
Example
Isolation of tannase producing microorganisms
Two strains of filamentous fungi Rhizopus oryzae (RO III RBI3, NRRL-
21498) and Aspergillus foe tidus (GMRB013) were isolated from the soil of
I.I.T., Kharagpur campus by a baiting technique. The organisms were
maintained on 2% malt extract agar slant.
SUBCULTURE OF MICROORGANISMS ON SLANTS:
100 ml of malt extract agar (MEA) medium was sterilized. After
sterilization, 5 ml of it was transferred into sterile test tubes and allowed to
cool in slanting position. After solidification, the sterile slants were
inoculated with pure cultures of Rhizopus oryzae (RO IIT RBI3, NRRL-
21498) and Aspergillus foetidus (GMRB 013) m a 1:1 ratio by streaking and
was kept in incubator at 30°C for (72-96) his.
The slant cultures were then used for further work or stored in fridge at 4°C.
PREPARATION OF INDUCED INOCULUM
Tannase being an adaptive enzyme, pre-induced inoculum is required to be
prepared. 50 ml of 2% tannic acid in Czapekdox medium was taken in
100ml conical flasks. It was then sterilised at 12i°C for 15 mins and
inoculated with 2ml of spore suspension prepared from the cultured slants.
They were then kept in BOD incubator at 30°C under shaking condition for
72 hrs. For subsequent studies of SSF, this induced inoculum was used.
MODIFIED SOLID STATE FERMENTATION USING
MIXED SUBSTRATES BY CO-CULTURE METHOD
Fermentation was carried out in batch process under modified solid state
fermentation in a modified tray type reactor.
The raw materials used as mixed substrates where myrobalan seed powder
{Terminalia chebula) and gallo seed cover powder (Caesalpinia digyna)
taken in a proportion of 1:3 to 3:1. This substrate mixture was placed on the
float of the tray reactor. Liquid Czapek dox medium in the ration of 0.2:1 to
0.8:1 (solid-liquid ratio) is taken beneath the float in the tray. This kind of
float with the substrate is kept above the liquid in the tray in order ro carry
out modified solid state fermentation. Thus, the mixed substrate placed on
the float comes in contact with the liquid medium in the tray. The modified
tray reactor is then autoclaved at 121°C for 15 mins. Fermentation of the
substrates on the float is carried out by adding 2 X 106 spores/ml of induced
inoculum of Rhizopus oryzae and Aspergillus foetidus per 20 gms of
substrate ie. By co-culture method. The microorganism added to the
substrate converts the substrate into the desired product, which leaches into
the liquid medium.
GALLIC ACID EXTRACTION
Gallic acid was isolated using the organic solvent ethyl acetate. The
fermented material was removed, water added, and heated to about (60-
70)°C because gallic acid is soluble at this temperature, and not in cold
water. Then it was cooled to room temperature. Organic solvent was then
added to it and the whole mixture was taken in a separating funnel. The
mixture was immediately mixed thoroughly by vigorous shaking. Gallic acid
being soluble in organic solvent, comes into the organic phase and the rest of
the matter remains in aqueous phase. The aqueous phase was discarded and
the organic layer was collected. This process was continued till the entire
gallic acid came out into the organic layer. The collected volume of organic
layer was now taken for separation of gallic acid from ethyl acetate in the
rotary vacuum evaporator. The pressure and temperature at which this
separation was done was 200 mbar and 70°C. Alternatively, the ethyl acetate
layer was extracted with diethyl ether, the ether layer was evaporated in a
rotary evaporator to obtain pure gallic acid. The yield of the gallic acid
obtained was found to vary from 65.4 to 94.8%. Studies were conducted for
optimization of various environmental parameters for obtaining maximum
gallic acid production. —_------------------------------------------------------_,
OPTIMIZATION OF PHYSICOCHEMICAL PARAMETERS FOR
TANNASE AND GALLIC ACID PRODUCTION BY MODIFIED SOLID-
STATE FERMENTATION:
The effect of the various environmental parameters on the production of
tannase and gallic acid upon carrying out MSSF by both the organisms using
mixed substrates under both pure culture and co-culture conditions was
studied:-
Effect of pH - The effect of pH on production of tannase was studied by
varying the intitial pH of the Czapekdox medium from 3.5 to 7.0. The
optimum pH was found to be 5, at which tannase activity was 35. lU/ml and
gallic acid produced was 91.81%.
Effect of temperature :- The effect of temperature on tannase and gallic
acid production was studied by varying the temperature in the humidity
cabinet from 25°C to 40°C where the optimum temperature was found to be
30°C. The amount of tannase and gallic acid produced was 36.4 U/ml and
93.25% respectively.
Effect of humidity :- The effect of humidity on tannase production and %
yield of gallic acid was studied by varying the humidity in the humidity
chamber from 70% to 90% where the optimum lies between 80%. The
amount of tannase and gallic acid produced was 36.4U/ml and 93.25%
respectively.
Effect of inoculum amount:- The optimum amount of inoculum required
for the maximum tannase production was obtained after varying the
amount of inoculum from 1 ml to 4 ml with optimum at 3 ml.
Effect of moisture:- To study the effect of moisture on production of
tannase, addition of Czapekdox medium to inyrobalan substrate was varied
from 0.2:1 to 0.8:1 where optimum lies to 0.4:1. Tannase and gallic acid
produced were 35.3 U/ml and 92.41 respectively at this moisture condition.
An incubation period of 48h was found to be the optimum for Gallic acid
production by co-culture method using mixed substrates, giving a tannase
activity of 33.1U/ml.
A comparison between pure culture and co-culture methods shows the
following advantages of the co-culture method as highlighted in Tab lei.
WE CLAIM:
1. A process for the preparation of gallic acid using co-
culture comprising providing s tannin-rich mixed substrate
comprising myrobaian fruit powder (Terminal ia chebula) and galla
seed cover powder (Caesalpinia digyna) in a proportion of 1:3
to 3:1 and a culture medium such as a modified CzapeKdox medium
in fluid communication with each other, adding an induced
innoculum comprising the fungi Rhsizapuss arysae and Aspergillus
foetidus in a ratio of .6.5: 2 to 1:1, to the substrate and
o
incubating at a temperature in the range of 25 to 40 C, to obtain
a fermented mass and gallic acid* extracting the gallic acid from
the culture meoiuas and the fermented jjass, using an organic
solvent such as ethyl .acetate, followed by evaporation of the
organic solvent to obtain gallic acid.
2. The process as claimed in cl3is lt wrsereirs said culture
medium is a modified Czapefcdox medium.
3. The process as claimed in. claia 1, wherein the modified
Czapekdox medium comprises tannic acid as the carbohydrate
source.
4. The process as claimed in claia 1, wherein the substrate
and culture meaxuffl are present in a ratio of 0.2:1 to 0.8:1.
5. The process as claimed in- claisa 1 wherein the fungi are !
maintained on a 27, malt extract agar slant.
6. The process as claimed in claiss i. wherein the induced
innoculum is obtained by providing a tannic acid substrate in
Czapekdox medium, sterilizing the same in a known manner fcilowed
by inoculation of the substrate with a spore suspension of the
fungi Rhizopus oryzae and Aspergillus foetidus, to obtain, an
inoculated substrate incubating tne inoculated substrate to
obtain an incuced innoculum.
7. The process as claimed in claiiH: i, wherein an induced
5 S
innoculum containing 1 X 10 spares/mi to 2 X IB spares per
ml is added to 20 gms of the mixed substrate.
8. The process as claimed in claim: 1, wherein gallic acid is
extracted from the liquid snediuffi with an organic solvent such as
ethyl acetate.
9. The process as claimed in dais 1, wherein gallic acid is
obtained from the fermented biomass fey adding water thereto,
o
followed by heating at a temperature in the range of 60-70
to obtain s.n aqueous solution of gallic: acid, extracting the
aqueous solution of gallic acid with an organic solvent to obtain
a solution of the gallic acid in the organic solvent, followed by
evaporation of the solvent to obtain gallic acid.
10. The process as claimed in ciaisR 9t therein the solution
of gallic acid in organic solvent is further extracted with a
solvent such as diethyl ether to obtain an ether solvent of
gallic acid followed by evaporation of ether to obtain pure
gallic acid.
11. A process for the preparation of gallic acid' using co-
culture substantially as herein described.

This invention relates to a process for the preparation of
gallic acid using co—culture comprising providing a tannin—rich
mixed substrate comprising myrobalan fruit powder (Terminalia
chebula) and gallo seed cover powder (Caesalpinia digyna) in a
proportion of 1:3 to 3:1, and a culture medium such as a modified
Czapekdox medium in fluid communication with each other, adding
an induced innoculum comprising the fungi Rhizopus oryzae and
Aspergillus foetidus in a ratio of 0.5:2 to 1:1, to the substrate
and incubating at a temperature in the range of 25 to 400 C, to
obtain a fermented mass and gallic acid,, extracting the gallic
acid from the culture medium and the fermented mass, using an
organic solvent such as ethyl acetate, fallowed by evaporation
of the organic solvent to obtain gallic acid.