www.ijapbc.com IJAPBC – Vol.

3(4), Oct - Dec, 2014 ISSN: 2277 - 4688

INTERNATIONAL JOURNAL OF ADVANCES IN

PHARMACY, BIOLOGY AND CHEMISTRY
Research Article

Optimization of process parameters for production

of Gallic acid by submerged fermentation using
Natural tannins of Anacardium occidentalis L. testa
Lenin Kumar Bompalli1*, Lokeswari Nallabilli2.
1
Department of Biotechnology, Dr. B. R. Ambedkar University, Etcherla, Srikakulam Dist.,
Andhra Pradesh, India - 532410.
2
Department of Biotechnology, Dr. B. R. Ambedkar University, Etcherla, Srikakulam Dist.,
Andhra Pradesh, India - 532410.

ABSTRACT
Gallic acid has enormous application in many fields and is regarded as a non toxic substance to man.
Furthermore, considerable health promoting effects have been ascribed to gallic acid. This work demonstrated
the ability of microorganism to utilize cashew testa and crude intermediate product to produce gallic acid in a
submerged fermentation system. Under optimal time and temperature tannins experiment was carried out, and
estimation of concern byproduct was estimated.

Keywords: Microorganism, Gallic acid, Submerged Fermentation and Tannins.

INTRODUCTION
In developing countries, huge quantities of agro about the role of some of the hydrolysable tannins
industrial residues are continuously being generated constituents on the synthesis of tannase. For instance,
and their disposal is associated with several gallic acid, one of the structural constituents of some
environmental problems. Utilization of these agro hydrolysable tannins, such as tannic acid, has been
residues and byproducts of agro industries as nutrient reported as an inducer of tannase synthesis under
sources for microbial tannase production may reduce submerged fermentation4. Gallic acid is used in the
the final enzyme production cost, which is one of the enzymatic synthesis of gallic acid esters, e.g., propyl
major challenges affecting the large scale production gallate, which is used mainly as an antioxidant in fats
of enzymes5. A number of reports given by different and oils, as well as in beverages2. Ethanolic extracts
workers showed the use of the liquid surface, (gallic acid and ellagic acid) of the leaves and stems
submerged (SmF) or solid state fermentation (SSF) of Banaba ( Lagerstroemia speciosa L.), has been
for the production of tannase. The submerged used as traditional medicines and are effective in
fermentation is mostly preferred for sterilization and controlling diabetes and obesity and inhibits HIV-1
process-control. But this technique is not only infection6. The present work is exploring towards the
expensive but also energy intensive, hence solid state utilization of waste cashew testa into useful industrial
fermentation (SSF) is the alternative method, since product by using microbial fermentation.
obtained levels of tannase are higher on solid Hence the present study was focused on (i) extraction
substrates9. Phenolic compounds such as gallic acid, of tannins from industrial waste Anacardium
pyrogallol, methyl gallate and tannic acid induces occidentalis testa by solvent extraction method (ii)
tannase synthesis. The induction mechanism has not Utilization of tannin extract for production of gallic
been demonstrated and there is some controversy acid through microbial submerged fermentation.

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MATERIALS AND METHODS Estimation of Gallic acid:

Sample Collection: Spectrophotometic method was used by using
Anacardium occidentale testae were collected from methanolic rhodanine for the estimation of gallic acid
the cashew industries in Palasa, srikakulam district at 520nm by spectrophotometer (Shimadzu UV-
and Visakhapatnam, Visakhapatnam district, Andhra 1800)8.
pradesh, India.
RESULTS & DISCUSSION
Sample Preparation: Capable of moving with high speed industrialization
Samples were collected fresh and they were and increasing population in India have contributed
conserved, freeze-drying is the gentlest method of to an increased generation of variety of solid waste.
preservation and is recommended instead of freezing Currently in India, about 960 million tones of solid
and air or oven drying. waste is being generated annually as by-products
during industrial, mining, municipal, agricultural and
Sample handling: other processes. In India more than 40% of solid
After collecting the samples, Samples were stored in waste generated annually is from organic and
the absence of heat, and placed in a dark container. agricultural sources. There is a growing concern for
these accumulating wastes as they are either being
Sample extraction: dumped in landfills, burnt, or left to rot in the open,
The dried testa of cashew husk was crushed using leading to severe environmental pollution. A
ball mill to get the particular size below 5mm. 4g of sustainable solution would be to utilize these solid
the powder was added to a bottle and extracted by wastes as an ‘economical’ alternative to costly raw
reflux in 50ml 60% methanol for 2 hours. The materials and produce industrially important products
mixture was filtered, and the filtrate was collected. of practical utility7. Tannase has now been
The extract was then Concentrated to dryness by extensively used in different biochemical industries.
rotary vaporization at 30ºC under reduced pressure The selected bacterium used in this study is able to
and a light brown powder was obtained. synthesize high amounts of tannase through
fermentation of crude tannin of A. Occidentale.
Submerged Fermentation: Exploitation of these plant extracts could be a source
Obtained extracted powder (sample) was used in of cheaper substrate for industrial production of
submerged fermentation by using modified Czapek’s microbial tannase.
Dox medium. By studying the parameters time and temperature that
enhancing the extract of tannins from testa, we found
Composition of modified Czapek’s dox medium: that the maximum tannins was extracted from solvent
(pH 5.5) extraction method (5.83 mg/ml) in the presence of
Glucose 1% w/v 60% methonol.
Pot. Di hydrogen phosphate 0.5% w/v The optimum time period for maximum tannin
Pot. Chloride 0.3% w/v extraction was 40 min (Table 1). Extraction of
Magnesium sulphate 0.1% w/v tannins has increased with increase in time up to 40
Ferrous sulphate 0.01% w/v minutes, with further increase in time decreased
Zinc sulphate 0.1% w/v tannins content.
Sodium Nitrate 0.1% w/v Temperature 50ºC was optimum for extraction of
5.83mg/ml concentration of tannin with the optimum
Effect of time of incubation: temperature condition (Table 2). High temperature
These experiments were conducted to find out the results less concentrations of tannin and it promotes
effect of time of incubation period on production of hydrolysis and unwanted extraction of hemicelluloses
tannase and gallic acid. After inoculation, the and cellulose materials. Obtained tannin extract was
different incubation periods were observed by used for the production of gallic acid by using
changing the incubation time between 12 hrs to 84 microbial fermentation. By using this method
hrs. The flasks were incubated at 35°C and samples 30.12mg/ml (Table 3) gallic acid was obtained for 48
were withdrawn periodically at every 12 hrs3. hours of fermentation with 8% substrate
concentration, 120 rpm agitation speed at room
Estimation of Total Tannins: temperature.
Tannin estimation was done by following the protein The optimum period 48hrs was found for the
precipitation method for the quantitative production of gallic acid (Table 3). Further extension
determination of tannins as described by Hagerman of time, the minimum production of gallic acid
and Butler1. concentration was observed at 84hrs incubation

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(Graph 1). The production of gallic acid has Thus this gallic acid production from this waste
increased with increase in incubation time up to cashew testa would help the country to produce gallic
48hrs, with further increase in the incubation time, acid from cheep resources. Plenty of such efficacious
decrease production was observed. Decreased yield raw materials ensured the possibility for exploitation
on prolonged incubation could also be due to of this microbial fermentation in large-scale
inhibition and denaturation of the intermediate production of gallic acid.
product in microbial submerged fermentation system.

Table 1
Optimisation of the time period for the extraction of tannins from cashew testa.

Time period (min.) Tannin (mg/ml)

15 4.79
20 4.82
25 4.99
30 5.14
35 5.47
40 5.82
45 5.65
50 5.41

Table 2
Optimisation of temperature for extraction of tannins from cashew testa.
Temperature (ºC) Tannin (mg/ml)
30 5.12
35 5.23
40 5.53
45 5.62
50 5.83
55 4.89
60 4.61

Table 3
Effect of incubation period on production of gallic acid.
Incubation period (hrs.) Gallic acid con. (mg/ml)
12 22.82
24 26.15
36 28.62
48 30.12
60 28.77
72 26.19
84 23.34

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35

30

25

20

15
Gallic Acid
10 Concentration (mg/ml)

0
12 24 36 48 60 72 84

Incubation Period (hrs.)

Graph 1
Effect of incubation period on gallic acid concentration.

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