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IOSR Journal of Pharmacy

Vol. 2, Issue 1, Jan-Feb.2012, pp. 109-112

IOSR

GALLIC ACID PRODUCTION AND TANNASE ACTIVITY OF

PENICILLIUM PURPUROGENUM STOLL EMPLOYING

AGROBASED WASTES THROUGH SOLID STATE

FERMENTATION : INFLUENCE OF CARBON AND NITROGEN

SOURCES.

S. BHASKAR REDDY and VANDANA RATHOD*

Department of Microbiology, Gulbarga University, Gulbarga 585 106.

ABSTRACT

The influence of various carbon sources and nitrogen sources on tannase and gallic acid production in SSF by P.
purpurogenum BVG7 was studied. Sugars were beneficial only up to 0.2% concentrations and higher levels inhibited tannase
and gallic acid production. Amongst the various N compounds (NH 4 N0 3 , NH 4 C1, NaN0 3 , and KN0 3 ) supplemented to the
substrates, NH 4 N0 3 even at much lower concentration was the most beneficial even at lower concentrations than others for
gallic acid production.

INTRODUCTION

Use of microorganisms for producing gallic acid through suspended solid and submerged fermentations has been of late
receiving more attention since the product finds wide applications in pharmaceutical and chemical industries due to its varied
biological activities (antioxidant, anti-apoptotic, antibacterial, antiviral, analgesic etc.) and also being precursor of
trimethoprim, propyl gallate and some dyes (Pourrat et al, 1987; Mondal, 2001). Gallic acid production through the
mediation of tannase enzyme of microbial origin through either SmF or SSF utilizes the agrobased wastes, thereby reducing
environmental pollution as well as harnessing wealth from waste. Solid state fermentation is considered to be more
advantageous over the submerged fermentation (Aguilar et al., 2001). Adapting SSF necessitates the process of optimization
of fermentation parameters including physico-chemical parameters. Once the basic fermentation parameters like fermentation
period, moisture content, inoculum size, substrate concentration, /?H, and temperature are optimized, it becomes necessary to
attempt increases in product yields. Since the agrobased wastes are generally ill-defined substrates, supplementation of
various carbon and nitrogen sources, other organic substances like fatty acids, alcohols, acids, vitamins etc., have proved to
be beneficial in achieving higher product yields. The present report deals with the evaluation of influence of certain C and N
sources on tannase and gallic acid production by Penicillium purpurogenum BVG7 from agrobased wastes like Acacia pods,
redgram husk, sorghum husk and spent tea powder through SSF.

MATERIALS AND METHODS

The fungal strain, P. purpurogenum BVG7, was isolated from the soil samples in the vicinity of leather industries,
agricultural fields and cobbler's places and maintained on 2% malt extract slants at 32°C. The slants were subcultured
routinely at an interval of 4-5 weeks and the freshly grown slant cultures were used for the experimental studies. The
substrates used in the present study were Acacia pods, redgram husk, sorghum husk and spent tea powder. Each substrate was
dried, finely powdered in a mixer mechanically and then 15 g of each substrate was taken in a 50 ml Ehrlenmeyer flask. The
initial culture conditions maintained were moisture level of 70%, pH 5.5, and inoculation with 4 ml inoculum (2 x 10 7
spores/ml) and incubated at 30° C for 96 hr. In the studies involving the influence of carbon sources on the process of tannase
and gallic acid production, glucose, fructose, lactose and sucrose were separately added to each substrate in the varying
concentrations of 0.01, 0.05, 0.1, 0.2, .05, 1.0, 3.0, and 5.0 g/100 g substrate. In the studies pertaining to the influence of
nitrogenous compounds, ammonium nitrate, ammonium chloride, sodium nitrate and potassium nitrate were separately added
to each substrate in varying concentrations of 250, 500, 750, 1000, 1500 and 2000 mg/100 g substrate. Suitable controls
were maintained with moisture level of 70%, pH 5.5, and inoculation with 4 ml inoculum (2 x 10 7 spores/ml) and incubated at
30° C for 96 hr for each substrate. The results are the mean of five replicates.

Tannic acid estimation was as done per the protein precipitation method of Haggerman and Butler (1978) employing bovine
serum albumin (BSA) as the standard protein. Tannic acid content of the substrates and the fermentation medium were

ISSN: 2250-3013 www.iosrphr.org 109 I P a g e

IOSR Journal of Pharmacy

Vol. 2, Issue 1, Jan-Feb.2012, pp. 109-112

IOSR

estimated as per Ibuchi et al. (1967) and the per cent yield of gallic acid was calculated based on the estimation of available
tannic acid in the fermented medium.

RESULTS

The results on the influence of sugar supplementation in different concentrations to the four substrates on tannase activity and
gallic acid production of P. purpurogenum is presented in Fig. 1 and 2, taking the example of glucose supplemenatation.
Tannase activity and gallic acid production tended to increase gradually up to 0.2 g glucose/100 g substrate, while higher
supplementations of glucose sharply decreased both the enzyme activity and acid production. At 5.0 g glucose level, enzyme
activity and gallic acid production were almost inhibited. Same trend was observed in all the substrates. Enzyme activity and
gallic acid production were maximal in Acacia pods, followed by redgram husk and sorghum husk, the least being in the
spent tea powder. The supplementation of lactose and sucrose also led to similar observations, maximum tannase activity and
gallic acid production being recorded at 0.2 g/100 g substrate. Only in case of fructose, higher level of fructose
supplementation (0.5 g fructose/100 g substrate) was necessary to yield maximum enzyme and acid production (Tables 1 and
2). Higher levels of fructose additions sharply decreased the enzyme activity and acid production, as in case of other sugars.

The results on the influence of nitrogen supplementation in different concentrations to the four substrates on tannase activity
and gallic acid production of P. purpurogenum are presented in Fig. 3 and 4, taking example of NH4NO3 addition. Tannase
and gallic acid production tended to increase up to 500 mg NH4NO3/IOO g substrate, while higher supplementations of the
salt sharply decreased both the enzyme activity and acid production. At 2000 mg NH4NO3/IOO g substrate, enzyme activity
and gallic acid production were almost inhibited. Similar trend was observed in all the substrates. Enzyme activity and gallic
acid production were maximal in Acacia pods, followed by redgram husk and sorghum husk, the least being in the spent tea
powder. The supplementation of NaN0 3 and KN0 3 also led to similar observations, maximum tannase activity and gallic
acid production being recorded at 1000 mg/100 g substrate. But in sorghum husk, higher level (1500 mg/100 g substrate) of
NH4CI4 was required to effect maximum enzyme activity and acid production. Only in case of NH4NO3, a lower level of 500
mg/100 g substrate was required to cause maximum enzyme and acid productions (Tables 3 and 4).

DISCUSSION

The present study indicates that amongst the various sugars, glucose was the most beneficial sugar for the production of
tannase activity and gallic acid production by P. purpurogenum BVG7. The next beneficial sugar was lactose, with fructose
closely following and sucrose the least. All sugars were beneficial only up to concentrations of 0.2 g/100 g substrate and
thereafter in their higher concentrations, enzyme activity and gallic acid production tended to decrease sharply (Table 1 and
2). Earlier workers (Hadi et al., 1994; Aguilar et al, 2001a; Van de Lagemaat and Pyle, 2001; Fumihiko and Kiyoshi, 1975)
had employed 0.06 to 7.0% glucose and sucrose to promote tannase production by various fungal species and reported that
excepting glucose other sugars had no beneficial effects on the activities and hence, glucose appeared to be the most favored
of the sugars. Lekha and Lonsane (1997) too reported that the presence of other readily utilizable carbon sources did not
enhance tannase production of A. niger PKL 104. The decrease in tannase and gallic acid production by higher concentrations
of sugars appear to be due to the osmotic stress (Bradoo et al, 1977).

The nitrogen sources supplemented to the various substrates in the present study were all equally slightly beneficial to P.
purpurogenum BVG7 as far as tannase production is concerned (Table 3). But their influence on gallic acid production was
more pronounced. NH4NO3 was most beneficial even at 500 mg/100 g substrate concentration. Other forms of N sources
(NH4CI, NaN0 3 and KN0 3 ) to were beneficial to the organism under study but considerably at higher levels of 2000 mg/100
g substrate for gallic acid production (Table 4). Earlier workers reported NH4NO3 to be a better nitrogen source for tannase
production since the fungal species were able to utilize N from both NH 4 and N0 3 (Nagib and Saddik, 1960; Bradoo et al.,
1997; Lekha and Lonsane, 1997; Shamina Begum, 2006). Battestin and Macedo (2007) too reported that NH4NO3 had
significant influence on tannase production than KN0 3 . In the present study too, NH4NO3 not only stimulated tannase and
gallic acid production at lower concentrations but also proved to be more beneficial than other inorganic N sources.

On the whole it can be concluded that glucose was the most favored C source and NH4NO3 was the most favored N source
for the tannase and gallic acid production respectively, by P. purpurogenum BVG7 from the various agrobased substrates
through suspended solid fermentation.

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Vol. 2, Issue 1, Jan-Feb.2012, pp. 109-112

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Table 1. Influence of C sources on Tannase activity in different substrates in SSF

Sugars, mg/100 g
substrate

Acacia pods

Redgram husk

Sorghum husk

Spent tea powder

Glucose, 0.2 mg

46.5+1.2

44.0+1.8

43.5+2.1

38.0+1.2

Fructose,0.2 mg

43.5+1.0

43.0+1.9

42.0+2.0

35.5+1.0*

Lactose, 0.2 mg

45.5+2.3

44.0+1.1

43.7+2.1

37.5+1.7

Sucrose, 0.2 mg

44.1+1.0

43.7+1.7

43.0+2.3

36.7+2.0

Control

40.5+1.4

39.0+1.4

38.5+2.1

30.0+1.2

* at 0.5 mg/100 g substrate

Table 2. Influence of C sources on Gallic acid production in different substrates in SSF

Sugars, mg/100 g
substrate

Acacia pods

Redgram husk

Sorghum husk

Spent tea powder

Glucose, 0.2 mg

85.7+2.3

83.0+1.2

82.0+1.5

78.5+2.2

Fructose,0.2 mg

79.7+1.9

77.0+1.2

75.0+1.5

65.8+2.5*

Lactose, 0.2 mg

82.7+1.2

82.0+1.9

81.0+2.5

73.5+2.6

Sucrose, 0.2 mg

80.7+2.5

79.0+1.2

77.0+1.9

69.5+2.0

Control

70.7+1.8

69.0+1.7

68.4+1.5

58.5+2.2

at 0.5 mg/100 g substrate

Table 3. Influence of N sources on Tannase activity in different substrates in SSF

mg/100 g
substrate

Acacia pods

Redgram husk

Sorghum husk

Spent tea powder

Amm. chloride,
1000 mg

42.5+1.4

41.0+1.4

40.5+1.2*

36.0+1.2

Amm. Nitrate,
500 mg

41.2+2.2

40.5+2.0

40.9+1.0

36.0+1.2

Sod. nitrate,
1000 mg

42.5+1.4

41.0+1.4

40.5+1.2

36.0+1.2

Pot. nitrate,
1000 mg

43.0+1.4

41.0+1.4

39.5+1.2

36.0+1.2

Control

40.5+1.4

39.0+1.4

38.5+2.1

30.0+1.2

at 1500 mg/100 g substrate

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Table 2. Influence of N sources on Gallic acid production in different substrates in SSF

mg/100 g
substrate

Acacia pods

Redgram husk

Sorghum husk

Spent tea powder

Amm. chloride,
1000 mg

77.7+1.8

75.0+1.7

74.3+1.9*

62.5+2.2

Amm. Nitrate,
500 mg

75.0+1.9

77.0+1.2

75.0+1.5

65.8+2.5

Sod. nitrate,
1000 mg

73.7+1.8

71.0+1.7

70.3+1.9

62.5+2.2

Pot. nitrate,
1000 mg

74.7+1.8

69.0+1.7

67.3+1.9

60.5+2.2

Control

70.7+1.8

69.0+1.7

68.4+1.5

58.5+2.2

at 1500 mg/100 g substrate

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