INTRODUCTION
Tannin acyl hydrolyase (EC, 3.1.1.20) commonly called tannase is present in
plants, animals and microorganisms, it is mainly produced by several microorganisms
like fungi (Aspergillus, Penicillium, Rhizopus sp.,
Trichoderma viride, Mucor sp.), yeast (Candida sp., Saccharomyces
cerevisiae, Mycotorula japonica) and bacteria (Bacillus sp.,
Corynebacterium sp., Streptococcus bovis, Klebsiella pneumoniae,
Selenomonas ruminantium) (Ayed and Hamdi, 2002;
Nishitani and Osawa, 2003). Tannase can be catalyzes
the hydrolysis of ester and depside bonds in hydrolysable tannins, releasing
glucose and gallic acid (Aguilar et al., 2007;
Aguilar and Gutierrez-Sanchez, 2001). Tannase is extensively
used in the industrial processes such as; clarification of fruit juices, manufacture
of coffee-flavored soft drinks, manufacture of instant tea and as an analytical
probe for determination the structure gallic acid esters (Seth
and Chand, 2000; Srivastava and Kar, 2010). Gallic
acid possesses wide range of biological activities, such as antioxidant, antibacterial,
antiviral, analgesic etc. As antioxidant gallic acid acts as an antiapoptotic
agent and helps to protect human cells against oxidative damage. Gallic acid
is also found to show cytotoxic activity against cancer cells, without harming
normal cells (Bajpai and Patil, 2008).
The present study, reports the production of tannase from Aspergillus niger
in liquid and solid culture medium by submerged fermentation and solid-state
fermentation technique. Some properties of tannase have also been studied.
MATERIALS AND METHODS
Fungal strain: Asperigillus niger was used for screening its
ability of utilizing tannic acid as main carbon source for production of fungal
tannase. Asperigillus niger was obtained from the Laboratory of Plant
Pathology of National Research Center, Cairo, Egypt.
Microorganism maintenance and inoculum preparation: Asperigillus
niger was grown on potato dextrose agar medium (PDA). It grows rapidly at
room temperature 25 to 37°C. The slant cultures were then used for further
work or stored in refrigerator at 4°C. Malt medium containing malt extract
(30 g L-1); yeast extract (5 g L-1) was used for preparing
the activated fungal inocula; Tannic acid Powder Medium (TAPM) recommended by
Lekha and Lonsane (1994) and Bradoo
et al. (1997) was used for growth and tannase production.
Two media were used for tannase enzyme production the first medium (submerged
fermentation), composition was as following (g L-1) malt extract
30.0, tannic acid 20.0 and adjusted to pH 5.0. The second medium (solid stat
fermentation), composition was as the following of ammonium sulphate 1.7 %,
sodium chloride 0.1 %, sodium phosphate 2.0 %, PKP 25% and adjusted pH to 5.0.
Spores of fungi were transferred from surface of the actively growing slants
of (PDA) medium to 250 mL. conical flasks which contained 50 mL of malt medium.
After incubation on a rotary shaker (120 rpm) at 32°C for 48 h, the grown
cultures were employed as inocula for experimental flasks (250 mL) contained
the previous media at rate of 2% (V/V) inoculum size.
Optimization of fermentation process for tannase enzyme production:
Enzyme production was carried out in 250 mL conical flasks containing 50 mL
each from the previous two media. Static cultures were used for studying fungal
tannase production under variable environmental condition as follows.
Effect of inoculum ratio: Inoculum ratios ranged from 1 to 8% (V/V)
were used with the tested fungal cultures.
Effect of incubation period: Tannase assay was performed after various
incubation periods i.e., 1, 2, 3, 4, 5 and 6 days (24 to 144 h) and the tannase
activity was determined according to Mondal et al.
(2001).
Effect of initial pH: The influence of different initial pH values was
studied through adjusting pH values at 3, 3.5, 4, 4.5, 5.5, 6 and 6.5 using
buffer citric.
Effect of nutrients sources
Effect of nitrogen sources: Various nitrogen sources were used separately
at an equivalent concentration of 0.33 g (N/L) media as recommended by Murad
(1998). The nitrogen source included three inorganic salts (ammonium sulphate,
ammonium chloride and sodium nitrate) and three organic sources (meat extract,
yeast extract and peptone). These sources replace the original nitrogen source
in the test medium.
Effect of carbon sources: Influence of various carbon sources on tannase
enzyme production were studied by testing different tannins containing waste
materials including banana wastes, rice straw, wheat straw, sugarcane baggase,
wheat bran and palm kernel powder in range between 5% to 30% (w/v) to the fermentation
media with or without addition tannic acid powder 2% (w/v). Pure tannic acid
powder was also used as a sole carbon source for comparison as a control.
Assay of tannase: Tannase enzyme activity was determined by the method
of Mondal et al. (2001). One unit of the tannase
enzyme was defined as the amount of enzyme which is able to hydrolyse 1 μmole
of ester linkage of tannic acid in 1 min at specific condition (pH 5.0 and 40°C).
|
| Fig. 1: |
Stander curve of tannic acid |
Stander curve: Stander curve was design according to Mondal
et al. (2001). Figure 1 represents the calibration
curve for tannic acid, presenting linearity between 100 and 1000 μg L-1.
RESULTS AND DISCUSSION
Effect of agitation on the production of tannase from A. niger:
Data presented in Fig. 2 illustrated the difference between
using shaked culture and static culture. Shaked culture exhibited the highest
tannase activity than that of the static culture, being 206.25 and 178.13 (U
L-1 min-1), respectively. These results are in agreement
with those reported by Purwanto et al. (2009)
who suggested that agitation speed is a very important factor in the fermentation
process since it will increase the amount of dissolved oxygen in the cultivation
medium.
Effect of inoculum size on the production of tannase from A. niger:
Fungal culture exhibited different responses to variations in inoculum size
from 1 to 8% (V/V). Data presented in Fig. 3 illustrated that
the production of tannase enzyme by A. niger was increased significantly
(p<0.05) by increasing inoculum ratio up to 2% (V/V) being (193.75 U L-1
min-1). Further increasing in inoculum ratio up to 8% led to decrease
in tannase production by fungal cultures. These results are in line with those
reported by Lokeswari and Raju (2007).
Effect of incubation period on the production of tannase from A. niger:
The effect of different incubation periods (24 to 144 h) on tannase production
is shown in Fig. 4. the tannase production was gradually increased
with a rise of incubation period until 96 h and then decreased. Maximum tannase
production was found after 96 h reaching 200.00 U L-1 min-1.
|
| Fig. 2: |
Effect of agitation on the production of tannase from A.
niger |
|
| Fig. 3: |
Effect of inoculum size on the production of tannase from
A. niger |
|
| Fig. 4: |
Effect of incubation period on the production of tannase from
A. niger |
These results are in agreement with those reported by Lekha
and Lonsane (1997) and Paranthaman et al. (2009b)
who reported that maximum extra-cellular tannase production by Aspergillus
oryzae was reached after 96 h of incubation. While, Lal
et al. (2012) found that maximum tannase production from A. niger
was shown at 7th day.
|
| Fig. 5: |
Effect of the initial pH values on the production of tannase
from A. niger |
Paranthaman et al. (2009a) reported that the
decreased enzyme yield on prolonged incubation could also be due to inhibition
and denaturation of the enzyme. It has been reported before that tannase was
produced during the primary phase of growth and thereafter the activity decreases
either due to the decrease in production or due to enzyme degradation.
Effect of the initial pH values on the production of tannase from A.
niger: The influence of different initial pH values (from 3.0-6.0) on
tannase production was studied. The highest value of tannase activity was recorded
at pH 5.0 (173.44 U L-1 min-1) (Fig. 5).
These results are in line with those reported by Barthomeuf
et al. (1994) who confirmed that the tannase from A. niger contained
both esterase and depsidase activity with the esterase and tannase activities
peaking at a pH of 5.0. Also, Iibuchi et al. (1968)
found that optimum pH for tannase isolated from A. niger was shown to
be between 5.0 and 6.0, with instability occurring at a pH above pH 6.0. In
addition, Lal et al. (2012) found that optimum
pH for tannase production from A.niger was shown at 5.0.
In the present study, a decrease in enzyme yield with increased pH values until
pH 5 was noted. This might be due to that tannase enzyme was active at acidic
pH and the activity decreased as the pH approached the alkaline range. And any
change of pH affects the protein structure and decline enzyme activation or
its instability (Lokeswari and Raju, 2007). It could
be concluded from the results that tannase from the A. niger needed an
acidic environment to be active. Lekha and Lonsane (1997)
reported that fungal tannase is an acidic protein in general.
Effect of nitrogen sources on the production of tannase from A. niger:
The effect of supplementation of different organic (meat extract, yeast extract
and peptone) and inorganic (ammonium chloride, ammonium sulfate and sodium nitrate)
nitrogen sources on tannase production was evaluated and results are shown in
Fig. 6.
|
| Fig. 6: |
Effect of nitrogen sources on the production of tannase from
A. niger |
The yeast extract was found to be the best organic nitrogen source producing
the highest level of tannase activity by A. niger being 215.63 U L-1
min-1. Similar results were reported by Reddy
and Kumar (2012) who found that the maximum enzyme production was observed
with yeast extract, but Kulkarni et al. (2012)
found that the addition of beef extract yielded the highest enzyme activity.
Ammonium chloride gave the highest level of tannase activity by A. niger
being 175.00 U L-1 min-1 as an inorganic nitrogen source,
compared with control (malt).
These results agree with those reported by Paranthaman
et al. (2009a) who stated that the maximum activity in all nitrogen
sources was observed when ammonium chloride and ammonium nitrate were used.
This data indicate that the source of nitrogen should be organic for better
results. Nitrogen can be an important limiting factor in the microbial production
of enzymes. The presence of an additional nitrogen source in the substrate may
have promoted cell growth and enzyme production (Sabu et
al., 2005).
Effect of substrate on the production of tannase from A. niger:
Data in Fig. 7 illustrated the use of available agro-industrial
residues as a carbon source in the growth medium with addition of Tannic Acid
Powder (TAP). It was know that using agro-industrial residues are generally
considered the best substrates for the process of enzyme production based on
SSF and reduce the costs of enzyme production (Ellaiah et
al., 2002). Among of the available substrate material tested palm kernel
powder (PKP) gave the maximum tannase production (931.27 U L-1 min-1)
when was fermented with A. niger flowed by wheat bran with activity reached
893.76 U L-1 min-1, while wheat straw produced the lowest
activity (6.25) followed by rice straw 131.25 U L-1 min-1.
|
| Fig. 7: |
Effect of substrate on the production of tannase from A.
niger |
|
| Fig. 8: |
Effect of PKP concentration in the growth medium on the production
of tannase from A. niger |
These results are in coincide with those reported by Sabu
et al. (2005) observed that the maximum tannase production was reached
with palm kernel cake as a substrate after 96 h of fermentation period by A.
niger ATCC 16620, using medium containing TAP, methylgallate and gallic
cid. Also, Reddy and Kumar (2012) who found that the
maximum enzyme production of tannase was achieved with using wheat bran using
medium containing 2% TAP.
In addition production of tannase enzyme was observed when these materials
were used with addition of 2% (w/v) TAP. While there was no production of tannase
enzyme was observed when these materials were used without addition of TAP,
except the case when PKP was used as substrate. The results in the present study
indicated that tannase production was varied with the type of by-product used
as substrate. This could be attributed to that solid materials have dual roles
supply of nutrients to the microbial culture (Reddy and
Kumar, 2012).
Effect of PKP concentration in the growth medium on the production of tannase
from A. niger: Among several factors affecting tannase production,
moisture level content is one of the most critical factors (Pandey
et al., 2000). Data in Fig. 8 showed the impact
of PKP concentration in the growth medium on the production of tannase from
A. niger with or without TAP supplementation. The general results showed
that maximum of tannase production were attained in medium containing TAP. The
maximum tannase production was observed at 25% (w/v) PKP in both media (with
or without TAP) being 931.26 and 243.75 U L-1 min-1, respectively.
CONCLUSION
The production of tannase from Aspergillus niger under solid-state fermentation
and submerged fermentation cultures were evaluated and standardized. These results
found that tannase production from solid-state fermentation cultures superior
over tannase production from submerged fermentation cultures under the optimum
conditions cultures.
