Download Determination of antimicrobial activity of tannic acid in pickling

Romanian Biotechnological Letters
Copyright © 2010 University of Bucharest
Vol. 15, No.3, 2010
Printed in Romania. All rights reserved
ORIGINAL PAPER
Determination of antimicrobial activity of tannic acid in pickling process
Received for publication, October 23, 2009
Accepted, June 30, 2010
SELİME MENTEŞ ÇOLAK1, BİNNUR MERİÇLİ YAPICI2, ALİ NAİL YAPICI3
1
Ege University, Engineering Faculty, Department of Leather Engineering, 35100
Bornova-Izmir, Turkey.
2
Canakkale Onsekiz Mart University, Faculty of Arts and Science, Biology Department, Basic
and Industrial Microbiology Section , 17020 Canakkale, Turkey.
3
Canakkale Onsekiz Mart University, Biga Vocational College, 17200 Biga-Çanakkale, Turkey.
Abstract
In the present study, the antimicrobial efficacy of tannic acid used in the pickling stage was
investigated against some microorganisms. Leather samples treated with various concentrations of
tannic acid in the stage were assessed against sixteen test microorganisms by Disc Diffusion Method.
The used microorganisms - bacteria, moulds and yeasts – were Bacillus cereus (ATCC
11778), Salmonella typhimurium CCM 5445, Proteus vulgaris (ATTC 6889), Enterobacter aerogenes
(ATCC 13048), Pseudomonas aeruginosa (ATTC 27853), Escherichia coli (ATTC 25922),
Staphylococcus aureus (ATCC 6538), Neisseria canis, Aspergillus niger, A. fumigatus, A. flavus,
Penicillium granulatum P. granulosum, Geotricum candidum, Yarrowia lypolitica and Rhodotorula
rubra (DSM 70403). The results showed that tannic acid exhibited antimicrobial activity against all
bacteria, moulds and yeasts tested. It was also determined that the most effective concentration of
tannic acid was 3 %.
Keywords: Antimicrobial efficacy, leather industry, microorganisms, tannic acid.
Introduction
Raw skin is a favourable medium for microbial growth. In the absence of appropriate
counter-measures, microorganisms present in the skin multiply and cause damage in its
structure. In raw skin, the microorganism load can be affected by various parameters,
including but not limited to water content, storage temperature and effectiveness of
conservation process.
During the manufacturing processes, the microbial load varies depending on many
other factors, such as pH values, temperature, duration of each process, and biocides and other
compounds of different chemical compositions used [1-3].
Fungicides are mainly used to protect chrome tanned leather from microorganisms
when post tanning processes are delayed [4]. While biocides inhibit basic metabolic activities
of the microorganisms, they may be toxic due to containing active compounds hazardous to
human and environmental health.
Recent studies have focused on reducing these risks by limiting the use of biocides or
identifying less hazardous alternative agents [5-9]. A study on investigation of wood
preservative activities of some plant extracts (Pinus brutia, Quercus ithaburensis, Quercus
infectoria, Rhus coriara) as fungicide, it has been found that the extracts have greatly
decreased the growth of some fungus mycelium in treated wood above 3% concentration
[10]. Many studies conducted before confirm that phenolic agents are effective against
pathogenic microorganism attacks [11-14].
Catechin and pyrogallol, found in vegetable tannins, are phenols toxic to
microorganisms. There are two hydroxyl groups in catechin and three in pyrogallol. The
number and location of hydroxyl groups in the phenolic compounds are the key factors in
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SELİME MENTEŞ ÇOLAK, BİNNUR MERİÇLİ YAPICI, ALİ NAİL YAPICI
their toxicity to microorganisms. As the number of the hydroxyl groups increases, so does the
toxicity [15].
Tannins are also toxic to fungi, bacteria and viruses and inhibit their growth [15-17].
A 1:50000 solution of acerin and similarly a 1:50000 solution of mimosa, quebracho,
canaigre and babul were found to destroy viruses within 5 minutes, while a milder effect was
observed for a chestnut wood, valonia and sumac solution [18]. With reference to Carson and
Frish, Gustavson stated that tannic acid (TA) and digallic acid inactivate influenza viruses. In
another resource, it is declared that gallic acid in carob has antibacterial, antifungal and
antioxidant properties [19].
Antibacterial properties of vegetable tannins have been exploited in various fields,
particularly in medicine. TA, whose phenolic structure is shown in Figure 1, exhibits
antioxidant, antimutagenic and anticarcinogenic properties [20-26].
The study was carried out to examine the antimicrobial properties of TA used in
different concentrations in the pickling process against bacteria, moulds and yeasts.
Figure 1. Molecular structure of tannic acid (TA).
Experimental Material
20 pieces of whole dry-salted hair sheep skins were used in the study.
Method
Methodology of leather processing
The detailed leather production process is presented in Table 1. Except for TA, all the
chemicals used were of technical grade. TA used was of analytical grade (Merck).
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Romanian Biotechnological Letters, Vol. 15, No. 3, 2010
Determination of antimicrobial activity of tannic acid in pickling process
Table 1. Processing of dry-salted hair sheep skin
PROCESS
Weighing
Pre-soaking
Drain
Main
Soaking
Unhairing
Liming
Fleshing
Trimming
Weighing
DelimingBating
Add
Wash
Degreasing
%
CHEMICALS
% based on raw skin weight.
500
Water
o
C
22
DURATION
(min.)
pH
MA*(rpm)
240
7.0-7.5
0
500
0.5
Water
22
Non-ionic
emulsifier
0.5
Bactericide
1080
7.0-7.5
4
(Run on automatic, run 10 minutes/stop 50 minutes for 24 hours).
After painting solution (18 0Bé Na2S, 26 0Bé Ca(OH)2, 28 0Bé china clay) was
applied onto flesh side, the skins were dehaired within 4 hours.
300
Water
20
2
Sodium sulphide
4
Lime
30
11.5-12
4
(Run on automatic, run 5 minutes/stop 55 minutes for 24 hours).
Recorded as pelt weight, % based on pelt weight.
150
Water
35
1
Ammonium
45
sulphate
0.8
Bating agents
30
150
20
10
100
Water
35
5
Degreasing agent
60
200
Water
35
35
200
Water
35
35
100
Water
22
Washing
Washing
PicklingTanning
Add
Add
7
1
Add
Add
x
0.5
Add
8
Add
1
NaCl
Formic acid
(diluted 1:10)
Tannic acid**
Sulphuric acid
(diluted 1:10)
Chrome
tanning
agent (Powder)
Basification agent
8.0
12
12
12
10
30
30
120
12
12
12
3.0
240
12
12
4.0
12
* MA: Mechanical action of leather processing drum.
**: Tannic acid was only used in trials for different concentration (0.1, 0.5, 1, 2 or 3%).
After soaking, dehairing-liming, deliming-bating and degreasing, TA was added to the
pickling float at five different ratio (0.1, 0.5, 1, 2 and 3%) based on pelt weight. The leather
pieces obtained were labelled as experimental groups (E). On the other hand, leather pieces
without TA added in the pickling float were marked as control group (C). At the end of the
chrome tanning, an 8.0 mm sterilized stainless steel template was used to obtain leather
samples from E and C groups for the microbiological analysis.
Microbial test organisms: In the present study; 8 bacteria (Bacillus cereus ATCC 11778,
Salmonella typhimurium CCM 5445, Proteus vulgaris ATTC 6889, Enterobacter aerogenes
ATCC 13048, Pseudomonas aeruginosa ATTC 27853, Escherichia coli ATTC 25922,
Staphylococcus aureus ATCC 6538, Neisseria canis); 5 moulds (Aspergillus niger, A.
fumigatus, A. flavus, Penicillium granulatum, P. granulosum) and 3 yeasts (Geotricum
candidum, Yarrowia lypolitica and Rhodotorula rubra DSM 70403) were used as test
Romanian Biotechnological Letters, Vol. 15, No. 3, 2010
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SELİME MENTEŞ ÇOLAK, BİNNUR MERİÇLİ YAPICI, ALİ NAİL YAPICI
microorganisms. Except for Rhodotorula rubra, all fungi were obtained from Basic and
Industrial Microbiology Culture Collection of Ege University.
Antimicrobial activity test: Nutrient agar was used for the test bacteria and Malt
Extract Agar for the test moulds and yeasts. All the bacteria were incubated at 35 ± 0.1 0C for
24 h by inoculation into Nutrient Broth (Difco Laboratories, MI, USA). One millilitre of
inoculum containing 106 bacterial cells/mL was spread on Mueller Hinton Agar (Oxoid Ltd.,
Hampshire, UK) plates. Mould and yeast cultures were incubated at 25 ± 0.1 0C for 48 h in
Malt Extract Broth (Difco Laboratories). Same quantity of inoculum containing 106 mould or
108 yeast cells/mL was spread on Malt Extract Agar [27, 28]. Then, E and C leather samples
were placed on the plates. After the plates were kept at +40C for 2 h, they were incubated at
35 ± 0.1 0C for 24 h for bacteria and at 25 ± 0.1 0C for 48h for moulds and yeasts. At the end
of the incubation period, the diameters of inhibition zones occurred on the medium were
evaluated in millimetres. The experiments were run in triplicate.
Results and discussion
In the study, all of the leather samples treated with different concentrations of TA (0.1,
0.5, 1, 2 and 3%) showed an inhibition zone against the bacteria, moulds and yeasts tested.
There was no inhibition zone in any of control samples.
Inhibition zone diameters of leather samples were determined to be larger for some
microorganisms at 3% TA concentration [Table 2]. The highest antimicrobial activity with 24
mm of inhibition zone was observed versus Proteus vulgaris in this concentration. Salmonella
typhimurium, Enterobacter aerogenes and Neisseria canis species also exhibited high
inhibition zone (16 mm). In terms of sensitivity, S.aureus, Bacillus cereus, Pseudomonas
aeruginosa, and E. coli respectively followed them.
Phenolic agents inhibit bacterial growth and protease activity by damaging its cell wall and
cytoplasm [29,15], causing the rapid destruction of the vegetative structure of bacteria and
more slowly of the spores [30]. Therefore, it was revealed that TA added into the pickling
process exhibited antibacterial activity against different bacteria species with various degrees.
Yeasts
Moulds
Bacteria
Table 2. Inhibition zones of bacteria, moulds and yeasts.
*
Bacillus cereus
Salmonella typhimurium
Proteus vulgaris
Enterobacter aerogenes
Pseudomonas aeruginosa
Escherichia coli
Staphylococcus aureus
Neisseria canis
Aspergillus niger
Aspergillus fumigatus
Aspergillus flavus
Penicillium granulatum
Penicillium granulosum
Geotricum candidum
Yarrowia lypolitica
Rhodotorula rubra
(0,1% TA)
12
14
21
11
12
11
14
15
10
9
10
10
10
10
11
11
Inhibition zones (mm)*
Experimental Groups (E)
(0,5% TA) (1% TA) (2% TA) (3% TA)
13
14
14
14
15
16
16
16
22
22
24
24
14
14
15
16
12
12
13
14
12
12
12
13
14
14
15
15
15
15
15
16
10
11
11
14
10
11
11
11
10
10
11
12
11
11
12
12
10
11
12
14
10
10
11
12
11
12
12
13
11
12
13
13
Control
Group** (C)
-***
-
:
Values, including diameter of the chrome tanned leather sample (8.0 mm), are means of three replicate
** : No tannic acid was used.
*** : No inhibition zone was observed.
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Determination of antimicrobial activity of tannic acid in pickling process
The efficacy of tannic acid against the test moulds was found out relatively similar to
each other. A. niger and P. granulosum, frequently encountered moulds in tanneries, were
determined as the most sensitive species against the tannic acid. In the 3% TA concentration,
their inhibition zone was found to be 14 mm in diameter. In addition, A.flavus and P.
granulatum were detected as sensitive moulds with a 12 mm inhibition zone in the
concentration. The inhibition zones for the yeasts were observed as similar to those of moulds
rather than those of bacteria. The most sensitive test yeasts were found as Yarrowia lypolitica
and Rhodotorula rubra with 13 mm inhibition zone in 3% TA concentration. It is well known
in literature that compounds found in the structure of plant extracts, such as tannic acid, gallic
acid, ellagic acid, catechin and essential oils have antimicrobial effect [31-36]. Actually, TA
exhibited antimicrobial activity not only against bacteria but also against moulds and yeasts
tested in our study.
In general, it was observed that the antimicrobial activity slightly increased for higher
TA concentrations. Therefore, it can be said that 3 % tannic acid concentration was more
effective than the other concentrations.
Conclusion
TA is a component existent together with the other phenolic compounds in the
structure of vegetable tanning agents and is not categorized as a toxic agent by the
67/548/EEC directive of the EU [37]. Many commercial biocides used in the leather industry
are classified as agents hazardous to human and environment health and recent research has
been carried out to reduce their rate of use or to find alternatives to them.
The inhibition zones of the leather samples – obtained by adding tannic acid at different ratios
(0.1, 0.5, 1, 2 or 3%) in the pickling process – against various microorganism groups
(bacteria, moulds and yeasts) were investigated in this study. When obtained experimental
samples were compared with the control samples, it was found out that TA is effective on
each of the three microorganism groups, bacteria in particular. As the amount of TA added
into the pickling float increased, so did its antimicrobial effect. It is found out that 3 % tannic
acid concentration was more effective than the others (0.1, 0.5, 1, 2%). Accordingly, it was
concluded that tannic acid can be used in leather industry as an alternative antimicrobial agent
in the pickling process.
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