Food Chemistry 156 (2014) 301–304
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Potential of tara (Caesalpinia spinosa) gallotannins and hydrolysates as natural antibacterial compounds Ana Aguilar-Galvez a, Giuliana Noratto b, Flor Chambi a,c, Frédéric Debaste c, David Campos a,⇑ a
Instituto de Biotecnología (IBT), Universidad Nacional Agraria La Molina (UNALM), Av. La Molina s/n, Lima, Peru School of Food Science, Washington State University, FSHN Bldg., Room 106, Pullman, WA 99164, USA c Transfers, Interfaces and Processes (TIPs), Chemical Engineering Unit, Université Libre de Bruxelles-(ULB), 50 Avenue F.D. Roosevelt, C.P. 165/67, 1050 Brussels, Belgium b
a r t i c l e
i n f o
Article history: Received 26 August 2013 Received in revised form 23 November 2013 Accepted 28 January 2014 Available online 6 February 2014 Keywords: Gallotannins Staphylococcus aureus Pseudomonas fluorescens
a b s t r a c t Gallotannins obtained from tara pod extracts (EE) and from the products of acid hydrolysis for 4 and 9 h (HE-4 and HE-9) were characterised for their composition, antioxidant activity, antimicrobial activity (AA) and minimum inhibitory concentration (MIC). Results of AA and MIC showed that EE exerted the highest inhibitory activity against Staphylococcus aureus, followed by Pseudomonas fluorescens; and among these bacteria, the antibacterial potency was enhanced after EE hydrolysis only against S. aureus. The lowest minimum inhibitory concentration (MIC) value (0.13 mg gallic acid equivalent (GAE)/ml) was exerted by HE-4 against S. aureus. These results indicate that tara gallotannins have the potential to inhibit pathogenic bacteria with potential application in foods as antimicrobials and their AA can be enhanced by acid hydrolysis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Caesalpinia spinosa (Molina) Kuntze known as tara, is a leguminous native to Peru (Galvez, Riedl, & Conner, 1997; Marien & Delaunay, 2010) and widely used in traditional medicine since pre-Hispanic times due to its effects as an antibiotic to fight respiratory related illness and skin infections (De-la-Cruz, Vilcapoma, & Zevallos, 2007). Tara pods and seeds have been used as source of tannins and gum (De La Cruz, 2004). Tara pods represent 62% of weight with a high percentage of tannins (between 40% and 60%) which are of the hydrolysable type, with gallic acid (GA) as the main constituent that can be isolated by acid hydrolysis (Mancero, 2008). Tannins in general are phenolic compounds with astringent, antiviral, antibacterial, antiparasitic, and antioxidant properties. Previous studies have reported that gallotannins extracted from Galla chinensis exerted antimicrobial activity (AA) and identified that 5–7 galloylglucopyranoses (GG) inhibited Salmonella typhimurium, 6–7 GG inhibited Bacillus cereus, whereas 2GG exerted a very low AA against the two strains (Tian, Li, Ji, Zhang, & Luo, 2009). Hydrolysable tannins and derivatives (GA and diverse molecular weight gallotannins) have been shown to possess bacteriostatic and bactericidal activities against Aeromonas hydrophila,
⇑ Corresponding author. Tel./fax: +511 3495764. E-mail address:
[email protected] (D. Campos). http://dx.doi.org/10.1016/j.foodchem.2014.01.110 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Enterobacter sakazakii, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhi and Salmonella Typhimurium (Kim, Silva, Kim, & Jung, 2010). Toxicity of phenolic compounds to microorganisms involves several mechanisms including the inhibition of enzymes by oxidised phenolics (Cowan, 1999). Such oxidised phenolics can react with sulphur groups of enzymes to produce covalent bonds, to increase the mass due to polymerisation and to the formation of non-covalent bonds (Scalbert, 1991). Gallotannins specifically exert their AA due to their ability to chelate iron and to make it unavailable for microorganisms (Chung, Lu, & Chou, 1998; Scalbert, 1991). In general, most tannins can chelate metallic ions such as iron and copper due to their o-diphenol groups; this feature allows the formation of metal–tannin complexes and decreases metalloenzymed activities (Scalbert, 1991). Our objective was to assess the AA of tannins extracted from tara pods and the products of their acid hydrolysis against gram positive and gram negative bacterial strains.
2. Materials and methods 2.1. Plant material, extraction and hydrolysis Tara was purchased from a local market in Lima – Peru, produced in the Ancash region and harvested when moisture of the tara pods reached approximately 8% and immediately distributed
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to local markets. Approximately 3 Kg of tara were purchased for the study and conditioned as follows: a composite of approximately 1 Kg was used and tara pods were subjected to seed removal, washing, drying at 55 °C to reach a final moisture of 3%, grinding to 80 mesh, and stored at 20 °C for future use. The tannin extraction was performed with distilled water in a ratio 1:100 (w:v) at 55 °C for 30 min, followed by centrifugation at 4000g for 15 min to obtain the supernatant which was kept at 4 °C overnight and centrifuged at 13,328g for 10 min to remove insoluble fiber and polysaccharides and obtain the clear supernatant that was used as the tara pods extracts (EE). Hydrolysis was performed as previously reported (Chambi et al., 2013). Briefly, EE containing 20 mg GAE/ml was subjected to hydrolysis with H2SO4 2N at 100 °C for 4 h (HE-4) or 9 h (HE-9). All extracts (EE, HE-4, and HE-9) were then subjected to liquid:liquid extraction with ethyl acetate (1:2 ratio, v/v) for two consecutive times. The ethyl acetate was removed under vacuum with a rotary evaporator at 38 °C until dry and solids were dissolved with 5% DMSO in Mueller Hinton Broth (HMB), stored at 80 °C with N2 gas for further analysis. Gallotannins, free and total GA were determined by the rodanine method (Inoue & Hagerman, 1988; Salminen, 2003), and phenolics were determined by the Folin Ciocalteu method (Singleton & Rossi, 1965). Antioxidant activity was determined by the ABTS (Arnao, Cano, & Acosta, 2001), FRAP (Benzie & Strain, 1996) and ORAC (Ou, Hampsch-Woodill, & Prior, 2001) assays. The degree of hydrolysis (DH) was determined after quantification of GA in the EE (GAEE), each one of the hydrolysates HE-4 and HE-9 (GAHE), and the EE were subjected to complete hydrolysis (GAT), as follow: DH (%) = [(GAHE GAEE)/(GAT GAEE)] 100. All analysis of tara extracts and hydrolysates were performed at least three times on different days before their use and results were consistent.
antimicrobial activity was determined by the diameter of the inhibition zone. 2.4. Minimum inhibitory concentration (MIC) The MIC was assessed by the macro-dilution method (Zhu, Zhang, & Lo, 2004). Briefly, 0.9 ml of twofold serial dilutions (0-50 mg GAE/ml for EE and 0–70 mg GAE/mL for HE-4) were prepared in MHB, inoculated with 0.1 ml of suspension of bacteria (106 CFU/ml), and incubated at 37 °C for 18 h. The MIC was the dilution at which visible growth was not observed. 2.5. Minimum bactericidal concentration (MBC) MBC was determined by inoculating 100 ll of broth culture from each one of the MIC test on MHA plates and incubated at 37 °C for 24 h. MBC was the concentration (GAE/ml) at which bacterial growth was inhibited in 99.9% (Alade & Irobi, 1993). 2.6. Statistical analysis Quantitative data represent mean values of three or more replicates with the respective standard deviation (SD). A one-way analyses of variance (ANOVA) followed by a Duncan test were performed with Stat Graphics Plus 5 (Statistical Graphics Corp., Herndon, VA, USA). A value of p < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Extract characterisation
2.2. Microorganisms and growth media Bacterial strains gram-positive (Bacillus subtilis (NRRL B-3384), Enterococcus faecium (CWBI-B1430), Listeria innocua (NRRL B-33198), Listeria monocytogenes (CWBI-B2232), Micrococcus luteus (NRRL B-1018) and S. aureus (ATCC 25923)), and gram-negative (Escherichia coli (NRRL B-2073), Pseudomonas fluorescens (NRRL B-2641) and Salmonella enteritidis (kindly provided by the National Health Institute – Peru) were used to assess the AA of EE, HE-4, and HE-9. Bacterial growth in Man, Rogosa, and Sharpe (MRS) agar (Enterococcus), Palcam agar (Listeria spp.), Manitol agar (Staphylococcus), McConkey agar (Escherichia), Brilliant Green agar (Salmonella), and Mueller Hinton agar (MHA) (for remaining bacterial strains) was assessed after 24 h incubation at 37 °C. Bacterial strains maintained in agar plates at 4 °C were replicated weekly. All experiments on antibacterial activity of tara extracts were conducted three times independently on different days, to evaluate the reproducibility and robustness of the assays.
Results showed a DH of 46% and 95.4% after 4 and 9 h of acid hydrolysis, HE-4 and HE-9, respectively (Table 1). This was accompanied by an increase in the content of free GA compared to EE (from 2.4 to 17.3 and 36.4 mg GA/ml for HE-4 and HE-9, respectively) and a decrease in the gallotannins content (from 34.4 to 19.4 and 1.7 mg GAE/ml for HE-4 and HE-9, respectively). The increase of DH to 46% was accompanied by an increase in antioxidant activity by 50% and 59% when assessed by the FRAP and ABTS methods, respectively, whilst the increase was only 16% when assessed by the ORAC method. Likewise, a DH of 95.4% was accompanied by an increase in antioxidant activity by 65%, 62%, and 27% (ABTS, FRAP, and ORAC methods, respectively). Moreover, HE-4 and HE-9 showed similar antioxidant activities (p < 0.05) as assessed by the ABTS, FRAP, and ORAC methods. Similar results showing the increase in antioxidant activity upon tannins processing have been previously reported (Chambi et al., 2013; Kim et al., 2010). 3.2. Antimicrobial activity (AA)
2.3. Antimicrobial activity The antimicrobial activity was assessed by the standard disk diffusion technique as previously reported (Bauer, Kirby, Sherris, & Turck, 1966). Briefly, bacterial strains were inoculated in test tubes containing Mueller Hinton Broth (MHB) and incubated for 4 h at 37 °C. Sterile paper disks (7.0 mm diameter, Whatman N°1) were impregnated with 20 ll of extracts (EE, HE-4, and HE-9) at a concentration of 35 mg GAE/ml and placed on MHA plates inoculated with 100 ll of bacterial suspension adjusted to 106 colony forming units (CFU)/ml using a sterile saline solution at 0.85%. Disks containing 5% DMSO were used as a negative control and disks containing ciprofloxacin or vancomycin (1 mg/ml) were used as positive controls. Plates were incubated at 37 °C for 24 h and
Results showed that among the tested bacteria, 7 out of 9 strains were inhibited by EE, HE-4 and HE-9 at 35 mg GAE/ml (Table 2). Only E. faecium and E. coli required a higher concentration of EE and HE-4 (80 mg GAE/ml) to be inhibited to a similar extent. In addition, the AA of EE was higher against B. subtilis, M. luteus and P. fluorescens compared to HE-4 and HE-9 (p < 0.05), whereas L. innocua, L. monocytogenes, S. aureus, and S. enteritidis were more sensitive to HE-4 and HE-9 than to EE (Table 2). However, at the concentrations of EE used, the low pH might have influenced the AA. These results are in agreement with a previous study demonstrating that hydrolysed tannic acid inhibited S. aureus and L. monocytogenes at higher extend than tannin acid (Kim, Silva, & Jung, 2011). In addition, previous studies have reported that origin,
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A. Aguilar-Galvez et al. / Food Chemistry 156 (2014) 301–304 Table 1 Phenolic compounds and antioxidant activity of tara pod extracts (EE) and the products of their acid hydrolysis (HE-4 and HE-9).
Degree of hydrolysis (DH) (%) Free gallic acid (mg GA/ml) Gallotannins (mg GAE/ml) Total phenolics (mg GAE/ml) Antioxidant activity (lmol TE/ml): ABTS FRAP ORAC
EE
HE-4
HE-9
0 2.4 ± 0.3 34.4 ± 0.1 36.8 ± 3.2
46.0 ± 0.8 17.3 ± 4.8 19.4 ± 0.2 39.7 ± 1.6
95.4 ± 0.4 36.4 ± 5.8 1.7 ± 0.2 41.3 ± 4.1
647.2 ± 7.4b A 632.5 ± 33.1b A 240.0 ± 15.8b B
1029.3 ± 23.8a A 945.9 ± 31.8a A 279.2 ± 22.0a B
1066.7 ± 24.8a A 1025.4 ± 70.3a A 304.0 ± 18.8a B
Tara pod extracts (EE) and their hydrolysates for 4 and 9 h (HE-4 and HE-9) were obtained as detailed in the materials and methods section. Results are the mean (n P 3)±SD. Different superscript letters within same row (lower case) or same column (capital letter) indicate significant difference p < 0.05.
Table 2 Antibacterial activity of tara pod extracts (EE) and the products of their acid hydrolysis for 4 and 9 h (HE-4 and HE-9). Strains
EE
HE-4
HE-9
(35 mg GAE/mL) Bacteria gram-positive: Bacillus subtilis Enterococcus faecium Listeria innocua Listeria monocytogenes Micrococcus luteus Staphylococcus aureus Bacteria gram-negative: Escherichia coli Pseudomonas fluorescens Salmonella enteritidis
Enterococcus faecium Escherichia coli
Ciprofloxacin
Vancomycin
(1 mg/mL)
11.7 ± 0.3c NA 7.3 ± 0.6d 15.3 ± 1.5d 11.0 ± 1.0c 18.2 ± 0.3d
9.3 ± 0.6d NA 8.8 ± 1.0c,d 16.3 ± 1.2c,d 9.7 ± 0.6c 26.3 ± 1.2b
9.7 ± 1.6d NA 9.3 ± 0.6c 19.3 ± 2.9c 9.8 ± 0.3c 26.7 ± 0.6b
33.3 ± 1.2a 30.3 ± 0.6a 38.7 ± 1.2a 33.7 ± 1.5a 34.0 ± 2.0a 35.0 ± 1.0a
21.3 ± 1.2b 30.0 ± 0.0a 27.3 ± 1.2b 29.3 ± 1.2b 29.3 ± 1.2b 22.3 ± 0.6c
NA 12.0 ± 0.0c 8.3 ± 1.5c
NA 9.0 ± 0.0d 10.3 ± 2.5c
NA 9.2 ± 0.3d 15.3 ± 2.1b
50.0 ± 2.00a 34.8 ± 0.8a 47.3 ± 1.2a
14.7 ± 1.2b 23.2 ± 0.3b N.A.
EE (80 mg GAE/ml)
HE-4 (80 mg GAE/ml)
11.0 ± 0.85a 9.0 ± 1.00a
11.0 ± 0.58a 9.0 ± 0.90a
EE, HE-4, and HE-9 at doses of 35, and 80 mg gallic acid equivalent (GAE)/ml were used to assess antimicrobial activity by the standard disk diffusion assay as detailed in materials and methods. Results are the mean (n = 3)±SD. NA = not active. Different superscript letters within same row indicate significant difference p < 0.05.
concentration, chemical structure, and therefore the products obtained from the hydrolysis of tannins influence their AA (Min et al., 2008). Likewise, tannin acid, but not gallic acid inhibited the growth of several bacterial strains including E. coli, S. aureus, and S. typhimurium, and the lactic acid bacteria L. acidophilus and Bifidobacterium infantis (Chung, Stevens, Lin, & Wei, 1993; Chung et al., 1998). E. coli however was found to be the most resistant to EE and their hydrolysates which is in agreement with previous studies (Kloucek, Polesny, Svobodova, Vlkova, & Kokoska, 2005; Min et al., 2008; Taguri, Tanaka, & Kouno, 2004). Since gramnegative bacteria are more resistant to antibiotics because of their relatively impermeable cell wall, the sensitivity of bacterial strains might be influenced by the cell membrane structure and by the degree of hydroxylation of phenolic compounds (Kim et al., 2011). In general, among all tested bacteria strains S. aureus was the most sensitive to all EE, HE-4, HE-9 (biggest inhibition halo), however when compared to positive controls (ciprofloxacin and vancomycin, antibiotics) their potency is exceeded by approximately 35-fold.
Table 3 Minimum inhibitory and bactericidal concentrations (MIC and MBC) of tara pod extracts (EE) or the products of their acid hydrolysis for 4 (HE-4).
3.3. MIC and MBC of tara extracts
and their hydrolysates inhibited bacteria growth, the pH was within 6–7; therefore pH did not influence their antibacterial activity. M. luteus, L. innocua and L. monocytogenes were not significantly inhibited (MIC and MBC P 25 mg GAE/mL). HE-4 was instead effective against S. aureus, E. faecium, and E. coli with MIC of 0.13, 1.13, and 1.46 mg GAE/ml, respectively, with MBC values of 14 and 4.05 for S. aureus and E. coli, respectively. The effect of HE-4 against E. faecium was bactericidal rather than bacteriostatic since it did not grow on the agar plate.
The extracts that exerted the highest AA were selected to assess the MIC and MBC, except for S. enteritidis, which showed to be more sensitive to EE than HE-9 in the culture medium used to test MIC. Results showed that P. fluorescens, B. subtilis and S. enteritidis were the most sensitive to EE with MIC of 0.53, 1.06, and 4.25 mg GAE/ml, respectively; while the MBC were 4.25, 8.5, and 17 mg GAE/ml, respectively (Table 3). At concentrations that EE
MIC
MBC
(mg GAE/ml) Gram positive strains Bacillus subtilis [EE] Enterococcus faecium [HE-4] Listeria innocua [EE] Listeria monocytogenes [EE] Micrococcus luteus [EE] Staphylococcus aureus [HE-4]
1.06 ± 0.0 1.13 ± 0.0 50.0 ± 0.0 50.0 ± 0.0 25.0 ± 0.0 0.13 ± 0.02
8.5 ± 0.0
