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Structure analysis of condensed tannin from rice straw and its inhibitory effect on Staphylococcus aureus
Industrial Crops & Products 145 (2020) 112130
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Structure analysis of condensed tannin from rice straw and its inhibitory effect on Staphylococcus aureus
T
Jie Shi, Yazhu Wang, Huanran Wei, Jiajun Hu, Min-Tian Gao* Shanghai Key Laboratory of Bio-energy Crops, School of Life Sciences, Shanghai University, 99 Shangda Road, Shanghai, 200444, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Staphylococcus aureus agricultural waste Tannins Biofilm Antimicrobial agent
To reduce the production cost of bioethanol and improve the utilization rate of straw resources, more high valueadded products have to be found and applied. In this study, tannins were considered as a high-value by-product. The extraction of condensed tannins from rice straw was studied, and the tannins were characterized. The crude tannins consisted mainly of tannins, phenolic acids and monosaccharides. The crude tannins were separated and purified with AB-8 macroporous resin and Sephadex LH-20. The matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) assay confirmed that catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid were the major components in the purified tannins. The tannins exhibited strong inhibitory effects on growth of Staphylococcus aureus. Higher concentrations of crude tannins resulted in lower ethanol concentration and intercellular ATP content, and higher residual glucose. Moreover, the tannins could regulate morphology of biofilms. The images from atomic force microscopy and confocal laser scanning microscopy showed that cells hardly adhered to the plate surface, and large aggregations of cells were observed in culture medium containing tannins, and almost all cells were dead. This study provides the feasibility of tannins derived from rice straw as new antimicrobial agents with a different inhibitory mechanism.
1. Introduction
antibiotics would make strains more drug tolerant (DeLeo and Chambers, 2009). In order to reduce the frequency of use of antibiotics on bacteria and prevent the development of drug resistance, it is now urgent to find a bacteriostatic agent to assist and replace antibiotics (Doernberg et al., 2017) (Usai et al., 2019). A number of papers reporting antimicrobial properties of condensed tannins, polyphenols and flavonoids isolated from higher plants have been published in recent years (Arora and Mahajan, 2019; Teanpaisan et al., 2017; Xu et al., 2015). The plant extracts have been documented to have the ability to effectively inhibit the growth of phytopathogenic fungi (Donadu et al., 2019), bacteria (Cannas et al., 2015) and tumor cells (Chaves-López et al., 2018; Donadu et al., 2017). A biofilm is a kind of surface-related bacterial aggregation within a generated extracellular polymer matrix, and can easily adhere to the surfaces of foods and medical instruments, which would lead to the increase in the resistance to common antibiotics and disinfectants. Condensed tannins are the most common polyphenols in plants, and are beneficial to human and animal nutrition and can promote health as antioxidants due to their antibacterial, antiviral and protein-binding activities (Salminen, 2018). Leaves and rhizomes of varied plants contain condensed tannins (Barnaba et al., 2018; Ismayati et al., 2017; Suvanto et al., 2017). However, few studies have investigated the
Rice straw is an abundant lignocellulosic waste, and consists mainly of cellulose, hemicellulose, lignin and silica, which could be used for the production of energy and materials (Linh et al., 2017). However, the bioconversion of rice straw suffers from low fermentation performance and high enzyme cost. Therefore, many studies have focused on improving conversion technology, feedstock production and conversion technology to minimize production costs (Diep et al., 2012). Besides cellulose, hemicellulose and lignin, rice straw contains many bioactive compounds, such as phenolic acids and flavonoids (Dong et al., 2005; Goufo and Trindade, 2014). During its bioconversion, the bioactive compounds can be released from rice straw, and can be considered as high value-added products. The integrated production of the bioactive compounds and ethanol is an effective strategy to improve economic competitiveness and reduce the cost of bioethanol (Ma et al., 2019). Staphylococcus aureus is a common pathogenic bacteria, which can cause suppurative inflammation and disease due to its exotoxins (Ward et al., 2018). This bacteria is widely found in air and water, causing bacterial infections in medical treatment and food poisoning, and is a great challenge to human health (Shi et al., 2017). Antibiotics are capable of killing infectious diseases. However, long-term use of ⁎
Corresponding author. E-mail address: [email protected] (M.-T. Gao).
https://doi.org/10.1016/j.indcrop.2020.112130 Received 16 October 2019; Received in revised form 9 January 2020; Accepted 13 January 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. Flow chart for extraction of condensed tannins from rice straw. Table 1 HPLC analysis of the different components of each fraction from rice straw. mg/g
Total phenols
tannins
A1 A2 B1 C1 D1 D2 D3 D4 D5
0.71 0.62 0.17 0.52 0.10 0.03 0.21 0.41 0.05
0.65 0.53 0.01 0.46 0.03 0.02 0.10 0.31 0.03
± ± ± ± ± ± ± ± ±
0.05 0.06 0.02 0.01 0.01 0.01 0.05 0.03 0.02
± ± ± ± ± ± ± ± ±
0.01 0.06 0.00 0.02 0.00 0.00 0.00 0.02 0.00
glucose
xylose
Vanillic acid
vanillin
Coumaric acid
Ferulic acid
19.60 ± 0.11 7.57 ± 0.24 11.06 ± 0.53 0.59 ± 0.04 ND ND ND ND ND
16.97 ± 0.24 6.36 ± 0.2 10.45 ± 0.12 0.61 ± 0.15 ND ND ND ND ND
0.18 ND ND 0.12 ND 0.05 0.04 0.01 0.01
0.15 ND ND 0.09 ND 0.01 0.06 0.03 ND
0.39 ND ND 0.30 ND ND 0.02 0.04 ND
0.09 ND ND 0.08 ND ND 0.01 0.01 ND
± 0.01
± 0.02 ± ± ± ±
0.00 0.00 0.00 0.00
± 0.00
± 0.00 ± 0.00 ± 0.00 ± 0.00
± 0.00
± 0.00
± 0.00 ± 0.00
± 0.00
± 0.00
± 0.00 ± 0.00
*The date represent averages ± standard deviations for duplicate experiments. ND = not detected.
colony-forming units (CFUs), metabolites and ATP, and observing the structural change in biofilm using confocal laser scanning microscopy (CLSM). To the best of our knowledge, this is the first study on the characterization of tannins derived from rice straw and the inhibitory mechanism on S. aureus. The results will be beneficial for utilization of rice straw in food and medical fields.
condensed tannins derived from rice straw. Previous research showed that tannins derived from rice straw had an inhibitory effect on fungi (Wang et al., 2017), bacteria (Ahn et al., 2016; Cai et al., 2017) and yeasts (Fang et al., 2015; Nakanishi et al., 2012), and the removal of tannins could improve fermentation efficiency. Based on these results, we proposed that condensed tannins derived from rice straw could be used as antimicrobial agents. To characterize the tannins derived from rice straw, we isolated and purified them using Sephadex LH-20. To investigate the chemical structure of the condensed tannins, further characterization was continued by means of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), which is considered a method of choice for analysis of compounds exhibiting large structural heterogeneity (Wang and Dai, 2018). Then, we investigated the inhibitory effect of the tannins and their fractions on S. aureus growth and their damaging effect on biofilm formation by S. aureus by determining
2. Material and methods 2.1. Preparation of condensed tannins from rice straw Rice straw was treated as previously described (Wang et al., 2017). 10 g of the treated straw was put into 100 mL of 70% acetone for extraction at 60 °C and 150 rpm for 2 h. Solid insoluble matter was filtered out and the extraction was repeated three times. 2
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Fig. 2. Typical MALDI-TOF analysis of rice straw tannin extract with molecular weight range of 500–4000 Da.
3
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Fig. 3. The main monomer unit structures of condensed tannins.
Sephadex LH-20 chromatography column. The Sephadex LH-20 was soaked in 60–70% ethanol overnight. Installed chromatography column of wet state with no gap, washed with water until ethanol could not be detected and the chromatography column was balanced. The Sephadex LH-20 was eluted with methanol–water system and reverse phase solvent. Then 10 mL of the crude extract of condensed tannins dissolved in methanol was added to the column and eluted with 30, 50, 70 and 100% methanol successively. The column was eluted at a flow rate of 1 mL/min.
2.2. Purification and separation of tannins 2.2.1. Adsorbed and eluted by AB-8 macroporous resin Macroporous resin (AB-8; Shanghai Yuanye S&T Co. Ltd, China) was used for the separation of tannins. Solvent solid–liquid extraction of the solution, using low-temperature rotation evaporation, remove organic solvents, added AB-8 macroporous resin according to the volume/mass ratio of 1:5, and placed in a shaking bed at room temperature for adsorption at 150 rpm for 30 min. The loaded column (250 mm × 50 mm) was carried out for the macroporous resin that was saturated with adsorption. After equilibrium of the column, 70% ethanol was used for elution until colorless. The elution solution was collected for low-temperature rotary evaporation to remove organic solvent. The remaining aqueous solution was freeze-dried to obtain dry powder of crude tannin.
2.3. Culture conditions The S. aureus (ATCC 6538) strain was used in this study. The strain was precultured in Luria–Bertani (LB) medium at 37 °C for 12 h. The initial optical density was set at OD600 = 0.05. The planktonic culture was carried out with 5 mL of LB medium at 200 rpm for 37 °C. The biofilm was grown in 96-well plates and six-well plates at 37 °C for 24 h. The liquid in the 96-well plates was then decanted and the plates washed three times with sterile water. In the six-well assays, 5 mL of LB medium was used to accommodate the larger bottoms of the
2.2.2. Separation and purification of tannins by Sephadex LH-20 Sephadex LH-20 (Shanghai Aladdin S&T Co. Ltd, China). The freezedried crude tannin powder was dissolved in methanol, and passed through a standard 0.45-μm sieve. It was further separated using a 4
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Table 2 Interpretation of the MALDI-TOF mass peaks for series crude tannins of rice straw extract. Experimental[M + Na+]
Calculated[M + Na+]
Monomeric unit type A
408.487 419.066 467.254 468.844 470.848 475.452 481.287 494.851 495.283 503.392 560.508 563.131 577.138 591.155 605.172 635.486 673.597 789.667 839.661 878.694 892.717 908.747 936.789 964.823 978.826 985.838 992.851 1002.684 1018.692 1046.728 1074.758 1103.917 1214.001 1396.221 1410.221
E+132 A+132 C+23 C+23 unknow unknow C+39 unknow unknow H+23 D + E+23 A+E A+A F+F C+F B + B+23 D + E+132 unknow A+E+E E + E+B+23 A + A+A+23 A + A+A+39 D + D + E+132 A + D + E+132 B + D + E+132 E + E+B+132 A + D + E+132 E + E+B+132 + 16 B + B + E+132 B + C+E+23 B+B+C unknow B + B+B + E+23 2A+3D+23 2A+3D+39
406.3 422.3 465.4 465.4 – – 481.4 – – 504.35 561.3 564.6 580.6 594.5 603.55 635.6 670.3 – 838.9 877.9 893.9 909.9 936.3 961.6 977.6 986.9 961.6 1018.9 1046 1055 – 1216.2 1398.6 1414.6
B
C
Degree of polymerisation D
E
F
G
H
1 1 1 1
1
1 1 2
1 1
1 1 2 1
1 2 1 1
1 2 2
1 3 3 2 1 1
1 1 1 1
1 2 1 2
1 1
1 1
3 2 2
1 1 1 2 1
1
3 3
1 1 1 1 – – 1 – – 1 3 2 2 2 2 2 2 – 3 3 3 3 3 3 3 3 3 – 3 3 3 – 4 5 5
Fig. 4. Inhibitory effects of crude tannins on S. aureus growth. (a.) OD600. (b.) CFU. (c.) Glucose consumption. (d.) Ethanol production. (e.) Acetic acid production. (f.) ATP. (g.) Membrane permeability. (h.) Aggregation of bacteria.
wells. The biofilm grown in 96-well plates was used for the assays of resazurin, crystal violet staining and CFUs, while the biofilm grown in six-well plates was used for CLSM observation.
modification (Makkar and Becker, 1993). Quantification was by means of comparison with a standard curve for catechin. The TP content was measured by Folin–Ciocalteu colorimetric method (Schmauch and Singleton, 1964). The TP content was quantified using a standard curve of gallic acid prepared in 80% methanol (v/v) (Matsuo and Itoo, 2006).
2.4. Analysis methods 2.4.2. Analysis of high performance liquid chromatography (HPLC) Glucose and acetic acid were analyzed by HPLC (LC-20AD,
2.4.1. Analysis of condensed tannins and total phenolics (TP) Condensed tannins was determined by vanillin method with minor 5
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Fig. 5. Influence of crude tannins on viability of biofilm cells and biofilm formation. (a.) Resazurin activity. (b.) OD550. (c.) Ethanol production. (d.) Glucose consumption. (e.) Acetic acid production.
Fig. 6. Inhibitory effects of purified tannins on S. aureus growth. (a.) OD600. (b.) CFU. (c.) Glucose consumption. (d.) Acetic acid production. (e.) Ethanol production. (f.) Aggregation of bacteria.
2.4.3. Analysis of MALDI-TOF MS The analysis of MALDI-TOF MS was carried out as previously described (Shu-Dong et al., 2010). The wavelength of pulsed nitrogen laser was set at 337 nm, and the laser pulse duration was 3 ns. Accelerating voltage was set at 20.0 kV and flectron voltage was set at 23.0 kV in the positive reflectron mode. The matrix was 2,5-Dihydroxybenzoic acid (10 mg/mL 30% acetone solution).
Shimadzu, Kyoto, Japan) with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) at 65 °C. 5 mM H2SO4 was used as the mobile phase and the flow rate was set at 0.6 mL/min. The phenolic compounds were analyzed by HPLC (UV detector at 280 nm, EX1600, Exformma, USA). An Eclipse XDB C18 column (250 mm × 4.6 mm, Agilent, USA) was used for separation, which carried out at 35 °C. Solvent A (water–acetic acid 99.5:0.5) and solvent B (methanol–water–acetic acid 95:0.5:0.5) were used as the mobile phases. Elution was carried out as follows: 0–5 min, 5% B–5% B; 5–10 min, 5% B–25% B; 10–30 min, 25% B–40% B; 30–45 min, 40% B–50% B; 45–55 min, 50% B–100% B; 55–60 min, 100% B–100% B; and 60–65 min, 100% B–5% B.
2.4.4. Quantitative analysis of planktonic cell and biofilm The planktonic cells were cultured at 37 °C for 12 h, and quantitatively analyzed by OD600 and CFU. The biofilm biomass was determined by a crystal violet staining assay (O’Toole (2011)). The biofilm was stained with 0.01% crystal violet, followed by dissolution with 30% 6
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Fig. 7. Influence of purified tannins on viability of biofilm cells and biofilm formation. (a.) Resazurin activity. (b.) OD550. (c.) Glucose consumption. (d.) Acetic acid and ethanol production.
3. Results and discussion
acetic acid (v/v). The solution was put to new clean plates to measure OD550, which indicated the biomass of biofilm.
3.1. Purification and component analysis of tannins derived from rice straw 2.4.5. CLSM observation The biofilm cultured on glass sheet was stained with SYTO 9 and PI successively for 30 min in darkness. After staining, sterile water was used to remove excess staining, and seal the film with fluorescent sealing agent (0.5 M carbonate buffer and glycerol, isovolumetric mixture) for observation with a CLSM (Olympus FV3000).
3.1.1. Separation and purification of tannins Plant tannins are principally recovered by solid–liquid extraction. Ethanol, acetone and methanol are commonly used as solvents for tannin extractions. However, these extracts also contain sugars, proteins, lipids and other phenolic compounds (Brown et al., 2017). Highly pure and well-characterized tannin samples are necessary for antibacterial studies or larger scale in vitro studies (Zeller, 2019). In this study, 1 kg of ball-ground straw was extracted with 70% acetone (w/v 1:10) at 60 °C and 150 rpm for 2 h, followed by centrifugation to obtain the crude tannin extract. The resulting solid residue could be used for saccharification and lactic acid production (Chen et al., 2018b; Wang et al., 2017; Xue et al., 2017). The tannin content in the crude tannin extract (named A1) was 0.65 g/kg ballground straw (Fig. 1 and Table 1). The crude tannins also contained small amounts of monosaccharides and phenolic acids, which could influence microorganism growth (Wang et al., 2017). Cui et al. reported that the phenolic acids derived from rice straw had a strong inhibitory effect on S. aureus and they could destroy biofilm structure and change the morphology of cells in biofilms (Cui et al., 2019). Wang et al. also reported that phenolic acids could damage cell membranes of Pichia stipites, resulting in low ethanol production (Wang et al., 2017). For that reason, the phenolic acids in the crude tannin extract have to be separated for better understanding the influence of tannins on S. aureus. Macroporous resin is commonly used to purify macromolecule compounds, such as tannins and flavonoids. In this study, AB-8 macroporous resin was used for tannin adsorption, and acetone was used as
2.4.6. Atom force microscope (AFM) observations An Agilent 5500 AFM/SPM (USA) atomic force microscope with 10 μm AFM probes was used for the observation of the biofilms. The images were analyzed with NanoScope Analysis software (Tang and Li, 2018)
2.4.7. Determination of membrane permeability The λex355/λem420 fluorescence value was immediately determined by taking 200 μL of sterile washed bacteria onto the blackboard and adding 10 g/L 1-n-phenylnaphthalamine dissolved in 2 μL of acetone. 2.5. Statistical analysis OriginPro 9.0 (OriginLab, Northampton, MA, USA) was used for assessing the significant differences of data. Statistical significance was defined as P < 0.05. Experiments performed in 96-well plates used six wells for each replication, while experiments in six-well plates used two wells. Experiments performed in tubes were replicated three times. 7
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Fig. 8. Observations of biofilm and planktonic cells with CLSM and AFM. (a.) CLSM image of the control biofilm. (b.) CLSM image of the control planktonic cells. (c.) AFM image of the control biofilm. (d.) CLSM image of biofilm treated with crude tannins. (e.) CLSM image of planktonic cells treated with crude tannins. (f.) AFM image of the planktonic treated with crude tannins. (g.) CLSM image of biofilm treated with purified tannins. (h). CLSM image of the planktonic treated with purified tannins. (i.) AFM image of planktonic treated with purified tannins.
purified by Sephadex LH-20, and analyzed by MALDI-TOF MS. Fig. 2 shows a typical MALDI-TOF analysis of tannins with MW range of 500–4000 Da. The peaks of the tannins in rice straw were mainly formed by monomer polymerization (Fig.3). According to the literature, the common monomers of tannins are catechin, epicatechin, epigallocatechin and epicatechin gallate with MW of 290.3, 290.3, 306.3 and 442.4 Da, respectively. Mangrove polyflavonoid tannins show the presence of a repeating structure, the MW of which is regular at 264.0–264.9 Da (structure D) (Oo et al., 2010). Calculation of the MALDI masses indicated that certain peaks could only be explained by the presence of epicatechin gallate units, from which the gallic acid residue had been removed, of 274.3 Da (structure E). There were two other monomer structures: fisetinidin [M + Na+]of 297.5 Da (structure F) and chebulic acid [M + Na+] of 379.23 Da (structure G) and 16 Da (OH−). Based on the existing monomers, we deduced the polymer composition of each peak pattern in the MALDI-TOF MS of crude tannins (Table 2). This indicates that the main structural elements of the tannins consisted of three component polymers: catechin, epicatechin and epigallocatechin. The main peaks were 419.066, 591.155, 878.694, 936.789, 1074.758 and 1410.221 Da; and 132 Da was the weight of Cs+. The 419-Da peak represented structure A and a Cs+. The peak at 591.155 Da was due to the F dimer structure. The 878.694-Da peak was composed of structure F dimer and structure B integrated with Na+. The 936.789-Da peak represented structure D dimer and structure E integrated with Cs+. The 1074.758-Da peak was due to structure B dimer and structure C. The 1410.22-Da peak was a pentamer composed of structure A dimer and D trimer integrating Na+ and OH−. The mean MWs of glucose and xylose were 180.16 and 150.13 Da, respectively. The common phenolic acids in straw mainly include
the organic solvent. Before the adsorption, the crude extract was evaporated to remove acetone and obtain the A2 fraction (0.53 g tannins/ kg straw). After purification of the A2 fraction by AB-8 resin, the B2 fraction was obtained and the tannin yield was 0.51 g tannins/kg straw. Since monosaccharides could not be adsorbed on the AB-8 resin, monosaccharides were separated from the tannin extract in this step (B1). However, the B2 fraction contained phenolic acids as well as tannins. Then, the B2 fraction was eluted with 70% acetone to obtain the C1 fraction (0.46 g tannins/kg straw). Sephadex LH-20 is a hydroxypropyl derivative of Sephadex G-25; its unique gel framework improves the hydrophilic properties of the gel filler and can be used in polar organic solvents or aqueous mixed solvents (Chen et al., 2018a; Ye et al., 2018). Therefore, the C1 fraction was freeze-dried, and then dissolved in methanol for isolation and purification by Sephadex LH-20. Ethanol eluents of 30, 50, 70 and 100% were respectively collected and named D01 (unabsorbed), D02, D03, D04 and D05. The tannin yields were 0.00, 0.02, 0.10, 0.31 and 0.03 g tannins/kg straw, respectively. In contrast, the phenolic acid content decreased with increasing ethanol concentration. At 70% ethanol, about 70% tannins could be recovered with few phenolic acids. Therefore, the 70% ethanol fraction (D04) was used as the purified tannins in further study, and D02, D03 and D05 were used for comparison.
3.1.2. Analysis of tannins by MALDI-TOF MS The tannins derived from rice straw are natural polymers that consist of various types of polyphenolic oligomers. Indeed, no characterization of condensed tannins in rice straw has been reported. Therefore, the tannins derived from rice straw were determined by MALDI-TOF-MS, and the spectra analyzed using the monomer structures previously found and studied. Four compounds were isolated and 8
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staining, which clearly showed a decrease in biofilm amount with increasing concentration of crude tannins (Fig. 5b). Meanwhile, the metabolites and glucose in the static culture were determined by HPLC. Higher concentrations of crude tannins resulted in lower ethanol and higher glucose concentrations (Fig. 5c and d). When the concentration of crude tannins was 1.1 g/L, little glucose was consumed. Moreover, no acetic acid was detected in the media for a crude tannin concentration of 0.6 g/L (Fig. 5e). The results demonstrated that the crude tannins reduced biofilm formation and had a marked inhibitory effect on cells in biofilm. Moreover, the change of morphology of biofilm was observed by CLSM in this study. The fluorescent dyes, SYTO 9 and PI, were used to stain biofilm cell. The SYTO 9 can specifically stain the DNA of live and dead cells, but PI cannot penetrate the complete membrane structure and only stains the DNA of damaged or dead cells. Because PI has a stronger affinity for DNA than SYTO 9, SYTO 9 can only stain the DNA of live cells. The live cells stained with SYTO 9 were green and dead cells stained with PI were red. A clear biofilm was observed with CLSM when crude tannins were not added, and there were few dead cells in the biofilm and few cells in the culture medium (Fig. 8a and b). The biofilm without tannin treatment consisted of regular spherical cells as shown by AFM (Fig. 8c). In contrast, few cells adhered to the plate surface (Fig. 8d), but large aggregations of cells were observed in the culture medium containing crude tannins, and almost all cells were dead (Fig. 8e). The AFM assay clearly showed that the surface of aggregation was uneven, and cell distribution was not uniform (Fig. 8f). It was obvious that many cells flocked together and seemed to be covered by unknown substances. The observations confirmed that crude tannins accelerated planktonic cell aggregation, and thus reduced the ability of cells to adhere to the surface, which was the main cause of crude tannins inhibiting biofilm formation. Research on the antimicrobial ability of plant polyphenols has mostly focused on inhibition of polyphenols on the tricarboxylic acid cycle pathway (Cui et al., 2019), and the relationship between antioxidant activity and antimicrobial ability (Muccilli et al., 2017). However, there have been less studies on changes in morphology due to tannins. Tannins have the ability to chelate proteins (Adnan et al., 2017). Bacterial cells form biofilms by aggregating amyloid proteins, and the physiological stress reaction of bacteria produces the secretion of extracellular matrix proteins (Lotti and Pollegioni, 2014). The essential oils inhibited the growth of P.aeruginosa due to the interaction between proteins and phenolic compounds (Amorese et al., 2018). Most plant extracts have bacteriostatic effect because they target proteins in cell membrane (Donadu et al., 2018). Therefore, we considered that the substances covering the cells would be an extracellular matrix containing proteins, which were chelated by tannins, resulting in acceleration of cell aggregation in the culture medium, and thus reducing biofilm formation.
vanillic acid, vanillin, coumaric acid and ferulic acid with average MWs of 168.15, 152.15, 140.09 and 194.19 Da, respectively. Thus, tannins derived from rice straw were mainly composed of dimers and trimers, and the main constituent units were catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid. Polyphenols have functional parts with antioxidant, antiviral, anticancer and antihypertension properties. For example, catechins containing gallate groups, especially epigallocatechin gallate, have shown the strongest antimicrobial activity by causing deactivation of the toxins produced by bacteria (Lotti and Pollegioni, 2014) (Adnan et al., 2017). Gallic acid and catechin alone or in combination have significant antimicrobial activity against Helicobacter pylori (Díaz-Gómez et al., 2013). Since the tannins derived from rice straw contain so many polyphenol monomers, these tannins should be functionally investigated. 3.2. Inhibitory effect of crude extract on S. Aureus 3.2.1. Growth inhibition of S. Aureus S. aureus is one species of Gram-positive cocci that can infect many animals. To investigate the influence of the crude extract on growth of S. aureus, cells were cultured with different tannin concentrations at 37 °C and 200 rpm for 12 h, and the influence first evaluated by OD600 value. The OD600 value decreased with increasing tannin concentrations (Fig. 4a). Especially when tannin concentrations exceeded 0.09 g/ L, the OD600 value increased significantly but slowly; the CFU value showed a similar trend (Fig. 4b). Moreover, glucose consumption was inhibited by the crude tannins (Fig. 4c). When tannin concentrations were 0.09 g/L or lower, glucose was completely consumed in 12 h, but residual glucose increased with increasing tannin concentrations exceeding 0.09 g/L. In contrast, higher concentrations of crude tannins resulted in lower acetic acid concentrations in the medium (Fig. 4e). In order to further confirm the inhibitory effect of the tannins on the growth of S. aureus, ATP content was determined in cells cultured for 12 h. The ATP content decreased with increasing tannin concentration (Fig. 4f), indicating that the energy metabolism of cells was affected and overall metabolic energy was reduced. Thus, the crude tannins had a significant inhibitory effect on S. aureus growth. Amorese et al. (2018) reported that phenolic compounds could affect permeability of membrane due to their hydrophobicity. The crude tannins also affected permeability of membrane. However, it should be noted that membrane permeability decreased with increasing concentrations of crude tannins (Fig. 4g). Furthermore, at high crude tannin concentrations, ethanol was detected in the medium (Fig. 4d). After the culture, the cells were uniformly present in the control medium, while large aggregation and precipitation of cells were observed in media containing the crude tannins (Fig. 4h). Based on the results, we hypothesized that tannins induced the aggregation and precipitation of cells, causing the decrease in membrane permeability. More importantly, the aggregation and precipitation of cells resulted in a reduction in specific surface area of cells, which could reduce mass transfer of oxygen into cells. Thus, some glucose was converted into ethanol rather than acetic acid.
3.3. Inhibitory effect of purified tannins To obtain purified tannins, the crude tannins were isolated and purified with Sephadex LH-20, and were eluted with 30, 50, 70 and 100% methanol in turn, and named D01(unabsorbed), D02, D03, D04 and D05, respectively. Since D01 did not contain any tannins and phenolic acids, we only investigated the inhibitory effect of other fractions on cell growth. All these fractions exhibited inhibitory effects on cell growth (Fig. 6a–d); and of these, D04 had the strongest inhibitory effect. The results indicate that the inhibitory effect of crude tannins did not result from phenolic acids. More importantly, the addition of D04 enhanced ethanol production (Fig. 6e) and resulted in the aggregation and precipitation of cells (Fig. 6f). The MALDI-TOF analysis clearly showed that the tannins derived from rice straw consisted mainly of catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid, which were rich in
3.2.2. Influence of crude tannins on biofilm formation by S. Aureus Biofilms are colonies of microorganisms with a polysaccharide matrix. S. aureus commonly form biofilm on the surface of medical devices form, causing systemic infection (Giannelli et al., 2017; Jian et al., 2017; Tango et al., 2018). In this study, we explored the influence of crude tannins on biofilm formation, and analyzed the biofilm activity since we found that tannins induced the aggregation and precipitation of cells. Cells were cultured in 96-hole plates to form biofilm. The biofilm activity in each culture hole was measured by resazurin reduction test in darkness. The crude tannins inhibited biofilm formation (Fig. 5a). The amount of the biofilm fixed on the cell culture plate was determined by crystal violet 9
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References
the D04 fraction. The purified tannins exhibited similar inhibitory effects to the crude tannins. Catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid are bioactive taxonomic compounds (Dhanani et al., 2017), and investigation into their deodorizing, antimicrobial and enzyme activity indicate that they play an important role in the metabolism and enzymatic activity of bacterial cells (Kim et al., 2017). Our results and the literature indicate that the inhibitory effect of the crude tannins resulted mainly from these tannins. The purified tannins also affected formation of biofilm. The D02, D03 and D04 fractions decreased biofilm formation (Fig. 7b) and lowered the viability of cells in biofilm (Fig. 7a,c,d) – as expected, D04 had the strongest effect. The CLSM images showed that cells hardly adhered to the plate surface (Fig. 8g), but large aggregations of cells were observed in the D04-containing culture medium, and almost all cells were dead (Fig. 8h). These phenomena were consistent with the case of the crude tannins, indicating that the inhibitory effect of the crude tannins on cells in biofilm resulted from tannins. More importantly, the surface of aggregation was more uneven than that for the crude tannins, and cells were clearly covered by unknown substances (Fig. 8i). The observation demonstrates that tannins could induce formation of some extracellular matrix or some macromolecular compounds in the medium due to the presence of tannins. The presence of the substances reduced adherence of cells to the plate surface, resulting in the reduction of biofilm.
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4. Conclusion In this study, tannins derived from rice straw were separated and purified. The MALDI-TOF-MS analysis indicated that catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid were the major unit components in the tannins. The tannins exhibited strong inhibitory effects on growth of S. aureus. Moreover, the tannins inhibited biofilm formation by accelerating planktonic cell aggregation, and thus reducing the ability of cells to adhere to the surface. The results provide a new approach to the production of low-cost antimicrobial agents and confirm that tannins can be separated from rice straw as a by-product in the process of biofuel production and their antimicrobial ability could have wide application prospects in medical and food fields. CRediT authorship contribution statement Jie Shi: Conceptualization, Methodology, Software, Investigation, Writing - original draft. Yazhu Wang: Data curation, Formal analysis, Visualization. Huanran Wei: Validation, Visualization. Jiajun Hu: Validation, Formal analysis, Visualization. Min-Tian Gao: Formal analysis, Resources, Writing - review & editing, Supervision. Declaration of Competing Interest This manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All study participants provided informed consent, and the study design was approved by the appropriate ethics review boards. No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. There are no conflicts of interest to declare. The manuscript does not contain experiments using animals and human studies. Acknowledgements This study was supported by grants from the Special Fund for Agroscientific Research in the Public Interest (No. 201503135-14). 10
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Structure analysis of condensed tannin from rice straw and its inhibitory effect on Staphylococcus aureus
T
Jie Shi, Yazhu Wang, Huanran Wei, Jiajun Hu, Min-Tian Gao* Shanghai Key Laboratory of Bio-energy Crops, School of Life Sciences, Shanghai University, 99 Shangda Road, Shanghai, 200444, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Staphylococcus aureus agricultural waste Tannins Biofilm Antimicrobial agent
To reduce the production cost of bioethanol and improve the utilization rate of straw resources, more high valueadded products have to be found and applied. In this study, tannins were considered as a high-value by-product. The extraction of condensed tannins from rice straw was studied, and the tannins were characterized. The crude tannins consisted mainly of tannins, phenolic acids and monosaccharides. The crude tannins were separated and purified with AB-8 macroporous resin and Sephadex LH-20. The matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) assay confirmed that catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid were the major components in the purified tannins. The tannins exhibited strong inhibitory effects on growth of Staphylococcus aureus. Higher concentrations of crude tannins resulted in lower ethanol concentration and intercellular ATP content, and higher residual glucose. Moreover, the tannins could regulate morphology of biofilms. The images from atomic force microscopy and confocal laser scanning microscopy showed that cells hardly adhered to the plate surface, and large aggregations of cells were observed in culture medium containing tannins, and almost all cells were dead. This study provides the feasibility of tannins derived from rice straw as new antimicrobial agents with a different inhibitory mechanism.
1. Introduction
antibiotics would make strains more drug tolerant (DeLeo and Chambers, 2009). In order to reduce the frequency of use of antibiotics on bacteria and prevent the development of drug resistance, it is now urgent to find a bacteriostatic agent to assist and replace antibiotics (Doernberg et al., 2017) (Usai et al., 2019). A number of papers reporting antimicrobial properties of condensed tannins, polyphenols and flavonoids isolated from higher plants have been published in recent years (Arora and Mahajan, 2019; Teanpaisan et al., 2017; Xu et al., 2015). The plant extracts have been documented to have the ability to effectively inhibit the growth of phytopathogenic fungi (Donadu et al., 2019), bacteria (Cannas et al., 2015) and tumor cells (Chaves-López et al., 2018; Donadu et al., 2017). A biofilm is a kind of surface-related bacterial aggregation within a generated extracellular polymer matrix, and can easily adhere to the surfaces of foods and medical instruments, which would lead to the increase in the resistance to common antibiotics and disinfectants. Condensed tannins are the most common polyphenols in plants, and are beneficial to human and animal nutrition and can promote health as antioxidants due to their antibacterial, antiviral and protein-binding activities (Salminen, 2018). Leaves and rhizomes of varied plants contain condensed tannins (Barnaba et al., 2018; Ismayati et al., 2017; Suvanto et al., 2017). However, few studies have investigated the
Rice straw is an abundant lignocellulosic waste, and consists mainly of cellulose, hemicellulose, lignin and silica, which could be used for the production of energy and materials (Linh et al., 2017). However, the bioconversion of rice straw suffers from low fermentation performance and high enzyme cost. Therefore, many studies have focused on improving conversion technology, feedstock production and conversion technology to minimize production costs (Diep et al., 2012). Besides cellulose, hemicellulose and lignin, rice straw contains many bioactive compounds, such as phenolic acids and flavonoids (Dong et al., 2005; Goufo and Trindade, 2014). During its bioconversion, the bioactive compounds can be released from rice straw, and can be considered as high value-added products. The integrated production of the bioactive compounds and ethanol is an effective strategy to improve economic competitiveness and reduce the cost of bioethanol (Ma et al., 2019). Staphylococcus aureus is a common pathogenic bacteria, which can cause suppurative inflammation and disease due to its exotoxins (Ward et al., 2018). This bacteria is widely found in air and water, causing bacterial infections in medical treatment and food poisoning, and is a great challenge to human health (Shi et al., 2017). Antibiotics are capable of killing infectious diseases. However, long-term use of ⁎
Corresponding author. E-mail address: [email protected] (M.-T. Gao).
https://doi.org/10.1016/j.indcrop.2020.112130 Received 16 October 2019; Received in revised form 9 January 2020; Accepted 13 January 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. Flow chart for extraction of condensed tannins from rice straw. Table 1 HPLC analysis of the different components of each fraction from rice straw. mg/g
Total phenols
tannins
A1 A2 B1 C1 D1 D2 D3 D4 D5
0.71 0.62 0.17 0.52 0.10 0.03 0.21 0.41 0.05
0.65 0.53 0.01 0.46 0.03 0.02 0.10 0.31 0.03
± ± ± ± ± ± ± ± ±
0.05 0.06 0.02 0.01 0.01 0.01 0.05 0.03 0.02
± ± ± ± ± ± ± ± ±
0.01 0.06 0.00 0.02 0.00 0.00 0.00 0.02 0.00
glucose
xylose
Vanillic acid
vanillin
Coumaric acid
Ferulic acid
19.60 ± 0.11 7.57 ± 0.24 11.06 ± 0.53 0.59 ± 0.04 ND ND ND ND ND
16.97 ± 0.24 6.36 ± 0.2 10.45 ± 0.12 0.61 ± 0.15 ND ND ND ND ND
0.18 ND ND 0.12 ND 0.05 0.04 0.01 0.01
0.15 ND ND 0.09 ND 0.01 0.06 0.03 ND
0.39 ND ND 0.30 ND ND 0.02 0.04 ND
0.09 ND ND 0.08 ND ND 0.01 0.01 ND
± 0.01
± 0.02 ± ± ± ±
0.00 0.00 0.00 0.00
± 0.00
± 0.00 ± 0.00 ± 0.00 ± 0.00
± 0.00
± 0.00
± 0.00 ± 0.00
± 0.00
± 0.00
± 0.00 ± 0.00
*The date represent averages ± standard deviations for duplicate experiments. ND = not detected.
colony-forming units (CFUs), metabolites and ATP, and observing the structural change in biofilm using confocal laser scanning microscopy (CLSM). To the best of our knowledge, this is the first study on the characterization of tannins derived from rice straw and the inhibitory mechanism on S. aureus. The results will be beneficial for utilization of rice straw in food and medical fields.
condensed tannins derived from rice straw. Previous research showed that tannins derived from rice straw had an inhibitory effect on fungi (Wang et al., 2017), bacteria (Ahn et al., 2016; Cai et al., 2017) and yeasts (Fang et al., 2015; Nakanishi et al., 2012), and the removal of tannins could improve fermentation efficiency. Based on these results, we proposed that condensed tannins derived from rice straw could be used as antimicrobial agents. To characterize the tannins derived from rice straw, we isolated and purified them using Sephadex LH-20. To investigate the chemical structure of the condensed tannins, further characterization was continued by means of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), which is considered a method of choice for analysis of compounds exhibiting large structural heterogeneity (Wang and Dai, 2018). Then, we investigated the inhibitory effect of the tannins and their fractions on S. aureus growth and their damaging effect on biofilm formation by S. aureus by determining
2. Material and methods 2.1. Preparation of condensed tannins from rice straw Rice straw was treated as previously described (Wang et al., 2017). 10 g of the treated straw was put into 100 mL of 70% acetone for extraction at 60 °C and 150 rpm for 2 h. Solid insoluble matter was filtered out and the extraction was repeated three times. 2
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Fig. 2. Typical MALDI-TOF analysis of rice straw tannin extract with molecular weight range of 500–4000 Da.
3
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Fig. 3. The main monomer unit structures of condensed tannins.
Sephadex LH-20 chromatography column. The Sephadex LH-20 was soaked in 60–70% ethanol overnight. Installed chromatography column of wet state with no gap, washed with water until ethanol could not be detected and the chromatography column was balanced. The Sephadex LH-20 was eluted with methanol–water system and reverse phase solvent. Then 10 mL of the crude extract of condensed tannins dissolved in methanol was added to the column and eluted with 30, 50, 70 and 100% methanol successively. The column was eluted at a flow rate of 1 mL/min.
2.2. Purification and separation of tannins 2.2.1. Adsorbed and eluted by AB-8 macroporous resin Macroporous resin (AB-8; Shanghai Yuanye S&T Co. Ltd, China) was used for the separation of tannins. Solvent solid–liquid extraction of the solution, using low-temperature rotation evaporation, remove organic solvents, added AB-8 macroporous resin according to the volume/mass ratio of 1:5, and placed in a shaking bed at room temperature for adsorption at 150 rpm for 30 min. The loaded column (250 mm × 50 mm) was carried out for the macroporous resin that was saturated with adsorption. After equilibrium of the column, 70% ethanol was used for elution until colorless. The elution solution was collected for low-temperature rotary evaporation to remove organic solvent. The remaining aqueous solution was freeze-dried to obtain dry powder of crude tannin.
2.3. Culture conditions The S. aureus (ATCC 6538) strain was used in this study. The strain was precultured in Luria–Bertani (LB) medium at 37 °C for 12 h. The initial optical density was set at OD600 = 0.05. The planktonic culture was carried out with 5 mL of LB medium at 200 rpm for 37 °C. The biofilm was grown in 96-well plates and six-well plates at 37 °C for 24 h. The liquid in the 96-well plates was then decanted and the plates washed three times with sterile water. In the six-well assays, 5 mL of LB medium was used to accommodate the larger bottoms of the
2.2.2. Separation and purification of tannins by Sephadex LH-20 Sephadex LH-20 (Shanghai Aladdin S&T Co. Ltd, China). The freezedried crude tannin powder was dissolved in methanol, and passed through a standard 0.45-μm sieve. It was further separated using a 4
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Table 2 Interpretation of the MALDI-TOF mass peaks for series crude tannins of rice straw extract. Experimental[M + Na+]
Calculated[M + Na+]
Monomeric unit type A
408.487 419.066 467.254 468.844 470.848 475.452 481.287 494.851 495.283 503.392 560.508 563.131 577.138 591.155 605.172 635.486 673.597 789.667 839.661 878.694 892.717 908.747 936.789 964.823 978.826 985.838 992.851 1002.684 1018.692 1046.728 1074.758 1103.917 1214.001 1396.221 1410.221
E+132 A+132 C+23 C+23 unknow unknow C+39 unknow unknow H+23 D + E+23 A+E A+A F+F C+F B + B+23 D + E+132 unknow A+E+E E + E+B+23 A + A+A+23 A + A+A+39 D + D + E+132 A + D + E+132 B + D + E+132 E + E+B+132 A + D + E+132 E + E+B+132 + 16 B + B + E+132 B + C+E+23 B+B+C unknow B + B+B + E+23 2A+3D+23 2A+3D+39
406.3 422.3 465.4 465.4 – – 481.4 – – 504.35 561.3 564.6 580.6 594.5 603.55 635.6 670.3 – 838.9 877.9 893.9 909.9 936.3 961.6 977.6 986.9 961.6 1018.9 1046 1055 – 1216.2 1398.6 1414.6
B
C
Degree of polymerisation D
E
F
G
H
1 1 1 1
1
1 1 2
1 1
1 1 2 1
1 2 1 1
1 2 2
1 3 3 2 1 1
1 1 1 1
1 2 1 2
1 1
1 1
3 2 2
1 1 1 2 1
1
3 3
1 1 1 1 – – 1 – – 1 3 2 2 2 2 2 2 – 3 3 3 3 3 3 3 3 3 – 3 3 3 – 4 5 5
Fig. 4. Inhibitory effects of crude tannins on S. aureus growth. (a.) OD600. (b.) CFU. (c.) Glucose consumption. (d.) Ethanol production. (e.) Acetic acid production. (f.) ATP. (g.) Membrane permeability. (h.) Aggregation of bacteria.
wells. The biofilm grown in 96-well plates was used for the assays of resazurin, crystal violet staining and CFUs, while the biofilm grown in six-well plates was used for CLSM observation.
modification (Makkar and Becker, 1993). Quantification was by means of comparison with a standard curve for catechin. The TP content was measured by Folin–Ciocalteu colorimetric method (Schmauch and Singleton, 1964). The TP content was quantified using a standard curve of gallic acid prepared in 80% methanol (v/v) (Matsuo and Itoo, 2006).
2.4. Analysis methods 2.4.2. Analysis of high performance liquid chromatography (HPLC) Glucose and acetic acid were analyzed by HPLC (LC-20AD,
2.4.1. Analysis of condensed tannins and total phenolics (TP) Condensed tannins was determined by vanillin method with minor 5
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Fig. 5. Influence of crude tannins on viability of biofilm cells and biofilm formation. (a.) Resazurin activity. (b.) OD550. (c.) Ethanol production. (d.) Glucose consumption. (e.) Acetic acid production.
Fig. 6. Inhibitory effects of purified tannins on S. aureus growth. (a.) OD600. (b.) CFU. (c.) Glucose consumption. (d.) Acetic acid production. (e.) Ethanol production. (f.) Aggregation of bacteria.
2.4.3. Analysis of MALDI-TOF MS The analysis of MALDI-TOF MS was carried out as previously described (Shu-Dong et al., 2010). The wavelength of pulsed nitrogen laser was set at 337 nm, and the laser pulse duration was 3 ns. Accelerating voltage was set at 20.0 kV and flectron voltage was set at 23.0 kV in the positive reflectron mode. The matrix was 2,5-Dihydroxybenzoic acid (10 mg/mL 30% acetone solution).
Shimadzu, Kyoto, Japan) with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) at 65 °C. 5 mM H2SO4 was used as the mobile phase and the flow rate was set at 0.6 mL/min. The phenolic compounds were analyzed by HPLC (UV detector at 280 nm, EX1600, Exformma, USA). An Eclipse XDB C18 column (250 mm × 4.6 mm, Agilent, USA) was used for separation, which carried out at 35 °C. Solvent A (water–acetic acid 99.5:0.5) and solvent B (methanol–water–acetic acid 95:0.5:0.5) were used as the mobile phases. Elution was carried out as follows: 0–5 min, 5% B–5% B; 5–10 min, 5% B–25% B; 10–30 min, 25% B–40% B; 30–45 min, 40% B–50% B; 45–55 min, 50% B–100% B; 55–60 min, 100% B–100% B; and 60–65 min, 100% B–5% B.
2.4.4. Quantitative analysis of planktonic cell and biofilm The planktonic cells were cultured at 37 °C for 12 h, and quantitatively analyzed by OD600 and CFU. The biofilm biomass was determined by a crystal violet staining assay (O’Toole (2011)). The biofilm was stained with 0.01% crystal violet, followed by dissolution with 30% 6
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Fig. 7. Influence of purified tannins on viability of biofilm cells and biofilm formation. (a.) Resazurin activity. (b.) OD550. (c.) Glucose consumption. (d.) Acetic acid and ethanol production.
3. Results and discussion
acetic acid (v/v). The solution was put to new clean plates to measure OD550, which indicated the biomass of biofilm.
3.1. Purification and component analysis of tannins derived from rice straw 2.4.5. CLSM observation The biofilm cultured on glass sheet was stained with SYTO 9 and PI successively for 30 min in darkness. After staining, sterile water was used to remove excess staining, and seal the film with fluorescent sealing agent (0.5 M carbonate buffer and glycerol, isovolumetric mixture) for observation with a CLSM (Olympus FV3000).
3.1.1. Separation and purification of tannins Plant tannins are principally recovered by solid–liquid extraction. Ethanol, acetone and methanol are commonly used as solvents for tannin extractions. However, these extracts also contain sugars, proteins, lipids and other phenolic compounds (Brown et al., 2017). Highly pure and well-characterized tannin samples are necessary for antibacterial studies or larger scale in vitro studies (Zeller, 2019). In this study, 1 kg of ball-ground straw was extracted with 70% acetone (w/v 1:10) at 60 °C and 150 rpm for 2 h, followed by centrifugation to obtain the crude tannin extract. The resulting solid residue could be used for saccharification and lactic acid production (Chen et al., 2018b; Wang et al., 2017; Xue et al., 2017). The tannin content in the crude tannin extract (named A1) was 0.65 g/kg ballground straw (Fig. 1 and Table 1). The crude tannins also contained small amounts of monosaccharides and phenolic acids, which could influence microorganism growth (Wang et al., 2017). Cui et al. reported that the phenolic acids derived from rice straw had a strong inhibitory effect on S. aureus and they could destroy biofilm structure and change the morphology of cells in biofilms (Cui et al., 2019). Wang et al. also reported that phenolic acids could damage cell membranes of Pichia stipites, resulting in low ethanol production (Wang et al., 2017). For that reason, the phenolic acids in the crude tannin extract have to be separated for better understanding the influence of tannins on S. aureus. Macroporous resin is commonly used to purify macromolecule compounds, such as tannins and flavonoids. In this study, AB-8 macroporous resin was used for tannin adsorption, and acetone was used as
2.4.6. Atom force microscope (AFM) observations An Agilent 5500 AFM/SPM (USA) atomic force microscope with 10 μm AFM probes was used for the observation of the biofilms. The images were analyzed with NanoScope Analysis software (Tang and Li, 2018)
2.4.7. Determination of membrane permeability The λex355/λem420 fluorescence value was immediately determined by taking 200 μL of sterile washed bacteria onto the blackboard and adding 10 g/L 1-n-phenylnaphthalamine dissolved in 2 μL of acetone. 2.5. Statistical analysis OriginPro 9.0 (OriginLab, Northampton, MA, USA) was used for assessing the significant differences of data. Statistical significance was defined as P < 0.05. Experiments performed in 96-well plates used six wells for each replication, while experiments in six-well plates used two wells. Experiments performed in tubes were replicated three times. 7
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Fig. 8. Observations of biofilm and planktonic cells with CLSM and AFM. (a.) CLSM image of the control biofilm. (b.) CLSM image of the control planktonic cells. (c.) AFM image of the control biofilm. (d.) CLSM image of biofilm treated with crude tannins. (e.) CLSM image of planktonic cells treated with crude tannins. (f.) AFM image of the planktonic treated with crude tannins. (g.) CLSM image of biofilm treated with purified tannins. (h). CLSM image of the planktonic treated with purified tannins. (i.) AFM image of planktonic treated with purified tannins.
purified by Sephadex LH-20, and analyzed by MALDI-TOF MS. Fig. 2 shows a typical MALDI-TOF analysis of tannins with MW range of 500–4000 Da. The peaks of the tannins in rice straw were mainly formed by monomer polymerization (Fig.3). According to the literature, the common monomers of tannins are catechin, epicatechin, epigallocatechin and epicatechin gallate with MW of 290.3, 290.3, 306.3 and 442.4 Da, respectively. Mangrove polyflavonoid tannins show the presence of a repeating structure, the MW of which is regular at 264.0–264.9 Da (structure D) (Oo et al., 2010). Calculation of the MALDI masses indicated that certain peaks could only be explained by the presence of epicatechin gallate units, from which the gallic acid residue had been removed, of 274.3 Da (structure E). There were two other monomer structures: fisetinidin [M + Na+]of 297.5 Da (structure F) and chebulic acid [M + Na+] of 379.23 Da (structure G) and 16 Da (OH−). Based on the existing monomers, we deduced the polymer composition of each peak pattern in the MALDI-TOF MS of crude tannins (Table 2). This indicates that the main structural elements of the tannins consisted of three component polymers: catechin, epicatechin and epigallocatechin. The main peaks were 419.066, 591.155, 878.694, 936.789, 1074.758 and 1410.221 Da; and 132 Da was the weight of Cs+. The 419-Da peak represented structure A and a Cs+. The peak at 591.155 Da was due to the F dimer structure. The 878.694-Da peak was composed of structure F dimer and structure B integrated with Na+. The 936.789-Da peak represented structure D dimer and structure E integrated with Cs+. The 1074.758-Da peak was due to structure B dimer and structure C. The 1410.22-Da peak was a pentamer composed of structure A dimer and D trimer integrating Na+ and OH−. The mean MWs of glucose and xylose were 180.16 and 150.13 Da, respectively. The common phenolic acids in straw mainly include
the organic solvent. Before the adsorption, the crude extract was evaporated to remove acetone and obtain the A2 fraction (0.53 g tannins/ kg straw). After purification of the A2 fraction by AB-8 resin, the B2 fraction was obtained and the tannin yield was 0.51 g tannins/kg straw. Since monosaccharides could not be adsorbed on the AB-8 resin, monosaccharides were separated from the tannin extract in this step (B1). However, the B2 fraction contained phenolic acids as well as tannins. Then, the B2 fraction was eluted with 70% acetone to obtain the C1 fraction (0.46 g tannins/kg straw). Sephadex LH-20 is a hydroxypropyl derivative of Sephadex G-25; its unique gel framework improves the hydrophilic properties of the gel filler and can be used in polar organic solvents or aqueous mixed solvents (Chen et al., 2018a; Ye et al., 2018). Therefore, the C1 fraction was freeze-dried, and then dissolved in methanol for isolation and purification by Sephadex LH-20. Ethanol eluents of 30, 50, 70 and 100% were respectively collected and named D01 (unabsorbed), D02, D03, D04 and D05. The tannin yields were 0.00, 0.02, 0.10, 0.31 and 0.03 g tannins/kg straw, respectively. In contrast, the phenolic acid content decreased with increasing ethanol concentration. At 70% ethanol, about 70% tannins could be recovered with few phenolic acids. Therefore, the 70% ethanol fraction (D04) was used as the purified tannins in further study, and D02, D03 and D05 were used for comparison.
3.1.2. Analysis of tannins by MALDI-TOF MS The tannins derived from rice straw are natural polymers that consist of various types of polyphenolic oligomers. Indeed, no characterization of condensed tannins in rice straw has been reported. Therefore, the tannins derived from rice straw were determined by MALDI-TOF-MS, and the spectra analyzed using the monomer structures previously found and studied. Four compounds were isolated and 8
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staining, which clearly showed a decrease in biofilm amount with increasing concentration of crude tannins (Fig. 5b). Meanwhile, the metabolites and glucose in the static culture were determined by HPLC. Higher concentrations of crude tannins resulted in lower ethanol and higher glucose concentrations (Fig. 5c and d). When the concentration of crude tannins was 1.1 g/L, little glucose was consumed. Moreover, no acetic acid was detected in the media for a crude tannin concentration of 0.6 g/L (Fig. 5e). The results demonstrated that the crude tannins reduced biofilm formation and had a marked inhibitory effect on cells in biofilm. Moreover, the change of morphology of biofilm was observed by CLSM in this study. The fluorescent dyes, SYTO 9 and PI, were used to stain biofilm cell. The SYTO 9 can specifically stain the DNA of live and dead cells, but PI cannot penetrate the complete membrane structure and only stains the DNA of damaged or dead cells. Because PI has a stronger affinity for DNA than SYTO 9, SYTO 9 can only stain the DNA of live cells. The live cells stained with SYTO 9 were green and dead cells stained with PI were red. A clear biofilm was observed with CLSM when crude tannins were not added, and there were few dead cells in the biofilm and few cells in the culture medium (Fig. 8a and b). The biofilm without tannin treatment consisted of regular spherical cells as shown by AFM (Fig. 8c). In contrast, few cells adhered to the plate surface (Fig. 8d), but large aggregations of cells were observed in the culture medium containing crude tannins, and almost all cells were dead (Fig. 8e). The AFM assay clearly showed that the surface of aggregation was uneven, and cell distribution was not uniform (Fig. 8f). It was obvious that many cells flocked together and seemed to be covered by unknown substances. The observations confirmed that crude tannins accelerated planktonic cell aggregation, and thus reduced the ability of cells to adhere to the surface, which was the main cause of crude tannins inhibiting biofilm formation. Research on the antimicrobial ability of plant polyphenols has mostly focused on inhibition of polyphenols on the tricarboxylic acid cycle pathway (Cui et al., 2019), and the relationship between antioxidant activity and antimicrobial ability (Muccilli et al., 2017). However, there have been less studies on changes in morphology due to tannins. Tannins have the ability to chelate proteins (Adnan et al., 2017). Bacterial cells form biofilms by aggregating amyloid proteins, and the physiological stress reaction of bacteria produces the secretion of extracellular matrix proteins (Lotti and Pollegioni, 2014). The essential oils inhibited the growth of P.aeruginosa due to the interaction between proteins and phenolic compounds (Amorese et al., 2018). Most plant extracts have bacteriostatic effect because they target proteins in cell membrane (Donadu et al., 2018). Therefore, we considered that the substances covering the cells would be an extracellular matrix containing proteins, which were chelated by tannins, resulting in acceleration of cell aggregation in the culture medium, and thus reducing biofilm formation.
vanillic acid, vanillin, coumaric acid and ferulic acid with average MWs of 168.15, 152.15, 140.09 and 194.19 Da, respectively. Thus, tannins derived from rice straw were mainly composed of dimers and trimers, and the main constituent units were catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid. Polyphenols have functional parts with antioxidant, antiviral, anticancer and antihypertension properties. For example, catechins containing gallate groups, especially epigallocatechin gallate, have shown the strongest antimicrobial activity by causing deactivation of the toxins produced by bacteria (Lotti and Pollegioni, 2014) (Adnan et al., 2017). Gallic acid and catechin alone or in combination have significant antimicrobial activity against Helicobacter pylori (Díaz-Gómez et al., 2013). Since the tannins derived from rice straw contain so many polyphenol monomers, these tannins should be functionally investigated. 3.2. Inhibitory effect of crude extract on S. Aureus 3.2.1. Growth inhibition of S. Aureus S. aureus is one species of Gram-positive cocci that can infect many animals. To investigate the influence of the crude extract on growth of S. aureus, cells were cultured with different tannin concentrations at 37 °C and 200 rpm for 12 h, and the influence first evaluated by OD600 value. The OD600 value decreased with increasing tannin concentrations (Fig. 4a). Especially when tannin concentrations exceeded 0.09 g/ L, the OD600 value increased significantly but slowly; the CFU value showed a similar trend (Fig. 4b). Moreover, glucose consumption was inhibited by the crude tannins (Fig. 4c). When tannin concentrations were 0.09 g/L or lower, glucose was completely consumed in 12 h, but residual glucose increased with increasing tannin concentrations exceeding 0.09 g/L. In contrast, higher concentrations of crude tannins resulted in lower acetic acid concentrations in the medium (Fig. 4e). In order to further confirm the inhibitory effect of the tannins on the growth of S. aureus, ATP content was determined in cells cultured for 12 h. The ATP content decreased with increasing tannin concentration (Fig. 4f), indicating that the energy metabolism of cells was affected and overall metabolic energy was reduced. Thus, the crude tannins had a significant inhibitory effect on S. aureus growth. Amorese et al. (2018) reported that phenolic compounds could affect permeability of membrane due to their hydrophobicity. The crude tannins also affected permeability of membrane. However, it should be noted that membrane permeability decreased with increasing concentrations of crude tannins (Fig. 4g). Furthermore, at high crude tannin concentrations, ethanol was detected in the medium (Fig. 4d). After the culture, the cells were uniformly present in the control medium, while large aggregation and precipitation of cells were observed in media containing the crude tannins (Fig. 4h). Based on the results, we hypothesized that tannins induced the aggregation and precipitation of cells, causing the decrease in membrane permeability. More importantly, the aggregation and precipitation of cells resulted in a reduction in specific surface area of cells, which could reduce mass transfer of oxygen into cells. Thus, some glucose was converted into ethanol rather than acetic acid.
3.3. Inhibitory effect of purified tannins To obtain purified tannins, the crude tannins were isolated and purified with Sephadex LH-20, and were eluted with 30, 50, 70 and 100% methanol in turn, and named D01(unabsorbed), D02, D03, D04 and D05, respectively. Since D01 did not contain any tannins and phenolic acids, we only investigated the inhibitory effect of other fractions on cell growth. All these fractions exhibited inhibitory effects on cell growth (Fig. 6a–d); and of these, D04 had the strongest inhibitory effect. The results indicate that the inhibitory effect of crude tannins did not result from phenolic acids. More importantly, the addition of D04 enhanced ethanol production (Fig. 6e) and resulted in the aggregation and precipitation of cells (Fig. 6f). The MALDI-TOF analysis clearly showed that the tannins derived from rice straw consisted mainly of catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid, which were rich in
3.2.2. Influence of crude tannins on biofilm formation by S. Aureus Biofilms are colonies of microorganisms with a polysaccharide matrix. S. aureus commonly form biofilm on the surface of medical devices form, causing systemic infection (Giannelli et al., 2017; Jian et al., 2017; Tango et al., 2018). In this study, we explored the influence of crude tannins on biofilm formation, and analyzed the biofilm activity since we found that tannins induced the aggregation and precipitation of cells. Cells were cultured in 96-hole plates to form biofilm. The biofilm activity in each culture hole was measured by resazurin reduction test in darkness. The crude tannins inhibited biofilm formation (Fig. 5a). The amount of the biofilm fixed on the cell culture plate was determined by crystal violet 9
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References
the D04 fraction. The purified tannins exhibited similar inhibitory effects to the crude tannins. Catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid are bioactive taxonomic compounds (Dhanani et al., 2017), and investigation into their deodorizing, antimicrobial and enzyme activity indicate that they play an important role in the metabolism and enzymatic activity of bacterial cells (Kim et al., 2017). Our results and the literature indicate that the inhibitory effect of the crude tannins resulted mainly from these tannins. The purified tannins also affected formation of biofilm. The D02, D03 and D04 fractions decreased biofilm formation (Fig. 7b) and lowered the viability of cells in biofilm (Fig. 7a,c,d) – as expected, D04 had the strongest effect. The CLSM images showed that cells hardly adhered to the plate surface (Fig. 8g), but large aggregations of cells were observed in the D04-containing culture medium, and almost all cells were dead (Fig. 8h). These phenomena were consistent with the case of the crude tannins, indicating that the inhibitory effect of the crude tannins on cells in biofilm resulted from tannins. More importantly, the surface of aggregation was more uneven than that for the crude tannins, and cells were clearly covered by unknown substances (Fig. 8i). The observation demonstrates that tannins could induce formation of some extracellular matrix or some macromolecular compounds in the medium due to the presence of tannins. The presence of the substances reduced adherence of cells to the plate surface, resulting in the reduction of biofilm.
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4. Conclusion In this study, tannins derived from rice straw were separated and purified. The MALDI-TOF-MS analysis indicated that catechin, epicatechin, epigallocatechin, epicatechin gallate and gallic acid were the major unit components in the tannins. The tannins exhibited strong inhibitory effects on growth of S. aureus. Moreover, the tannins inhibited biofilm formation by accelerating planktonic cell aggregation, and thus reducing the ability of cells to adhere to the surface. The results provide a new approach to the production of low-cost antimicrobial agents and confirm that tannins can be separated from rice straw as a by-product in the process of biofuel production and their antimicrobial ability could have wide application prospects in medical and food fields. CRediT authorship contribution statement Jie Shi: Conceptualization, Methodology, Software, Investigation, Writing - original draft. Yazhu Wang: Data curation, Formal analysis, Visualization. Huanran Wei: Validation, Visualization. Jiajun Hu: Validation, Formal analysis, Visualization. Min-Tian Gao: Formal analysis, Resources, Writing - review & editing, Supervision. Declaration of Competing Interest This manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All study participants provided informed consent, and the study design was approved by the appropriate ethics review boards. No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. There are no conflicts of interest to declare. The manuscript does not contain experiments using animals and human studies. Acknowledgements This study was supported by grants from the Special Fund for Agroscientific Research in the Public Interest (No. 201503135-14). 10
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