Adsorption mechanism of tenuazonic acid using inactivated lactic acid bacteria

Food Control 82 (2017) 274e282

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Adsorption mechanism of tenuazonic acid using inactivated lactic acid bacteria Na Ge b, Jingjing Xu b, Bangzhu Peng a, *, Siyi Pan a a b

Key Laboratory of Environment Correlative Dietology, Ministry of Education, Huazhong Agricultural University, 430070, Wuhan, Hubei, China College of Food Science & Technology, Huazhong Agricultural University, 430070, Wuhan, Hubei, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 March 2017 Received in revised form 10 June 2017 Accepted 9 July 2017 Available online 10 July 2017

Tenuazonic acid (TeA) is a fungal secondary metabolite that is produced by a number of Alternaria species and is therefore a natural contaminant of food and feed samples. The aim of this study was to investigate the adsorption mechanism of TeA using various modified inactivated lactic acid bacteria (LAB). The bacterial cells were characterized by Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectroscopy (SEM-EDS) and Transmission Electron Microscopy (TEM). The results indicated that increasing the surface area of the cell wall improves the adsorption capacity of TeA, and an ion-exchange reaction may occur during the adsorption process. Fourier Transform Infrared (FTIR) Spectroscopy and X-ray Diffraction (XRD) analysis indicated that CeO, OeH and NeH groups, which are related to protein and carbohydrate components, were obviously involved in the adsorption of TeA. The zeta potential indicated that TeA adsorption was related to the surface charge of the bacteria cells. Above all, polysaccharides and protein were demonstrated to be important components of the LAB cell wall and are involved in TeA removal. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Lactic acid bacteria Tenuazonic acid Adsorption mechanism

1. Introduction Tenuazonic acid ((5S, 8S)-3-acetyl-5-sec-butyltetramic acid, TeA) is a toxic metabolite produced by Alternaria spp., Phoma sorghina and Pyricularia oryzae (Meronuck, Steele, Mirocha, & Christensen, 1972; Umetsu, Kaji, & Tamari, 1972). TeA is thought to be a hybrid of 1 isoleucine and 2 acetates (Yun, Motoyama, & Osada, 2015). It can inhibit protein biosynthesis by suppressing the release of new proteins from the ribosome (Shigeura & Gordon, 1963). TeA has been reported to exert antiviral, antitumor, antibacterial, cytotoxic and phytotoxic properties and to be acutely toxic in mammals (Lou, Fu, Peng, & Zhou, 2013; Rychlik, Lepper, € Doymaz, & Alta, Weidner, & Asam, 2016; Yekeler, Bçitmçis, O, 2001). The oral medium lethal dose (LD50) of TeA was shown to be 225 mg/kg body weight (bw) in mice and 100e150 mg/kg bw in Macaca fascicularis; thus, TeA is considered to be the most toxic Alternaria mycotoxins (Ostry, 2008). TeA-producing fungi are ubiquitous in many biological environments and are able to infest most plant species (Gross, Curtui, Ackermann, Latif, & Usleber, 2011). Consequently, TeA was found in foods derived from plants,

* Corresponding author. E-mail address: [email protected] (B. Peng). http://dx.doi.org/10.1016/j.foodcont.2017.07.009 0956-7135/© 2017 Elsevier Ltd. All rights reserved.

especially in flour and bakery products (Janic et al., 2016; Siegel, Rasenko, Koch, & Nehls, 2009; Zhao, Shao, Yang, Li, & Zhu, 2015), tomatoes and their processing products (Da Motta & Valente Soares, 2000; Siciliano et al., 2015), beverages (Abramson, Delaquis, & Smith, 2007; Siegel, Merkel, Koch, & Nehls, 2010), and even infant food (Asam & Rychlik, 2013; Gross, Asam, & Rychlik, 2017; Rychlik et al., 2016). Mycotoxin decontamination by physical and chemical methods has been reviewed extensively elsewhere (Jouany, 2007; Kabak, Dobson, & Var , 2006). Although there are many different approaches available for mycotoxin decontamination, most of them are not popular due to high costs or practical difficulties involved in the detoxification process. In recent years, using biotechnology to control and remove mycotoxin contamination has received much attention and has gradually become a hot topic in the field of mycotoxin decontamination because of its potential applications , Deschamps, & Richard-Forget, 2010; Ji, (Bata & L asztity, 1999; Dalie Fan, & Zhao, 2016; Shetty & Jespersen, 2006). Bacteria and yeasts have been studied for their potential ability to reduce the levels of mycotoxins, including aflatoxin B1, ochratoxin A, zearalenone, Fusarium toxins, fumonisin, and patulin (Corassin, Bovo, Rosim, & Oliveira, 2013; El-Nezami, Kankaanpaa, Salminen, Ahokas, & Ahokas, 1998; El-Nezamiy, Chrevatidis, Auriola, Salminen, & Mykkanen,

N. Ge et al. / Food Control 82 (2017) 274e282

2002; Fuchs et al., 2008; Guo, Yuan, Yue, Hatab, & Wang, 2012, 2013; Hernandez-Mendoza, Garcia, & Steele, 2009; Joannis-Cassan, Tozlovanu, Hadjeba-Medjdoub, Ballet, & Pfohl-Leszkowicz, 2011; Sangsila, Faucet-Marquis, Pfohl-Leszkowicz, & Itsaranuwat, 2016; Shetty, Hald, & Jespersen, 2007). Lactic acid bacteria (LAB), due in large part to their Generally Recognized as Safe (GRAS) status and use as probiotics, are of particular interest for reducing mycotoxins (Elsanhoty, Salam, Ramadan, & Badr, 2014; Haskard, El-Nezami, A P E KSalminen, & Ahokas, 2001; Hatab, Yue, & Mohamad, 2012). Recently, various physical, chemical and enzymatic treatments have been used to ascertain the potential mycotoxin adsorption sites on bacterial cells. Although the LAB mechanism for binding mycotoxins is not fully understood, some authors have supposed that the primary cellular components involved in this phenomenon are parietal poly-saccharides (Luo et al., 2015; Ringot et al., 2007; Vijayaraghavan & Yun, 2008; Wang et al., 2015). The LAB cell wall consists of a peptidoglycan matrix that forms a major structural component of the cell wall, which houses other components, such as teichoic and lipoteichoic acid, a proteinaceous S layer and neutral polysaccharides (Delcour, Ferain, Deghorain, Palumbo, & Hols, 1999). These components have various functions, including adhesion and macromolecular binding, especially the fibrillar network of teichoic acids and neutral polysaccharides. Adsorption on the cell wall surface is an interaction between the toxins and functional groups of the cell surface, based on physical adsorption, ion exchange and complexation. The cell walls harbouring polysaccharides (glucan, mannan), proteins and lipids exhibit numerous different and easily accessible adsorption centers as well as different binding mechanisms including hydrogen bonds, ionic or hydrophobic interactions (Faucet-Marquis, Joannis-Cassan, Hadjeba-Medjdoub, Ballet, & Pfohl-Leszkowicz, 2014). Yiannikouris et al. (2006) found that hydroxyl, ketone, and lactone groups are involved in the formation of both hydrogen bonds and van der Waals interactions between aflatoxins B1, deoxynivalenol, ZEA and patulin, and b-D-glucans. The aim of this work was to further characterize the possible mechanisms involved in the removal of TeA by inactived LAB used as a biological adsorbent in an aqueous system. Fourier transform infrared (FTIR) spectroscopy analysis was used to search for the potential binding sites and possible functional groups of the tested strains. Then, Energy Dispersive X-ray Spectroscopy (SEM-EDS), transmission electron microscopy (TEM), zeta potential, and X-ray Diffraction (XRD) were used to assess the inactivated LAB before and after TeA adsorption. 2. Materials and methods 2.1. Chemicals and media Lactobcillus brevis CICC 20023 (LAB-20023) was obtained from the China Center of Industrial Culture Collection (CICC) (Beijing, China). Stock cultures were maintained at
80  C in 25% (v/v) glycerol. Tenuazonic acid, copper (II) salt (purity 98%), was purchased from Toronto Research Chemicals (TRC, Canada). (CAS Registry No. 610-88-8; M.W. 197; Molecular formula C10H15O3N). HPLC gradient grade acetonitrile (ACN) was purchased from Fisher Chemical (Fisher Scientific China); all other chemicals utilized in this study were of analytical-reagent grade, purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing China). 2.2. Preparation of TeA working solution Commercial TeA copper (II) salt was dissolved in acetonitrile to obtain an approximate concentration of 100 mg/mL as standard

275

stock solutions. Working solutions (500 mg/L) were prepared by further dilution with acidified water (pH4.0). All solutions were stored in the dark at
20  C to ensure stability. 2.3. Bacterial cells preparation Lactobacillus brevis 20023 (LAB-20023) was selected as the test strain because it has a higher specific surface area and cell wall volume (Wang et al., 2015) and showed higher capacity to adsorb TeA from the aqueous solution based on our report (Ge, Xu, Li, Peng, & Pan, 2017). LAB-20023 was cultured in a liquid medium and incubated at 30  C and 120 rpm for 24 h. The composition of the culture medium for LAB-20023 was glucose, 10% (w/v); yeast extract, 0.75% (w/v); peptone, 0.75% (w/v); potassium hydrogen phosphate, 0.2% (w/v); and polysorbate 80, 0.05% (v/v). The pH of the solution was adjusted to 7 using 1 N NaOH. Then, a first round shaking culture was transferred into a new liquid medium as a second shaking culture under the same conditions as the first shaking culture. After the second incubation, bacterial cells were collected by centrifugation (5804R, Eppendorf Ltd., Germany) at 6000g for 10 min, and the collected biomass was washed at least three times with distilled water. 2.4. Physical and chemical treatments of LAB cells Heat treatment: LAB-20023 cells were killed at 121  C for 20 min. Cell viability was detected with methylene blue staining. A drop of methylene blue (1%, w/v) was mixed with a drop of cells onto a microscope slide and viewed by light microscopy after 10 and 30 min (Guo et al., 2013). Lysozyme treatment: LAB-20023 cells were chemically treated in vitro with glycine and lysozyme to induce their conversion to the cell wall defective (CWD). Briefly, mid log-phase cells were pretreated with a 1% glycine solution in a liquid medium for 16 h at 30  C. Glycine is an analog of D-alanine (an amino acid of the cell wall structure) that, when incorporated by bacteria, leads to the formation of leaky cell walls (pre-CWD cells). Pre-CWD forms were then harvested by centrifugation at 6000  g for 10 min at room temperature and washed 2 times with phosphate buffered saline (PBS). Cells were re-suspended in a PBS solution supplemented with 20 mg/mL lysozyme. The lysozyme treatment was achieved for 2 h at 37  C. Cells were then centrifuged at 6000g for 6 min to remove intact bacillary forms and an autoclave (121  C, 20 min) (Rosu, Bandino, & Cossu, 2013). Formaldehyde treatment: 10.0 g of inactivated (121  C, 20 min) LAB-20023 cells was suspended in a 250-mL Erlenmeyer flask containing 50 mL of formaldehyde and 100 mL of formic acid under magnetic stirring for 6 h at room temperature. The main reaction is as follows:

RCH2 NH2

HCHOþHCOOH

ƒƒƒƒƒ!

RCH2 NðCH3 Þ þ CO2 þ H2 O

The chemical reaction is mainly to shield the amino group of the cell wall to reveal the other functional groups. Acetone treatment: 10.0 g of inactivated bacteria was suspended in 100 mL of acetone under magnetic stirring for 6 h. Acetone treatment was performed to dissolve organic matter inside cells to obtain cell walls. Caustic treatment: Caustic treated cells were prepared by mixing 10.0 g of inactivated cells with 100 mL of 0.1 M NaOH under magnetic stirring for 6 h. NaOH treatment can hydrolyze the ester groups of the cell wall to form carboxyl and hydroxyl groups. Methanol treatment: The 10.0 g of inactivated bacteria was mixed with 100 mL of anhydrous methanol and 2 mL of concentrated hydrochloric acid under magnetic stirring for 6 h. The main

276

N. Ge et al. / Food Control 82 (2017) 274e282

reaction is as follows: Hþ

RCOOH þ CH3 OHƒƒƒ!RCOOCH3 þ H2 O Methanol treatment under acid conditions was performed to consume the carboxyl groups of the cell wall to form ester groups according to the esterification reaction (Guo et al., 2013; Han, Yang, Zhang, Bao, & Shi, 2006). All bacterial samples were washed at least three times with distilled water and then collected for freeze drying at
54  C for 26 h using a vacuum freeze dryer (BTP-8ZL00X, Vitrices Group Co. USA). The dried biomass was grounded and screened through a set of sieves to obtain geometrical sizes of approximately 100e120 mesh and stored in desiccators for future use. 2.5. TeA binding assay Inactivated LAB powder samples were taken as adsorbents to test the percentage of TeA bound to bacteria. First, 0.50 g of adsorbents was washed with 20 mL of deionized water twice to avoid unnecessary interference and then added into a 50-mL centrifugal tube containing 10 mL of a 500 mg/L TeA test solution. The control was prepared without the addition of adsorbents. The control and test solutions were placed on a shaker-incubator at 120 rpm and 30  C for 12 h. After the incubation period, the biomass was centrifuged and collected for release experiments. The supernatants of each strain were collected for the detection of TeA levels by HPLC. All assays were performed in triplicate; negative controls (bacterial cells suspended in acetic acid solution) and positive controls (TeA working solution) were also performed.

structure, morphology and elementary composition of the LAB20023 cells were evaluated through SEM (8010, Techcomp Ltd. China). Before and after the TeA binding experiment, the bacterial cell was fixed with 2.5% (v/v) glutaraldehyde with 1% osmium tetroxide. Dry pellets were embedded in epoxy resins, and the completed block was cut to a 50 nm thickness using the Ultramicrotome (UC6, LEICA, Germany) (Teemu, Seppo, Jussi, Raija, & Kalle, 2008). Thin sections were viewed under transmission electron microscopy (TEM) (H-7650, Hitachi Ltd. Japan). 2.8. FTIR analysis The FTIR technique was used to identify chemical groups of unknown composition and intensity of absorption spectra associated with the molecular composition of the chemical group. FTIR analysis was performed with a NEXUS 470 FTIR spectrophotometer (USA). Bacterial cell samples were mixed and grounded with KBr (Spectral) (Sample: KBr ¼ 1:100 by dry weight) in an agate mortar, and then, each dried KBr-Sample mixture was pressed into a transparent disc. All IR spectra were recorded at room temperature, and the IR spectra range was 4000e400 cm
1. An average of 120 scans has been reported for the analysis. The FTIR experiments were performed in triplicate (Lin, Ye, Li, Xu, & Wang, 2011). 2.9. XRD analysis X-ray diffraction (XRD) data were collected by a diffractometer (D8 ADVNCE, Malvern Instruments, UK) equipped with Cu Ka, using a radiation at 40 kV and 30 mA. 2.10. Estimation of the zeta potential of the bacterial cells

2.6. HPLC conditions The HPLC equipment used was the Waters e2695HPLC system (WATERS, USA), including 2690/5 four binary gradient pumps, a 2998 UVevis photodiode array detector, and Empower2 real-time analysis software. The analysis of TeA was accomplished with a Waters C18 column (250  4.6 mm, i.d., 5 mm). The mobile phase consisted of the following: Solvent A was water, solvent B contained 90% vol ACN and 10% vol water, and both A and B were adjusted for pH with 0.1% formic acid. A gradient program with a constant flow rate of 1 mL/min was used, starting with 20% B, increasing to 100% B in 7 min, then to 50% B in 3 min, and holding at 50% B for 5 min and 20% B for 1 min. An additional 4 min equilibration time at 20% B was added between runs; the injection volume was 20 mL, and the column was thermostated to 40  C. All samples were filtered through a 0.22-mm pore membrane before analysis. The effluent from the column was monitored by DAD detection (200e400 nm). All compounds showed a UV-spectrum identical to that of TA with the absorption maximum at 277 nm (Schwarz, Kreutzer, & Marko, 2012). The TeA adsorption ratio, which is the percentage of TeA bound to the bacteria, was calculated according to following equation:

   TeA peak area of sample %Adsorption ratio ¼ 100  1
TeA peak area of control

2.7. TEM-EDS and SEM measurements The morphology and elementary composition of bacterial cells were determined by Scanning Electron Microscopy coupled with Energy Dispersive X-Ray Spectroscopy (SEM-EDS) and Transmission Electron Microscopy (TEM). In this study, the physical

The Zeta potential was measured with the help of the microelectrophoretic apparatus Zeta Plus (Zetasizer APA2000, Malvern Instruments, UK), equipped with a HeliumeNeon laser (633 nm) as a source of light, with the detection at a 90 scattering angle at room temperature (25  C). Each of the experiments was carried out under identical experimental conditions (n ¼ 10). The pH of the suspension was adjusted using 0.1 M HCl and 0.1 M NaOH (Jastrze˛ bska, Karwowska, Olszyna, & Kunicki, 2015). 3. Results and discussion 3.1. TeA binding assay The TeA adsorption ratios were compared with LAB-20023 cells that were treated with six different treatments. As shown in Fig. 1, all of the tested treatments affected the TeA adsorption capability to some extent. The methanol-treated sample (F) had the highest TeA adsorption ratio of 90.12%, followed by the formaldehyde-treated sample (C), with a TeA adsorption ratio of 87.07%. The lysozymetreated sample (B) showed the lowest removal percentage (12.47%) compared with other treatments because of the dissolution of intact cell walls with the action of lysozyme. The results demonstrated that TeA adsorption was most likely caused by cell wall surface groups and the adsorption reaction mainly occurred on the cell wall. This is in agreement with the results obtained by Luo et al. (2015). Compared with the LAB-20023 biomass inactivated by autoclaving (A), the TeA adsorption rate of the formaldehyde-treated biomass (C) showed a sharp increase. It is possible that the number of methyl group increased after the hydrogen atoms (eH) in the amino (eNH2) groups on the cell wall were substituted by methyl and the binding site of TeA to the methyl group increased significantly. The TeA adsorption rate of the acetone-treated biomass (D)

N. Ge et al. / Food Control 82 (2017) 274e282

277

100

a

b

c

d

e

f

Removal rate of TeA (%)

80

60

40

20

0

A

B

C

D

E

F

Fig. 1. Adsorption rate of TeA by various inactivated lactic acid bacteria prepared by different treatments. (A: Autoclave-inactivated LAB-20023 biomass, B: Lysozymetreated biomass, C: Formaldehyde-treated biomass, D: Acetone-treated biomass, E: Caustic-treated biomass, and F: Methanol-treated biomass).

also showed an obvious increase, possibly because the contents of the cell wall per gram of dried biomass treated by acetone increased significantly. The results indicated that the cell wall plays an important role in the adsorption of TeA by lactic acid bacteria. The TeA adsorption rate of the caustic-treated biomass (E) reduced slightly, which may be because the amide and ester bonds of organic compounds on the cell wall were hydrolyzed under alkaline conditions. These results indicated that the amide and ester bonds on the surface of the cell wall were involved in the adsorption reaction of TeA. The TeA adsorption rate of the methanol-treated biomass (F) under acidic conditions showed a sharp increase, which suggests that the ester bond, which increased with the esterification reaction, plays a significant role in the binding of TeA and that the TeA adsorption capacity of the ester bond is much higher than that of the carboxyl group. 3.2. SEM-EDS and TEM analysis SEM plays an important role in microbiology due to its high resolution. It has been a key provider of cellular micro-structure observations and analyses in microorganisms. SEM images (10,000) of the LAB-20023 biomass with different treatments are shown in Fig. 2. Fig. 2 shows that the morphologies of cells modified by different chemical methods (b-f) were distinctly broken to some extent compared to cells treated by autoclaving (a). However, this cell breakage after chemical modification could significantly increase the surface area of the cell wall and thus improve the adsorption capacity of TeA. EDS spectra analyses before and after TeA was loaded are shown in Fig. 3a and b, respectively. The EDS analysis showed the presence of C, N, O, P, K, Mg and Al in bacterial cells (Fig. 3a). For strain LAB20023, C, N, O, P and K constituted the major elements of the bacterial cells. The concentration of C was the highest, followed by that N, O, P and K. An important parameter derived from the EDS analysis of bacterial cells is the nitrogen-to-carbon (N/C) ratio. LAB20023 showed a N/C ratio of 0.2259. A high value indicates the presence of large amounts of proteins or other amino compounds in LAB-20023 (Wang et al., 2015). Compared with TeA-unloaded bacterial cells (Fig. 3a), the EDS of the TeA-loaded bacterial cells (Fig. 3b) exhibited some changes: K, Mg and Al disappeared, possibly because of the ion-exchange reaction during the

Fig. 2. SEM images of heat inactivated cells and various modified inactive cells at 10,000  magnification.(a: Autoclave-inactivated LAB-20023 biomass, b: Lysozymetreated biomass, c: Formaldehyde-treated biomass, d: Acetone-treated biomass, e: Caustic-treated biomass, and f: Methanol-treated biomass).

adsorption process. Guo et al. (2012)reported that the ionic interaction was not the mechanism of adsorption of patulin by heated yeast cells. The ultra-structures of inactivated LAB-20023 before and after TeA binding are shown in the TEM images in Fig. 4. The bi-layer structure of the cell wall is observed. The cell has no adhesion material, and the cell wall after TeA loading is somewhat blurred. The surface folds. The images revealed the presence of a layer or shell-like structure on the cell wall of the bacteria. The outer wall regions have an apparently looser structural arrangement, leading to a gel-like response on indentation.

3.3. FTIR analysis The functional groups present on the cell surface can be identified by Fourier transform infrared (FTIR) spectroscopy because each group has a unique energy absorption band (Jin & Bai, 2002). The assignments of FTIR bands and detailed wavenumber shifts for different treatments are summarized in Table 1, whereas the average FTIR spectra obtained before and after TeA loading of inactivated, formaldehyde-treated and methanol-treated biomasses are shown in Fig. 5. From Fig. 5, we can see that the peak shapes of each sample were basically the same. Apparently, TeAloaded bacterial cells did not completely lose their original structure. The FTIR spectrum of the inactivated biomass (Fig. 5A) shows strong band (3301.59 cm
1) in the region of 3600e3200 cm
1 and there is a possibility of overlap of the NeH and the OeH stretching vibrations. The absorption band at 3301.59 cm
1 may be due to NeH or OeH stretching vibrations (Zouboulis, Loukidou, & Matis, 2004). The microbial cell wall contains different biopolymers, such as chitin, chitosan, glucan, and mannan, in addition to proteins and lipids (Tripathi et al., 2012). Due to the complex nature of the

278

N. Ge et al. / Food Control 82 (2017) 274e282

Intensity/a.u.

a

Intensity/a.u.

b

Energy/KeV Fig. 3. EDS spectra of inactivated cells before and after TeA loading. (A: lnactivated LAB-20023 biomass and B: Inactivated cellsLAB-20023 biomass after TeA loading).

b

a

Fig. 4. TEM images of inactivated LAB-20023 before and after TeA binding. (a: Inactivated LAB-20023 biomass and b: Inactivated LAB-20023 biomass after TeA loading).

is centered at 1724.08 cm
1 and is due to lipid material. The amide I band is primarily a C]O stretching mode and is centered at 1639.22 cm
1, whereas the amide II band is a combination of NeH bending and CeN stretching, centered near 1535.09 cm
1. The peak at 1280.52 cm
1 can be attributed to the COOe of the carboxylate group present in the biomass. The bands in the region of 1052.96e979.68 cm
1 are due to the presence of a polysaccharide skeleton in the biomass. The FTIR spectrum of the TeA-loaded biomass (Fig. 5B) shows many differences compared to the inactivated biomass. The transmittance at 3301.59 and 2935.18 cm
1 shifted to 3286.16 and

Table 1 FTIR bands observed for the various modified biomasses before and after TeA adsorption. Functional groups

Wave number (cm
1) Inactivated

Lysozyme-treated

Formaldehyde-treated

Acetone-treated

Methanol-treated

Caustic-treated

Stretching (OeH/NeH) Stretching (CeH) Stretching esters (C]O) Amide I (C]O)þ(NeH) Amide II (NeH)þ(CeN) (C]O) Stretching (COOe) Stretching carboxylic acids (CeO) Stretching polysaccharides (CeO)

3301.59 (3286.16)a 2935.18 (2931.32) 1724.08 (1724.08) 1639.22 (1627.65) 1535.09 (1531.23) 1280.52 (1276.67) 1052.96 (1060.67) 979.68 (979.68)

3290.02 (3282.30) 2927.46 (2931.32) 1729.86 (1724.08) 1660.44 (1644.65) 1535.09 (1529.30) 1280.52 (1280.52) 1056.82 (1054.89) 979.68 (979.68)

3293.88 (3290.02) 2931.32 (2931.32) 1726.01 (1724.08) 1641.15 (1646.94) 1537.01 (1527.37) 1278.59 (1288.24) 1056.82 (1052.96) 977.75 (975.82)

3284.23 (3286.16) 2931.32 (2931.32) 1722.15 (1726.01) 1635.37 (1629.58) 1533.16 (1535.09) 1278.59 (1276.67) 1058.75 (1054.89) 979.68 (981.61)

3288.09 (3290.02) 2929.39 (2929.39) 1724.08 (1727.93) 1645.01 (1637.29) 1535.09 (1537.01) 1280.52 (1278.59) 1052.96 (1056.82) 979.68 (979.68)

3288.09 (3286.16) 2933.24 (2933.32) 1727.93 (1724.08) 1635.37 (1654.65) 1531,23 (1535.09) 1284.38 (1276.67) 1056.82 (1060.67) 979.68 (979.68)

a

The wave numbers obtained from TeA-loaded bacterial cells are shown in parentheses.

cell wall composition, it is not possible to determine the exact group responsible for this stretching vibration. The absorption peak at 2935.18 cm
1 is mainly from the extension of the CeH bonds of proteins and lipids. The C]O bond vibration absorption of carboxyl

2931.32 cm
1, respectively, after the adsorption process, suggesting an interaction of TeA with NeH or OeH. Further, the peaks at 1639.22 and 1535.09 cm
1 changed to 1627.65 and 1531.23 cm
1, respectively, which shows that TeA interacted with the amine and

N. Ge et al. / Food Control 82 (2017) 274e282

0 4000

3500

1500

512.98 501.41 509.12

979.68

1056.82 979.68

1000

511.05 512.98 512.98

979.68 979.68

1052.96

1060.67

1056.82 977.75

1052.96

975.82

1052.96

1278.59

1537.01 1452.16 1380.81

1380.81 1280.52 1288.24 1276.67

1627.65

1537.01 1452.16 1380.81 1278.59

1727.93

2000

1531.23 1454.09 1380.81

1646.94

1724.08

1726.01 1641.15

2500

1527.37 1454.09 1384.66

1645.01 1535.09 1452.16

1724.08

2929.39 2931.32 2931.32

3000

1639.22 1535.09 1454.09 1380.81 1280.52

3301.59

100

1724.08

3286.16

A

1637.29

2929.39 3290.02 3288.09

B

1724.08

200

C

2931.32

300

D

2935.18

Transmittivity(%)

400

E

3290.02

500

F

3293.88

600

279

500

-1 Wavenumber ( cm ) Fig. 5. FTIR absorption spectra of the inactivated LAB with various modifications before and after TeA adsorption. (A, C and E are inactivated, formaldehyde-treated and methanoltreated cells, respectively, before TeA adsorption; B, D and F are inactivated, formaldehyde-treated and methanol-treated cells, respectively, after TeA adsorption).

carboxyl groups of the biomass. In addition, the absorption peak of the polysaccharide skeleton vibration (1052.96 cm
1) shifted to 1060.67 cm
1 after adsorption, indicating that the CeO of carbohydrates had some contribution to the adsorption. The formaldehyde-formic acid treatment provides a good method for methylation of primary and secondary amines. This reagent also reversibly replaced the labile H-atoms of the COOH and SH groups of proteins. The FTIR spectrum of the formaldehydetreated biomass (Fig. 5C) shows that all groups underwent displacement compared with the inactivated biomass (Fig. 5A). These changes may be related to the exposure of the organic functional groups of LAB. The FTIR spectrum after TeA adsorption of

the formaldehyde-treated biomass (Fig. 5D) changed; 3293.88, 1726.01 and 1641.15 cm
1 changed to 3290.02, 1724.08 and 1646.94 cm
1, respectively, which suggests that amine groups and C]O are mainly responsible for TeA adsorption. A comparison of the results of the TeA adsorption tests confirmed that the cell wall plays an important role in the adsorption process. Majumdar et al. (2008) reported that NH2 groups are the major binding sites for the adsorption of copper by MRB. Methanol-hydrochloric acid esterifies carboxylic acid groups and, being a strong alkylating agent, might also displace the H atoms of NH2 and OH groups present on the biomass. This can be observed in Fig. 5A and E; the H-atoms obviously change at 3288.09

280

N. Ge et al. / Food Control 82 (2017) 274e282

and 2929.39 cm
1. The FTIR spectrum after TeA adsorption of the methanol-treated biomass (Fig. 5F) also changed. From the result of the TeA adsorption tests, it can be seen that protein, carbohydrate and lipid on the cell wall were involved in the adsorption reaction. Guo et al. (2013) report patulin biosorption by acetone-treated yeast and NaOH-treated yeast increased significantly, whereas the methylation of amino groups and esterification of carboxylate functionalities of the yeast cell surface caused a decrease in patulin binding. This result is quite different from ours. One reason for this difference may be that there are differences in the cell wall of yeast and LAB. Another may be the difference in the structure of patulin and TeA. Ji et al. (2016) found that the adsorption of toxins by yeasts and bacteria was predominantly related to some protein and carbohydrate components in the cell wall. Our experimental data for FTIR support the conclusion that polysaccharides and proteins of the cell walls are important components in the adsorption of TeA. Thus, the FTIR study clearly delineates the involvement of functional groups, such as eNH2, CeO and eOH, that are present on the biomass surface during TeA adsorption.

and affected the crystallinity. Moreover, the crystal structures of the bacterial cells after the treatment of formaldehyde (Fig. 6A f) and esterification (Fig. 6B f) were more damaged after adsorption, which may be related to the stronger adsorption capacity of formaldehyde and esterification. These results are consistent with the adsorption rate data, possibly because the hydrogen atoms (eH) on the cell wall were substituted by methyl (eCH3) during formaldehyde treatment and that there were more ester bonds formed during the esterification treatment, with a greater amount of chemical bonds present the adsorption spectra. In contrast, formaldehyde may have generated new chemical bonds, creating a new crystallization peak. Therefore, we can conclude that the diffraction peaks of cells after TeA adsorption underwent large changes, indicating the interaction between the biosorption agent and TeA molecules, and because various cell wall modification reactions are not the same, the crystallization properties have an effect on the adsorbent and thus affect the removal of TeA. 3.5. Zeta potential analysis

3.4. XRD analysis

B 21.46

6000

25.69

A

5000

5000

f

4000

e d

3000

c

2000

f

4000

Intensity

Intensity

The zeta potential investigation results obtained for the analyzed inactivated cells and various modified lactic acid bacteria are presented in Fig. 7, and the Zeta potential before and after TeA adsorption of cells with a pH ¼ 4.0 acetic acid solution is summarized in Table 2. Fig. 7 shows that the zeta potentials of the biomass depends on the pH: the zeta potentials are positive at low pH values, go through zero and become negative as the pH increases, reaching a maximum at a pH of 3.0 (þ8.55e26.37 mV). The zeta potentials of various modified lactic acid bacteria were approximately
30 mV at pH 7. Perhaps the cell surface contains various functional groups, such as carboxylate, phosphate, hydroxylate and amino moieties, thereby conferring a negative electrostatic charge to the cell periphery, notably at physiological pH (Bundeleva et al., 2011; Jastrze˛ bska et al., 2015). When the pH is low, the surface groups of lactic acid bacteria adsorbed Hþ in solution, and the surface of lactic acid bacteria had a positive charge, which enhanced the effect of adsorption for TeA. When the pH

13.49 17.14

22.23 25.51

6000

13.57 16.88

The cells’ crystal structure before and after TeA adsorption was confirmed by powder XRD. The XRD results of various modified lactic acid bacteria are presented in Fig. 6. As shown in Fig. 6A a, the XRD pattern of inactivated lactic acid bacteria has a weak crystallization peak at 2q ¼ 22 and broad and less intense peaks compared to cells after TeA adsorption (Fig. 6A b) due to the presence of strong hydrogen bonding adsorption within and between molecules that have strong crystallinity; sharp crystallization peaks at 2q ¼ 13.5 , 16.8 and 22.2 ; and an intense strength enhancement, with the formation of a new crystallization peak at 2q ¼ 25.5 . All of the results showed that there was a strong interaction between TeA and bacteria cells after TeA adsorption and that the structure changed, which made the crystallinity change. Compared with cells before TeA adsorption, the crystallinities of after TeA adsorption were obviously enhanced, which indicated that the crystal structure of cells was destroyed after adsorption

b

e 3000

d 2000

c b

1000

1000

a

a 0

0 10

20

30

40

2

50 (°)

60

70

80

90

10

20

30

40

2

50

60

70

80

90

(°)

Fig. 6. X-ray spectra of various modified inactivated LAB before and after TeA adsorption. (A: a, c and e are spectra of inactivated, lysozyme-treated and formaldehyde-treated cells, respectively, before TeA adsorption; b, d and f are spectra of inactivated, lysozyme-treated, formaldehyde-treated cells, respectively, after TeA absorption. B: a, c and e are spectra of acetone-treated, caustic-treated and methanol-treated cells, respectively, before TeA adsorption; b, d and f are spectra of acetone-treated, caustic-treated, methanol-treated cells, respectively, after TeA absorption.)

N. Ge et al. / Food Control 82 (2017) 274e282

formaldehyde-treated acetone-treated lysozyme-treated methanol-treated caustic-treated inactived biomass

40 30 20

Zeta potential (mv)

281

10 0 -10 -20 -30 -40 2

3

4

5

6

7

pH Fig. 7. Zeta potentials (mV) of the various modified LAB with different pH values.

Table 2 Zeta potential (mV) before and after TeA adsorption of cells with pH ¼ 4.0. Treatment

Heat treated Lysozyme-treated Formaldehyde-treated Acetone-treated Caustic-treated Methanol-treated

Zeta potential (mV) before TeA adsorption

after TeA adsorption


7.46 ± 0.32 8.48 ± 0.91
15.78 ± 0.11
2.89 ± 0.07
11.14 ± 0.38 8.79 ± 0.07


1.77 ± 0.06 19.77 ± 0.46
18.74 ± 0.25 0.54 ± 0.06
16.13 ± 0.22 12.53 ± 0.33

The data are the means ± the standard deviations (n ¼ 5).

increased, the surface groups released Hþ, so that the surface of lactic acid bacteria had a negative charge, which is conducive to the adsorption of TeA. This intersection pH corresponds to the point of zero salt effect, where the surface charge is not affected by a change in the background electrolyte concentration. According to the zeta potential results plotted in Fig. 7, the pH of the isoelectric point (pHiep) of various modified lactic acid bacteria is between 3 and 5. Previous zeta potential studies of intact heterotrophic bacteria report a pHiep of 2.2 and 2.5 for the Gramþ species Bacillus subtilis and Lactobacillus casei, respectively (Martinez, Pokrovsky, Schott, & Oelkers, 2008), which is most likely linked to the partial physical removal of negatively charged moieties of most surface carboxylate layers after autoclaving and extensive rinsing. According to the zeta potential results summarized in Table 2, the Zeta potentials before TeA adsorption varied between
15.78 mV and 8.79 mV. This difference may be explained in terms of the bacteria cell wall structure (Martinez et al., 2008). Compared with bacterial cells before TeA adsorption, the Zeta potentials of bacterial cells after TeA adsorption varied between
16.13 mV and 19.77 mV. These data show the transfer in the surface charge potential of bacterial cells before and after TeA adsorption. According to the abovementioned investigation results, we can conclude that TeA adsorption is caused not only by the integrity of the cell wall but also by the bacteria cell surface charge. It can be

assumed that the surface charge is formed separately on the sorbent particle surface and bacteria cell surface and may change significantly in real time during the sorption process. 4. Conclusions To determine the TeA absorption mechanism of inactivated LAB cells, SEM-EDS, TEM, FTIR spectra, XRD and zeta potential analyses were performed. The results showed that LAB-20023 cells had the capacity to adsorb TeA from aqueous solution and that biomass treated with formaldehyde and methanol significantly increased the TeA adsorption rate. The results of SEM-EDS and TEM show that increasing the surface area of the cell wall improves the adsorption capacity of TeA and that an ion-exchange reaction may occur during the adsorption process. From the results of FTIR and XRD, it can also be inferred that CeO, OeH and NeH groups, which are related to some protein and carbohydrate components, are obviously involved in the adsorption of TeA. The zeta potential results show that TeA adsorption is caused not only by the cell wall but also by the bacterial cell surface charge. To better describe the adsorption mechanism, further studies are required to identify the exact structures and composition characteristics of the cell wall that are responsible for the adsorption of TeA. Acknowledgments This research was partially supported by the Major Scientific and Technological Innovation Project of Hubei Province (2015ABA035) and by Fundamental Research Funds for the Central Universities (2662016PY027). The authors appreciate the anonymous reviewers very much for their valuable comments and improvement suggestions. References Abramson, D., Delaquis, P., & Smith, D. (2007). Assessment of ochratoxin A and tenuazonic acid in Canadian ice-wines. Mycotoxin Research, 23(3), 147e151. Asam, S., & Rychlik, M. (2013). Potential health hazards due to the occurrence of the mycotoxin tenuazonic acid in infant food. European Food Research and

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