New Levan-Type Exopolysaccharide from Bacillus amyloliquefaciens

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Cite This: J. Agric. Food Chem. 2019, 67, 8029−8034

New Levan-Type Exopolysaccharide from Bacillus amyloliquefaciens as an Antiadhesive Agent against Enterotoxigenic Escherichia coli Guolin Cai,†,‡,§,∥ Yifan Liu,† Xiaomin Li,*,‡ and Jian Lu*,†,‡,§,∥

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†

The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, P. R. China ‡ National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, P. R. China § Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, P. R. China ∥ School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, P. R. China S Supporting Information *

ABSTRACT: A special levan-type exopolysaccharide (EPS) from Bacillus amyloliquefaciens JN4 with antiadhesive activity against enterotoxigenic Escherichia coli (ETEC) was purified and identified. Chemical analysis indicated that EPS-JN4 with a low molecular weight of 8 kDa is composed of fructose and glucose with a molar ratio of 46.1:1. Structural analysis clarified that EPS-JN4 contains a main chain of β-(2,6)-linked Fruf residues and intensive branches of a single 2-linked Fruf at every six residues. Furthermore, the superior antiadhesive activity of EPS-JN4 against ETEC showed its potential usage as an antiadhesive agent for diarrhea prevention. EPS-JN4 is a specific type of levan family, for its small molecular size and intensive branches. The results expand the knowledge on structural types of levan and illustrate its potential as an antiadhesive agent for diarrhea prevention, which will be conducive to elucidate the relation between structure and function. KEYWORDS: exopolysaccharide, levan, antiadhesive, enterotoxigenic Escherichia coli, Bacillus amyloliquefaciens

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INTRODUCTION For a weaning piglet, approximately 80% of acute diarrhea that accounts for exceeding 10% mortality is deemed to be caused by enterotoxigenic Escherichia coli (ETEC).1,2 The normal treatment, antibiotic therapy, is the main incentive of genetic progression of antibiotic-resistant strains.3 Besides, it also results in an unbalance of the intestinal microbiota and disorder of the immune system, which subsequently leads to weight loss and poor growth of the surviving piglets and finally an unfortunate economic loss.2 Seeking an effective and safe substitution of antibiotics is therefore of great importance for animal farming. ETEC by means of surface colonization factors attaches to the intestinal epithelium and then colonization occurs, after which enterotoxins are produced to disrupt intestinal fluid homeostasis and thereafter cause diarrhea.4 Based on this infection mechanism, adherence of ETEC to host cells has been considered to be an essential step during infection.5 Therefore, many antiadhesive components, which can block the interactions between ETEC adhesins and host receptors by compactly adhering to ETEC cells, have been developed.6 Among them, exopolysaccharides (EPSs) from lactic acid bacteria (LAB) have been suggested as safe alternatives for antibiotic treatment.7−10 Most of these antiadhesive components contain galactose units, which can adhere to one type of colonization factor, such as fimbriae F4. In addition, some glucans from LAB can also bind to ETEC fimbriae F4, while fructan has been rarely reported.11 However, ETEC carries multiple adhesins with distinctive receptor binding specificities, © 2019 American Chemical Society

which make it impossible to block all adhesins by a single antiadhesive component.6 This encouraged us to find the different types of polysaccharides for binding with multiadhesins and preventing ETEC infections more effectively. Furthermore, most LAB strains yield a mite amount of EPS, less than 1 g/L, and thus did not arouse the interests for commercial exploitation.12 By contrast, Bacillus has been found to produce a high yield of EPS, which unfortunately has not been shown to be used as an antiadhesive agent for ETEC. In our previous work, a high EPS yielding (21.4 g/L) probiotic, Bacillus amyloliquefaciens JN4, was found to show antiadhesive activity against ETEC.13 The present study therefore purified the EPS from B. amyloliquefaciens JN4 for determination of its monosaccharide constitution and characterization of its chemical structure as well as for evaluation of its antiadhesive property against ETEC.

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MATERIALS AND METHODS

Extraction and Purification of EPS. B. amyloliquefaciens JN4 were cultured with LB medium containing 50 g/L sucrose at 37 °C for 72 h and then centrifuged at 12 000g for 30 min at 4 °C to collect the supernatant containing EPS. After precipitation with 2 volumes of chilled ethanol at 4 °C for 48 h, the EPS was collected by centrifugation at 25 000g for 30 min at 4 °C, followed by dissolution with distilled water and digestion with a papain (Shenggong Biotech., Received: Revised: Accepted: Published: 8029

May 24, 2019 June 25, 2019 June 27, 2019 June 27, 2019 DOI: 10.1021/acs.jafc.9b03234 J. Agric. Food Chem. 2019, 67, 8029−8034

Article

Journal of Agricultural and Food Chemistry China). The EPS was finally dialyzed against deionized water for 3 days to remove small molecules and lyophilized for further purification. The lyophilized sample was dissolved in deionized water, then loaded on a DEAE-Sepharose Fast Flow column (26 mm × 200 mm, GE Healthcare), and eluted with gradient NaCl solutions (0−1 M) at a flow rate of 1 mL/min. Fractions containing carbohydrates were detected, pooled, and concentrated for further purification using a Sepharose CL-6B column (16 mm × 500 mm, GE Healthcare) with 50 mM PBS (pH 7.0) at a flow rate of 0.5 mL/min. The main fractions were ultimately detected, pooled, dialyzed, lyophilized, and designated as EPS-JN4. Monosaccharide Composition Analysis. The purified EPS-JN4 was hydrolyzed with 0.1 M TFA to a final concentration of 10 mg/mL at 105 °C for 1 h. Monosaccharides were detected using HPAECPAD (Thermo Scientific) and identified by comparing the retention time with those of standard sugars, including arabinose, galactose, xylose, glucose, mannose, and fructose.14 All the standard sugars were purchased from Sigma-Aldrich. Molecular Weight Determination. The molecular weight (Mw) of the EPS-JN4 was determined by high-performance gel filtration chromatography (HPGFC) using a refractive index detector (Waters).15 The samples were passed through a 0.22 μm filter, injected into Ultrahydrogel Linear tandem columns (300 mm × 7.8 mm, Waters), and eluted with 0.1 M NaNO3 at a flow rate of 0.9 mL/ min. Standard curves for Mw determination were generated using dextran standards (1 000, 5 000, 12 000, 25 000, 50 000, 80 000, and 150 000 Da). Fourier-Transform Infrared Spectral Measurement. Infrared spectra (IR) of EPS-JN4 were recorded with a Nexus 470 FT-IR spectrophotometer (Thermo Nicolet) in the range of 4000−400 cm−1 using the KBr-disk method as described.16 Prior to testing, EPS-JN4 was mixed with KBr at a ratio of 1:100 and then ground and pressed into a thin sheet via evacuation. Methylation Analysis. Methylation of the EPS was analyzed according to the method described by Liu et al.17 After permethylation, hydrolysis, reduction, and acetylation, the partial methylated anhydroalditol acetates were determined with Trace GC 1310-ISQ MS (Thermo Scientific) with a capillary column (30 m × 0.25 mm, 0.25 μm) programmed from 160 to 210 °C at 2 °C/min, and then 5 °C/min to 240 °C. NMR Spectroscopy Assay. The EPS-JN4 sample was dissolved directly in D2O for NMR spectra recoding at a probe temperature of 295 K on a Bruker Avance 500.13-MHz spectrometer (BrukerBiospin, Coventry, U.K.) (500.13 MHz for 1H NMR and 125.77MHz for 13C NMR). Standard homonuclear and heteronuclear correlated 2D techniques were used for assignments of EPS-JN4, including correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond coherence (HMBC). Antiadhesive Activity Determination. Hemagglutination assay against ETEC strain with or without a dosage of EPS-JN4 was performed as described previously.10 The ETEC strain E. coli O78:K80 culture, from the China Center of Industrial Culture Collection, was suspended with PBS to 2.0 × 1010 CFU/mL and loaded in a 96-well V-bottom microplate, with 25 μL for each well. The E. coli cells were then dosed with a serial amount of test polysaccharides (2, 4, 6, 8, 10, and 12 mg/mL), including EPS-JN4, galactooligosaccharide (GOS), levan, and inulin, then incubated for 5 min at 37 °C. The same volume of PBS buffer was added as a blank. Finally, each well was mixed with 25 μL of 5% erythrocyte for 2 h at 4 °C. The hemagglutination against ETEC strain in the microplate was observed visually, and the minimum concentration of tested polysaccharides that could inhibit hemagglutination was defined as the titer of its activity. The GOS with degree of polymerization between 2 and 7 was purchased from Quantum Hi-Tech Biological, China; levan from Erwinia herbicola with a Mw higher than 1000 kDa was purchased from Sigma-Aldrich; inulin from chicory with a Mw of ∼10 kDa was purchased from Fuyun Biotech., China.

The cell experiment to determine the antiadhesive activity of EPSJN4 was performed using monolayer HT-29 cell as described previously.7 ETEC cells were cultured and washed three times with PBS (pH 7.2) and then resuspended in RPMI 1640 medium (SigmaAldrich) to a final concentration of 108 CFU/mL. A volume of 1 mL of the ETEC suspension plus EPS-JN4 or/and GOS was dosed to HT-29 cells in the six-well plates and then incubation at 37 °C for 30 min. After incubation, the unattached bacteria were washed three times with PBS, and the adhering cells were lysed with 0.1% (v/v) Triton-X100 (Sigma-Aldrich), then counted by plating serial dilutions on LB agar plates. The antiadhesive activity was calculated as the number of adherent bacterial cells per 50 HT-29 cells. The results are given as means ± SD. Analysis of variance and significant differences among means were determined by one-way ANOVA, using SPSS Statistics 17.0 (SPSS Inc., Chicago, IL).

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RESULTS AND DISCUSSION Molecular Weight and Monosaccharide Composition of EPS-JN4. The recovery of EPS-JN4 purified by using DEAE-Sepharose Fast Flow and Sepharose CL-6B column was 90.2%. A single peak appeared in HPGPC suggested that EPSJN4 was homogeneous, and the Mw of EPS-JN4 was calculated to be 8 kDa with a polydispersity index of 3.19, lower than EPS-2 (19.8 kDa) from the same species B. amyloliquefaciens C-1 and levan (5.82 × 103 kDa) from B. licheniformis NS032, indicating that the Mw of EPS is highly dependent on the strains.18,19 The low Mw may decrease the steric hindrance for adherence to bacterial adhesins. EPS-JN4 is primarily constituted of fructose and glucose in a molar ratio of 46.1:1. The monosaccharide composition of EPS-JN4 is similar to inulin or levan, although the molar ratio of them are different at 14:1 and 30.2:1, respectively.20,21 The different monosaccharides constitution of EPS-JN4 and its low Mw may contribute to its particular structure and properties, such as the antiadhesive activity against ETEC that has not been found in fructan and other polysaccharides from Bacillus species. However, this hypothesis of a relationship between structure and function of polysaccharides needs further data and evidence. Structure Characteristics of EPS-JN4. The FT-IR spectrum of EPS-JN4 showed its typical signals of fructan (Figure 1). For example, the strong band around 3405 cm−1 and two peaks at 2939 cm−1 arise from the O−H stretching

Figure 1. Fourier transform-infrared spectrum of EPS-JN4. 8030

DOI: 10.1021/acs.jafc.9b03234 J. Agric. Food Chem. 2019, 67, 8029−8034

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Figure 2. 1D NMR spectrum of EPS-JN4 recorded at 295 K: 1H NMR spectrum (500.13 MHz) (A) and 13C NMR spectrum (125.77 MHz) (B).

3,4,6-tri-O-methyl-D-mannitol and 6-O-acetyl-2,5-anhydro1,3,4-tri-O-methyl-D-glucitol indicate the (2 → 6)-linked β-DFruf residues, and 1,6-di-O-acetyl-2,5-anhydro-3,4-di-O-methyl-D-mannitol and 1,6-di-O-acetyl-2,5-anhydro-3,4-di-O-methyl-D-glucitol result from (1, 2 → 6)-linked β-D-Fruf residues as branching points.24,25 The three types of linkages occupied 14.6%, 70.2%, and 13.6%, respectively. The similar portions of terminal 2-linked β-D-Fruf residues and (1, 2 → 6)-linked β-DFruf residues and none of the (1 → 2)-linked β-D-Fruf residues indicated that only one terminal residue was attached to each branching point. This is significantly different from the levan structure reported so far, which has slightly long chain branches or no branches.23 This special structure of EPS-JN4 may be an important reason for its potential usage as an antiadhesive agent. The 1H NMR spectrum of EPS-JN4 (Figure 2A) showed seven main proton signals in the ring proton region (3.5−4.2

vibration and the C−H stretching vibration, respectively; the other two peaks at 1417 and 1068 cm−1 correspond to COOH stretching vibration and C−O−C asymmetric variable angle vibration, respectively. The intense peak at 1645 cm−1 that evoked by the bending vibration of O−H suggested the existence of bound water.22 In addition, the spectrum presented characteristic absorption peaks located at 928 and 818 cm−1 on behalf of symmetrical stretching vibration and bending vibration of D-type furanose, respectively, which further confirmed the existence of a furan ring in the polysaccharide chain.23 The methylation analysis was performed by using the reductive-cleave method, and GC-MS was used to identify the methylated, reductive-cleavage and acetylated derivatives. The presence of 2,5-anhydro-1,3,4,6-tetra-O-methyl-D-mannitol and 2,5-anhydro-1,3,4,6-tetra-O-methyl-D-glucitol correspond to the 2-linked β-D-Fruf residues, and 1-O-acetyl-2,5-anhydro8031

DOI: 10.1021/acs.jafc.9b03234 J. Agric. Food Chem. 2019, 67, 8029−8034

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the signal at 0.4 ppm downfield from the major C3 signal can be used to distinguish between branched levan and linear levan, indicating that EPS-JN4 was a branched levan.28 In addition, the signals were not weak enough to omit, indicating the presence of a large amount of branches. 2D NMR was used to assign the chemical shifts of the sugar units presented in the repeating unit of EPS-JN4 (Table 1). The COSY spectrum (Figure 3B) showed cross peaks between H3/H4, H4/H5, H5/H6a, H5/H6b, H6a/H6b, and H1a/H1b and exhibited similarity to those of levan. The difference in chemical shifts between H6a and H6b distinguishes levan and inulin by the shielding effect of oxygen and indicates EPS-JN4 as a levan.19 The HSQC spectrum (Figure 3C) indicated direct C−H correlations between protonated carbon atoms. There was no cross peaks between C2 and any other H, which confirmed its quaternary anomeric carbon character. The two cross peaks of H5/C5 (δ 3.89/82.9 and δ 3.93/82.9) were specific to levan, which illustrates the two types of linkages as → 6)-β-D-Fruf-(2 → and → 1,2)-β-D-Fruf-(6 →.29 The two diagnostic cross peaks H6a/C6 and H6b/C6 and the rest of the cross peaks including H1a/C1, H1b/C1, H3/C3, and H4/C4 were also in accordance with the data from levan.30 Combined with the chemical analysis and structural analysis, the 8 kDa EPS-JN4 is mainly composed of fructose and glucose, which constitutes the β-(2,6)-linked Fruf main chain and a single 2-linked Fruf branch at every six Fruf residues (Figure 4). As a levan-type polysacchride, EPS-JN4 is unique due to its residue numbers in the repeating unit, intensive branches, as well as low molecular weight. Antiadhesive Activity against ETEC. The antiadhesive activity against ETEC of EPS-JN4 was validated by a hemagglutination assay, which is a generally accepted model for inspecting bacterial adhesive properties (Table 2).6 Galactooligosaccharides (GOS) has been reported as an effective antiadhesive agent and was chosen as a positive control.8 The activities of inulin and levan, which are the two main types of fructans and have similar monosaccharide compositions to EPS-JN4 and the levan mainly with β-2,6 linkages while the inulin with β-2,1 linkages, were also tested. It was found that, except inulin, both EPS-JN4 and levan could prevent the hemagglutination induced by ETEC at the test concentration, which may result from the differences in linkage between inulin and levan. EPS-JN4 showed equal antiadhesive activity to GOS, while the comparative levan from Erwinia herbicola, with a Mw higher than 1000 kDa and a low degree of branching, showed less antiadhesive activity than them. It was hypothesized that the small molecular size and intensive branches contribute to the high affinity of EPS-JN4 to ETEC adhesins, leading to its superior activity. Most antiadhesive agents, including glucan and mannan, as well as GOS, can adhere to colonization factor fimbriae F4.31 Besides fimbriae F4, there are other possible colonization factors on the cell surface of ETEC, due to the fact that the GOS treated ETEC cells can still colonize the intestinal mucosa.7 Fructan has been rarely reported to be capable of inhibiting the adherence of ETEC to epithelial cells and neither the type of colonization factors it binds to.11 A cell experiment was performed to further evaluate the capability of EPS-JN4 to inhibit ETEC adherence to HT-29 cells (Figure 5). It was found that EPS-JN4 acted as a fimbriae receptor analogue as GOS,32 which inhibited the adherence of ETEC to the HT-29 cell. However, when EPS-JN4 and GOS were used together, the inhibition degree was greater than

ppm), which are mainly attributed to the fructose moieties in EPS-JN4. The 13C NMR spectrum of EPS-JN4 contained chemical shifts in the range of 62−107 ppm (Figure 2B). According to the literature, major signals at around 62.4, 106.7, 78.8, 77.4, 82.9, and 66.0 ppm can be attributed to C-1, C-2, C-3, C-4, C-5, and C-6, respectively.26 The low magnetic field characteristic signals of C-6, C-2, and C-4, combined with the relative spacing of C atoms, were similar to those assigned to levan rather than those of inulin, indicating that EPS-JN4 was a levan-type fructan.27 The cross peak of C2/H6 at δ 106.7/3.83 and δ 106.0/3.87 from the HMBC spectrum (Figure 3A) confirmed the existence of β-(2 → 6) linkages. Furthermore,

Figure 3. 1H/1H and 1H/13C correlation spectrum of EPS-JN4: 1 H/13C HMBC (A); 1H/1H TOCSY (B); 1H/13C HSQC correlation spectrum (C) correlation spectrum. 8032

DOI: 10.1021/acs.jafc.9b03234 J. Agric. Food Chem. 2019, 67, 8029−8034

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Journal of Agricultural and Food Chemistry Table 1. 1H and

13

C Chemical Shifts of EPS-JN4 δ13C/1H (ppm)

residue → 6)-β-D-Fruf-(2 → → 1,6)-β-D-Fruf-(2 → β-D-Fruf-(2 →

1

2

3

4

5

6

62.4 3.62, 3.74 62.4 3.65, 3.71 62.7 3.64, 3.74

106.7

78.8 4.16 79.2 4.20 78.8 4.14

77.7 4.07 76.8 4.07 77.7 4.07

82.9 3.89 82.9 3.93 83.6 3.84

66.0 3.49, 3.87 65.8 3.52, 3.83 64.7 3.84

106.0 106.3

Figure 4. Proposed structure of the repeating units of EPS-JN4.

Table 2. Antiadhesive Activity of EPS-JN4 against ETECa

superior antiadhesive activity against ETEC, which shed light on the development of a new therapeutic strategy of diarrhea caused by ETEC, taking place of the existing precarious antibiotic treatment. Moreover, the results expand the knowledge on structural types of levan and compare their antiadhesive activities against ETEC, which will be conducive to elucidate the relation between structure and function.

concentration (mg mL−1) group

0

2

4

6

8

10

12

EPS-JN4 galactooligosaccharide levan inulin

− − − −

− − − −

+ − − −

+ + − −

+ + + −

+ + + −

+ + + −

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“+” indicates antihemagglutination; “−” indicates hemagglutination.

a

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b03234.

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Figure S1, high-performance gel filtration chromatography of EPS-JN4; Figure S2, gas chromatogram of partial methylated anhydroalditol acetates of EPS-JN4; Figure S3, 1D NMR spectra of commercial levan from Erwinia herbicola: 1H NMR spectrum (500.13 MHz) and 13C NMR spectrum (125.77 MHz) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Phone: +86 510 8591 8191. Fax: +86 510 8591 8191.

Figure 5. Adherence inhibition of ETEC to HT-29 cells in the presence of EPS-JN4 and/or GOS: EPS/GOS-4/8 means supplemented with 4 or 8 mg/mL of EPS-JN4 or GOS, while EPS4+GOS4 for 4 mg/mL of EPS-JN4 plus 4 mg/mL GOS. The locations for each group marked by the different letters are significantly different (p < 0.05).

ORCID

Jian Lu: 0000-0002-8827-5943 Funding

This work was supported by the National Natural Science Foundation of China (Grant 31871785); the Natural Science Foundation of Jiangsu Province, China (Grant BK20181207); the Program of Introducing Talents of Discipline to Universities (111 Project) (Grant 111-2-06); and the Fundamental Research Funds for the Central Universities (Grant JUSRP21914).

using EPS-JN4 or GOS alone, which indicated that they may bind to different fimbriae in ETEC cells. Previous studies have reported the capability of cranberry juice to inhibit the type 1 fimbriae-mediated E. coli adhesion.33 The main component of the juice was fructose, the same as EPS-JN4, which suggested the possibility of EPS-JN4 to bind with colonization factor fimbriae F1, a different colonization factor. Therefore, we hypothesize that EPS-JN4 binds to colonization factor fimbriae F1 on the ETEC cell surface, while GOS interferes with the commonly known colonization factor fimbriae F4. In this study, levan-type polysaccharide EPS-JN4 containing a β-(2,6)-linked Fruf residues main chain and intensive branches of a single 2-linked Fruf at every six residues was purified from B. amyloliquefaciens JN4. The EPS-JN4 exhibited

Notes

The authors declare no competing financial interest.

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Journal of Agricultural and Food Chemistry

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DOI: 10.1021/acs.jafc.9b03234 J. Agric. Food Chem. 2019, 67, 8029−8034