Antibacterial thyme oil-loaded organo-hydrogels utilizing cellulose acetoacetate as reactive polymer emulsifier

Journal Pre-proof Antibacterial thyme oil-loaded organo-hydrogels cellulose acetoacetate as reactive polymer emulsifier

utilizing

Liduo Rong, Xin Shen, Bijia Wang, Zhiping Mao, Xueling Feng, Xiaofeng Sui PII:

S0141-8130(19)40184-0

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.052

Reference:

BIOMAC 14356

To appear in:

International Journal of Biological Macromolecules

Received date:

10 December 2019

Revised date:

31 December 2019

Accepted date:

6 January 2020

Please cite this article as: L. Rong, X. Shen, B. Wang, et al., Antibacterial thyme oilloaded organo-hydrogels utilizing cellulose acetoacetate as reactive polymer emulsifier, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/ j.ijbiomac.2020.01.052

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© 2018 Published by Elsevier.

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Antibacterial thyme oil-loaded organo-hydrogels utilizing cellulose acetoacetate as reactive polymer emulsifier Liduo Rong1, Xin Shen1, Bijia Wang1,2, Zhiping Mao1,2, Xueling Feng1,2, Xiaofeng Sui1,2*

1 Key Lab of Science and Technology of Eco-textile, Ministry of Education, College of

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Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai,

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201620, People’s Republic of China.

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2 Innovation Center for Textile Science and Technology of DHU, Donghua University,

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*Corresponding authors:

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Shanghai, 201620, People’s Republic of China.

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Address: No. 2999 North Renmin Road, Shanghai 201620, China. Tel.: +86 21 67792605. Fax: +86 21 67792707. E-mail: [email protected]

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Abstract Organo-hydrogel are widely used in various fields, due to functional organic ingredients immobilized by the gel network or stored and protected by the gels. Herein, cellulose acetoacetate (CAA) served as reactive natural polymer emulsifier to stabilize thyme oil-in-water (O/W) emulsions. Hydroxypropyl chitosan (HPCS) was

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added to the continuous phase in emulsions to achieve the organo-hydrogel via the enamine bonds under mild conditions. The thyme@CAA emulsion with different

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loadings of the inner phase (up to 50%) displayed uniform droplets distribution (3-5

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μm) and favorable stability. The organo-hydrogel was systematically analyzed by

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Fourier transform infrared spectroscopy, optical microscope, rheology analyses. The

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emulsion droplets evenly dispersed in the three-dimensional network. The modulus of

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organo-hydrogels depended on the viscosity of precursor emulsions and the crosslinking density. The resulting organo-hydrogel displayed favorable antibacterial

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activity against E. coli and S. aureus. CAA, as the reactive emulsifier and crosslinking agent, was a promising alternative candidate to fabricate a series of organo-hydrogel.

Keywords: organo-hydrogel; cellulose acetoacetate; antibacterial; enamine bonds; thyme oil

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1. Introduction Organo-hydrogels (Emulsion hydrogels) were mainly made up of both oil and water, two immiscible phases in one system; meanwhile, polar and non-polar phases were required within the same microenvironment system [1]. Emulsifying agents were used to stabilize two phases with opposite character distributed in the gels.

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Organo-hydrogels have attracted increasing attention due to it had the inherent properties of the hydrogels and different characteristic functional ingredients were

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distributed and immobilized by the gel network to suit a variety of applications [2-5].

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Various approaches were employed to fabricate functional organo-hydrogels. For

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example, Wang et al. reported that the cinnamon oil (CMO) loaded composite

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organo-hydrogels were effectively prepared via free radical polymerization of

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acrylamide in the continuous phase of cinnamon oil-in-water emulsion, and gelatin was used as the favorable macromolecular emulsifier to stabilize the oil-in-water

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concentrated emulsions [6]. The prepared organo-hydrogel could be acted as promising long-term antibacterial materials. Zhao et al. reported that shape memory organo-hydrogels were obtained utilizing in situ polymerization of an emulsion system that included hydrophilic monomers (i.e., acrylamide) in the continuous phase and oleophilic monomers in oil phase. The elastic hydrogel framework (poly acrylamide) and phase-transition micro organo-gels (molten paraffin) acted synergistically to provide favorable thermomechanical performance and shape memory effect [7]. Besides, Zhang et al. investigated that the oil phase stabilized by cellulose nanofibrils (CNF) were dispersed in the cross-linked alginate/Ca2+ network, 3

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achieving homogeneous and closely packed microstructures in organo-hydrogel [8]. This organo-hydrogel would be applied in cold chain logistics. Thus, it would be a new avenue to design high-performance gels by introducing two entirely different physiochemical characteristics and immiscible phase components and designing the suitable cellular network [5, 7, 9-11].

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It could be concluded that emulsifiers stabilizing two opposite phases were vital to construct organo-hydrogels. Cellulose acetoacetate [12, 13], as an amphiphilic

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biopolymer, was also one of the multifunctional cellulose derivatives. The acetoacetyl

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group was prone to a series of reactions via the active carbonyl carbons and

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methylene [14-16]. The enamine bonds could be formed by reacting primary amino

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groups with acetoacetyl groups under mild condition without any catalyst [17-19].

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Herein, our aim is to leverage the water-soluble cellulose acetoacetate (CAA) as an efficient and reactive emulsifier to stabilize oil-in-water emulsion. Meanwhile, the

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CAA macromolecules in the continuous phase could react with amino groups of the hydroxypropyl chitosan (HPCS) via the enamine bonds [20-22] to form the organo-hydrogel (Scheme 1). Thyme oil was chosen as the typical inner phase of emulsion; and it had all kinds of promising applications on account of its excellent antibacterial performance [23-26]. The stability, morphology, mechanical properties and microstructure of the organo-hydrogel were characterized in detail. Additionally, their antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was confirmed by the disc diffusion method.

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CAA-stabilized thyme-in-water emulsions.

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2. Experimental section 2.1 Materials

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Scheme 1. Schematic illustration of the preparation of thyme-loaded organo-hydrogel based on

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Cellulose was purchased from Xinxiang Natural Chemical Co., Ltd. The

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viscosity-average degree of polymerization was estimated as 870 according to measurements using an Ubbelohde viscometer in cupriethylenediamine hydroxide solution (CUEN). Hydroxypropyl chitosan (HPCS, degree of substitution was 0.85, and degree of deacetylation was 90%) was supplied by Nantong Green God Bioengineering Co., Ltd. The ionic liquid 1-allyl-3-methylimidazolium chloride (AMIMCl) was supplied by the Center for Green Chemistry and Catalysis (CGCC), LICP, CAS. tert-Butyl acetoacetate (t-BAA, 99%) was purchased from Sinopharm Chemical Reagent Co., Ltd. The thyme oil (thymol content ≥ 30.0%) was purchased from Jiangxi Xuesong Natural Medicinal Oil Co., Ltd. All chemicals were of 5

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analytical grade and used without further purification. 2.2 Synthesis of CAA CAA (Fig. S1, degree of substitution (DS) was 0.80) was prepared referring to a previously reported method [22]. Briefly, cellulose powder (1.0 g) was dispersed in ionic liquids (1-allyl-3-methylimidazolium chloride (AMIMCl) (19 g)). Then, the

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mixture system was stirred at 90 °C until cellulose was dissolved completely. As diluent, dimethylformamide (DMF) (20.0 mL) was added to the above solution. When

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the solution was heated to 110 °C, tert-butyl acetoacetate (t-BAA, 6 g, 37.9 mmol)

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was added dropwise to the reaction mixture under nitrogen with magnetic stirring for

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4.5 h. At the end of reaction, the yielded products were precipitated into methanol,

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then washed and dried at 60 °C in vacuum for 12 h.

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2.3 Preparation of thyme@CAA emulsions A certain amount of CAA was dissolved in phosphate buffered saline (PBS, pH =

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7.4) to obtain CAA solution at the concentration of 1.5 wt.%. Next, the thyme oil was added to CAA solution. Emulsions with gradient O/W ratios of 0.2:1, 0.4:1, 0.6:1, 0.8:1, and 1:1 w/w were prepared by using an ultrasonic cell pulverizer (SCIENTZ, JY92-IIDN, China) operating at 720 W for 2 min in the ice-water bath. Table 1. Compositions and codes of different samples. Emulsion sample

Code

Organo-hydrogel sample

Code

CAA

1.5%CAA 2 g, 1.5%HPCS 2g

H-0

1.5%CAA 2 g, thyme 0.4 g

E-1

E-1 2 g, 1.5%HPCS 2 g

H-1

1.5%CAA 2 g, thyme 0.8g

E-2

E-2 2 g, 1.5%HPCS 2 g

H-2

1.5%CAA 2 g, thyme 1.2g

E-3

E-3 2 g, 1.5%HPCS 2 g

H-3

1.5%CAA 2 g, thyme 0 g

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E-4

E-4 2 g, 1.5%HPCS 2 g

H-4

1.5%CAA 2 g, thyme 2.0 g

E-5

E-5 2 g, 1.5%HPCS 2 g

H-5

2.4 Preparation of organo-hydrogels The preparation process of thyme-loaded organo-hydrogels was carried out as the following procedure. Typically, the aqueous solution of HPCS was obtained by

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dissolving the HPCS in phosphate buffered saline (PBS, pH = 7.4) (1.5 wt.%). The

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organo-hydrogel was acquired by mixing different ratio concentrations of

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thyme@CAA emulsions with the HPCS solution with gentle shaking at room

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temperature. The mixture system was placed for 1 h to finish the gelation process at 37 °C. The formation of the organo-hydrogels was verified by placing the vial upside

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2.5 Characterization

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summarized in Table 1.

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down. The different organo-hydrogels were coded according to their composition, and

Fourier transform infrared (FTIR) spectroscopy were acquired on a PerkinElmer Spectrum Two (USA) which was equipped with an attenuated total reflectance (ATR) accessory. All spectra were obtained in the range of 4000 - 400 cm−1 and at a resolution of 4 cm−1. 1

H NMR spectra were carried out using a Bruker Avance 400 MHz spectrometer

(Bruker, USA) in the proton noise-decoupling mode with a standard 5 mm probe. The DS of the acetoacetate groups in CAA was calculated according to Equation (1): DS =

I1 × 7 I2 × 3

(1)

where I1 is the integration value of the methyls of acetoacetate, and I2 is the 7

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integration value of the backbone of the anhydroglucose ring of CAA. The laser particle size analyser (S3500, Microtrac, America) was utilized to measure the volume mean droplet diameters of thyme@CAA emulsions at ambient conditions. Before measurement, the emulsions were diluted with deionized water. The diluted emulsions were dropped into the instrument, and the deionized water was

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acting as a solvent. Each measurement was repeated three times and the mean values were calculated.

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The thyme@CAA emulsions were diluted with deionized water, and the

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organo-hydrogels were sliced into the thin. Then, an optical microscope (ECLIPSE

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80i, NIKON, Japan) was used to observe the emulsion droplets and the

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organo-hydrogel sample under ambient conditions.

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Rheology analyses of emulsions was performed utilizing a Thermo HAAKEMARS 60 rheometer (Thermo, USA) equipped with a conical plate with a diameter of 35/1°

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mm at 25 °C. Shear viscosity was recorded at shear rates from 0.1 to 100 s−1. Rheology analyses of organo-hydrogels were performed with a parallel plate set-up and gaps set at 2 mm. Silicone oil was utilized to seal the platform to prevent the evaporation of water. The storage and loss moduli (G′ and G″, respectively) of the samples were recorded versus strain and frequency sweeping. 2.6 Antibacterial performance Antibacterial

performance

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the

thyme-loaded

organo-hydrogels

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demonstrated against two different bacterial strains namely E. coli as the model Gram negative bacteria and S. aureus as the model Gram positive bacteria by Agar disc 8

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diffusion method. The hydrogels without thyme oil were chosen as the control samples, which were obtained by directly mixing the CAA solution and HPCS solution. In this method, the antibacterial performance of the organo-hydrogels was verified using the Luria-Bertani (LB) medium solid agar Petri dish ,which were prepared with respective culture medium for both bacterial strains [27]. The thyme-loaded organo-hydrogels and control hydrogels were prepared with 1.5 cm in

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diameter and 5 mm in height. For the antibacterial test, bacterial suspension (100 μL)

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was coating uniformly onto the surface nutrient agar plate. Then, the

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organo-hydrogels and control hydrogels were placed and covered on the surface of the

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nutrient agar plate. Afterwards, the above-mentioned nutrient agar plates were placed

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in the incubator and incubated for 18 h at 37 °C. At the end, the samples were

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observed to investigate whether the conspicuous inhibition zone appeared or not. The existence of inhibition zone on the nutrient agar plates suggested that the

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organo-hydrogels possessed favorable antibacterial performance against the corresponding microbials. Finally, the inhibition zones were measured.

3. Results and discussion

3.1 Preparation of thyme@CAA emulsions and organo-hydrogels In this work, the thyme-loaded organo-hydrogels were simply prepared based on the O/W emulsion template stabilized by reactive CAA. The thyme oil acted as oil phase was mixed with the CAA aqueous solution. The clear two-phase solution turned into white turbid mixture after ultrasonic dispersion (Fig. 1a). The prepared thyme emulsions stabilized by CAA were uniformly distributed. As shown in the 9

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micrographs (Fig. 1b), The size of droplets in all the emulsion was within a few microns (2-5 μm). Optical micrographs of freshly-prepared emulsions and emulsions after storing for seven days are displayed in (Fig. S2a). After storing for seven days, all the emulsions remained to be uniform and the droplet size of emulsion did not change significantly (Fig. S2b). The stability of the emulsions could be due to the

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amphiphilic CAA molecules at the oil-in-water interface, served as a mechanical barrier to emulsion droplet aggregation [28].

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The emulsion viscosity on the basis of a function of frequency with various O/W

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ratios was displayed in Fig. 2a. It could be demonstrated that thyme@CAA emulsions

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were viscous, and the viscosity of thyme@CAA emulsions increased with increasing

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the internal phase fraction. Additionally, the increasing the shear rate resulted in the

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decrease of the viscosity of thyme@CAA, suggesting a typical shear-thinning behavior [29, 30]. Moreover, the emulsion stability may also be related to the high

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viscosity of the continuous phase, that could hamper the movement of CAA during the process of emulsification and its final aggregation at oil-water interface [31].

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Fig. 1 (a) The preparation of thyme oil-in-water emulsions of thyme oil stabilized by CAA (1.5

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wt.%) with different O/W ratios. (b) Optical micrographs and mean droplet diameters of thyme

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oil-in-water emulsions freshly prepared (E-1 ~ E-5). The scale bars correspond to 20 μm.

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For the preparation of organo-hydrogels, the thyme@CAA emulsion was mixed with a certain amount of HPCS solution under room temperature with mild vortex

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mixing. After 10 min, the organo-hydrogels were formed by the reversed vial test method. As shown in Fig. 2b, the organo-hydrogel appeared milky compared with the transparent CAA/HPSC hydrogel. FTIR spectra were used to characterize the resulting hydrogels (Fig. 3a, b). The peaks at 1700 and 1760 cm−1 were belonged to the double carbonyl stretching in spectrum of CAA [22]. For the CAA/HPCS hydrogel (H-0), the new peaks at 1650 and 1605 cm−1 were the typical peaks of enamine bonds [32], and the peaks of the acetoacetyl groups at 1700 - 1760 cm−1 on the CAA appeared lower (Fig. 3a). Besides, the spectrum of the organo-hydrogel not only showed the same typical peaks with the CAA/HPCS hydrogel, and also the 11

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characteristic peaks of the residue thyme essential oil (Fig. 3b). These results

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indicated the organo-hydrogel was achieved by the crosslinking of enamine bonds.

Fig. 2 (a) Flow properties of 1.5% CAA solution and emulsion (E-1 ~ E-5). (b) Photograph

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demonstrates the organo-hydrogel formed between thyme@CAA emulsion and HPCS.

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The mechanical characteristics of the organo-hydrogels were assessed by the

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rheology tests. The elasticity of the organo-hydrogels was analyzed using strain

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amplitude sweeps. As shown in Fig. 4a, the G′ value significantly declined over the critical strain region, suggesting the collapse of the 3D cross-linked network of

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organo-hydrogels with excessive strain. The G′ and G″ curve intersected at the strain of approximately 300 - 400%; this result indicated that the state of the organo-hydrogels was between solid and fluid around this critical point [33]. The frequency sweep of the organo-hydrogel (Fig. 4d) was analyzed according to the strain amplitude sweep results. The organo-hydrogels with different inner phase ratios were prepared. As the internal phase increased, the corresponding changes of the modulus occurred. The maximum G′ value of H-3 could reach up to be 800 Pa, which was attributed to the crosslink density of the organo-hydrogel and the viscosity of the precursor thyme@CAA emulsion. It was suggested that the modulus of 12

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organo-hydrogels could be easily adjusted by changing the solid content or the viscosity of the emulsion to meet specific applications. To verify the stability of storage at room temperature, the organo-hydrogel was placed and kept for 7 day at 25 °C. The appearance of the organo-hydrogel did not show significant change after 7 day (Fig. S3a). It was demonstrated that the enamine bonds in the organo-hydrogel

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had favorable stability.

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Fig. 3 (a) FT-IR spectra of CAA, HPCS, CAA/HPCS hydrogel. (b) FT-IR spectra of

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CAA/HPCS hydrogel, thyme@CAA/HPCS organo-hydrogel, and thyme oil.

Fig. 4 Rheology analyses of (a) the storage modulus G′ (filled symbols) and loss modulus G″ (empty symbols) on strain sweep (37 °C, frequency = 1.0 Hz), (b) G′ and G″ versus frequency for the all the samples (37 °C, strain: 1%).

The organo-hydrogels were sliced, and observed utilizing an optical microscope 13

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(Fig. S3b). The related optical images with blue background indicated that all organo-hydrogel displayed well-organized emulsion droplets dispersed in the hydrogel system indicating the gelation process jus occurred in continuous phase, and did not affect the state of the emulsion. 3.2 Antibacterial Performance

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The antibacterial performance of the thyme oil loaded organo-hydrogels against Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus was determined

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using the inhibition zone method. The zones of inhibition (ZOI) were recorded, which

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displayed the regions where the bacterial could not grow around the organo-hydrogels

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in the agar disc [34]. Herein, the organo-hydrogel with different inner phase ratio

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were used to evaluate the antibacterial activity, while the CAA/HPCS hydrogel

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without thyme was chosen as a control. It was found from Fig. 5 that there were no any ZOI against E. coli or S. aureus in CAA/HPCS hydrogels, suggesting that had no

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antibacterial activity. The organo-hydrogels loaded with thyme oil produced visible ZOI against E. coli and S. aureus, suggesting that the organo-hydrogel possessed clearly favorable antibacterial performance against E. coli and S. aureus. To be specific, the conspicuous ZOI against E. coli and S. aureus appeared the average radius of not less than 2 cm in all organo-hydrogels after 18 h. Besides, it was suggested that the antibacterial activities of the organo-hydrogels against S. aureus were slightly higher than those against E. coli. This was because there was an outer membrane existing outside the peptidoglycan layer of E. coli, and it could prevent attacking by extraneous compounds to a certain degree [35, 36]. 14

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4. Conclusions Antimicrobial organo-hydrogel was prepared by crosslinking thyme@CAA and HPCS via formation of enamine bonds. As one of the functional cellulose derivatives, CAA can stabilize oil-in-water emulsions efficiently. Besides, the thyme@CAA emulsion containing up to 50% of thyme oil as inner phase displayed the uniform

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emulsion droplets (3 - 5 μm) and storage stability at least 7 day. Due to the active reactivity of CAA, the acetoacetate groups in continuous phase could react with the

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amino groups of HPCS via the enamine bonds to build the three-dimensional network

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of organo-hydrogel. Meanwhile, it was demonstrated that the uniform emulsion

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droplets dispersed well in the organo-hydrogel system. The modulus of

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organo-hydrogel could be related with emulsion viscosity and crosslinking density.

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Additionally, the results suggested that the thyme-loaded organo-hydrogel held an excellent and durable antimicrobial effect against E. coli. and S. aureus. Overall, it

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was envisioned that CAA as the reactive emulsifier to design various functional materials and the prepared organo-hydrogel could be served as promising multifunctional antibacterial materials.

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Fig. 5 Antibacterial activity of CAA/HPCS hydrogel and organo-hydrogel against (a) E. coli and

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(b) S. aureus. The scale bars correspond to 2 cm.

Acknowledgements

This work was financially supported by the Fundamental Research Funds for the Central Universities (No.2232019A3-06), the Fundamental Research Funds for the Central Universities and Graduate Student Innovation Fund of Donghua University (No. CUSF-DH-D-2019062).

Reference 16

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[1] M.E. Helgeson, S.E. Moran, H.Z. An, P.S. Doyle, Mesoporous organohydrogels from thermogelling photocrosslinkable nanoemulsions, Nat. Mater. 11(4) (2012) 344-352. [2] Y. Lu, L. Mao, Z. Hou, S. Miao, Y. Gao, Development of emulsion gels for the delivery of functional food ingredients: from structure to functionality, Food Eng. Rev. (2019) 1-14. [3] V.K. Singh, S. Ramesh, K. Pal, A. Anis, D.K. Pradhan, K. Pramanik, Olive oil based novel

of

thermo-reversible emulsion hydrogels for controlled delivery applications, J. Mater. Sci.: Mater. Med.

ro

25(3) (2014) 703-721.

[4] Q. Rong, W. Lei, L. Chen, Y. Yin, J. Zhou, M. Liu, Anti‐ freezing, Conductive self‐healing

-p

organohydrogels with stable strain‐sensitivity at subzero temperatures, Angew. Chem. Int. Ed. 56(45)

re

(2017) 14159-14163.

lP

[5] H. Gao, Z. Zhao, Y. Cai, J. Zhou, W. Hua, L. Chen, L. Wang, J. Zhang, D. Han, M. Liu, Adaptive

na

and freeze-tolerant heteronetwork organohydrogels with enhanced mechanical stability over a wide temperature range, Nat. Commun. 8 (2017) 15911.

Jo ur

[6] J. Wang, Y. Li, Y. Gao, Z. Xie, M. Zhou, Y. He, H. Wu, W. Zhou, X. Dong, Z. Yang, Cinnamon oil-loaded composite emulsion hydrogels with antibacterial activity prepared using concentrated emulsion templates, Ind. Crops Prod. 112 (2018) 281-289. [7] Z. Zhao, K. Zhang, Y. Liu, J. Zhou, M. Liu, Highly Stretchable, Shape memory organohydrogels using phase‐transition microinclusions, Adv. Mater. 29(33) (2017) 1701695. [8] X. Zhang, Y. Wang, X. Luo, A. Lu, Y. Li, B. Li, S. Liu, O/W pickering emulsion templated organo-hydrogels with enhanced mechanical strength and energy storage capacity, ACS Appl. Bio Mater. 2(1) (2018) 480-487. [9] S. Kovačič, M.S. Silverstein, Hydrogels through emulsion templating: sequential polymerization 17

Journal Pre-proof

and double networks, Polym. Chem. 8(40) (2017) 6319-6328. [10] P. Sriamornsak, P. Asavapichayont, J. Nunthanid, M. Luangtana-anan, S. Limmatvapirat, S. Piriyaprasarth, Wax-incorporated emulsion gel beads of calcium pectinate for intragastric floating drug delivery, AAPS PharmSciTech 9(2) (2008) 571-576. [11] W. Xu, L. Huang, W. Jin, P. Ge, B.R. Shah, D. Zhu, J. Jing, Encapsulation and release behavior of

of

curcumin based on nanoemulsions-filled alginate hydrogel beads, Int. J. Biol. Macromol. 134 (2019)

ro

210-215.

[12] K.J. Edgar, K.M. Arnold, W.W. Blount, J.E. Lawniczak, D.W. Lowman, Synthesis and properties

-p

of cellulose acetoacetates, Macromolecules 28(12) (1995) 4122-4128.

re

[13] H. Würfel, M. Kayser, T. Heinze, Efficient and catalyst-free synthesis of cellulose acetoacetates,

lP

Cellulose 25(9) (2018) 4919-4928.

na

[14] L. Rong, M. Zeng, H. Liu, B. Wang, Z. Mao, H. Xu, L. Zhang, Y. Zhong, J. Yuan, X. Sui, Biginelli reaction on cellulose acetoacetate: a new approach for versatile cellulose derivatives, Carbohydr.

Jo ur

Polym. 209 (2019) 223-229.

[15] Y.B. Kim, H.K. Kim, H. Nishida, T. Endo, Synthesis and Characterization of Hyperbranched Poly (β‐ ketoester) by the Michael Addition, Macromol. Mater. Eng. 289(10) (2004) 923-926. [16] E. Esen, M.A. Meier, Modification of starch via the biginelli multicomponent reaction, Macromol. Rapid Commun. (2019) 1900375. [17] L. Rong, H. Liu, B. Wang, Z. Mao, H. Xu, L. Zhang, Y. Zhong, J. Yuan, X. Sui, Enamine approach for versatile and reversible functionalization on cellulose related porous sponges, ACS Sustainable Chem. Eng. 6(7) (2018) 9028-9036. [18] H. Liu, L. Rong, B. Wang, Z. Mao, R. Xie, H. Xu, L. Zhang, Y. Zhong, X. Sui, Facile synthesis of 18

Journal Pre-proof

cellulose derivatives based on cellulose acetoacetate, Carbohydr. Polym. 170 (2017) 117-123. [19] L. Rong, H. Liu, B. Wang, Z. Mao, H. Xu, L. Zhang, Y. Zhong, X. Feng, X. Sui, Durable antibacterial and hydrophobic cotton fabrics utilizing enamine bonds, Carbohydr. Polym. 211 (2019) 173-180. [20] H. Liu, L. Rong, B. Wang, R. Xie, X. Sui, H. Xu, L. Zhang, Y. Zhong, Z. Mao, Facile fabrication

of

of redox/pH dual stimuli responsive cellulose hydrogel, Carbohydr. Polym. 176 (2017) 299-306.

ro

[21] H. Liu, C. Li, B. Wang, X. Sui, L. Wang, X. Yan, H. Xu, L. Zhang, Y. Zhong, Z. Mao, Self-healing and injectable polysaccharide hydrogels with tunable mechanical properties, Cellulose 25(1) (2018)

-p

559-571.

re

[22] H. Liu, X. Sui, H. Xu, L. Zhang, Y. Zhong, Z. Mao, Self‐healing polysaccharide hydrogel based on

lP

dynamic covalent enamine bonds, Macromol. Mater. Eng. 301(6) (2016) 725-732.

na

[23] V. Ryu, D.J. McClements, M.G. Corradini, L. McLandsborough, Effect of ripening inhibitor type

104-111.

Jo ur

on formation, stability, and antimicrobial activity of thyme oil nanoemulsion, Food Chem. 245 (2018)

[24] I. Rasooli, M.B. Rezaei, A. Allameh, Ultrastructural studies on antimicrobial efficacy of thyme essential oils on Listeria monocytogenes, Int. J. Infect. Dis. 10(3) (2006) 236-241. [25] D. Altiok, E. Altiok, F. Tihminlioglu, Physical, antibacterial and antioxidant properties of chitosan films incorporated with thyme oil for potential wound healing applications, J. Mater. Sci.: Mater. Med. 21(7) (2010) 2227-2236. [26] E. Abdollahzadeh, M. Rezaei, H. Hosseini, Antibacterial activity of plant essential oils and extracts: The role of thyme essential oil, nisin, and their combination to control Listeria monocytogenes inoculated in minced fish meat, Food control 35(1) (2014) 177-183. 19

Journal Pre-proof

[27] M. Ul-Islam, T. Khan, W.A. Khattak, J.K. Park, Bacterial cellulose-MMTs nanoreinforced composite films: novel wound dressing material with antibacterial properties, Cellulose 20(2) (2013) 589-596. [28] T. Winuprasith, M. Suphantharika, Properties and stability of oil-in-water emulsions stabilized by microfibrillated cellulose from mangosteen rind, Food Hydrocolloids 43 (2015) 690-699.

of

[29] R. Pal, Shear viscosity behavior of emulsions of two immiscible liquids, J. Colloid Interface Sci.

ro

225(2) (2000) 359-366.

[30] Q. Li, Y. Wang, Y. Wu, K. He, Y. Li, X. Luo, B. Li, C. Wang, S. Liu, Flexible cellulose nanofibrils

-p

as novel pickering stabilizers: The emulsifying property and packing behavior, Food hydrocolloids 88

re

(2019) 180-189.

lP

[31] Y. Jiang, L. Liu, B. Wang, X. Yang, Z. Chen, Y. Zhong, L. Zhang, Z. Mao, H. Xu, X. Sui,

na

Polysaccharide-based edible emulsion gel stabilized by regenerated cellulose, Food hydrocolloids 91 (2019) 232-237.

Jo ur

[32] A. Sanchez-Sanchez, D.A. Fulton, J.A. Pomposo, pH-responsive single-chain polymer nanoparticles utilising dynamic covalent enamine bonds, Chem. Commun. 50(15) (2014) 1871-1874. [33] Z. Wei, J.H. Yang, Z.Q. Liu, F. Xu, J.X. Zhou, M. Zrínyi, Y. Osada, Y.M. Chen, Novel biocompatible polysaccharide‐ based self‐ healing hydrogel, Adv. Funct. Mater. 25(9) (2015) 1352-1359. [34] M.S. Islam, N. Akter, M.M. Rahman, C. Shi, M.T. Islam, H. Zeng, M.S. Azam, Mussel-inspired immobilization of silver nanoparticles toward antimicrobial cellulose paper, ACS Sustainable Chem. Eng. 6(7) (2018) 9178-9188. [35] T. Wu, A.-G. Xie, S.-Z. Tan, X. Cai, Antimicrobial effects of quaternary phosphonium salt 20

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intercalated clay minerals on Escherichia coli and Staphylococci aureus, Colloids Surf., B 86(1) (2011) 232-236. [36] Y. Ouyang, X. Cai, Q. Shi, L. Liu, D. Wan, S. Tan, Y. Ouyang, Poly-l-lysine-modified reduced graphene oxide stabilizes the copper nanoparticles with higher water-solubility and long-term

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additively antibacterial activity, Colloids Surf., B 107 (2013) 107-114.

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Author statement Liduo Rong: Conceptualization,

Methodology, Validation, Formal analysis,

Investigation, Writing -Original Draft, Writing Review & Editing. Xin Shen: Formal analysis, Writing -Original Draft. Bijia Wang: Conceptualization, Writing -Review & Editing.

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Zhiping Mao: Methodology, Resources. Xueling Feng: Resources, Supervision, Funding acquisition.

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Xiaofeng Sui: Conceptualization, Resources, Supervision, Funding acquisition.

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Journal Pre-proof Highlights

1. CAA served as reactive polymer emulsifier to prepare thyme oil-in-water emulsions. 2. Antimicrobial organo-hydrogel was prepared by mixing thyme@CAA emulsion and HPCS. 3. CAA was a promising alternative candidate to prepare a series of

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organo-hydrogel.

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