beta-cyclodextrin proteoliposomes

Accepted Manuscript Title: Antibacterial poly(ethylene oxide) electrospun nanofibers containing cinnamon essential oil/beta-cyclodextrin proteoliposomes Authors: Lin Lin, Yajie Dai, Haiying Cui PII: DOI: Reference:

S0144-8617(17)31068-8 http://dx.doi.org/10.1016/j.carbpol.2017.09.043 CARP 12788

To appear in: Received date: Revised date: Accepted date:

30-6-2017 12-9-2017 12-9-2017

Please cite this article as: Lin, Lin., Dai, Yajie., & Cui, Haiying., Antibacterial poly(ethylene oxide) electrospun nanofibers containing cinnamon essential oil/beta-cyclodextrin proteoliposomes.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.09.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antibacterial poly(ethylene oxide) electrospun nanofibers containing cinnamon essential oil/beta-cyclodextrin proteoliposomes

Lin Lin, Yajie Dai, Haiying Cui*

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

*Corresponding

author. Tel.: +86 51188780201

E-mail address: [email protected] (H. Y. Cui)

Hightlights 

The cinnamon essential oil/β-cyclodextrin (CEO/β-CD) proteoliposomes were prepared.



The proteoliposomes had high encapsulation efficiency of CEO.



The controlled release of CEO in nanofibers was realized via bacterial protease.



The nanofibers containing CEO/β-CD proteoliposomes showed high antibacterial activity.

Abstract A novel antibacterial packaging material was engineered by incorporating cinnamon essential oil/β-cyclodextrin (CEO/β-CD) proteoliposomes into poly(ethylene oxide) (PEO) nanofibers by electrospinning technique. Herein, PEO was a stabilizing polymer and used as electrospinning polymeric matrix for the fabrication of CEO/β-CD proteoliposomes nanofibers. The nanoliposomes were inlaid with protein are defined as proteoliposomes. Taking advantage of bacterial protease secreted from Bacillus cereus (B. cereus), the controlled release of CEO from proteoliposomes was achieved via proteolysis of protein in proteoliposomes. The CEO/β-CD inclusion complex was prepared by the aqueous solution method and characterized by Raman and FTIR spectroscopy. After the treatment of CEO/β-CD proteoliposomes nanofibers packaging, the satisfactory antibacterial efficiency against B. cereus on beef was realized without any impact on sensory quality of beef. This study demonstrated that the CEO/β-CD proteoliposomes nanofibers can significantly extend the shelf life of beef and have potential application in active food packaging. Keywords: CEO/β-CD proteoliposomes; Bacillus cereus; stimulated release; electrospinning nanofibers; beef.

1

1. Introduction B. cereus is identified as a source of contamination in food industry (Ribeiro et al., 2017). It can cause food spoilage and lead to diseases in humans as it produces degradation enzymes and cytotoxic factors in their idiophase (Kaboré et al., 2013). B. cereus is widely distributed in soil and animal gut, and is one of the most important spoilage microorganisms in meat products due to its rich nutrition. In addition, B. cereus can adapt extreme environment (acidic, alkaline and high temperature) (Kumari & Sarkar, 2016). So an effective control of B. cereus in meat process is still a challenging task. In recent years, various natural antimicrobial are employed for meat products, especially essential oils (EOs) (Ayari et al., 2013). CEO has been reported to exhibit a broad spectrum of antimicrobial activity against a wide range of microorganisms due to the inhibition of cell wall biosynthesis, membrane function and specific enzyme activities (Faikoh et al., 2014). CEO can protect physicochemical properties of food without affecting the nutrition value. In addition, the application of CEO in meat products, not only has high effective antibacterial function, but also can improve the meat flavor and antioxidant effect. However, the present application of CEO is restricted due to its volatility, poor water-solubility and chemical instability in the presence of air, light, and high temperature (Makwana et al., 2014). Hence, many nanotechnologies have been introduced to enhance the chemical stability and solubility of EOs, such as nanoliposomes (Wu et al., 2015; Cui et al., 2017a) and nanoemulsions (Chen et al., 2016). Nanoliposomes are microscopic vesicles consisting of a central compartment surrounded by one or more concentric phospholipid bilayers. They can encapsulate hydrophilic substances in the interior aqueous compartment, lipophilic drugs within lipid bilayers and amphiphilic molecules at the lipid/water interface (Sebaaly et al., 2016). Due to their biocompatibility, biodegradability, and low toxicity, potential applications of liposomes as pharmaceutical carriers and sustained-releasing capability are well recognized (Neethirajan & Jayas, 2011). With the development of nanoliposomes technology, many researchers began to exploit innovative nanoliposomes to achieve controlled-release. A new generation of stimuli-sensitive nanoliposomes has been developed, which depends on different environmental factors (including pH, magnetism, and temperature) to trigger the release of bioactive molecule (Mufamadi et al., 2011). Lin et al. (2015) reported one stimulated nanoliposomes which used the 2

toxin protein secreted from infectious bacteria as a stimulating pore-forming agent. Cui et al. (2016) engineered an intelligent-released proteoliposomes via stimulation of bacterial protease secreted from bacteria. The nanoliposomes were inlaid with protein are defined as proteoliposomes. The difference between proteoliposomes and nanoliposomes is that the phospholipid bilayer of proteoliposomes is inlaid with protein. In detail, the antibacterial agents in proteoliposomes will released due to the degradation of the protein which inlaid into the proteoliposomes. In other words, the antibacterial agents in proteoliposomes will not be released until the proteoliposomes meet the infective target bacteria, which could secrete the protease (Cui et al., 2016). In this way, the proteoliposomes are able to deliver antibacterial agents accurately to targeted sites which are affected with bacteria. The released antibacterial agents will then exert its antibacterial activity rapidly and locally, which is the advantage of the proteoliposomes. Nevertheless, the encapsulation efficiency (EE) of EOs in nanoliposomes is low due to their hydrophobicity. In order to further improve the EE and stability of CEO in proteoliposomes, β-CD is introduced in this system. β-CD is made up of 7 glucopyranose units with hydrophobic cavity interior and hydrophilic exterior (Shrestha et al., 2017). Because of their unique chemical structure, β-CD is able to form inclusion complexes with EOs, enhancing their solubility, chemical stability, and bioavailability. Moreover, researchers have confirmed that β-CD further improve the aqueous solubility of EOs and protected them from oxidation (Sebaaly et al., 2016). Thus, β-CD has been widely applied in drug delivery and controlled drug release systems (Tao et al., 2014). In addition, proteoliposomes are easily agglomerate and shedding when they directly contact with food surface, resulting in the reduction of bioactivity of encapsulated substances. Recently, electrospinning nanofibers have been gained interest as novel food packaging material because of their large specific surface area, high drug encapsulation efficiency and high mechanical strength (Yang et al., 2016). Hence, nanofibers can act as the fixed support of proteoliposomes in active packaging. PEO is a suitable hydrophilic polymer for electrospinning due to its no toxicity, which has been safely used in the fields of medicine and food. And chitosan/PEO nanofibers had been prepared for Cinnamaldehyde delivery vehicles that potentially eradicate pseudomonas infections (Rieger, & Schiffman, 2014). Based on the above analyses, the aim of the present study is to engineer nanofibers incorporating CEO/β-CD proteoliposomes by electrospinning technology. Schematic of 3

electrospinning for CEO/β-CD proteoliposomes incorporate PEO nanofibers was shown in Fig. 1. As an innovative food packaging material, the physicochemical properties of CEO/β-CD proteoliposomes nanofibers were characterized and its antibacterial activity against B. cereus on beef was evaluated as well. 2. Materials and methods 2.1 Materials and culture CEO was purchased from J.E International (Caussols plateau, France). β-CD was bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Soy lecithin and cholesterol were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). PEO (average Mv~900000) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo, USA). Fresh beef was obtained from the local supermarket. All the other chemicals were of analytical grade. The B. cereus ATCC 14,579 strain was provided by China General Microbiological Culture Collection Center (Beijing, China). 2.2 Preparation and characterization of CEO/β-CD inclusion complex 2.2.1 Preparation of CEO/β-CD inclusion complex The CEO/β-CD inclusion complex was prepared in aqueous solution as described by Kfoury et al (2014) with slight modification. A certain amount of β-CD was dissolved in distilled water, and stirred for 2 h at 60 °C to obtain β-CD solution. Then a required amount of CEO was added into the β-CD solution to obtain CEO and β-CD mixture (CEO: β-CD ratio was 1:6, 1:7, 1:8, 1:9, or 1:10, w/w), and continuously stirred at 200 rpm for 2.5 h at 50 °C. Subsequently, the mixture was treated by ultrasonic wave for 0.5 h at 60 W and then stored at 4 °C for 24 h. The precipitation was collected after filter using a 0.22 μm membrane and dried at 30 °C to obtain CEO/β-CD inclusion complex. Simultaneously, the effect of temperature (40 °C, 50 °C, 60 °C, 70 °C, and 80 °C) and reaction time (1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h) on the inclusion efficiency were also investigated to obtain the optimal condition according to the inclusion rate (IR) of CEO/β-CD inclusion complex and the embedding rate (ER) of CEO (Wen et al., 2016). The evaluation index of inclusion efficiency was calculated using the following equations: IR (%)=

CEO/β-CD inclusion complex (g)  100% Initial CEO (g) + β-CD(g) 4

ER (%)=

Embedded CEO (g)  100% Total CEO/β-CD inclusion complex (g)

2.2.2 Characterization of CEO/β-CD inclusion complex 2.2.2.1 Raman spectra analysis CEO, β-CD and CEO/β-CD inclusion complex were characterized by Raman spectrum (DXR, Thermo Electron, Massachusetts, US). Raman measurements consisted of acquiring multiple spectral windows in the range of 0-3500 cm-1 (Stokes shifts). 2.2.2.2 Fourier transform infrared spectroscopy (FTIR) analysis FTIR spectra of CEO/β-CD inclusion complex were captured using a Nicolet is50 FTIR spectrometer (Thermo, Electron, Massachusetts, US) to analyze the sample composition. IR spectra were recorded in the 500-4000 cm−1 range with a resolution of 4 cm−1. The room was kept at a controlled ambient temperature (25 °C) and relative humidity (30%). 2.3 Preparation and characterization of CEO/β-CD proteoliposomes 2.3.1 Preparation of CEO/β-CD proteoliposomes The preparation of CEO/β-CD proteoliposomes was performed using thin-lipid film evaporation-ultrasonic hydration-freeze and thaw technique based on the method reported by Cui et al (2016). In brief, soy lecithin and cholesterol (5:1, w/w) were dissolved in trichloromethane, and mixed with a certain amount of CEO/β-CD inclusion complex (0 mg/mL, 10 mg/mL, 20 mg/mL, 30mg/mL, and 40mg/mL). The mixture was evaporated until a thin film was formed and dried in vacuum oven at 30 °C for 24 h. Then, the dry lipid film was mixed with phosphate buffer solution (PBS, 0.03 M, pH 7.2 ） and homogenized in cell ultrafine grinding instrument (Ymnl-1000Y, NanJing Immanuel Instrument Equipment Co., Ltd., Nanjing, China) for 20 min at 400 W. Finally, the mixture was centrifuged at 4000 rpm for 10 min and filtered using a 0.45 μm membrane. Thus, the CEO/β-CD nanoliposomes were obtained. After all these, casein (0.1 mg/mL) was dissolved in PBS containing 1.0 mg/mL surfactant Brij-35 and incubated for 3 h at room temperature. Subsequently, the nanoliposomes and casein were mixed, followed by 10 min bath sonication. The mixture was frozen at -18 °C for 2 h, and then slowly thawed at 0 °C for 2 h. Then, caseins were embedded into the nanoliposomes, followed by mild sonication to promote the sealing of proteoliposomes to obtain the CEO/β-CD proteoliposomes.

For

comparison,

CEO

proteoliposomes 5

without

β-CD

and

blank

proteoliposomes were prepared according to the above-mentioned method as well. 2.3.2 Characterization of CEO/β-CD proteoliposomes 2.3.2.1 The particle size, polydispersity index (PDI) and Zeta potential of CEO/β-CD proteoliposomes The particle size, PDI and Zeta potential of CEO/β-CD proteoliposomes were measured using a dynamic light scattering Zeta sizer (Nano ZS90, Malvern Instruments, Worcester, UK). The formulations were appropriate diluted with distilled water in purpose of avoiding the multi scattering phenomena. The samples were diluted 10 times to obtain the concentration of 10% (v/v). Each sample was measured in triplicate at 25 °C. 2.3.2.2 Determination of EE of CEO in CEO/β-CD proteoliposomes Firstly, a standard curve of a range of CEO concentrations (0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL, 1.2 mg/mL, and 1.6 mg/mL) was analyzed using a gas chromatography-mass spectrometer (GC-MS, Agilent 6890N/5973N, Agilent, California, USA). The standard curve was drawn depended on the peak area of eugenol, which is the main composition of CEO. The linear equation of CEO standard curve was obtained (Li et al., 2013). Subsequently, a certain amount of CEO/β-CD proteoliposomes was centrifuged at 12,000 rpm for 1 h using a desktop high speed centrifuge (H1850, HuNan xiangyi centrifuge instrument Co., Ltd., HuNan, China). The vesicles were resuspended in same volume ethanol, and treated by ultrasonic wave of 100 W for 3 h at 30 °C. Finally, the mixture was centrifuged at 6000 rpm for 15 min. The supernatant was filtered by 0.22 μm membrane and analyzed through GC-MS. The concentration of CEO in CEO/β-CD proteoliposomes was calculated using above standard curve. Then, the EE CEO in CEO/β-CD proteoliposomes was calculated as follows: EE (%)=

CEO encapasluted in CEO/β-CD proteoliposomes (mg/mL)  100% Initial CEO added (mg/mL)

2.3.2.3 The storage stability evaluation of CEO/β-CD proteoliposomes The prepared CEO/β-CD proteoliposomes were divided into four groups and stored at 4 °C, 12 °C, 25 °C, and 37 °C for 60 days, respectively. At predetermined time, a certain amount of CEO/β-CD proteoliposomes were removed to measure the particle size, PDI，and Zeta potential. Further, turbidity of CEO/β-CD proteoliposomes was detected by turbidimeter (WGZ-2000, Shanghai, China). 6

2.4 Antibacterial evaluation of CEO/β-CD proteoliposomes 2.4.1 Antibacterial activity of CEO/β-CD proteoliposomes We investigated the antibacterial activity of CEO/β-CD proteoliposomes against B. cereus using the plate colony counting method (Lin et al., 2016). Firstly, B. cereus was cultured at 37 °C for 24 h. Subsequently, the CEO/β-CD proteoliposomes (30%) was added to PBS with B. cereus (105-6 CFU/mL). The samples contained B. cereus (105-6 CFU/mL) without CEO/β-CD proteoliposomes was used as a control. All the samples were divided into four groups and cultured at 4 °C, 12 °C, 25 °C, and 37 °C, respectively. At predetermined times, the numbers of residual bacteria were observed at 0 d, 1 d, 2 d, 3 d and 4 d, respectively. 2.4.2 Antibacterial mechanism analysis of CEO/β-CD proteoliposomes The possible antibacterial mechanisms of CEO/β-CD proteoliposomes against B. cereus were explored as follows. The B. cereus samples treatment with CEO/β-CD proteoliposomes (30%) at 37 °C for 24 h. The sample without CEO/β-CD proteoliposomes treatment was used as a control. Firstly, the morphologies of B. cereus were examined with Transmission Electron Microscopy (TEM, JSM-2100F, JEOL, Tokyo, Japan) to observe the level of B. cereus membrane damage caused by CEO/β-CD proteoliposomes. In detail, the bacterial samples after treatment were collected by centrifugation at 8000 rpm for 10 min and washed thrice with PBS. Then the bacterial suspension were immobilized on copper grids dyed with 3% (w/v) phosphate tungsten acid for 3 min and dried at room temperature, followed by microscopic examinations. Secondly, the B. cereus were collected by centrifugation at 6000 rpm for 10 min at 4 °C, and resuspended in PBS. The absorption of supernatant was determined by a microplate reader (Infinite 200 PRO, Tecan Austria GmbH Untersbergstr, Grodig, Austria) at 260 nm to analyze the loss of 260 nm absorbing materials (Lee et al., 2014). The cellular DNA was extracted with TIANamp Bacteria Genomic DNA Kit (TIANGEN Biotech Co., Ltd, Beijing, China) and the DNA quantification was conducted by Nanodrop 2000 (Thermo Scientific, USA) (Cui et al., 2016). The cellular ATP concentration was determined by the CleanSenseTM Surface Hygiene Test Kit (LEYU Biotechnology, Shanghai, China), which is based on the detection of light generated by the ATP dependent enzymatic conversion of D-luciferin to oxyluciferin by firefly luciferase (Finger et al., 2012). The intracellular protein concentration was determined by the BCA (bicinchoninic acid) Protein Assay Kit (Jiancheng Bioengineering Institute, Jiangsu, China), which 7

is based on absorbance at 562 nm by using bicinchoninic acid method (Cui et al., 2016). Lastly, the precipitate of B. cereus (after centrifuged at 6000 rpm for 10 min at 4 °C) was discarded and the supernatant was diluted 20 times to determine its Electrical conductivity using a portable multi-parameter analyzer (DZS-718, Shanghai Precision Science Instrument Co., Ltd, Shanghai, China). In addition, the activity of β-galactosidase is an important parameter indicative of cell membrane permeability (Wang et al., 2017). The β-galactosidase activity was analyzed by measuring the absorbance values at 405nm using ultraviolet spectrophotometer (NANODROP 2000, Thermo Fisher Scientific, Waltham, MA, USA) after adding the β-galactosidase reaction buffer and the ONPG to the supernatant. 2.5 Preparation and characterization of CEO/β-CD proteoliposomes nanofibers 2.5.1 Preparation of CEO/β-CD proteoliposomes nanofibers Firstly, a certain amount of PEO powder was dissolved in distilled water under magnetic stirring at room temperature for 3 h to obtain concentrations of 25% (w/v). Then the PEO solution was mixed with CEO/β-CD proteoliposomes at a volume ratio of 2:8 (v/v). The solution was stirred at room temperature for another 12 h to obtain CEO/β-CD proteoliposomes and PEO mixture solution. In the final mixture solution system, the concentration of PEO was 5% (w/v), and the concentration of CEO/β-CD proteoliposomes was 80% (v/v). The electrospinning apparatus (SNAN-01, Electrospinning Setup, MECC Co., Ltd., Fukuoka, Japan) was employed to spin nanofibers. The electrospinning conditions are as follows, the capacity of the syringe was 5.0 mL with a hypodermic needle with an inner diameter of 0.9 mm. The applied high voltage power was adjusted at 25 kV. Nanofibers were collected on electrical connection of aluminum foil placed at a 12 cm vertical distance to the needle tip. The flow rate of spinning solution was regulated to 0.6 mL/h by a syringe driver (Ge et al., 2012). For comparison, PEO nanofibers were also prepared as described above. 2.5.2 Characterization of CEO/β-CD proteoliposomes nanofibers 2.5.2.1 The morphology of CEO/β-CD proteoliposomes nanofibers The incorporation of CEO/β-CD proteoliposomes into PEO nanofibers was achieved by electrospinning technology. The surface morphologies of CEO/β-CD proteoliposomes nanofibers were characterized by scanning electron microscope (SEM, JSM-7001F, JEOL, Tokyo, Japan). For comparison, the surface morphologies of PEO nanofibers were also surveyed as described above. 8

2.5.2.2 In vitro release kinetics of CEO from CEO/β-CD proteoliposomes nanofibers A certain amount of CEO/β-CD proteoliposomes nanofibers was added into ethanol (10 mL) treated by ultrasonic wave for 3 h, then centrifuged for 20 min at 6000 rpm/min. The supernatant was analyzed using GC-MS to determine the concentration of CEO to obtain the total amount of CEO in nanofibers. The same quantity of CEO/β-CD proteoliposomes nanofibers were added into 10mL PBS mixture with B. cereus (107-8 CFU/mL), and shaking cultured at 4 °C, 12 °C, 25 °C, and 37 °C, respectively. A certain amount of supernatant was removed at different time intervals to determine the concentration of CEO using GC-MS. As a control, the sample cultured without B. cereus was also examined according to the above method. Then, the release rate (RR) of CEO was calculated using the following equations: RR (%)=

Wrelease ×100% Wtotal

Where Wrelease (mg/mL) was amount of CEO released from CEO/β-CD proteoliposomes nanofibers, and Wtotal (mg/mL) was the total amount of CEO in CEO/β-CD proteoliposomes nanofibers. 2.6 Antibacterial evaluation of CEO/β-CD proteoliposomes nanofibers 2.6,1 Antibacterial activity of CEO/β-CD proteoliposomes nanofibers in vitro In order to evaluate the antibacterial activity of CEO/β-CD proteoliposomes nanofibers against B. cereus in vitro, the nanofibers were cut in 20 × 20 mm square and sterilized by UV irradiation for 30 min. Subsequently, the CEO/β-CD proteoliposomes nanofibers were placed into PBS containing B. cereus (105-6 CFU/mL). The bottles with PEO nanofibers (20 × 20 mm) were used as control groups. All the samples were divided into four groups and cultured at 4 °C, 12 °C, 25 °C, and 37 °C, respectively. The numbers of residual bacteria were observed at 0 d, 1 d, 2 d, 3 d and 4 d using the plate colony counting method. 2.6.2 The antibacterial application of CEO/β-CD proteoliposomes nanofibers on beef The CEO/β-CD proteoliposomes nanofibers were cut into 80 × 80 mm square, and then sterilized by UV irradiation for 30 min. Fresh beef samples (30 × 20 × 5 mm) were inoculated with B. cereus (105-6 CFU/g) and packaged with CEO/β-CD proteoliposomes nanofibers. The packaged beef samples were divided into four groups, stored at 4 °C, 12 °C, 25 °C, and 37 °C, respectively. The numbers of residual bacteria were observed at 0 d, 1 d, 2 d, 3 d and 4 d using the 9

plate colony counting method. The beef samples packaging with PEO nanofibers were used as control groups. 2.7 Color and texture evaluation The fresh beef samples were packaged with CEO/β-CD proteoliposomes nanofibers and PEO nanofibers and stored at 4 °C, 12 °C, 25 °C, and 37 °C, respectively. After 4 d, the changes of beef surface color were measured with a Chromatic meter (Color Quest XE, Hunter Lab Co., Reston, Virginia, USA). Hunter color values, L* (lightness), a* (redness), and b* (yellowness) were determined. Surface texture measurement of the beef was performed using a TA.XT. Plus (Stable Micro Systems Ltd, Godalming, Surrey, UK). Hardness, springiness and chewiness were used to evaluate the texture quality of the beef. 2.8 Statistical Analysis Except the SEM and TEM images, all experiments were carried out in triplicates. And dates are presented as means  standard deviations (SD), and all statistical tests were performed using the SPSS software (SPSS19.0 for Windows). Statistical significance was ascertained when P˂0.05. 3. Results and discussion 3.1 Characterization of CEO/β-CD inclusion complex 3.1.1 The optimization of CEO/β-CD inclusion complex β-CD due to its special structure was used to prepare CEO/β-CD inclusion complex. The effect of some parameters on its inclusion efficiency was investigated and the results are presented in Fig. 2. The ER of CEO had reached the maximum value (82.50%) when CEO: β-CD ratio was 1:9, while the IR had reached the maximum value (68.25%) when CEO: β-CD ratio was 1:8 (Fig. 2A). The IR and ER had reached the maximum value (60.89% and 82.58%, respectively) at 50 °C (Fig. 2B). Lower temperature was unfavorable for dissolution of β-CD, while higher temperature would result in a remarkable decrease of CEO loading content due to the volatilization of CEO (Wen et al., 2016). As shown in Fig. 2C, the IR and ER both increased with the extension of time and reached a maximum at 2.5 h. These results indicated that the CEO was embedded into β-CD effectively. Under the optimized preparation conditions, the IR and ER was 67.12% and 82.23%, respectively. 3.1.2 Raman spectrum analysis 10

In Raman spectra, the variation of the shape, shift, or intensity of absorption peaks corresponds to the vibrational or rotational energy levels of molecules. As shown in Fig. 2D, compared to the Raman spectrum of CEO, evident changed of CEO/β-CD inclusion complex characteristic peaks were observed. It can be observed from the Raman spectra of CEO/β-CD inclusion complex that the typical signatures of CEO not appear, such as 795 cm-1, 1208 cm-1, and 3005 cm-1. Although the typical peak of CEO (1629 cm-1) was appears, but the peak intensity was weakened due to the inclusion function of β-CD. Compared the Raman spectra of CEO/β-CD inclusion complex with β-CD, the similar positions and shapes of these peaks in β-CD were detected. In addition, the original characteristics peak of the β-CD was shift (477 cm-1 to 490 cm-1, 859 cm-1 to 866 cm-1, 1124 cm-1 to 1137 cm-1), which further proved the formation of CEO/β-CD inclusion complex. Therefore, the results implied that CEO was embedded into β-CD successfully (Chowdhry et al, 2015; Seiça et al., 2016). 3.1.3 FTIR analysis FTIR spectrum was employed to provide information about the occurrence of inclusion complex formation and the functional groups of the components. Changes in the shape, position and intensity of the absorption bands of the different samples were observed and presented in Fig. 2E. FTIR spectrum of pure CEO exhibited the characteristic peaks at 1646 cm-1 and 1735 cm-1 corresponding to skeletal vibrations relating -C=C- stretching in the benzene ring and the carbonyl group (-C=O) (Wen et al., 2016). Additionally, the peaks at 1150 cm-1 (-C-O-C-), 1020 cm-1 (-C-C-), 853 cm-1 and 796 cm-1 (-C-H) were also observed. As for pure β-CD, the peaks were as follows: 3306 cm-1 (-OH), 2934 cm-1 (-C-H), 1652 cm-1 (-O-H), 1150 cm-1 (-C-O-C-), 1020 cm-1 (-C-O-) (Lakkakula et al., 2017). For CEO/β-CD inclusion complex, characteristic peaks of CEO and β-CD were observed, confirming the presence of both CEO and β-CD in CEO/β-CD inclusion complex almost (Fig. 2E). But the typical signatures of CEO (1646 cm-1) not appear in the CEO/β-CD inclusion complex, this is due to the embedding of β-CD to cover this characteristic peaks. In addition, the characteristic peaks of CEO at 1510 cm-1, 2928 cm-1 shifted to 1515 cm-1, 2934 cm-1 in CEO/β-CD inclusion complex, respectively. The peaks of CEO (1150 cm-1 and 1510 cm-1) were remarkably weakened due to the inclusion function of β-CD. However, the characteristics peak of physical mixture of CEO and β-CD was the simple superposition of the peaks of CEO and β-CD, 11

and the intensity of the peak was not significantly different from the peaks of CEO and β-CD. The results further indicated that the CEO was embedded into β-CD successfully. 3.2 Characterization of CEO/β-CD proteoliposomes 3.2.1 The particle size, PDI and Zeta potential of CEO/β-CD proteoliposomes The physicochemical properties of the CEO/β-CD proteoliposomes containing different concentration of CEO/β-CD inclusion complex were determined as shown in Table 1. The average particle size of CEO/β-CD proteoliposomes was slightly enlarged with the increase of CEO/β-CD inclusion complex concentration, and ranged from 317 nm to 364 nm. This means that more CEO/β-CD inclusion complexes are encapsulated into proteoliposomes. As a parameter of particle size distribution, low PDI value (˂0.3) indicated that the nanoliposomes had a narrow size distribution (Tan et al., 2013). The PDI value of different types of proteoliposomes ranged from 0.075 to 0.198, suggesting the proteoliposomes were largely homogenous. The presence of CD in the liposomal formulations did not affect the PDI, which is in agreement with other studies (Gharib et al., 2017). The Zeta potential is an important parameter indicating the stability of nanoliposomes systems. A relatively high Zeta potential (>30 mV) might provide a repelling force between the particles, thus increasing the stability of the nanoliposomes (Zhong et al., 2012). As shown in Table 1, Zeta potential of different types of proteoliposomes ranged from -29.1 mV to -48.9 mV. The results indicated that the proteoliposomes have high stability. 3.2.2 The EE of CEO in CEO/β-CD proteoliposomes The EE of CEO in proteoliposomes ranged from 29.40% to 49.07% as displayed in Table 1. With the increasing of CEO/β-CD inclusion complex concentration, the EE of CEO in CEO/β-CD proteoliposomes reached the maximum value (49.07%) at 30.0 mg/mL of CEO/β-CD inclusion complex, while slightly declined at a concentration of 40.0 mg/mL. This phenomenon could be explained by the saturation of CEO/β-CD inclusion complex content in CEO/β-CD proteoliposomes. Based on the above-mentioned analysis, the CEO/β-CD proteoliposomes prepared at 30.0 mg/mL of CEO/β-CD inclusion complex was chosen for further studies, due to their desirable properties. In addition, compared with the CEO proteoliposomes, the increase of EE of CEO in CEO/β-CD proteoliposomes was observed. The result verified that the EE of CEO in proteoliposomes can be enhanced by forming of CEO/β-CD inclusion complex. 12

3.2.3 The storage stability evaluation of CEO/β-CD proteoliposomes The storage stability of CEO/β-CD proteoliposomes was investigated and displayed in Table 2. The particle size of CEO/β-CD proteoliposomes had no significant changes with the extension of time and the increase of temperature. Table 2 revealed that there was no significant difference about PDI of proteoliposomes when stored at different temperature for 60 d. Low PDI value (˂0.3) indicated that the nanoliposomes had a narrow size distribution. PDI values for all the proteoliposomes formulations were less than 0.3, suggesting the proteoliposomes were largely homogenous and had good dispersibility. The surface Zeta potential is an important parameter indicating the stability of liposomes systems. As shown in Table 2, Zeta potential of CEO/β-CD proteoliposomes ranged from -30.7 mV to -33.2 mV (>30 mV). Therefore, the change of Zeta potential of CEO/β-CD proteoliposomes was extremely small when they were stored at different temperature for 60 d, indicating the stability of proteoliposomes. The EE of CEO in CEO/β-CD proteoliposomes slightly decreased with the extension of time and the increase of temperature. This phenomenon was due to the natural leakage of CEO from CEO/β-CD proteoliposomes. Besides, the leakage of EOs from nanoliposomes during the storage will also lead to the slightly increase of turbidity. As shown in Table 2, after 60 d, the turbidity of CEO/β-CD proteoliposomes slightly increased with the extension of time and the increase of temperature. This phenomenon also was due to the natural leakage of CEO from CEO/β-CD proteoliposomes. In summary, the results showed that the CEO/β-CD proteoliposomes possessed high stability at different storage temperature for 60 d. 3.3 Antibacterial evaluation of CEO/β-CD proteoliposomes 3.3.1 The antibacterial activity of CEO/β-CD proteoliposomes As shown in Fig. 3, the antibacterial activity of CEO/β-CD proteoliposomes against B. cereus was investigated at different temperature. Compared with the control groups, the number of viable B. cereus treated with CEO/β-CD proteoliposomes showed a gradual and consecutive decrease. After 4 d, 99.6% and 99.9% reduction in population was investigated at 4 °C and 12 °C (Fig. 3A and 3B), respectively. And 99.99% and 99.999% reduction in population was investigated at 25°C and 37 °C (Fig. 3C and 3D), respectively. The results showed that the antibacterial efficiency of CEO/β-CD proteoliposomes against B. cereus was positively associated with temperature. The results corresponded to above-mentioned release kinetics studies describing CEO release from 13

CEO/β-CD proteoliposomes. In addition, the schematic of B. cereus proteinase-triggered CEO release from proteoliposomes was shown in Fig. 1. The CEO/β-CD proteoliposomes can accurately inhibit bacterial multiplication by the stimulus-response of casein to bacterial protease under the present of B. cereus. 3.3.2 Evaluation of antibacterial mechanism of CEO/β-CD proteoliposomes TEM images were carried out to visualize the efficacy of CEO/β-CD proteoliposomes on the morphological and physical changes to B. cereus. The untreated B. cereus had a normal and intact cell structure. (Fig. 3E), but B. cereus treated with CEO/β-CD proteoliposomes exhibited irregularly edges (Fig. 3F). There was obvious breakage of the B. cereus cellular membrane. The optical density at 260 nm was detected to evaluate the influence of CEO/β-CD proteoliposomes to the cellular membrane permeability. The OD260 nm of B. cereus was increased from 0.063 to 0.292 after treatment (Fig. 3G), which indicated that CEO/β-CD proteoliposomes treatment resulted in the permeability of cell membrane changed, leading to the leakage of the intracellular substances. The measurement of the quantification of DNA revealed that DNA content in B. cereus was significantly reduced by 63.73% after treatment, which implied CEO/β-CD proteoliposomes treatment led to the decrease of DNA. Results of ATP bioluminescence assay showed the intracellular ATP contents of B. cereus after treatment dropped by 78.89%. Simultaneously, the protein concentration in B. cereus was reduced by 64.18% (Fig. 3G). In addition, the electrical conductivity of B. cereus suspension increased from 0.187 mS·cm-1 to 0.252 mS·cm-1 after treatment. The change is caused by cell membrane permeability, followed by leakage of intracellular K+, Na+, thus increasing the electrical conductivity. The effect of CEO/β-CD proteoliposomes on cell membrane permeability was further examined by the determination of β-galactosidase activity. As shown in (Fig. 3G), the OD405nm of β-galactosidase increased from 1.533 to 1.924 after treatment for 4 h. Thus, the rapid increase of OD405nm of β-galactosidase indicates a change in cell membrane permeability. All of these changes resulted in cell decomposition eventually, which was directly reflected in the reductions of bacteria numbers. These results indicated that significantly severe damages to the microbial cytoplasmic membrane and cellular features, lead to cell disruption cellular disintegration (Lin et al., 2016). 3.4 Characterization of CEO/β-CD proteoliposomes nanofibers 14

3.4.1 The morphology of CEO/β-CD proteoliposomes nanofibers The SEM images of PEO nanofibers and CEO/β-CD proteoliposomes nanofibers were compared in Fig. 4. As shown in Fig. 4A that the morphology of pure PEO nanofibers were smooth and uniform, and the diameter mostly ranged between 350 nm and 450 nm (Fig. 4C). Conversely, the morphology of CEO/β-CD proteoliposomes nanofibers was more rough (Fig. 4B), and the diameter mostly ranged between 500 nm and 650 nm (Fig. 4D). In addition, the surface of CEO/β-CD proteoliposomes nanofibers has some small protrusions (Fig. 4B). This phenomenon can be explained by the introduction of CEO/β-CD proteoliposomes in PEO nanofibers. A similar phenomenon was also observed for PLA/CEO/β-CD nanofibers and nisin-loaded γ-PGA/CS nanoparticles-embedded PEO nanofibers (Wen et al., 2016; Cui et al., 2017b). The results indicated that the CEO/β-CD proteoliposomes was incorporated into PEO nanofibers successfully. 3.4.2 In vitro release kinetics of CEO from CEO/β-CD proteoliposomes nanofibers As shown in Fig. 4E-4H, with the B. cereus treatment for 48 h, the release rate of CEO from CEO/β-CD proteoliposomes nanofibers at different temperature was 38.60%, 46.90%, 72.90%, and 78.60%, respectively. And after 96 h, the cumulative release rate of CEO can reach to 46.54%, 58.48%, 78.48%, and 87.04%, respectively. The slow release of CEO can last for more than 48 hours under the present of B. cereus. And it is obvious that the release rate of CEO increased as increasing temperature. The main reason is that the relatively high temperature is more suitable for the life activities of B. cereus. In addition, CEO/β-CD proteoliposomes nanofibers only have minor amounts of CEO release under the absence of B. cereus. The results further indicated that the controlled release of CEO from CEO/β-CD proteoliposomes nanofibers was realized via bacterial protease secreted from B. cereus. 3.5 Antibacterial evaluation of CEO/β-CD proteoliposomes nanofibers 3.5.1 The antimicrobial activity of CEO/β-CD proteoliposomes nanofibers in vitro The antibacterial activity of CEO/β-CD proteoliposomes nanofibers against B. cereus in PBS were evaluated at 4 °C, 12 °C , 25 °C and 37 °C，respectively. From Fig. 5, the control samples showed rapid growth of B. cereus. But the number of B. cereus in the CEO/β-CD proteoliposomes nanofibers were decreased or remained unchanged with the prolongation of the time. And it is obvious that the antibacterial efficiency of CEO/β-CD proteoliposomes nanofibers increased with the increasing of temperature, about 99.999% population reduction of B. cereus at 15

25 °C and 37 °C. (Fig. 5C and 5D) However, when the samples were cultivated at 4 °C and 12 °C, the number of B. cereus in the CEO/β-CD proteoliposomes nanofibers were slightly changed with the extension of the time (Fig. 5A and 5B). The results can be explained that the antibacterial efficiency of CEO/β-CD proteoliposomes nanofibers mainly relied on the release rate of CEO. 3.5.2 The antibacterial activity of CEO/β-CD proteoliposomes nanofibers on beef Considering the possible application of proteoliposomes in the future, the antibacterial effects of CEO/β-CD proteoliposomes nanofibers against B. cereus on beef was investigated and the results were shown in Fig. 8. After 4 d, almost 99.999% reduction in population of B. cereus was observed after the treatment of CEO/β-CD proteoliposomes nanofibers at 25 °C and 37 °C (Fig. 5G and 5H). And almost 87.0% and 99.9% reduction in population of B. cereus was observed after the treatment with CEO/β-CD proteoliposomes nanofibers at 4 °C and 12 °C, respectively (Fig. 5E and 5F). The results were corresponding to the antibacterial efficiency of CEO/β-CD proteoliposomes nanofibers in vitro. The results demonstrated that CEO/β-CD proteoliposomes nanofibers had a significant antibacterial efficiency against B. cereus on beef. In view of their favorable antibacterial activity, CEO/β-CD proteoliposomes nanofibers would be a promising antimicrobial agent in meat products preservation field. 3.6 Color and texture evaluation The color and texture evaluation of beef samples packaged with CEO/β-CD proteoliposomes nanofibers at different storage duration were showed in Table 3. Although the parameters of beef treated with PEO nanofibers and CEO/β-CD proteoliposomes nanofibers have both changed significantly after 4 d, but the parameters changes of beef treated with CEO/β-CD proteoliposomes nanofibers were relatively small. The results indicated that the CEO/β-CD proteoliposomes nanofibers have no impact on the sensorial property of beef, and could maintain sensory quality of beef samples to a certain degree. This may be due to the fact that CEO has the function of preventing the oxidation of beef. 4. Conclusions In conclusion, the CEO/β-CD proteoliposomes nanofibers were prepared successfully, and the physicochemical stability and the EE of CEO proteoliposomes were significantly improved by introducing β-CD. Additionally, the antibacterial efficiency of CEO/β-CD proteoliposomes against B. cereus was enhanced, accompanying the improvement of their stability via nanofibers 16

encapsulation. From the perspective of safety and practicability, this novel antibacterial packaging has broad prospect in the field of meat production preservation. Acknowledgments The authors acknowledge the financial support from National Natural Science Foundation of China (grant no. 31470594), Natural Science Foundation of Jiangsu Province (grant no. BK20170070), Jiangsu Province Foundation for talents of six key industries (grant no. NY-013), Innovation Fund Designated for Graduate Students of Jiangsu Province (grant no. SJLX16-0444), Jiangsu University scientific research project for student (grant no. 15A185) and Jiangsu University Research Fund (grant no. 11JDG050).

17

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21

Fig. 1 Schematic of electrospinning for CEO/β-CD proteoliposomes incorporated into PEO nanofibers. And schematic of B. cereus proteinase-triggered CEO release from CEO/β-CD proteoliposomes.

Fig. 2 The effect of CEO:β-CD rate (A), temperature (B) and time (C) on CEO/β-CD inclusion complex. Raman spectra of CEO, β-CD and CEO/β-CD inclusion complex (D). FTIR spectra of CEO, β-CD, CEO/β-CD inclusion complex, and CEO and β-CD physical mixture (E).

22

Fig. 3. The antibacterial activity of CEO/β-CD proteoliposomes against B. cereus in vitro at different temperature 4 ℃ (A), 12 ℃ (B), 25 ℃ (C), and 37 ℃ (D). TEM images analysis of B. cereus before (E) and after (F) CEO/β-CD proteoliposomes treatment. The analysis of B. cereus cell constituents’ release and the cell membrane permeability before and after CEO/β-CD proteoliposomes treatment (G).

Fig. 4 The SEM-micrographs of pure PEO nanofibers (A) and CEO/β-CD proteoliposomes nanofibers (B). Diameter distribution of pure PEO nanofibers (C) and CEO/β-CD proteoliposomes nanofibers (D). The release rate of CEO/β-CD proteoliposomes nanofibers stored at different temperature 4 °C (E), 12 °C (F), 25 °C (G), and 37 °C (H) for 4 d.

23

Fig. 5 The antibacterial activity of CEO/β-CD proteoliposomes nanofibers against B. cereus in vitro at 4 °C (A), 12 °C (B), 25 °C (C), and 37 °C (D) for 4 d. And on beef at 4 °C (E), 12 °C (F), 25 °C (G), and 37 °C (H) for 4 d.

24

Table 1 The characterization of different type of proteoliposomes. Parameter Zeta Liposome type

Particle size PDI

potential

EE (%)

(nm) (mV) 0.075



159  13.5

Proteoliposomes

-48.9  1.09

0

0.037 0.099



203  23.2

CEO Proteoliposomes

29.40

0.011 CEO/β-CD

proteoliposomes

(10

0.146

1.428 

317  30.8 mg/mL) CEO/β-CD

(20

0.163

mg/mL)



(30

0.182

mg/mL)



49.07

(40

0.198

1.521 

364  36.7 mg/mL)

The data in parentheses represent the concentration of CEO/β-CD inclusion complex.

25

36.24 -29.1  1.71

0.054

Data represent the mean  SD.



-32.2  2.11 0.042

proteoliposomes



1.592

349  35.8

CEO/β-CD

44.53 -33.2  2.16

0.031 proteoliposomes



1.446

306  33.8

CEO/β-CD

42.32 -37.2  2.06

0.033 proteoliposomes



-45.0  2.25

1.502



Table 2 The properties changes of CEO/β-CD proteoliposomes during the storage at 4 °C, 12 °C, 25 °C, and 37 °C. Storage time (d)

Temperat Parameters ure Particle

0

15

30

45

60

349  33.7

348  30.9

347  29.3

347  31.1

345  33.2

size

(nm) 0.183



0.189



0.188



0.182



0.196



PDI 0.031 4 °C

Zeta potential (mV)

-32.2

0.011 

2.16

-32.7

0.036 

1.88 49.08



-32.8

0.028 

2.09 49.11



-33.0

0.039 

1.93 49.09



-33.2



48.72



2.18 49.01



EE (%) 1.59

1.66

1.49

1.28

1.42

Turbidity 526  9.30

529  12.4

531  10.7

539  9.30

543  6.50

349  30.1

348  28.7

348  22.9

346  21.7

344  27.6

(NTU) Particle

size

(nm) 0.185



0.181



0.182



0.185



0.189



PDI 0.072 12 °C

Zeta potential (mV)

-32.3

0.031 

2.07

-32.1

0.082 

1.93 49.09



-32.8

0.081 

1.47 49.91



-32.5

0.029 

1.86 48.83



-32.0



48.65



612



1.48 48.71



EE (%) 1.33

1.71

2.09

1.43

1.62

Turbidity 527  9.32

531  14.1

543  7.80

586  9.60 12.3*

(NTU) Particle

size 349  29.3

347  27.1

346  24.1

344  26.0

342  31.4

(nm) 25 °C 0.187



0.189



0.192



0.195



0.197



PDI 0.032 Zeta potential

-32.9

0.059 

-32.4 26

0.031 

-31.4

0.053 

-31.2

0.033 

-31.3



(mV)

3.01

2.27 49.05



1.51 49.01



1.76 48.97



1.29 48.71



47.62



647



EE (%) 1.59

1.82

2.13

2.45

Turbidity

621 527  9.30

541  7.90



549  13.6 9.70*

(NTU) Particle

1.44

17.1*

size 350  27.2

348  26.3

345  23.9

342  25.5

341  28.6

(nm) 0.185



0.182



0.183



0.197



0.199



PDI 0.044 37 °C

Zeta potential (mV)

-32.3

0.054 

2.93

-31.9

0.024 

2.08 49.09



-31.4

0.049 

1.42 49.01



-31.7

0.033 





1.12*

1.46 48.99

-30.9

48.42



47.23



EE (%) 1.66

1.75

2.04

1.04*

2.19

Turbidity

628 528  9.11

548  7.10

12.1*

(NTU)



662

564  6.50 14.9*

Data represent the mean  SD. *

The values were considered to be significant (P<0.05 compared to the value obtained immediately after

preparation, 0 days).

27



Table 3 Sensory evaluation of beef samples after treatment during the storage at 4 °C for 4 d. CEO/β-CD Temperature

Fresh beef

Control

(0 d)

(4 d)

Parameters

proteoliposomes nanofibers (4 d)

L*

35.17  0.48

32.02  0.17*

33.97  0.26*

a*

27.49  0.27

29.98  0.78*

28.92  0.18*

b*

14.32  0.23

16.41  0.82*

15.12  0.31*

Hardness (N)

7.82  0.45

7.01  0.53*

7.31  0.67*

Spring (%)

78.48  0.59

73.39  0.66*

76.87  0.37*

Chewiness (mJ)

26.39  0.72

23.12  0.46*

25.71  0.32

L*

35.17  0.48

30.85  0.28*

33.51  0.37*

a*

27.49  0.27

32.98  0.18*

28.44  0.19*

b*

14.32  0.23

17.85  0.27*

15.86  0.09*

Hardness (N)

7.82  0.45

6.47  0.17*

7.04  0.13*

Spring (%)

78.48  0.59

72.76  0.82*

73.01  0.20*

Chewiness (mJ)

26.39  0.72

21.03  0.72*

24.06  0.78*

L*

35.17  0.48

29.96  0.37*

31.17  0.16*

a*

27.49  0.27

34.61  0.19*

30.34  0.47*

b*

14.32  0.23

18.20  0.47*

16.03  0.19*

Hardness (N)

7.82  0.45

6.01  0.40*

6.97  0.34*

Spring (%)

78.48  0.59

68.27  0.17*

74.75  0.17*

Chewiness (mJ)

26.39  0.72

19.78  0.11*

23.08  0.24*

L*

35.17  0.48

26.22  0.19*

30.43  1.34*

a*

27.49  0.27

37.52  0.37*

31.07  0.92*

b*

14.32  0.23

20.35  0.80*

18.47  0.52*

Hardness (N)

7.82  0.45

5.13  0.73*

6.28  0.07*

Spring (%)

78.48  0.59

61.25  1.31*

70.40  2.52*

Chewiness (mJ)

26.39  0.72

16.41  0.89*

21.59  1.12*

4 °C

12 °C

25 °C

37 °C

28

Data represent the mean  SD. * The

values were considered to be significant (P<0.05 compared to fresh beef (0 d)).

29