The use of bacterial cellulose from kombucha to produce curcumin loaded Pickering emulsion with improved stability and antioxidant properties

2023-01-21 05:02ZhiyuLiWenxiuHuJijiDongFielisAziXioXuChunhiTuSijieTngMingshengDong
食品科学与人类健康(英文) 2023年2期

Zhiyu Li,Wenxiu Hu,Jiji Dong,Fielis Azi,Xio Xu,Chunhi Tu,Sijie Tng,Mingsheng Dong,*

a College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,China

b College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China

c School of Life Science,Shaoxing University,Shaoxing 312000,China

d College of Food Science and Pharmacy,Zhejiang Ocean University,Zhoushan 316022,China

Keywords:Bacterial cellulose Curcumin Pickering emulsion Stability Antioxidant activity

ABSTRACT Curcumin is a bioactive molecule with limited industrial application because of its instability and poor solubility in water.Herein,curcumin-loaded Pickering emulsion was produced using purified bacterial cellulose from fermented kombucha (KBC).The morphology,particle size,stability,rheological properties,and antioxidant activities of the curcumin-loaded Pickering emulsion were investigated.The fluorescence microscope and scanning electron microscopy images showed that the curcumin-loaded Pickering emulsion formed circular droplets with good encapsulation.The curcumin-load Pickering emulsion exhibited better stability under a wide range of temperatures,low pH,sunlight,and UV-365 nm than the free curcumin,indicating that the KBC after high-pressure homogenization improved the stability of the CPE.The encapsulated curcumin retained its antioxidant capacity and exhibited higher functional potential than the free curcumin.The study demonstrated that the KBC could be an excellent material for preparing a Pickering emulsion to improve curcumin stability and antioxidant activity.

1.Introduction

In recent years,numerous studies have been carried out to stabilize curcumin.Curcumin is a polyphenolic compound with low intrinsic toxicity but possesses multiple biological activities,including an antidiabetic ability [1],antiviral [2],anti-inflammatory,antimicrobial [3],and anti-carcinogenic properties [4].However,the use of this bioactive molecule is limited because of its poor water solubility,low stability,and low bioavailability at target sites [5].Severalin vitroandin vivoresearches have been carried out to improve curcumin’s stability and water solubility to retain its bioactivity.An emulsion has been highly researched as a delivery system for functional food because of its potential to encapsulate the various micro-ingredients and protect against the degradation and improvement of the bioavailability [6,7].

Many research studies have reported that the Pickering emulsion can be a potential delivery system.It is formed by solid colloidal particles and form a thick barrier that is rigid and porous [8].In contrast to the conventional emulsion that uses surfactant to form a stable system that may be toxic,the Pickering emulsion is much safer.Different types of particles have been used to form Pickering emulsions.These include inorganic particles (silica and silicabased particles) [9],carbohydrate-based particles (modified starch,micr°Crystalline cellulose,chitin nan°Crystals,c°Coa powder/fibers,chitosan-coated alginate particles,hydrophobized cellulose nan°Crystals),protein-based particles (plant protein or dairy protein),and lipid-based particles (fat crystals) [10].Cellulose is widely sourced and non-toxic,and its crystallinity ratio can be manually controlled [11,12].Currently,many reports have proved that Pickering emulsions using cellulose as an emulsifier retain the excellent stability of surfactant emulsions [7],with strong stability,good bi°Compatibility,and low toxicity [13].Traditionally,cellulose as an emulsion stabilizer is obtained by hydrolysis of sulfuric acid or hydr°Chloric acid [14].However,it has been reported cellulosebased Pickering emulsions are typically prepared using chemical reagents.The low yield,high cost,and strong acid use limit the application of this method in the food industry.Recently,mechanical methods have gained interest to reduce cellulose size by grinding [15],microfluidization [16],and high-pressure homogenization (HPH) [17].HPH treatment is an effective technology that promotes particle size reduction of the cellulose fibrils to the nanoscale via crushing forces efficiently and simply [18].

Kombucha (KBC) is a slightly sweet and acidic fermented beverage produced using bacteria and yeast symbiotic consortium [19].The primary substrate for KBC production is tea leaves;hence the fermented beverage contains many biologically active molecules,including antioxidants,polyphenols,glucuronic acid,and vitamins.Several reports have demonstrated that the beverage can detoxify the human biological system [20],fortify the immune system [21],facilitate the treatment of gastric ulcers [22],etc.During the beverage fermentation,the acetic acid bacteria produce a biofilm that floats on the surface of the KBC beverage.This biofilm is a cellulosic pellicle layer,a by-product of the fermentation pr°Cess.This pellicle layer is also known as KBC fermented bacterial cellulose,and excellent water retention capacity,high crystallinity,and thermostability.Compared to the traditional cellulose from plant sources,which needs strong acids,alkali to be hydrolyzed from the hemicellulose,pectin,and lignin,the KBC has higher purity and is easier to be extracted [11].

Meanwhile,the KBC has high biodegradability,bi°Compatibility,flexibility,non-toxicity,and mechanical strength.It is an important bio-molecule with a wide range of potential applications in the pharmaceutic field,medical field,and food industry [23].Previous studies on KBC have only f°Cused on the fermented tea beverage,but less attention has been paid to its by-product KBC.According to the study,the properties such as the basic structure of KBC are similar to that of BC produced by single BC producing bacteria because the cellulose is produced by the bacterial genera only even though KBC is a symbiotic culture [11,24].Therefore,to effectively expand and improve the utilization of KBC,it is critical to explore other potential areas of application of KBC.

We hypothesize that the Pickering emulsion prepared using purified bacterial cellulose from fermented KBC could improve curcumin’s stability and bioactivity.Thus,the stability of the curcumin encapsulated in the Pickering emulsion under sunlight and ultraviolet (UV) and at different pH and temperatures were evaluated.In this approach,KBC structure and the morphology,particle size distribution,curcumin stability were comprehensively examined.The work explores the efficacy of the Pickering emulsion as a prospective delivery system for bioactive molecules.

2.Materials and methods

2.1 Materials and reagents

KBC was obtained from the st°Ck preserved in our laboratory.Olive oil was purchased from a commercial supermarket.Black tea was produced from Yunnan Zhongcha Tea Industry Co.,Ltd.Methanol (≥ 99.5% purity) was purchased from Sinoph arm Chemical Reagent Cooperation.Curcumin (C7090,95% purity) was purchased from Beijing Solarbio Science&Technology Co.,Ltd.,Beijing,China.2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO,98% purity) was purchased from Macklin Cooperation.D-α-T°Copherol(97% purity) was purchased from Aladdin Cooperation.Sodium bromide (NaBr,≥ 99.0% purity) was purchased from Lingfeng Cooperation.Sodium hydroxide (NaOH,≥ 96% purity) was purchased from Nanjing Reagent Cooperation.All the reagents were of analytical grade,and all solutions were prepared with doubledistilled water.

2.2 Purification of the bacterial cellulose from KBC

To prepare the KBC,5 g black tea was boiled in 1 L water for 20 min,and then 100 g/L sucrose was added to the tea broth.Then,the broth was sterilized at 121 °C for 20 min.After cooling the sweetened tea broth,10% (V/V) of previous fermented KBC tea was added,and the black tea medium was cultured at 28 °C for 7 days.After that,the KBC was collected and was washed by running tap water overnight.Then the brown translucent KBC was mixed with double distilled water at a ratio of 1:20 (V/V) and then ground with a blender at 20 000 r/min for 4 min.Then excess water was removed by centrifugation at 12 000 r/min for 10 min.The sediment was collected and purified with 3% H2O2at a ratio of 1:20 (V/V);the 1 mol/L sodium hydroxide solution was used to adjust the pH to 9.5.Then the mixture was put in an ultrasonic bath at 100 W for 60 min.After ultra-sonication treatment,the mixture was centrifuged at 12 000 r/min for 10 min to remove excess water.And the sediment was washed using distilled water at a ratio of 1:20 (V/V)and was centrifuged at 12 000 r/min for 10 min.Repeated washing with distilled water was done until the pH was about 7.The collected samples were coded as KBC [11,25].

2.3 Preparation of high-pressure homogenized KBC

A colloidal recirculation mill (BME 100LX,Shanghai Weiyu,China) and high-pressure homogenizer (D-3L,PhD-Tech,America)were used to reduce the dimension of the KBC.The cellulose gel of KBC was centrifuged,collected,and then milled by a colloidal recirculation mill for 20 min.To further reduce the dimension of the KBC membrane,the milled gel of the KBC was passed through a high-pressure homogenizer at 100 MPa for 3 cycles.Then the KBC membrane was made to pass through a rotary evaporator to concentrate the KBC.After that,the samples were freeze-dried and coded as HPH-KBC.

2.4 Characterization of the bacterial cellulose from KBC

The surface morphological structure of KBC and HPH-KBC was obtained by scan electron microscopy (SEM,EVO-LS10,Zeiss,Germany).Before observation,the freeze-dried KBC and HPH-KBC were coated with approximately 20 nm gold-palladium.All samples were examined with an accelerating voltage of 10 kV with a sputtered time of 30 s.The surface photomicrographs of KBC were taken at 10 000 × and the HPH-KBC at 30 000 ×.

The Fourier transformed infrared spectroscopy (FTIR) spectra of KBC and HPH-KBC were acquired using a Nicolet iS50 FTIR spectrometer (Thermo Nicolet,the United States).For each sample,the spectrum was collected over the range of 4 000 to 500 cm-1with an accumulation of 64 scans.

The KBC and HPH-KBC were investigated using an X-ray diffractometer (Bruker,D2 PHASER,the United States) with a Ni-filtered Cu-Kα (α=0.154 18 nm) at a voltage of 40 kV and a filament emission of 40 mA.The freeze-dried BC were scanned radiation in the 2θrange from 5° to 60° with 0.02° (2θ) step and 5 °/min scanning rate.

The crystallinity index (CrI) of the KBC and HPH-KBC was calculated according to the classical Segal et al.[26]method by the equation as follows:

whereI200is the maximum diffraction intensity of the lattice peak at 2θaround 22° andIamis the minimum diffraction intensity of the amorphous peak at 2θaround 18°.

A simultaneous thermal analyzer SDT-650 (TA Instruments Co.,the United States) with 99.99% of 100 mL/min nitrogen as the purge gas and 99.99% of 30 mL/min nitrogen as the protective gas was used to analyze the thermal stability of the KBC and HPH-KBC.With an empty aluminum pan as a reference,5-10 mg BC powder was weighted and sealed with a hydraulic device.After being equilibrated at 30 °C,the system was heated linearly to 800 °C with a 10 °C/min scanning rate.

2.5 Particle size distribution and contact angle measurement of KBC

The KBC and HPH-KBC’s particle size distribution was acquired using a nanoparticle potentiometer (Zetasizer Nano ZSE-03,Malvern Panalytical,China).Before measurement,the sample to be tested was diluted with deionized water to 1 g/L,and a 1.5 mL sample was added to the sample tank.

Contact angle measurement was applied to test the particle wettability of the KBC and HPH-KBC.Firstly,the suspension of different KBC was evenly spread over the glass and evaporated at 40 °C until completely dry.Then the contact angles of different samples were tested by a KRUSS contact angle measuring instrument(DSA100,Kruss GmbH,Germany).3 μL distilled water was dropped on the film’s surface with a spread of 2 μL/s,and the average value was calculated from data detected at three different positions.

2.6 Preparation of curcumin-loaded Pickering emulsion (CPE)

CPE was produced via a two-step homogenization pr°Cess.First,the curcumin-loaded oil was prepared according to the method of Marefati et al.[27],and then mixed with 0.4% (m/m) suspensions of the HPH-KBC at a ratio of 20:80 (V/V).After that,the mixture was further subjected to a high-shear mixer homogenization (IKA T18 digital ULTRA TURRAX homogenizer,IKA Inc,Germany) at 10 000 r/min for 3 min.Then the emulsion was passed through a highpressure homogenizer at 100 MPa for 3 times with an ice bath to cool down.Then the CPE was collected and stored in a refrigerator at 4 °C for further analysis.Fig.1 summarizes the CPE preparation pr°Cess.

Fig.1 Schematic diagram of curcumin-loaded Pickering emulsion.

2.7 Rheological measurements

Complete strain scan tests (strain range 0.01% -100%) were applied at the frequency ƒ of 1 Hz,using a rheometer (Discovery HR10,TA Instruments,the United States).The scanning results selected a strain value in the linear viscoelastic region following total frequency and shear stress scanning tests.The selection of strain value should be based on the structure of the sample and depend on the sample viscosity and gel state.

The strain value was determined according to the strain scanning test results,and the frequency variation range was 1-100 Hz.A fullfrequency scanning test was conducted to obtain the relationship between the energy modulus (storage modulusG’ and loss modulusG”) and the applied frequency of the emulsion.

The strain value was determined according to the strain scan test results,the frequency ƒ was 1 Hz,and the shear rateγvaried from 0.01 s-1to 1 000 s-1.The relationship between shear viscosityηand shear rateγwas obtained from flow sweep tests.

2.8 Microscopy observation of CPE

The fluorescence microscopy of the CPE was performed according to the method described by Yan et al.[14].Briefly,the olive oil containing curcumin could produce green fluorescence by itself at 465 nm excitation wavelength;therefore,there was no need to stain the olive oil further.The 0.4% (m/m) suspensions of the KBC(4 mg/mL) were stained by 1 mg/mL Congo red for 24 h.Then,a droplet of CPE was dropped on the surface of the glass slide and was applied to the glass.Then the slide was observed at the same view with a fluorescence microscope at 464 nm and 540 nm separately to get two pictures of curcumin-loaded oil and HPH-KBC.The two photos were further merged using the image pr°Cessing software(NIS Elements F.3.0) to illustrate the Pickering emulsion structure comprehensively.

The SEM observation was performed according to the previous method [28].The CPE was dipped in nitrogen gas for 10 min.Then dried Pickering emulsion was coated with gold and visualized using a Hitachi S-3000N (HITACHI,Japan).

2.9 Particle size distribution of CPE

Different influencing factors (pH and temperature) to the particle size distribution of the CPE were assessed in this study.The pH of the CPE was respectively adjusted to 2,4,6,8,10,and 12 by using 0.1 mol/L HCl/NaOH and letting them sit for 1 h at 25 °C.The CPE was put in different temperatures (0,20,40,60,80,and 100 °C) for 1 h and then waited for the diameter measurements.The average particle size of the CPE was determined by a laser particle analyzer(Mastersizer 3000,Malvern Instruments,UK),and the volume means diametersD[3,4]were recorded.The samples at different pH and temperatures were added to the distilled water flow system and then pumped through the optical chamber.Then in the optical chamber,the particle size was determined.The refractive index of samples and the continuous phase were separately set to 1.590 and 1.330.The obscuration was set between 10% and 20% .All emulsions were performed five times and washed in triplicate.

2.10 Evaluation of the stability of curcumin in CPE

pH stability: The CPE was adjusted to different pH (2,4,6,8,10,and 12,respectively) using 0.1 mol/L HCl/NaOH.Then the emulsion samples were stored in the dark for 1,3,5,and 7 h,and the retained curcumin content of CPE was determined.

Thermal stability: The CPE was stored at different temperatures (0,20,40,60,80,and 100 °C) for 1,3,5,and 7 h,and then the content of retained curcumin was determined.

Ultra-Violet stability: The CPE and the same concentration of curcumin methanol solution were put in a UVP EC3 Darkroom (EC3 310,UVP,America) with a 365 nm ultraviolet;samples were pipetted for further analysis at 1 h interval within 12 h.

Sunlight stability: The Pickering emulsion at the same concentration of curcumin methanol solution was put in sunlight;samples were pipetted further at 1 h interval within 72 h.

Storage stability: The Pickering emulsion and free curcumin at the same concentration of curcumin methanol solution were kept in the dark place at 4 °C for 15 days.

2.11 Determination of curcumin content

The preparation of free curcumin was performed by dissolving curcumin into the olive oil (1 mg curcumin per milliliter of olive oil).The curcumin content was determined by UV-vis spectrophotometry at 430 nm wavenumber after incubated at 80 °C (stirring for 10 min) and centrifuged at 12 000 r/min for 10 min.With the standard calibration curves of free curcumin,the concentration (μg/mL) of curcumin was determined,using the mathematical formulation shown as below:

Further,to determine the curcumin content encapsulated in olive oil,200 μL oil was mixed vigorously with 1 mL methanol.Then the mixture was centrifuged at 13 500 r/min for 15 min to separate the olive oil.After that,800 μL supernatant was collected and mixed with 200 μL methanol.The absorbance of the mixture was measured at 430 nm.The blank was prepared by substituting the olive oil with deionized water.

2.12 In vitro antioxidant activities

2.12.1 Iron ion chelating assay

Different concentrations (1,2,4,8,and 16 μg/mL) of the CPE was mixed with 0.2 mL Ferrozine (5 mmol/L),0.05 mL FeCl2(2 mmol/L),and 1.25 mL of deionized water.The mixture was incubated at 25 °C in a water bath for 20 min.Then the samples were centrifuged at 13 500 r/min for 10 min to remove the KBC and olive oil.The absorbance of the samples was read at 562 nm.The blank was prepared by substituting CPE with distilled water.The same series of concentrations of free curcumin were prepared.The Pickering emulsion without curcumin (PE) was prepared with the same dilution of CPE.The standard calibration curves were prepared using determined concentrations of EDTA-2Na.The calculation of the chelation capacity was calculated as EDTA-2Na·equivalent (μg/mL).The chelating capacity increases when the EDTA-2Na·equivalent increases.

2.12.2 Ferric reducing antioxidant power (FRAP)

At low pH,the antioxidant agent can reduce the orange Fe3+-TPTZ to blue Fe2+-TPTZ,and this compound can be read at 593 nm to determine the total antioxidant activities.Briefly,0.1 mL of different concentrations (1,2,4,8,and 16 μg/mL) of CPE were mixed with 3 mL FRAP which is a mixture of acetate buffer (300 mmol/L pH3.6),TPTZ (10 mmol/L) and FeCl3(20 mmol/L) with a ratio of 10:1:1 (V/V).Then the mixture was incubated at 37 °C in a water bath for 5 min.Then the samples were centrifuged at 13 500 r/min for 5 min to remove the KBC and olive oil.The absorbance of the samples was read at 593 nm.The blank was prepared by substituting CPE with water.The same series of concentrations of free curcumin were prepared.In addition,the PE was prepared with the same dilution of CPE.Different concentrations of FeSO4·7H2O were prepared to obtain the standard calibration curves.The calculation of the chelation capacity was expressed as FRAP value (μmol/L Fe2+).

2.12.3 ABTS+ radical scavenging ability

The ABTS+was achieved by mixing 7 mmol/L ABTS aqueous solution with 2.45 mmol/L K2S2O8solution with a ratio of 1:2 (V/V).Then the ABTS+solution was conserved in the dark over 16 h before use.The ABTS+solution was further diluted by ethanol to obtain a good absorbance at 734 nm.0.3 mL of different concentrations (1,2,4,8,and 16 μg/mL) of PEC were mixed with 1.2 mL diluted ABTS+solution for 20 min,and then the mixture was centrifuged at 13 500 r/min for 5 min.The supernatant was collected,and the absorbance was read at 734 nm.The same series of concentrations of free curcumin were prepared.In addition,the PE was prepared with the same dilution of curcumin-loaded Pickering emulsion.The control was prepared by substituting the samples with water.The following formula was used to calculate the capacity of the samples to scavenge the ABTS+:

whereAcontrolis the absorbance of the ABTS+solution with distilled water.Asampleis the absorbance of the ABTS+solution with different samples.

2.13 Statistical analysis

All experiments were conducted in triplicate.Significant differences were determined using one-way analysis of variance(ANOVA) by Duncan’s test atP<0.05.

3.Results and discussion

3.1 Structure and thermal stability of KBC and HPH-KBC

The morphology of the KBC and HPH-KBC were shown in Fig.2.The KBC had densely packed ultrafine fibrils interwoven in a threedimensional net structure,as recorded by SEM micrographs in Fig.2A,which was consistent with the results reported the BC fromAcetobacter xylinum[14],Komagataeibacter rhaeticus[29], andgluconacetobacter xylinus[30].The KBC hydrogels were dispersed and sheared into nanofibers by high-pressure homogenization.As shown in Figs.2C,the BC homogenized under high pressure presents a loose network structure,and some of the fibers have broken.In addition,the mean diameter calculation of KBC and HPH-KBC was determined by manually calculating 100 random fibers from the SEM image (Figs.2B and 2D).The fiber ribbon diameter of KBC was distributed in the diameter range at 10-120 nm,where its mean diameter was (60.97 ± 19.35) nm.However,the fiber ribbon diameter of HPH-KBC was distributed in the lower diameter range (10-40 nm),where its mean diameter was (19.11 ± 5.68) nm,which was similar to the nan°Cellulose from ginkgo seed shells using the high-pressure homogenization [31].

Fig.2 The morphological characterization of bacterial cellulose from fermented KBC (A) and diameter distribution of KBC (B),the morphological characterization of HPH-KBC (C) and diameter distribution of HPH-KBC (D)

The heating behavior of KBC and HPH-KBC were shown in Fig 3A.Thermogravimetric (TG) involved the loss of sample weight following increasing temperature in the form of programmed heating,and the differential scanning calorimetry (DSC) measured the heat energy absorbed or released by a material as a function of temperature or time [32].The TG curves of the KBC and HPH-KBC exhibited two main mass loss stages.The first loss stage of KBC was ascribed to the degradation of media and other impurities at around 200 °C,while the counterpart of HPH-KBC was at a low temperature around 50-100 °C for the losses of physically adsorbed water.The second loss stage for these two samples was similar and ascribed to the decomposition of polymer moieties at a higher temperature around 280-380 °C.It is worth noting that the weight retentions of KBC and HPH-KBC at 650 °C were respectively 22.43% and 16.27%,which was similar to the previous report [14].Meanwhile,two endothermic peaks were shown in DSC corresponding to a glass transition temperature and crystalline melting temperature of KBC and HPH-KBC,respectively [33].

The FTIR spectra of KBC and HPH-KBC were displayed in Fig.3B.Typical characteristic peaks ass°Ciated with the cellulose structure were found in the two samples.The absorption peaks at 3 340,3 342,and 3 232 cm-1were ass°Ciated with O-H stretching.In addition,a strong absorption peak at around 2 945,2 892,and 2 849 cm-1corresponded to the C-H bond of typical cellulose type-I [34].Moreover,the absorption peak at around 1 456 cm-1was ass°Ciated with symmetric CH2bending vibration,known as the‘crystallinity band’ [35].The other absorption peaks at around 1 161-999 cm-1were ascribed to carbohydrate monomers connected into a polymer with the -C°C and -CO stretching bands.Lastly,the peak at around 897 cm-1was antisymmetric out-of-phase ring stretching ofβ-glucosidic linkages between glucose units,which could be caused by the oxidization of 3% H2O2in the purified KBC (ass shown in Fig.1).It was worth noting that the additional peak at around 1 732 cm-1in HPH-KBC was assigned for the bending vibration of-COOH,indicating the oxidation of the pyranose ring [14].

Fig.3 Structural characterization of bacterial cellulose from fermented KBC and bacterial cellulose from HPH-KBC.DSC-TGA (A),FTIR (B),and XRD (C).

The XRD analysis of the KBC and HPH-KBC were presented in Fig.3C.In ordered crystal regions,four characteristic peaks 2θangles of approximately 14.7°,16.5°,22.6°,and 34.5° were found in XRD spectra which corresponded to the primary diffraction of (100),(010),(002) and (004) planes of native BC type I [12,36].According to the classical Segal method,the crystallinity index of the KBC and HPHKBC was 75.98% and 76.28%,respectively.

3.2 Particle size distribution and wettability of high-pressure homogenized cellulose

The particle size distribution of KBC and HPH-KBC were presented in Fig.4A.The size distribution of KBC was between 70-950 nm,while the HPH-KBC was 32-110 nm,which was lower with the bacterial cellulose nan°Crystals fromAcetobacter xylinumby sulfuric acid hydrolysis [14].This result indicated that repeated high-pressure homogenization could destroy the network structure of BC and result in the formation of smaller and more uniform nanofibers.

As the emulsion stabilizer,solid particles need to be partially infiltrated by the water phase and partially infiltrated by the oil phase,so they need certain wettability.The contact angle can describe the wettability of solid particles.The emulsion was most stable for solid particles with a contact angle close to 90o,and it was easy to form an O/W emulsion at 70°-86°[37].As shown in Fig.4B and 4C,the contact angle of KBC without high-pressure homogenization was(34.2 ± 2.5) °,while the contact angle of HPH-KBC was (72.7 ± 3.8) °.The increase of contact angle might be related to the hydrophobic domains formed after partial degradation of crystalline regions under the intense homogenization pressure.Ni et al.[31]reported similar results,that the contact angle of the nan°Cellulose from ginkgo seed shells increased from 41.5° to 65.6° after multiple highpressure homogenization.In addition,the hydrophobicity of cellulose nanofibers was also improved by high ultrasound power treatment,according to a previous report [38],suggesting that physical methods could effectively improve the hydrophobicity of cellulose and then cause the increased contact angle.The hydrophobicity was improved by high-pressure homogenization,and the treated KBC was more suitable for stabilizing the Pickering emulsion.

3.3 Morphology of Pickering emulsion

The structure of the Pickering emulsion was made into three layers: the curcumin-loaded oil was encapsulated in KBC to form two layers.Then the KBC encapsulating olive oil was dispersed in the water phase.To verify the structure of the Pickering emulsion and the absorption of the KBC at the oil/water interface in the Pickering emulsion,the fluorescent microscope,and scanning electron microscopy were used to observe the microstructure of the Pickering emulsion.

As shown in Fig.5A and 5B,the green droplets representing the curcumin-loaded droplets of olive oil were dispersed in the continuous phase under the excitation wavelength of 465 nm.In contrast,bacterial cellulose stained with Congo red could fluoresce a red light at an excitation wavelength of 540 nm.In Fig.5B,the red hollow circle represented the mechanical barrier formed outside the curcumin-loaded olive oil because of the absorption of KBC on the oil/water interface.To further explore the microstructure of the droplet of Pickering emulsion,Fig.5A and Fig.5B were merged.The green droplets representing the curcumin-loaded olive oil were well encapsulated in the red hollow circle,which meant a good encapsulation of curcumin-loaded oil (Fig.5C).The droplets of Pickering emulsion were well separated by KBC,which formed a mechanical barrier outside the oil droplet.The formed mechanical barrier protected the Pickering emulsion from coalescence,fl°Cculation,and gravitational separation.This further improved the stability of the Pickering emulsion and the curcumin.Some bigger droplets might have been formed due to the application of the Pickering emulsion on the glass,which deconstructed the structure of the Pickering emulsion.However,most of the Pickering emulsion particle size was a dozen micrometer,which was confirmed by the mean volume of the determined particle size.The result aligned to the report of Yan et al.[14],where the emulsion droplets were shown to have a good spherical shape.The emulsion was further confirmed to be an oil-in-water emulsion with good stability.

Fig.5 The fluorescent images of curcumin-loaded oil (A),bacterial cellulose from KBC by high pressure homogenization (B) and the curcumin-loaded Pickering emulsion (C).The SEM of the curcumin-loaded Pickering emulsion (D)and the bridging phenomenon between droplets (E).

The SEM of the Pickering emulsion and the bridging phenomena among the droplets of the Pickering emulsion were presented in Figs.5D and 5E.The droplets of the Pickering emulsion were uniformly dispersed in the continuous phase.The purified KBC was filiform and coated with the curcumin-loaded oil to form a mechanical and porous barrier.The droplet diameter was about a dozen micrometres,which agreed with the mean volume particle size determination.The two droplets of Pickering emulsion were connected by KBC to form the bridging phenomena (Red frame).This phenomenon could have resulted from the entanglement of the different lengths of the purified KBC [28].

Based on these results,the HPH-KBC could be used to produce a Pickering emulsion.The purified cellulose was coated outside the olive oil to form the mechanical barrier to protect the Pickering emulsion from being destroyed by coalescence,fl°Cculation,and gravitational separation.Further,to verify whether the curcuminloaded Pickering emulsion produced by cellulose could be a stable delivery system,the particle size distribution of Pickering emulsion and the stability of curcumin encapsulated in Pickering emulsion were investigated.

3.4 Rheological properties of emulsions

The energy storage modulusG’ refers to the amount of energy stored in the system when deformation °Ccurs while the loss modulusG” is the amount of energy consumed in the form of viscosity loss when deformation °Ccurs.Fig.4D shows the variation of the energy storage modulusG’ and the loss modulusG” of emulsion with strain.The emulsion’sG’ andG” was stable in the strain range of 0.01% -1%,indicating that the PE and CPE sample structures were not damaged within this linear viscoelastic zone.Thus,the strain value of 0.1% in the linear viscoelastic region was selected for the oscillation frequency and flow sweep tests.

BothG’ andG” of the emulsion under 0.1% strain showed increasing patterns as a function of frequency,as shown in Fig.4E.Within the applied frequency range,theG’ values of PE and CPE samples were higher than theG” values and exhibited a typical gellike characteristic.Interestingly,theG’ andG” of the CPE emulsion were significantly higher than those of KBC,which implies that the addition of curcumin increased the energy in the emulsion system.The result further signifies that the curcumin increased the stability and structural complexity of cross-linked CPE emulsion.

The degree of aggregation between droplets and the shear stability of the PE and CPE emulsion system was shown in the viscosity curves in Fig.4F.PE and CPE samples exhibited high viscosities at a low shear rate (0.011 s-1).With the increase in the shear rate,the viscosity of both samples showed downward trends.With the further increase of the shear rate (100-1 000 s-1),the downward trend of the emulsion viscosity gradually slowed down,which was defined as a “shear thinning” phenomenon [39].Both PE and CPE samples showed the characteristics of a typical non-Newtonian fluid,which explains why the increment of shear rate overcame the disordered Brownian motion of the droplets in PE and CPE,resulting in the orderly flow of the droplets and decreased viscosities [39].

3.5 Particle size distribution of Pickering emulsion

Due to the possible ionization of the side functional groups of biopolymers,pH is always considered the main factor determining its complexation [40].For this reason,the effect of different pH (2,4,6,8,10,and 12) on the CPE was studied.There was no significant difference among the mean volume particle size of CPE samples at pH 2,pH 4,and pH 8 (Fig.6A),while all of them were lower than that of CPE at pH 6.This might be due to the isoelectric point of the droplets,which was close to pH 6.At the isoelectric point of pH 6,the molecule had relatively weak electrostatic repulsion [41],which meant less power was needed to cross the energy barrier.

Fig.6 Effects of pH (A) and temperature (B) on the mean volume particle size of the curcumin-loaded Pickering emulsion.

Meanwhile,the energy barrier was the main force that kept the droplets from aggregating.Hence the reduction in this force resulted in the coalescing of the droplets.Furthermore,the decrease in the average particle of the Pickering emulsion size at pH 8 was due to the increased electrostatic repulsion.Then the increase of the particle size at pH 10 and 12 was due to the excess negative charge density generated by the hydroxyl group.The negative charge species promoted swelling in the polymeric network and subsequent aggregation of smaller particles that collided to form larger particles.The same phenomenon was observed by Yan et al.[14].

The role of temperature in the particle size of CPE was also evaluated.It was found that temperature played a minor role in the particle size variation of the CPE system.As shown in Fig.6B,the mean volume particle size of the CPE kept stable with the increase in temperature of 0-80 °C,i.e.,there was no significant difference(P>0.05) among these samples treated at 0-80 °C in the mean volume particle size,and only CPE treated at 100 °C showed a significant difference.This meant that a wide range of temperatures below 100 °C seldom altered the particle size of CPE,which was consistent with the finding of Zhai et al.[42].

3.6 Stability of curcumin

3.6.1 pH stability

The percentage of retained curcumin in the CPE and free curcumin under different pH (2,4,6,8,10,and 12) for 1,3,5,7 h was presented in Fig 7A.The curcumin content of CPE decreased slowly to around 75% from pH 2-6 within 7 h,while the free curcumin to around 55% .The curcumin content of CPE and free curcumin in the 7thh were (59.68 ± 1.31)% and (32.32 ± 1.15)%,respectively.The stability of curcumin in CPE was remarkably higher than that of free curcumin from pH 2-8,which attribute to the mechanic barrier formed by KBC.However,the stability of curcumin decreased sharply at pH 10 and pH 12 both in CPE and free curcumin,indicating the KBC had no protective effect on curcumin.This is mainly due to the decreased stability of BC-based emulsion at pH exceeding 8.15 [14]and the rapid degradation of curcumin under alkaline conditions [43].

3.6.2 Temperature stability

The percentage of the retained content of the curcumin in the CPE and free curcumin under different temperatures (0,20,40,60,80,100 °C) after 1,3,5,7 h is presented in Fig.7B.The retained curcumin in the CPE and free curcumin at 0,20,40,and 60 °C did not change significantly within 1h.The retained curcumin in the CPE was about 85% after 7 h in 0 °C to 40 °C and had little difference between CPE and free curcumin in 0 °C.However,The retained curcumin content of CPE was higher than the free curcumin in 20 °C to 40 °C,which attributed to the protective effect of KBC on curcumin.After 7 h at 80 °C and 100 °C,the whole curcumin was almost wholly degraded quickly,indicating the KBC had no protective effect on curcumin at high temperatures.

Fig.7 The retained content of curcumin in the curcumin-loaded Pickering emulsion and free curcumin under different pH(A) and different temperature (B),The retained content of curcumin in the curcumin-loaded Pickering emulsion and free curcumin under 365 nm Ultra Violet (C) and sunlight at a function of time (D).The retained content of curcumin in the curcumin-loaded Pickering emulsion and free curcumin at a function of time (E).

3.6.3 Ultra violet stability

The retained content of the curcumin in the Pickering emulsion under UV-365 nm within 12 h is demonstrated in Fig.7C.In general,the content of the retained curcumin decreased in the CPE and in free curcumin solution with the increase in time.However,curcumin encapsulated in the CPE decreased slower than the free curcumin under UV-365 nm.After 6 h under UV-365 nm,the retained content of the free curcumin decreased drastically from 100.00% to(6.96 ± 0.95)%,which represented almost a total degradation of the curcumin.While for CPE,the retained curcumin after 6 h was about(64.86 ± 3.88)% .Interestingly,for 50% curcumin degradation to°Ccur,the curcumin encapsulated in Pickering emulsion needed about 8.5 h,while the curcumin in the free curcumin solution needed only 2.5 h.

3.6.4 Sunlight stability

Fig.7D represented the amount of retained curcumin encapsulated in CPE and free curcumin solution under sunlight within 72 h.A similar phenomenon with the UV-365 nm was observed.This implies that the retained curcumin content decreased with the increase in time in the CPE and the free curcumin solution.However,the curcumin encapsulated in the CPE decreased slower than the free curcumin.After 72 h under sunlight,the curcumin in free curcumin solution had just (1.94 ± 0.58)% left,while the retained curcumin was(24.68 ± 1.92)% for emulsion.The curcumin wrapped in the CPE needed about 40 h before it could 50% of the curcumin could be degraded under sunlight compared to the curcumin dissolved in methanol which needed 25 h to get to the same level of degradation.In general,the results signified that the Pickering emulsion encapsulation of the curcumin significantly improved the stability.

These results displayed that the mean volume particle size of Pickering emulsion produced by KBC was uniform with a wide temperature range.However,the pH significantly influenced the mean volume particle size due to the electrostatic repulsion and the activation of hydroxyl groups.Conclusively,the curcumin encapsulated in CPE was more stable in an acid environment and at low temperatures (0-20 °C).Furthermore,curcumin in CPE had more excellent stability under sunlight and UV-365 nm than curcumin in solution.Therefore,to further verify whether the Pickering emulsion has an inhibitory effect on the functional characteristics of the curcumin produced encapsulated in the KBC,the antioxidant activity of the curcumin was examined.

3.6.5 Storage stability

The percentage of the retained content of the curcumin in the CPE and free curcumin stored in 4 °C dark condition as a function of time was displayed in Fig.7E.The degradation rate of curcumin in CPE was significantly lower than the free curcumin,and the curcumin encapsulated in the CPE exhibited better stability with less than 50% degraded after 15 days’ storage,which was superior to the curcumin-loaded Pickering emulsions based on glycated proteins and chitooligosaccharides [44].This result indicated curcumin encapsulated in the CPE exhibited improved stability and high retention.

3.7 In vitro antioxidant activities

The antioxidant activity of CPE,Pickering emulsion (without curcumin),and free curcumin was evaluated using metal ion chelating of curcumin and FRAP assay.Different concentrations were carried out to find whether the curcumin encapsulated in the Pickering emulsion could retain its antioxidant activity.

The metal ion chelating capacity of the different samples was presented in Fig.8A.It was observed that the metal ion chelating capacity increased with the increased concentration of curcumin.The final concentration of Pickering emulsion with curcumin was 32 μg/mL.It was noted that the Pickering emulsion encapsulated the curcumin with a low concentration (1 μg/mL) of curcumin significantly enhanced the metal ion chelating capacity compared to the free curcumin.In contrast,the metal ion chelating capacity of Pickering emulsion without curcumin and the free curcumin was equal or even lower than curcumin-loaded Pickering emulsion.It meant that the curcumin encapsulated in the Pickering emulsion retained its antioxidant activity even at a low concentration.

Similar trends were observed in Fig.8B,the increased curcumin concentration in the samples increased the FRAP.The FRAP of the CPE at 8 μg/mL was more substantial than the combined reducing antioxidant power of the Pickering emulsion without curcumin and the free curcumin.The result signified that the KBC used as a mechanical barrier did not reduce the functional activity;instead,it improved the antioxidant activity of the curcumin.Fig.8C schematically illustrates the increase of ABTS radical scavenging activity (%) of CPE,PE,and free curcumin with increasing concentrations.

Fig.8 The Metal ion chelating capacity (A),ferric ion reducing antioxidant power (B) and ABTS radical scavenging ability (C) of the curcumin-loaded Pickering emulsion,Pickering emulsion and free curcumin.

The study data thus show that the curcumin encapsulated in the Pickering emulsion retains its antioxidant activity even at a low concentration.Its antioxidant activity was higher than free curcumin.A similar phenomenon has been reported by other researchers [45,46].The observed increase in the antioxidant activity of the curcumin encapsulated in CPE could have been due to the improved solubility of the micellar curcumin compared to free curcumin [47].

4.Conclusion

Bacterial cellulose synthesized from KBC was used to produce curcumin-loaded Pickering emulsion.The homogenized KBC formed a mechanical barrier outside the emulsion droplet,which protected the Pickering emulsion from coalescence,fl°Cculation,and gravitational separation.Curcumin-load Pickering emulsion had better stability under a wide range of temperatures,low pH,sunlight,and UV-365 nm than free curcumin.Furthermore,the curcumin-load Pickering emulsion had the highestin vitroantioxidant activity than the free curcumin and Pickering emulsion without curcumin.The study demonstrated that the KBC could be an excellent material for preparing a stable Pickering emulsion system to improve curcumin stability and antioxidant activity.

Conflict of interest

The authors declare there is no conflict of interest.

Acknowledgments

This project was supported by the earmarked fund for the Priority Academic Program Development of Jiangsu Higher Education Institutions (080-820830).In addition,We want to thank Ms.Sha Yang from College of Food Science&Technology,Nanjing Agricultural University for providing writing assistance.