Ultrasonic treatment of α-chitin regenerated from a NaOH/urea solvent with tunable capacity for stabilization of oil in water emulsion

Abstract

α-chitin cannot be dispersed directly with ultrasonic treatment because of the strong intermolecular forces. However, in the current work, when α-chitin was first regenerated from a NaOH/urea solvent, the regenerated α-chitin can then be easily dispersed with ultrasonic treatment under weak acidic conditions. The morphology, size, zeta potential, structure and optical transmittance of the dispersed α-chitin solution were characterized by TEM, DLS, XRD and UV spectroscopy respectively. The dispersed chitin was then used for stabilization of oil in water emulsion. It was found that the emulsifying capacity of regenerated chitin was tunable by ultrasonic treatment. The dispersed chitin exhibited excellent emulsifying ability and the resulting emulsion was stable after long-term storage. In short, in this paper, a novel way to prepare dispersed chitin with excellent emulsifying ability was presented, and its emulsifying properties were further evaluated.

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1. Introduction

Research on natural, safe and highly effective emulsifiers accords with the practical requirements of food manufacturing, and is also a positive response to the highly concerning public issue of food safety.¹ Recently, particle stabilizers have aroused the interests of scientists because of their excellent properties, such as perfect interfacial stability and wide range of application.² Particles derived from natural polymers that can be used to prepare food grade Pickering emulsions consist of chitin nanocrystals,³ cellulose nanocrystals,⁴ hydrophobic modified starch,⁵ soybean protein nanoparticles,⁶ whey protein nanoparticles⁷ and zein colloidal particles.⁸ However, limitations of these particles, such as low yield in the preparation process,⁹ low emulsifying capacity,⁵ sensitivity to environmental stress (pH, temperature) hindered their practical application.⁸ In this context, research on new polymeric particles to overcome the above shortcomings is of great significance.

Chitin is the second most abundant polymer on earth after cellulose and derived from food processing byproducts such as shrimp and crab shells.¹⁰ But the strong intermolecular forces make it difficult to dissolve in common solvents.¹¹ Therefore, there is little research based on chitin in the past though its deacetylated product, chitosan has been given attention by scientists around the world. New green solvent developed by professor Zhang containing NaOH/urea provided a new option for the utilization of chitin and chitin materials based on this solvent is considered to be a hot scientific spot.¹² However, as one kind of natural and eatable polymer with a wide range of functionalities such as immune modulator and anti-inflammatory agent,¹³ there is little research on this polymer as food ingredients. Though chitin nanocrystal, prepared by acid hydrolysis to remove the amorphous region, has been developed as a kind of emulsifier for stabilization of oil in water emulsion.³ The yield of chitin nanocrystal by this method is only 55% as the high temperature and strong acid condition destroyed most part of crystalline region at the same time, which lead to the decrease in yield.⁹ Usually, chitin nanofiber can be obtained by high pressure homogenization of chitin under weak acidic condition, but relatively more energy input is required for this method.¹¹ Recently, chitin fiber was obtained through dissolution and regeneration from concentrated phosphoric acid,¹⁴ but large amount of phosphoric acid and long time were required to prepare chitin nanofiber. Hence improvement of these methods for the preparation of chitin derivatives with particular properties is necessary for efficient utilization of chitin.

Therefore, in this study, α-chitin was first dissolved and regenerated from a cost saving and green solvent containing NaOH/urea. Then the dispersed chitin was obtained through ultrasonic treatment of regenerated α-chitin under weak acidic condition. The yield of dispersed chitin in this process is as high as 90%. The structure and characteristics of the dispersed chitin were characterized. Then regenerated chitin containing crystalline region and amorphous region was researched as a new kind of emulsifier for stabilization of oil in water emulsion. It was found that the dispersed chitin exhibited excellent emulsifying ability and the emulsifying capacity of regenerated chitin can be adjusted with ultrasonic time. Moreover, the obtained emulsion was stable after long time storage. Therefore, the regenerated chitin obtained by this economic and eco-friendly method turned out to be a kind of highly efficient emulsifier.

2. Materials and methods

2.1. Materials

Chitin powder was purchased from Golden-Shell Biochemical Co. Ltd (Zhejiang, China) with molecular weight (M_w) of 4.0 × 10⁵ Da. Soybean oil (the Arawana brand, made in 2014) was obtained from the local supermarket in Wuhan without further purification. All other common chemical regents were of analytical grade and used without further purification.

2.2. Dissolution of chitin

Dissolution of chitin was carried out according to our previous study with slight modification.¹⁵ Briefly, 3 g chitin powder was added into 100 g mixture of NaOH, urea and distilled water in the ratio of 11 : 4 : 85 by weight. Subsequently, the mixture was frozen at −30 °C for 4 h, and then vigorously stirred at room temperature (25 °C). The freezing/thawing cycle was repeated twice to obtain a transparent chitin solution (3 wt%). The resultant viscous chitin solution was centrifugated at 6000 rpm under 0 °C for 10 min to eliminate the undissolved part.

2.3. Regeneration of chitin

The dissolved chitin was regenerated by adding 10 times (v/v) of deionized water under vigorous stirring, followed by precipitation and separation by centrifugation at 5000 rpm for 10 min. NaOH and urea from the precipitated chitin were removed by further washing for 10 times with deionized water. 5 wt% of regenerated chitin (RC) was stored at 4 °C until further use.

2.4. Dispersion of regenerated chitin (RC) in deionized water

12 g of the above regenerated chitin was added into 108 g deionized water in a 150 mL glass bottle, and then the pH was adjusted to desired value with HCl and NaOH. Then, the mixture was ultrasonically treated using a 2 mm probe at 90% of amplitude (a repeating cycle of 0.5 s ultrasonic treatment and 0.5 s pauses) with an output power of 600 W for desired time. Regenerated chitin ultrasonically treated under pH 3 for 10 min, 20 min, 40 min, 60 min were coded as RC10, RC20, RC40, and RC60 respectively.

2.5. Characterization of the ultrasonically treated regenerated chitin

The dispersed chitin particles were analyzed using JEOL 1010 transmission electron microscope (TEM). Samples were prepared by diluting each CNC sample with deionized water. A carbon-coated and glow-discharged copper grid was immersed into the diluted chitin dispersion and taken out. The grid was then placed on a piece of a filter paper for the removal of excessive water and dried at room temperature until analysis. Grids were analyzed at 100 kV under the standard conditions.

The particle size (z-average diameter) and ζ-potential of the regenerated chitin were determined using the dynamic light scattering (DLS) technique (Malvern Instruments Ltd, Worcestershire, U.K.). Regenerated chitin (0.5 wt%) was shaken severely to be evenly dispersed before determination. All measurements were carried out at 25 °C in triplicate.

The crystallinity of the dispersed chitin after freeze drying was analyzed using wide-angle X-ray diffraction (WAXD). Measurements were conducted on a Siemens D5000 diffractometer (Germany) equipped with a Cu Kα radiation source (λ = 0.1542 nm). Scans were taken over the 2θ range from 5° to 30° at a scanning speed of 2° min⁻¹. Crystallinity of the sample was determined by plotting the peak baseline on the diffractogram and calculating the area using the software (Jade6.0). The area above and under the curve referred to the crystalline domains and the amorphous regions, respectively. Ratio of upper area to total area was taken as the relative crystallinity: the crystallinity index (%) was calculated by the following equation

Crystallinity index (%) = area under the peaks/total curve area × 100.¹⁶

For Fourier transform infrared spectroscopy (FTIR) tests, regenerated chitin was grinded together with potassium bromide (weight ratio of 1 : 50) and pressed into disks for scanning with a Nexus 470 spectrometer (Nicolet, USA). Samples were scanned at 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹.

Optical transmittance of diluted chitin suspension (0.1 wt%) was measured with a Hach DR 2800 spectrophotometer (U.S.A.). Measurements were conducted for two times at wavelength ranging from 400 to 800 nm and the results were averaged.

The interfacial tension between the two phase (water and soybean oil) was measured using the Kruss K100 (Kruss, Germany) at 25 °C. The interfacial tension between the aqueous phase (pure water, regenerated chitin) and oil phase (soybean oil) was measured with the Wilhelmy plate method.¹⁷ Firstly, the Wilhelmy plate, made of platinum with a length, width and thickness of 19.9 mm, 10 mm and 0.2 mm, respectively was immersed in 20 g of aqueous phase to a depth of 3 mm under a surface detection speed of 15 mm min⁻¹. Subsequently, 50 g of the oil was carefully pipetted over the aqueous phase to create an interface between the aqueous phase and oil phase. The test was conducted over 2500 s and the temperature was maintained at 25 °C throughout the test.

2.6. Preparation and characterization of oil in water emulsion stabilized by regenerated chitin

16 mL of dispersed chitin (0.5 wt%) was taken in a 50 mL glass beaker. Then, 4 mL of soybean oil was mixed with the above chitin dispersion and emulsified using a high speed homogenizer (IKA T18, IKA-Werke GmbH & Co., KG, Germany) at 11 000 rpm for 2 min with 20 s as intervals. 10 mL of the prepared emulsion was placed in a sealed glass test tube. Storage stability of the emulsion was evaluated by determining the emulsion volume after storage for 12 h, 3 d, 10 d and 30 d at room temperature (25 °C). At the same time, another 10 mL of the emulsion was placed in a colorimetric tube, and the photographs were taken after storage at room temperature for 30 days with a digital camera (Digital Ixus 115 HS, Canon, Japan).

The morphological images of the O/W emulsion were captured by optical microscopy (Nikon 80i, Japan). A drop of emulsion was diluted with water on a microscopic slide and carefully covered with a cover slip. The size of the emulsion was determined at 25 °C with a Mastersizer APA2000 (Malvern, England). For each measurement, a given volume of emulsion was dropped in 1000 mL of distilled water with the concentration of 2–10% (v/v). Measurements were carried out for three times and the results were averaged. All tests were conducted at room temperature. Distilled water was used as the dispersion medium. The stirring rate was 600 rpm and the speed of circulation pump was 3000 rpm.

Rheological properties of the oil in water emulsion stabilized by regenerated chitin were performed at 25 °C using a controlled-stress AR2000ex rheometer (TA instrument Co. Ltd, UK) (diameter of 40 mm, gap height of 1000 mm). For the steady shear measurements, the shear rate was increased from 0.1 s⁻¹ to 10 s⁻¹, and the apparent viscosity (η) was recorded correspondingly. To obtain the linear viscoelastic region, oscillation strain sweep experiments were performed in the oscillation strain range from 0.01 to 10% (frequency, 1 Hz; temperature, 25 °C). For the dynamic oscillation measurements, the frequency sweep was conducted at 25 °C with frequency ranging from 1 to 10 Hz (all of the measurements were conducted within the linear viscoelastic region) and the elastic (G′) and loss (G′′) were recorded. All the determination were conducted on separate samples at least in duplicate.

3. Results and discussion

3.1. Characterization of the dispersed chitin

It can be seen from Scheme 1 that the original α-chitin could not be dispersed with ultrasonic treatment at any pH because chitin molecules are tightly bound to each other through an endless number of hydrogen bonds. However, β-chitin can be easily dispersed with ultrasonic treatment within short time because of the much weaker intermolecular forces.¹⁸ While α-chitin deserves more research work as its output is far larger than that of β-chitin. Interestingly, stable chitin dispersion can be easily obtained when α-chitin was first regenerated from the NaOH/urea solvent, and then ultrasonically treated under weak acidic condition. More importantly, regenerated α-chitin subjected to centrifugation and drying process can also be dispersed easily with ultrasonic treatment under pH ≤ 4. This phenomenon is different from previous reports that presented cellulose from mangosteen rind subjected to drying treatment could not be microfibrillated.¹⁹ Such particular characteristics of chitin made it convenient for fabrication of dispersed chitin on a large scale instantly. This is because the intermolecular hydrogen bonds of α-chitin after the regeneration process were weakened²⁰ and less energy input was enough to break the intermolecular forces of regenerated α-chitin. Moreover, the regenerated chitin after ultrasonic treatment under pH ≤ 4 was stable without any visible clumps or precipitate after standing for 24 h, while that under higher pH precipitated out. The zeta potential of regenerated chitin were shown in Fig. 4a, and it can be seen that α-chitin carried more positive charge under lower pH, leading to stronger repulsive forces among chitin molecules, which further contributed to the higher stability of chitin dispersion. Therefore, pH 3–4 is necessary to prepare relatively stable regenerated chitin/water dispersion. This process is very simple where no toxic chemicals were used, no complicated procedures were required and no much energy was consumed as that much needed in the preparation course of chitin nanofiber using high pressure homogenization.¹¹ Therefore, this process can be called as a cost saving and green process. The yield of dispersed chitin in this process is as high as 90%, which is higher than the yield of chitin nanocrystal (55%)⁹ because high temperature, strong acid condition used to remove the amorphous region destroyed the crystalline region at the same time in the preparation course of chitin nanocrystal.

Scheme 1 Representation of conversion of α-chitin into regenerated chitin (RC) and depiction of conditions suitable for dispersion of RC in deionized water.

Fig. 1 TEM images and size distribution of RC10 (a), SRC20 (b), RC40 (c) and RC60 (d).

Fig. 2 XRD (a) and FTIR (b) spectra of regenerated chitin.

Fig. 3 Optical transmittance of ultrasonically treated regenerated chitin (0.1 wt%).

Fig. 4 (a) Zeta potential of regenerated chitin (0.5 wt%) under different pH (b) interfacial tension between water and soybean oil as a function of ultrasonically treated regenerated chitin (0.5 wt%).

From the TEM image of dispersed chitin in Fig. 1, it can be seen that the shape of dispersed chitin was irregular and the size of the dispersed chitin was not even. For the regenerated chitin after ultrasonic treatment for 60 min (RC60), smaller particles were in the majority, but there were still large particles. The average size of RC10, RC20, RC40 and RC60 were 1183 nm, 892 nm, 670.9 nm and 640.6 nm with a PDI of 0.253, 0.347, 0.368 and 0.305 respectively. The dispersed chitin became relatively smaller with increasing of ultrasonic time on the whole. This can be understood that more energy input breaks regenerated chitin into much smaller particles. XRD of dispersed chitin was shown in Fig. 2a. Compared with the original regenerated chitin, the peak of ultrasonically treated regenerated chitin at 2θ = 12.8°, 26.5° gradually became weaker with increasing of ultrasonic time, while other peaks at 2θ = 9.3°, 19.2° remained almost unchanged.¹² Therefore, it can be easily inferred that the crystalline degree of chitin gradually decreased with increasing of ultrasonic time taking into the consideration that the crystalline degree can be obtained by calculating ratio of area for the crystallization peak and that of the total area.¹⁶ This indicated that the intermolecular hydrogen bonds of regenerated chitin were further broken, resulting in the damage of crystal structure during the ultrasonic process. Similar results were also found for the isolation of microfibrillated cellulose from sugarcane bagasse²¹ and prickly pear fruit peels²² with high pressure homogenization treatment. It can be seen from Fig. 2b that the FTIR spectra of the original regenerated α-chitin and the regenerated α-chitin ultrasonically treated for 60 min (RC60) are nearly identical, which means that the chemical structure of the chitin was not changed in the ultrasonic process.

The fact that high optical transmittance from dilute (0.1 wt%) chitin suspension (Fig. 3) were achieved at visible light wavelength ranging from 400 to 800 nm also confirmed the presence of small particles.²³ Moreover, the transmittance gradually became higher with increasing of ultrasonic time, which further confirmed that smaller chitin particles were obtained after ultrasonic treatment for longer time, and this was consistent with TEM results. Additionally, these results also suggested that larger fractions were present since the transmission was not yet above 90%, and this can also be confirmed by TEM in Fig. 1.

Fig. 4b presented the interfacial tension between water and soybean oil as a function of regenerated chitin. It can be seen that the interfacial tension of all system decreased with increasing of time and the presence of chitin particles lowered the interfacial tension values between water and soybean oil significantly. Moreover, the interfacial tension values obtained at equilibrium state were lower for regenerated chitin ultrasonically treated for longer time, indicating that emulsifying ability of smaller particles will be superior. This can be explained that smaller particles are easier to be adsorbed to the oil–water interface with a much faster speed and the theoretical maximum adsorbed amount of particles for a close-packed monolayer in the oil–water interface will be larger if the particles are smaller,²⁴ thus lowering the interfacial tension to a greater extent.

3.2. Evaluation of the emulsion

Although chitin nanocrystal has long been proved to be able to stabilize oil in water emulsion,³ it remains unknown whether regenerated chitin containing both crystalline region and amorphous region can stabilize oil in water emulsion. In this study, it was proved that the original regenerated chitin cannot stabilize emulsion because of the large particle size, while the ultrasonically treated regenerated chitin was a kind of excellent emulsifier. Fig. 5 showed the morphology of emulsion stabilized by RC10, RC20, RC40 and RC60 with average size of 46.8 μm, 31.6 μm, 23.2 μm and 19.1 μm respectively. Obviously, it was found that the size of emulsion prepared with regenerated chitin after ultrasonic treatment for longer time was smaller than that of the rest. At the same time, it can also be seen from Fig. 6 that the volume of emulsion stabilized by RC, RC10, RC20, RC40 and RC60 were 0 mL, 6 mL, 6.8 mL, 7 mL, 7.2 mL and 7.2 mL respectively. So ultrasonic treatment was necessary to prepare regenerated chitin particles with excellent emulsifying properties and the emulsifying capacity was larger for chitin with much smaller size. This is in agreement with the report of other researchers that particles with smaller size possess much better emulsifying ability.¹⁹ This further means that the emulsifying capacity of the regenerated chitin was tunable through the adjustment of ultrasonic time. Moreover, the emulsion volume changed little with increasing of storage time, suggesting excellent emulsion stability. The unique amphiphilic structure of chitin made it suitable for stabilization of oil in water emulsion directly without any chemical modification,¹³ while this process could not be achieved by other kinds of polysaccharide such as starch and konjac. Furthermore, the emulsifying capacity of regenerated chitin was even larger than that of octenyl succinic anhydride modified starch under the same condition.^5,25 This indicated that natural chitin after ultrasonic treatment was one kind of excellent emulsifier.

Fig. 5 Typical optical micrographs and size distribution of emulsion stabilized with ultrasonically treated regenerated chitin (0.5 wt%).

Fig. 6 Micrographs and volume of the O/W emulsion stabilized with ultrasonically treated regenerated chitin (0.5 wt%) after storage for 30 days.

pH has a strong effect on the surface charge and stability of colloidal particles, which will significantly affect the stability of emulsion. The influence of pH on the stability of emulsion was shown in Fig. 7, and it can be seen that the emulsion was stable in a wide range of pH from 3 to 9, indicating excellent pH stability. This further indicated that emulsion stabilized by insoluble polysaccharide can withstand extreme environment stress compared with that of other polymer particles such as protein.⁸ The emulsion volume is smaller and the emulsion size is larger in both the acidic and alkaline condition, while the emulsion was the most stable in neutral condition, with the smallest emulsion size and largest emulsion volume. The pK of chitin is reported to be around 6.3,³ so as the pH reached lower values, e.g. pH 3.0, or higher values e.g. pH 9.0, the chitin will be positively or negatively charged. The electrostatic repulsive forces at these pH levels and a low chitin concentration (0.5 wt%) would prevent the formation of a strong network. In contrast, when the pH of the dispersion is around 6.3, the repulsive electrostatic forces are minimized and the dispersed chitin could then easily aggregate and form a gel. This will further stabilize the emulsion towards creaming, which will result in better stability of the emulsion.

Fig. 7 Volume and average size of the O/W emulsion prepared with RC20 (0.5 wt%) after storage for 30 days.

The rheological properties of the Pickering emulsion were shown in Fig. 8. Pronounced shear-thinning behavior of the emulsion can be observed at shear rates higher than 0.1 s⁻¹. In all cases, shear viscosity of emulsion was high at low shear rate, and then progressively decreased with increasing of shear rate. This phenomenon is a typical flow behavior of oil-in-water emulsion,²⁶ which corresponds to the deflocculation of droplets upon shearing. But there was not obvious viscosity difference among emulsions stabilized by regenerated chitin treated for different time. Frequency sweep was conducted with a constant strain of 0.1%, which was within the linear viscoelastic region of emulsions. It was found that G′ was considerably higher than G′′ at any test frequency, indicating a predominantly elastic gel-like behavior.²⁷ Furthermore, G′ and G′′ gradually increased with frequency, suggesting the gel-like network might be maintained through non-covalent physical cross-links in nature.²⁸ Also there was not significant modulus difference between emulsions stabilized by regenerated chitin after different treatment. Similar observation was also reported in gel-like O/W emulsion stabilized by phytosterol colloidal particles²⁶ and hydrophobic starch particles.²⁵

Fig. 8 (a) Shear-rate dependence of viscosity and (b) dynamic frequency sweep of O/W emulsions stabilized by ultrasonically treated regenerated chitin (0.5 wt%).

4. Conclusion

The dissolution and regeneration process from NaOH/urea solvent weakened the intermolecular forces of α-chitin, making α-chitin easily dispersed with ultrasonic treatment under weak acidic condition. Ultrasonic treatment of regenerated chitin for longer time produced smaller chitin particles, which further resulted in larger emulsifying capacity of α-chitin. The emulsifying ability of α-chitin is tunable through ultrasonic treatment. Moreover, the emulsion is stable after long time storage. Therefore, this paper presented a novel method to prepare α-chitin dispersion and the α-chitin dispersion turned out to be a kind of excellent emulsifier. It was anticipated that this study may trigger deeper research into water-insoluble polysaccharide such as cellulose acting as particle emulsifiers, as this promising class of polymer is underutilized, environmentally friendly and scalable.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 31371841) and (No. 51403024). The authors would like to express their sincere gratitude to many conveniences offered by their colleagues at the Key Laboratory of Environment Correlative Dietology of Huazhong Agricultural University.

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