Modulation of starch nanoparticle surface characteristics for the facile construction of recyclable Pickering interfacial enzymatic catalysis

Article abstract of DOI:10.1039/c9gc00779b

In this work, maize starch (MS) was successively modified via an esterification reaction with acetic anhydride (AA) and phthalic anhydride (PTA). Combined with the gelatinization-precipitation process, the formed starch nanoparticles at an AA/PTA ratio of 2 (MS-AP (2)) and 3 (MS-AP (3)) had similar regular spheres but distinct surface characteristics. In order to enhance the activity of lipase B from Candida antarctica (CALB) in an organic solvent, we designed an oil-in-water (o/w) and a water-in-oil (w/o) Pickering interfacial catalytic system simultaneously by utilizing MS-AP (2) and MS-AP (3) as robust Pickering emulsion stabilizers. Impressively, during the esterification of 1-butanol and vinyl acetate, the specific activity of CALB in the o/w (0.0843 U μL^-1) or w/o (0.0724 U μL^-1) Pickering interfacial catalytic system was much higher than that of free enzymes in the monophasic (0.0198 U μL^-1) and biphasic (0.0282 U μL^-1) system. Moreover, after preliminarily elaborating mass transfer discrepancies between the o/w and w/o Pickering interfacial catalytic systems and calculating their mass transfer resistance, we clarified the effects of the location of these two phases on the catalytic capacity of the Pickering emulsion. Impressively, both Pickering interfacial catalytic systems exhibited high effectiveness in product separation. It was found that the w/o Pickering emulsion enabled the organic product to be facilely isolated through a simple decantation, while the o/w Pickering emulsion achieved similar results after adjusting the system temperature. The bio-based nanomaterials and simple protocol, in conjunction with the stability to simultaneously achieve high catalysis efficiency and excellent recyclability, makes us believe that this starch nanoparticle-based Pickering interfacial catalytic system is a promising system for meeting the requirements of green and sustainable chemistry.

Full text of DOI:10.1039/c9gc00779b

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Modulation of starch nanoparticles surface characteristics
for facile construction of recycling Pickering interfacial
enzymatic catalysis
Liang Qi , Zhigang Luo1,2,3*, Xuanxuan Lu4
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1. School of Food Science and Engineering , South China University of
Technology, Guangzhou, 510640, China.
2.South China Institute of Collaborative Innovation, Dongguan, 523808, China.
3.Overseas Expertise Introduction Center for Discipline Innovation of Food
Nutrition and Human Health (111 Center), Guangzhou 510640, China.
4.Department of Food Science, Rutgers, The State University of New Jersey, 65
Dudley Rd, New Brunswick, New Jersey 08901, USA.
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*Corresponding author:
Zhigang Luo, Tel: +86-20-87113845, Fax: +86-20-87113848. E-mail address:
zhgluo@scut.edu.cn
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9 Abstract
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In this work, maize starch (MS) was successively modified via the esterification
reaction with acetic anhydride (AA) and phthalic anhydride (PTA). Combined with
the gelatinization-precipitation process, the formed starch nanoparticles at AA/PTA
ratio of 2 (MS-AP (2)) and 3 (MS-AP (3)) had the similar regular spheres but distinct
surface characteristics. In order to enhance the activity of lipase
B from Candida antarctica (CALB) in organic solvent, we designed oil-in-water (o/w)
and water-in-oil (w/o) Pickering interfacial catalytic system simultaneously by
utilizing MS-AP (2) and MS-AP (3) as robust Pickering emulsion stabilizers.
Impressively, in the esterification of 1-butanol and vinyl acetate, the specific activity
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of CALB in the o/w (0.0843U·μL ) or w/o (0.0724U·μL ) Pickering interfacial
catalytic system was much higher than that of free enzymes in the monophasic
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(0.0198U·μL ) and biphasic (0.0282U·μL ) system. Meanwhile, after preliminarily
elaborating mass transfer discrepancies between o/w and w/o Pickering interfacial
catalytic system and calculating their mass transfer resistance, we clarified the effects
of the location of two phases on the catalytic capacity of Pickering emulsion.
Impressively, both Pickering interfacial catalytic systems exhibited high effectiveness
in product separation. It was found that w/o Pickering emulsion enabled the organic
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product to be facilely isolated through a simple decantation, while o/w Pickering
emulsion achieved the same results after adjusting system temperature. The biobased
nanomaterials and simple protocol, in conjunction with the stability to simultaneously
achieve high catalysis efficiency and excellent recyclability, makes us believe that this
starch nanoparticles-based Pickering interfacial catalytic system is promising for
meeting the requirements of green and sustainable chemistry.
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Key words: Starch nanoparticles, Pickering emulsion, interfacial enzymatic catalysis,
recyclable catalysis, green and sustainable chemistry.
5 Introduction
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Enzymes catalyze a broad variety of organic reactions with high chemo-, stereo-,
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and regioselectivity under mild conditions . However, the application of free enzymes
in organic media is often hampered by the fact that, in many cases, enzymes have low
stability and poor recyclability. A simple, gentle, and general way to solve these
problems is to create a microenvironment to maintain the enzyme activity in biphasic
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aqueous-organic system . In recent years, such researches have advanced in a
promising direction toward the use of nanoparticles as building blocks for the
construction of capsules (often referred to as Pickering emulsions) as enzyme
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carriers . The presence of Pickering emulsion droplets in the reaction systems can
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significantly accelerate organic/aqueous biphasic reactions because a large
liquid-solid interfacial area is created. To meet above requirements, it takes
hydrophobic modification of nanoparticle surface to construct water-in-oil (w/o)
Pickering emulsion. Meanwhile, high interfacial activity is necessary for
nanoparticles to be closely packed at the oil-water interface. Moreover, the
biocompatibility of nanoparticles to enzymes as another crucial parameter has to be
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considered . As a result, elaborately modified synthetic stabilizers, involving hybrid
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and surface functionalized inorganic particles11-13, have
micelles , polymersomes
been reported in the Pickering interfacial enzymatic catalysis.
As we know, once the particles are anchored onto the emulsion droplet surface,
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they cannot optionally depart from the droplet surface due to a high energy barrier .
Therefore, the applications of oil-in-water (o/w) Pickering emulsion are hampered in
enzymatic catalysis because existing methods for product separation, such as heating
and centrifugation, are bothersome due to enzyme activity loss, high time- and
energy-consumption. Inspired by interfacial metal nanoparticles catalysis15-17, we
propose developing stimulus-responsive o/w Pickering emulsions is a potential way,
because stimulus-responsive demulsification/emulsification cycle or phase inversion
provides a good recyclability and a facile separation. To meet this requirement,
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surface charge, wettability and even size that governs the stabilizer performance, are
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required to be easily tuned by adjusting the surrounding environment stimulation
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factors . However, since the transformation of environment stimulation factors is
dramatic in these reports, which may not be suitable for preserving the enzyme
activity, few stimulus-responsive o/w Pickering emulsions have been reported in
interfacial enzyme catalysis until now. Therefore, this limitation urges the
development of modified nanoparticles that are sensitive to the environment
stimulation factors.
In recent years, starch nanoparticles have acquired a reputation as having
potential as biobased nanomaterials since they are easily processed, renewable,
biocompatible, and nontoxic.19 Notably, they have been intensively studied as
stabilizers for Pickering emulsions owing to their regular sphere shape and excellent
tolerance in both organic and water phase. 20, 21Moreover, the abundant hydroxyls
endows starch with a reaction activity, thus esterifying agent, alkyl chains and
polymer chains can be easily introduced on the starch chains through
hydroxyls-derived reactions.22, 23 In this way, it is easy to functionalize the starch
nanoparticles to acquire the interfacial activity and structural characteristics as
requested.
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The starch modification with acetic anhydride (AA) has been reported to reduce
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surface energy of starch nanocrystals or cellulose nanofibers and shows a good
solubility in organic solvents such as acetone, chloroform and ethyl acetate. Because
of unique hydrophobicity, acetylated starch nanocrystals are useful in applications in
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packaging industry and as nano-reinforcements . Meanwhile, phthalic anhydride
(PTA) is a preferred esterifying agent, which displays the pH-responsive property
with sensitive protonation-deprotonation transition of carboxyl20, 26. By adjusting the
pH values, its charged-uncharged interconversion impacts the surface wettability of
attached substrates. Based on this, through the adjustment of substitution degrees of
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00 AA and PTA, it is expected to modulate the surface characteristics of starch
01 nanoparticles and achieve the hydrophilic and hydrophobic starch nanoparticles with
02 high interfacial activity simultaneously. Accordingly, hydrophobic starch nanoparticle
03 can be a potential candidate for the interfacial enzymatic catalysis in w/o Pickering
04 emulsion. In the meantime, the presence of PTA gives an opportunity for hydrophilic
05 starch nanoparticles to construct stimulus-responsive o/w Pickering emulsions
06 applying to the interfacial enzymatic catalysis.
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Besides, it is worth noting that, for the synthesis of starch nanoparticles, the
08 traditional acid hydrolysis is not an option under the ever-growing environmental
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09 pressure. Delightfully, the process of nanoprecipitation has been investigated as a new
10 procedure for the generation of nanoparticles27, 28. This process involves the
11 successive addition of a dilute solution of polymer to a nonsolvent, which leads to
12 polymer precipitation at a nanoscale. Benefiting from this, starch possesses a higher
13 chemical activity by liberalizing the molecular chains to expose more active hydroxyl
14 groups, and the usage of large amounts of toxic solvents and external energy sources
15 are avoided, which is in accordance with environment-friendly concept.
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Herein, through optimizing the rational adjustments of AA and PTA substitution
17 degree and cooperation with the gelatinization-precipitation method, we synthesized
18 two starch nanoparticles with the similar regular spheres but distinct surface
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19 characteristics. The structure of starch nanoparticles was clarified using H nuclear
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20 magnetic resonance ( H NMR), the results from X-ray diffraction (XRD) and
21 scanning electron microscopy (SEM) disclosed the change of crystallinity,
22 morphology and size of nanoparticles at different degree of esterification. For
23 enhancing lipase activity in organic solvent, we designed oil-in-water (o/w) and
24 water-in-oil (w/o) Pickering interfacial catalytic system simultaneously. The location
25 of enzymes was directly confirmed by laser scanning confocal microscopy (LSCM)
26 with fluorescent dye labeled lipase. Through comparison experiments with
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27 monophasic and biphasic system, the merits of these two Pickering interfacial
28 catalytic systems, such as high catalysis efficiency, facile products separation and
29 enzymes recycling were intensively demonstrated with esterification of 1-butanol and
30 vinyl acetate. Meanwhile, their underlying principles were preliminarily elaborated.
31 To the best of our knowledge, it is the first report on starch nanoparticles participating
32 in the Pickering emulsion for the recyclable interfacial enzymatic catalysis. The
33 excellent performance makes us believe that this system will attract widespread
34 interest from both fundamental and industrial researchers because it unites some key
35 advantages in terms of green and sustainable chemistry.
36 Experimental Section
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Materials
Native maize starch (MS) was provided by Qin Huangdao LiHua Maize
39 Products Co. (Qin Huangdao, China), the moisture content of starch was 12.2% (w/w),
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40 weight-average molecular weight was 2.634×10 g/mol, number-average molecular
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41 weight was 1.29×10 g/mol. Acetic anhydride (AA), phthalic anhydride (PTA),
42 dimethylsulfoxide (DMSO), N, N-dimethylaminopyridine (DMAP), pyridine,
43 n-heptane were purchased from Aladdin Chemical Reagent Company (Shanghai,
44 China). 1-butanol, vinyl acetate, agarose (Type VΙΙ-A, low gelling temperature),
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45 lipase B from Candida sp. expressed in Aspergillus niger (CALB, liquid form),
46 Tris(hydroxymethyl)aminomethane (Tris) and fluorescein isothiocyanate (FITC) were
47 purchased from Sigma-Aldrich (Shanghai, China).
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Preparation of starch amphiphilic nanoparticles
3 g of maize starch was added into 30 mL of DMSO in a 50 mL conical flask.
50 Then the temperature was raised to 40 C to initiate gelatinization process. After 15
51 min, the completely gelatinized starch paste was heated to 80 C followed by the
52 addition of a certain amount of PTA, 10 mL of pyridine and 0.05 g of DMAP,
53 respectively. This esterification reaction was carried out for 3 h under stirring of 200
54 rpm. After that, a certain amount of AA was added and the mixture was allowed to
55 react for another 3 h at 80 C under stirring.
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The nanosized granulation process was conducted using an water precipitation
57 process. Specifically, 10 mL of composite starch paste was firstly sucked with a
58 disposable syringe (ID 0.3×8 mm). Then, it was added dropwise into the 200mL of
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59 water at 1 mL·min rate. The resulting suspensions were stirred under 600 rpm for 20
60 min. Finally, the product was obtained after washing/centrifugation (with water for 3
61 times) and vacuum drying overnight.
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Preparation of Pickering emulsion
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Typically, 20 mg of hydrophobic nanoparticles were firstly dispersed in 2 mL of
64 n-heptane under ultrasonication for 10 min, followed by addition of the water phase
65 with the same volume. The resulting mixture was vigorously stirred using an
66 Ultraturrax T25 homogenizer (IKA, Germany) at a stirring rate of 15,000 rpm for 1
67 min, giving rise to a uniform emulsion composed of a large number of nanoparticle
68 capsules. The formation of Pickering emulsion stabilized by hydrophilic nanoparticles
69 was similar to that of MS-AP (3), except that nanoparticles firstly dispersed in 2 mL
70 of water phase.
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Preparation of lipase-loading Pickering emulsion
In a typical experiment, a 40 μL aqueous stock solution of CALB was diluted to
73 2 mL of Tris-HCl buffered saline (10 mM, pH 7.2) and mixed under shaking for 3
74 min. This enzyme buffered saline was taken as the water phase, the other procedures
75 followed the same procedures in the preparation of Pickering emulsion as mentioned
76 above.
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Assessment of the Catalytic Performance of Lipase
The catalytic performances of CALB were determined via the transesterification
79 of 1-butanol and vinyl acetate in a heptane medium by three different catalytic
80 systems. For Pickering interfacial system, 2 mL of heptane solution containing 150
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181 mmol·L 1-butanol and 150 mmol·L vinyl acetate was taken as the organic phase to
182 form a lipase-loaded Pickering emulsion. For biphasic system, except for the
183 nanoparticles, the other conditions, including substrate concentration, solvent
184 composition, and lipase content, were the same as given before. For monophasic
185 system, 40 μL aqueous stock solution of CALB was directly added into organic phase
186 with same substrate concentration to initiate the transesterification. All of
187 esterification reactions were carried out with stirring rate of 200 rpm at room
188 temperature. At different time points, A 60 μL aliquot of n-heptane in Pickering
189 interfacial system, biphasic system and monophasic system could be directly
190 extracted. For o/w Pickering interfacial system, after increasing the temperature of
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91 system to 40 C and stirring, the emulsion could be inversed accompanying with
92 organic phase transferring into the upper layer, product could be isolated accordingly.
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93 The concentration of the product analyzed via H NMR . One unit of lipase activity
94 (U) was defined as 1 μmol of product produced within 1 min. The specific activities
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95 (U·μL ) of lipase were determined under the same condition within 40 min. All
96 reactions were repeated at least three times.
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Characterization of starch amphiphilic nanoparticles
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The H NMR spectra were recorded using an AVANCE digital 400 (Brukev,
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99 Germany) operating at 400 MHz for H NMR. The H spectra were collected in 128
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00 individual scans with a sweep width of 16 ppm and a delay time of 1 s. Prior to H
01 NMR analysis, nanoparticles were dissolved in the deuterium dimethyl sulfoxide
02 (DMSO-d ) at 40 C to obtain clear solutions.
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Morphology observations were performed using a Merlin scanning electron
04 microscope (Zeiss Co., Germany) at an accelerating voltage of 3 kV. The samples
05 were dispersed in the distilled water (0.05 wt%) under mechanical agitation of 600
06 rpm for 2 min. One drop of dispersion was carefully placed on a glass slide and dried
07 at room temperature. After that, the samples were coated with Au for conductivity and
08 put under SEM for observations.
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The average size of starch nanoparticles was determined through statistical
10 analysis of the geometric parameters including the area, perimeter, diameter which
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11 were derived from SEM images using Image-pro Plus 6.0 .
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XRD patterns were obtained using a D/max-IIIA fully automatic XRD
13 instrument (Rigaku, Japan). Diffractograms were collected at 40 kV and 30 mA with
14 nickel-filtered Cu Kα radiation (λ=1.5405 Å). Powdered sample was scanned from 5
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15 to 60°(2θ) at a scanning rate of 4°·min . Before XRD analysis, starch nanoparticles
16 were sealed in a vessel at 75% relative humidity using saturated sodium chloride.
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Characterization of Pickering Emulsion
The emulsion droplets were visualized using a MB11 polarizing microscope
19 (Shanghai optical instrument factory, China) equipped with a video camera. Emulsion
20 droplets were placed directly onto a glass slide and captured under 50-400
21 magnification.
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The morphology of mircocapsules derived from self-assembly of starch
23 nanoparticles at oil-water interface was measured by SEM. For MS-AP (2) capsules, a
24 few drops of o/w Pickering emulsion were diluted to 10 times with distilled water,
25 then they were added on culture dish and lyophilized. The resulting powders were
26 placed onto a sample stage with an aluminum tape and then coated with Au and put
27 under SEM for observations. For easy characterization of MS-AP (3) capsules, the
28 aqueous core of w/o Pickering emulsion was solidified by addition of agarose (1.5
29 wt% to the water phase) prior to emulsification. Then the jellified Pickering
30 emulsions were lyophilized and imaged by SEM.
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N adsorption/desorption isotherm was measured at 77 K using a Flowsorb Ⅲ
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32 2310 Surface Characterization Analyzer (Micromeritics Instrument Co., USA) after
33 mircocapsules were first degassed at 45 °C overnight. Pore size distribution was
34 determined using the adsorption branch of N isotherms. Mesopore size distribution
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35 was calculated using the Barrett-Joyner-Halenda method (BJH).
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Zeta potential measurements of nanoparticles at different pH values were
37 measured using a nano ZS instrument (Malvern Instrument Ltd., UK) equipped with a
38 dip cell. The concentration of nanoparticles was maintained at 0.1 wt % and the
39 experiments were conducted at room temperature.
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Water contact angles were measured by analyzing the image of each water
41 droplet on the sample films using a DCa40 MICRO system (DATA physics,
42 Germany). Prior to measurement, the samples were pressed into the films. The films
43 were swollen in the solution at certain pH and then oven-dried at 40 C for 1 h. Water
44 contact angles were obtained by depositing water droplets of a given pH onto the film
45 that had been previously swollen at the same pH.
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To confirm the location of lipase within Pickering emulsion, the lipase molecules
47 were labeled with fluorescein isothiocyanate (FITC) and then were used for formation
48 of Pickering emulsion by following the same procedure as mentioned before. The
49 resulting lipase-loading emulsion were then directly observed with confocal laser
50 scanning microscopy (CLSM, TCS SPE, Leica, America) with an excitation
51 wavelength of 488 nm.
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Far-UV circular dichroism (CD) spectra of lipase solutions were measured on a
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53 J-810 CD spectropolarimeter (Jasco Inc., Tokyo, Japan) over a wavelength range of
54 200-280 nm with a resolution of 0.5 nm. A quartz cell with 1 mm path length was
55 used for CD measurements. Spectra were recorded three times using a scanning speed
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57 Results and discussion
58 Synthesis and characterization of starch nanoparticles
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The synthesis of starch nanoparticles is illustrated in Fig.1. The gelatinization
60 process was firstly performed so as to endow the starch with a high chemical activity
61 by liberalizing the molecular chains to expose more active hydroxyl groups. In this
62 condition, AA and PTA directly participated in the esterification reaction with starch
63 molecules catalyzed by the DMAP and pyridine. Subsequently, the modified starch
64 paste went through the granulation process by water precipitation to obtain the final
65 nanoparticles. Thus, the whole process was performed in an environment that was
66 simple and practical to implement.
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It is well established that the interfacial activity of nanoparticles is mainly
68 dictated by their surface characteristics. To screen out an optimal starch nanoparticle,
69 we have prepared a set of samples by adjusting additive amount of AA and PTA.
70 FT-IR spectra of samples are shown in Fig.2A. By contrast with MS, the formed
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71 starch products with different substitutions showed the carbonyl antisymmetry
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72 deformation vibration at 1751 cm , which was a strong ester signal. Besides it
73 increased in intensity with the rise of substitution degree. At the same time, new
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74 absorption bands at 1580-1600, 1372, and 1239 cm could be assigned to the
75 stretching vibration of benzene rings, symmetrical deformation vibration of methyls
76 and stretch vibration of carbonyls, respectively. The appearance of these new
77 absorptions confirmed the combination of AA and PTA with starch molecules during
78 the esterification process.
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1H NMR spectra (Fig.2B) gave a clearer composition picture of obtained
80 products. Peaks arising from the anhydroglucose in starch, AA and PTA were
81 assigned according to the literatures20, 31. The area of OH-2,3,6 and H-1 protons for
82 anhydroglucose, H-1’proton for AA and H-1’’ for PTA are calculated in Table 1. On
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83 the basis of the method by Chi et al. , we estimated the degree of substitution for AA
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84 and PTA (DSAA and DSPTA).
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From the relatively high values of DSAA in different samples, it suggested that
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86 AA as a highly reactive micromolecular agent could steadily graft onto starch chains
87 through esterification. In contrast, due to the steric effects of benzene ring, PTA had a
88 limited reactivity with starch chains by observing its much lower degree of
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89 substitution. Meanwhile, when the additive amount of AA kept constant and PTA
90 increased from 0 to 4.5, a clear decrease was observed for DSAA (2.2 to 0.9). This
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91 inferred although AA had a prior reactivity to hydroxyls of anhydroglucose, it still
92 suffered a competition with PTA in the reaction with active sites of starch chains. As
93 a result, it clearly confirmed that chemical composition of starch-based nanoparticles
94 could be easily altered by adjusting additive amount of each ingredient.
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In the meantime, the reactive sites were also specified from area of protons (S) in
96 1H NMR spectra. As expectedly, a decrease was found in the SOH-2,3 of MS-A and
97 MS-P, which meant hydroxyls in C-2 and 3 of anhydroglucose readily participated in
98 the esterification under the employed conditions. In the meantime, the new signals at
99 4.75, 5.25 and 4.25 ppm corresponding H-2 and H-3 and H-6 signals of
00 anhydroglucose unit in the sugar ester structure are detected. Besides, these signals
01 slightly varied at different of DSAA and DSPTA. However, there was a great
02 discrepancy in the SOH-6 between MS-A and MS-P, in which SOH-6 of MS-A was
03 decreased to 0 whereas SOH-6 of MS-P barely changed. This disclosed the hydroxyls in
04 C-6 were only involved in the AA esterification, showing the hydroxyl heterogeneity
05 of anhydroglucose. The similar phenomenon was also verified by other starch
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06 esterification and copolymerzation . Given the above, possible structures of MS-AP
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07 are listed in Fig.1.
08
In this study, starch nanoparticles were prepared by gelatinization-precipitation
09 process, in which the gelatinization contributed to the regularity destruction of native
10 starch granules. Therefore, the obtained products were mainly in amorphous forms, as
11 proved by mostly diffuse peaks in the corresponding XRD diffraction spectrum
12 (Fig.S1). Furthermore, since the change of composition directly affected the
13 molecular weight, polarity, and charge, etc. of dissolved polymer, there would be
14 differences in the granulation process for different samples in terms of formation of
15 aggregations in the supersaturation region and subsequent particle growth process
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16 within a poor solvent . From Figure.3A, nano-granulation of native starch could not
17 be achieved by only finding a block of deformed sedimentations in MS. This was
18 because hydroxyl-rich unmodified starch chains (water contact angle of 41° for MS)
19 possessed a similar polarity to water, which had a difficulty in entangling with each
20 other in the solvent conversion from DMSO to water. In stark contrast, MS-A
21 exhibited a regular spherical nanoparticle with average size of 421nm (Figure.3B and
22 Figure S2). We attributed this improvement to the “bridging action” brought by
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23 introduced acetyls. Since the carbonyls could form hydrogen bonds with hydroxyls ,
24 acetyls played a role of “bridge” that facilitated to connect other starch chains, thus
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25 more cross-linking occurred between modified starch chains in DMSO. In this
26 condition, exposed methyls played an important role in generating a hydrophobic
27 surface (water contact angle of 112° for MS-A), which not only facilitated the
28 formation of aggregations during the solvent conversion, but also avoided the collapse
29 and adhesion of obtained nanoparticles in particle growth process.
30
Moreover, when PTA was introduced to the synthesis of starch nanoparticles,
31 residual carboxyls of phthalic ester were of great importance in adjusting the charge
32 of modified starch chains. From zeta potential of MS-APs (Table.S1), it was clear
33 that dissociated carboxyls increased the negative charge of modified starch chains in
34 employed condition. This enhanced electrostatic repulsion between modified starch
35 chains possibly induced more localized aggregations formed by a smaller number of
36 modified starch chains in the supersaturation region, thus resulting in a limited size of
37 formed particles (Figure.3C-E). With rational DSAA and DSPTA, amphipathic MS-AP
38 (2) and (3) achieved ideal wettablities (water contact angle near to 90°) and presented
39 regular spheres with average size of 197 and 185nm, respectively. However, for
40 MS-AP (1), higher DSPTA induced more hydrophilicity of surface, thus particles
41 adhered to each other to form clusters in the end. With only PTA grafting onto starch
42 chains, MS-P (Figure.3F) exhibited highest zeta potential (-26.93mV) and lowest
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43 water contact angle (13°). As expectedly, MS-P was still in the form of starch paste
44 due to the increased electrostatic repulsion and excessive hydrophilicity.
45 Characterization of o/w and w/o Pickering emulsions
46
Before the formation of emulsion, we firstly observed the dispersibility of these
47 samples with different chemical characteristics in water/n-heptane solution. As shown
48 in Fig.4, hydroxyl-rich MS only dispersed in the aqueous phase, while MS-A after
49 hydrophobic modification dispersed well in n-heptane. For MS-AP (3), they initially
50 dispersed in organic phase, but transferred to the n-heptane-water interface within 10
51 min, suggesting their excellent amphiphilicity. Interestingly, a completely opposite
52 phenomenon was observed for MS-AP (2), where nanoparticles firstly dispersed well
53 in aqueous phase and subsequently transferred to the interface. When it came to
54 MS-AP (1) and MS-P, both samples steadily dispersed in the aqueous phase. However,
55 it was worth noting that dispersion of MS-P in the aqueous phase exhibited more
56 transparent because starch was mainly present in the form of dissolved molecular
57 chain.
58
Subsequently, we applied these samples to form Pickering emulsion and then
59 investigated the status of the emulsion. In specific experiment, based on the surface
60 characteristics, 1 wt% of sample was firstly dispersed in n-heptane or water, followed
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61 by addition of the other phase with the same volume. After homogenization (12000
62 rpm, 60 s), the resulting emulsion was allowed to stand at room temperature for 10
63 min without any disturbance. The emulsion type was determined by the conductivity
64 analysis. As shown in Fig.5, a demulsification was observed as soon as the emulsion
65 was formed in the emulsions stabilized by MS, MS-AP (1) and MS-P, which
66 confirmed these samples were unable to stabilize the emulsion mainly due to their
67 deformed morphology, non-uniform size and high hydrophilicity. Furthermore, the
68 use of hydrophobic MS-A as stabilizers led to the formation of w/o emulsion, but it
69 had low stability, as evidenced by the obvious increase in droplet size after standing
70 for 10 min. Noteworthily, because of regular morphology and excellent
71 amphiphilicity, MS-AP (2) and MS-AP (3) both had better emulsifying capacities,
72 leading to the long-lasting stable emulsion layers. The only difference was that, for
73 MS-AP (3) stabilized w/o Pickering emulsion, since the emulsion droplets with water
74 core possessed a higher density compared to continuous phase n-heptane, the
75 emulsion layer would sink to the bottom. In contrast, because of oil core, droplets in
76 o/w Pickering emulsion stabilized by MS-AP (2) possessed a lower density compared
77 to continuous phase water, the emulsion layer would float on the top.
78
Fig. 6A, D shows microscopic images of the droplets acquired from the emulsion
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379 stabilized by MS-AP (3) and MS-AP (2). The results indicated that both droplets had
380 a similar diameter range of 5-30 μm, which was related to the similar size of
381 stabilizers. However, in contrast to MS-AP (2) stabilized o/w emulsion that possessed
382 a uniform distribution of intact droplets, MS-AP (3) stabilized w/o emulsion had an
383 unsatisfactory status, in which the macroscopic agglomerations of stabilizers were
384 dispersed in the continuous phase and adhered to the surface of droplets. Scanning
385 electron microscopy (SEM) imaging confirmed this discrepancy. It clearly revealed
386 that the droplets of o/w Pickering emulsion stabilized by MS-AP (2) were regular
387 spheres (Fig.6E, F). The shell of the intact sphere was firm and remained unbroken
388 even in the freeze-drying process. After the formed spheres were greatly grinded with
389 a mortar, the broken spheres revealed the hollow interior, confirming the
390 microcapsule structures (Fig.6J, K). Moreover, their shells were thin and consisted of
391 one or two layers of starch-based nanoparticles (Fig.6L). In contrast, although most
392 droplets in Pickering emulsion stabilized by MS-AP (3) presented intact hollow
393 capsules as well (Fig.6B, C, G, H), excessive particles adhering to the surface greatly
394 increased the thickness of the shell (Fig.6I). This phenomenon was mainly due to the
395 discrepancy in dispersibility of starch nanoparticles in different solvent. In this study,
396 hydrophilic MS-AP (2) easily achieved regular arrangement when immersed in the
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97 water phase. The rational electrostatic repulsion brought by surface charges ensured
98 the stabilization of MS-AP (2) dispersion. Its corresponding polydispersity index (PDI)
99 was 0.125 (calculated by size distribution), which suggested the monodispersity of
00 particles. In contrast, although MS-AP (3) had a better wettability to organic phase
01 with higher DS , the residual hydroxyls and carboxyls made inevitable repulsions
AA
02 with n-heptane molecules, which induced MS-AP (3) to form the agglomeration in
03 organic phase. Because of this, there were easy particle sedimentations on the surface
04 of droplets in the formation of Pickering emulsion. The similar phenomenon was also
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05 found in our previous report .
06 Biocatalytic performances of Pickering interfacial catalytic systems
07
For construction of interfacial enzymatic catalytic system, we introduced CALB
08 as the target enzyme. Then Tris-HCl buffered saline (10 mM, pH 7.2) with CALB
09 was taken as the water phase of Pickering emulsion. Since the continuous phase and
10 dispersed phase were reversed for Pickering emulsion stabilized by MS-AP (2) and
11 MS-AP (3), CALB should be distributed inside and outside the shell of capsules,
12 respectively. To confirm this assumption, CALB molecules were firstly labeled with
13 FITC and then used for the formation of Pickering emulsion by following the same
14 procedure as mentioned before. As shown in Fig.7A, B, CLSM imaging clearly
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15 revealed that the emulsion stabilized by MS-AP (3) was composed of green capsules
16 and thus indicated that the interior of capsules was loaded with FITC-labeled lipase
17 molecules. But for MS-AP (2), the green fluorescent was observed around the blank
18 capsules, which confirmed the lipase molecules dispersed outside the capsules
19 (Fig.7C, D). Fig.7E, F illustrated the forming process of these two interfacial
20 enzymatic catalytic systems.
21
Furthermore, to determine whether the lipase was destroyed during the
22 emulsification process, we used CD spectra to identify the secondary structure of the
23 free lipase before emulsification and after release from the Pickering emulsions via
24 centrifugation (10000rpm, 5min). As shown in Fig.S3, the CD spectra showed little
25 changes in terms of secondary structure, suggesting that the emulsification process
26 insignificantly impacted the enzyme.
27
The biocatalytic performances of these two CALB loaded Pickering interfacial
28 catalytic systems were then investigated by transesterification of 1-butanol and vinyl
29 acetate. To confirm their superiorities, two other catalytic systems, biphasic and
30 monophasic enzymatic reactions containing same amount of free CALB, were carried
31 out as negative controls. As shown in Fig.8A, the conversion rate of the reaction in
32 o/w Pickering interfacial catalytic system reached 100% within 8 h. In the meantime,
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33 92% conversion was observed for w/o Pickering interfacial catalytic system. In stark
34 contrast, a significant reaction inhibition was observed for biphasic and monophasic
35 catalytic system, where only 60% and 32% conversion rate were achieved at the same
36 time. For directly comparing the catalytic activity of enzyme, the specific activity of
37 CALB for each system was calculated according to the conversion rate obtained
38 within 40 min. As shown in Fig.8B, free lipase dissolved in n-heptane only had a
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39 specific activity of 0.0198U·μL , which clearly indicated that long-term exposure of
40 free enzymes to an organic solvent caused reduced enzyme activity. Comparatively,
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41 the increased specific activity (0.0282U·μL ) in the water/n-heptane biphasic system
42 suggested that enzymes typically preferred an aqueous environment, which protected
43 them effectively from the organic media. However, inefficient mass transfer between
44 two phases resulted in the low conversion rate even after a long reaction time.
45 Delightfully, the formation of Pickering emulsion significantly improved the catalytic
46 activity of CALB by observing higher specific activities of 0.0724U·μL-1 and
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47 0.0843U·μL for w/o and o/w Pickering interfacial catalytic system, respectively. In
48 accordance with previous report, the higher catalytic activity could be attributed to the
49 nanoparticles-assembled capsules, which not only functioned as an idea screen for
50 preserving enzyme activity in organic solvent, but also provided increased interfacial
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51 areas that improved mass transfer and accessibility of enzyme molecules. More
52 importantly, by comparing specific activities of CALB between o/w and w/o
53 Pickering interfacial catalytic system, we proposed that the location of two phases in
54 Pickering emulsion also played a key role in the catalytic capacity.
55
To give a deeper insight of this proposal, the whole reaction processes for
56 transesterification of 1-butanol and vinyl acetate in o/w and w/o Pickering interfacial
57 catalytic system are illustrated in Fig.9. Given the same phases (water and organic
58 phases) and elements (substrates, enzymes and stabilizers), the reaction processes in
59 both two systems mainly composed of five steps: (1) enzymes dissolved in the water
60 phase and substrates dissolved in the organic phase transferred to the emulsion droplet
61 surface simultaneously, (2) enzymes and substrates both accessed to the
62 n-heptane-water interface, (3) the catalytic reaction took place on the interface, (4)
63 after the end of reaction, products and enzymes departed from the interface, (5)
64 products entered the organic phase and enzymes entered the water phase, respectively.
65 Clearly, step (1) and (5) mainly involved mass transfer in the phase, whereas step (2)
66 and (4) were closely related to the mass transfer on the interface.
67
As described in Fig.9A, the continuous phase of the o/w Pickering emulsion
68 served as an avenue for enzyme transfer. Because of good water solubility, enzymes
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69 could be uniformly distributed around the droplets in the water phase. Densely
70 dispersed oil droplets as microreactors efficiently confined substrates in tight spaces.
71 The resulting decreased transfer distance in organic phase was beneficial for
72 substrates to contact with enzymes distributed on the interface and thus resulted in a
73 higher specific activity. In stark contrast, the continuous phase of the w/o Pickering
74 emulsion was n-heptane (Fig.9B), so that it served as an avenue for substrates
75 transport. Given that enzymes were uniformly distributed within the droplets, thus
76 enzymatic reaction was determined by the transfer of substrates in bulk continuous
77 phase. Since transfer distance outside droplets was much larger, it was reasonable that
78 specific activity of CALB in w/o Pickering emulsion was relatively low.
79
In view of increasing stirring rate could enhance the mass transfer in the phase,
80 we compared the catalysis efficiency of these two systems under different stirring
81 input powers in Fig.10A (a larger stirring input power corresponds to a higher stirring
82 rate; the relationships were reflected in Table.S2). For MS-AP (2) stabilized o/w
83 Pickering interfacial catalytic system, specific activity of CALB slightly increased
84 with increasing stirring input power and then leveled off after the stirring input power
85 exceeded 12.5 W. The maximum specific activity for transesterification reached
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86 0.0868U·μL , which was only 4.8% higher than original value. This limited increase
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87 clearly confirmed a higher stirring rate had little impact on the catalysis efficiency of
88 o/w Pickering interfacial catalytic system, which was due to the short transfer distance
89 of substrates brought by droplets that had already promoted the interfacial reaction as
90 much as possible. When it came to MS-AP (3) stabilized w/o Pickering interfacial
91 catalytic system, specific activity of CALB underwent a sharp increase with
92 increasing stirring input power. Notably, its maximum catalysis efficiency could reach
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93 0.0853U·μL after the stirring input power exceeding 22.5 W, which was close to o/w
94 emulsion system. As expectedly, elevating stirring input power was more effective in
95 improving the catalysis efficiency of w/o Pickering interfacial catalytic system by
96 accelerating the transfer of substrates in bulk continuous phase. The similar results
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97 were also reported by Huo in the Pt-catalyzed interfacial reaction.
98
As mentioned above, the shell of hollow microcapsules was composed by the
99 layers of starch nanoparticles, whose status was closely related to the mass transfer on
00 the interface. For the o/w Pickering emulsion stabilized by MS-AP (2), self-assembled
01 starch nanoparticles at the n-heptane-water interface yielded a stable shell with one or
02 two nanoparticle thickness. Pore-size-distribution curves were plotted based on the
03 nitrogen adsorption-desorption isotherms using the BJH method (Fig.S4). The result
04 revealed microcapsules formed by MS-AP (2) had a board pore distribution ranging
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05 from 3 nm to 120nm. (Fig.10B). This confirmed that, in contrast to the uniform
06 distribution of inorganic particles at the oil-water interface, the starch nanoparticles
07 presented an irregular arrangement. Pores with size of less than 10 nm likely arose
08 from the interspaces between the starch nanoparticles closely arranged at the oil-water
09 interface. In the meantime, pores with larger size were mainly due to the gaps
10 between the layers of starch nanoparticles. As the hydrate diameters of the substrate
11 and the product molecules were about 0.6–0.8 nm (calculated by Material Studio
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12 software, Fig.S5) and CALB had dimensions of 3×4×5 nm , it could be deduced that
13 the shell of the microcapsules could allow the free diffusion of enzymes, substrates
14 and products. When it came to microcapsules formed by MS-AP (3), the data showed
15 an increase in the pore size of 5~10 nm whereas decrease in the pore size of 10~100
16 nm. This result indicated that excessive adhesion of starch nanoparticles to the surface
17 of droplets brought about more serious agglomeration, which resulted in not only
18 closer arrangement between MS-AP (3) at the oil-water interface, but also more
19 compressed space in the multilayer of starch nanoparticles(Fig.10B).
20
To quantificationally clarify the discrepancy in mass transfer on the interface
21 between these two systems, we designed a pure Pickering emulsion by tripling the
22 concentration of nanoparticles prior to emulsification (Fig.11), in which the emulsion
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