Purification of phosvitin phosphopeptides using macro-mesoporous TiO₂

Abstract

Macro-mesostructured titanium dioxide (MMTD) was synthesized, characterized and applied as an adsorbent in the purification of phosvitin phosphopeptides (PPPs) from egg-yolk protein hydrolysates successfully. The synthesized material was analyzed using Fourier transform infrared spectroscopy (FT-IR), nitrogen adsorption–desorption analysis and scanning electron microscopy (SEM). The resultant MMTD exhibited a uniform macroporous structure with worm-like mesoporous walls, and the particle sizes were around 22 μm with the facility to be separated from aqueous solution. In the purification process, the adsorption of PPPs onto MMTD fitted well with the Freundlich model and pseudo-second-order kinetics model, and the maximum adsorption capacity reached 36.47 mg g⁻¹ in 20 min when the crude polypeptide concentration was 16 mg mL⁻¹. The purity of the obtained PPPs was relatively high with a nitrogen/phosphorus molar ratio (N/P) of 5.4. Moreover, the reusability of MMTD was satisfying in the twenty repeated adsorption–desorption cycles. Hence, the purification of PPPs using MMTD was highly efficient and could be scaled-up for practical application.

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1. Introduction

Bioactive peptides from various sources have been explored since they have beneficial effects on human physiology. Among these, phosphorylated peptides were considered as health promoting agents due to their strong affinity with bivalent metals such as calcium, magnesium and ferric ions.^1–4 Egg yolk-derived phosvitin phosphopeptides (PPPs) which were separated from phosvitin hydrolysates, contained clusters of phosphoserines and could bind calcium effectively.^5,6 It has also been reported that PPPs of 1–3 kDa were superior to commercial casein phosphopeptides in terms of their calcium binding capacity.^7,8 Therefore, PPPs could potentially be used as nutraceutical agents to promote calcium absorption and prevent osteoporosis.

Conventional purification methods, such as membrane separation, calcium ethoxide precipitation and ion exchange chromatography have been applied in the separation of PPPs.^9–11 However, there were obvious disadvantages in these traditional methods including membrane fouling, large amounts of organic reagents and high energy consumption, leading to diseconomy and inefficiency in their practical application. Lately, due to tedious steps and low efficiency, immobilized metal affinity nanoparticles and functional immobilized metal affinity magnetic carboxymethyl chitosan nanoparticles were prepared to adsorb PPPs from crude polypeptides.^12,13 Although the purification process has been greatly simplified through using the novel nanoparticles above, the stability of the magnetic carboxymethyl chitosan nanoparticles was relatively low and the N/P of the obtained PPPs obviously increased when the nanoparticles were used three times. Moreover, solvents of imidazole which were used in the elution process are restricted in the food industry.

Titanium dioxide (TiO₂), with low cost, benign thermal stability and chemical inertness, is considered as a multifunctional material and is used in various areas.^14–19 As an amphoteric metal oxide, TiO₂ could act as an anion or cation exchange material according to the pH of the medium. Under acidic conditions TiO₂ acts as a Lewis acid with positive charges of Ti⁴⁺, which has a strong affinity with phosphate groups.^20–22 Based on the specificity of TiO₂ to phosphate peptides, a TiO₂/diatomite composite was prepared and used in the purification of PPPs in our previous report.²³ The stability of the TiO₂/diatomite composite in the repeated experiments was better than the nanoparticles prepared by Zhang.¹² However, the maximum adsorption capacity of the material was 8.15 mg g⁻¹, which was relatively low and thus limited its practical applications.

To solve the problems above, hierarchically ordered macro-mesostructured TiO₂ (MMTD) was prepared and applied in the purification of PPPs in this paper. The multimodal porous structures of MMTD not only provided a readily accessible pore-wall system, but also exposed more active sites for the binding of PPPs. Simultaneously, fully accessible surfaces and a specific affinity with phosphate groups were conducive to improving the binding capacity. In addition, PPPs with different molecular sizes and quantities of phosphate groups were suitable for the hierarchical porous structures. Therefore, MMTD with different pore sizes would be of appreciable interest as a promising adsorbent in the purification of PPPs. At the same time, hierarchically macro-mesoporous materials such as TiO₂ are widely used in other areas due to their highly interconnected and accessible pore structures.^24–30 For example, Li et al. synthesized hierarchically ordered macro-mesoporous anatase titanium dioxide flakes using a bio-template of natural rose petals, and the degradation of rhodamine B by the obtained TiO₂ flakes was slightly better than the commercial catalyst Degussa P25.²⁴ Yu et al. reported that the calcination temperature greatly influenced the structures and photocatalytic activities of MMTD, and the high photocatalytic activities were mainly due to the large specific surface areas and bimodal porous structure.²⁶ Qi et al. fabricated hierarchically macro-mesoporous Pt/TiO₂ which was used for the decomposition of formaldehyde and the results showed that this structured catalyst exhibited higher activities than P25 TiO₂.²⁸

In this study, MMTD with a hierarchical structure was prepared using a facile self-assembly process and used as an affinity adsorbent to purify PPPs from the hydrolysates of egg yolk protein (EYP), the by-product of egg-yolk lecithin. The adsorption properties and purification parameters of MMTD were investigated in detail, and the reusability of MMTD was also studied to assess its potency in practical applications.

2. Materials and methods

2.1 Materials and reagents

Tetrabutyl titanate (TBT, C₁₆H₃₆O₄Ti, Aldrich, USA), trypsin (E.C.3.4.21.4.3 × 10⁶ IU g⁻¹) and defatted egg yolk powders were obtained from Guangzhou Hanfang Pharmaceutical Co., Ltd (Guangzhou, China). The composition of the powders was 84.00% protein (%N × 6.25), 6.88% moisture and 5.17% fat. All chemicals used were of analytical reagent grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Water used was prepared using a Milli-Q system (Millipore, Bedford, MA, USA).

2.2 Preparation and characterization of MMTD

Hierarchically ordered MMTD was synthesized using a facile template-free method and the synthesis process was as follows: firstly, citric acid (0.64 g) and 95% ethanol (80 mL) were mixed and stirred at 25 °C until the citric acid dissolved completely. Secondly, 25% ammonia (40 mL) was added at a uniform speed, followed by further stirring for 15 min. Subsequently, TBT (12 mL) was added dropwise into the above solution prior to keeping the reaction system still for 12 h. After that, the resulting precipitates were filtered and rinsed with deionized water repeatedly. The obtained materials were then dried at 60 °C for 10 h. Finally, the as-prepared MMTD was calcined at 550 °C for 4 h in air with a heating rate of 1 °C min⁻¹.

SEM images were collected on a Hitachi S-4800 instrument in order to characterize the surface morphology of macro-mesoporous TiO₂. FT-IR spectra were recorded on a Nicolet iS10 FT-IR thermoscientific spectrometer to detect the functional groups of the materials. N₂ adsorption–desorption experiments were undertaken isothermally at −196 °C on automatic ASAP 2020 Micromeritics apparatus. Before the measurements, the as-prepared and calcined MMTD were respectively out-gassed at 95 °C and 200 °C for 4 h. Specific surface areas were calculated using the BET method and pore size distributions from the desorption branches of the isotherms were analyzed using the supplied BJH software package. The zeta-potential of the material was measured using a laser particle size and zeta potential analyzer.

2.3 Preparation of crude polypeptides from defatted EYP

The defatted EYP was obtained through supercritical fluid extraction technology and the fat content was about 5.17%. Therefore, the degreasing step was omitted.

At first, the defatted EYP was dissolved in 0.1 mol L⁻¹ NaOH and incubated at 37 °C for 3 h to free some of partial phosphorus.⁷ After alkaline treatment, the mixture was adjusted to neutral pH and centrifuged at 8000 rpm for 15 min. In order to remove the free phosphate anions, the suspension was ultra-filtered and the precipitate was washed with deionized water three times. Then the intercepted fluid and washed precipitate were digested by trypsin in an enzymatic bioreactor using the following conditions. The ratio of enzyme/substrate was 1 : 10 and the pH was constantly maintained at 8.0 and adjusted using NaOH (0.1 mol L⁻¹). The incubation temperature and time were 37 °C and 4 h, respectively. The enzymatic reaction was terminated by raising the temperature to 95 °C and maintaining it for 10 min. Then the digested solution was lyophilized and stored at 4 °C for further study.

2.4 Adsorption of PPPs from aqueous crude polypeptides

MMTD was applied in the purification of PPPs using the following process: firstly the synthesized MMTD (0.50 g) and crude polypeptide solution (15 mL) were mixed in a 50 mL centrifuge tube. Then the tube was shaken on an immersion oscillator (180 rpm) at 25 °C for 20 min. Once the adsorption equilibrium was reached, MMTD–PPPs were isolated from the solution using filtration with a sand core funnel and the precipitate was washed three times with distilled water. Elution was performed by incubating MMTD–PPPs for 40 min with NaOH solution (15 mL, 0.05 mol L⁻¹) on an immersion oscillator (180 rpm). The supernatants from the elution step were collected to measure the content of nitrogen and phosphorus. At the same time, the prepared material was regenerated using NaOH (0.1 mol L⁻¹). Every experiment was performed in triplicate.

The effect of the solution pH (1.0–6.0) on the purity of the obtained PPPs was analyzed in this experiment. Simultaneously, the influence of the incubation time (5–60 min) and crude polypeptide concentration (6–22 mg mL⁻¹) was also investigated to study the adsorption model. The micro-Kjeldahl and Molybdenum blue colorimetric method (GB/T 5009.87-2003) were used to determine the content of N and P, respectively. The content of protein was calculated with the N-protein conversion factor of 6.25. In order to assess the purity of the obtained PPPs, the nitrogen and phosphorus molar ratio (N/P) was calculated using formula (1). At the same time, the adsorption capacity of MMTD was calculated using formula (2).

where q is the molar ratio of N and P of the final obtained PPPs; C_N and C_P are the quality content (mg g⁻¹) of N and P in the PPPs which were purified using MMTD. 31 is the relative molecular mass of P, while 14 is the relative molecular mass of N.where Q_e is the adsorption capacity at equilibrium (mg g⁻¹), C_o and C_e are the initial and equilibrium concentrations (mg mL⁻¹), respectively. V_o (mL) is the initial volume of the solution and W (g) is the mass of MMTD.

2.5 Reusability of MMTD in the process of purification

In order to evaluate the reusability of MMTD, the adsorption and desorption cycles were repeated twenty times using the same batch of MMTD at its optimal enrichment conditions. The recycling process was as follows: when the adsorption equilibrium was reached, the composites of MMTD–PPPs were washed with distilled water to remove foreign peptides and then eluted using NaOH (0.05 mol L⁻¹, 15 mL). After the desorption equilibrium, the obtained supernatant which contained PPPs was separated using a sand core funnel and MMTD was regenerated with NaOH (0.1 mol L⁻¹). The content of N and P in the purified PPPs were calculated, this process was repeated three times to assess the performance of MMTD, together with the adsorption capacity and recovery of MMTD. The values of the N/P and adsorption capacity were calculated using formula (1) and (2), respectively. The recovery was measured by the weight of MMTD.

3. Results and discussion

3.1 The characteristics of MMTD

In order to identify the surface chemistry, FT-IR spectra of pure TiO₂, as-prepared MMTD without calcination and calcined MMTD were all collected. In the spectrum of pure TiO₂ (Fig. 1), only one broad peak, between 532 cm⁻¹ and 700 cm⁻¹, was detected which typically corresponds to the vibration of Ti–O–Ti.^31,32 According to the spectrum of MMTD without calcination, the peaks at 3170 cm⁻¹ and 1624 cm⁻¹ were attributed to the stretching vibration of the hydroxyl and carbonyl groups, and the peak at 1400 cm⁻¹ was ascribed to the bending vibration of C–H. Moreover, the broad peak from 540 cm⁻¹ to 700 cm⁻¹ which corresponds to the Ti–O–Ti vibration was also observed. Compared with the spectrum of the as-prepared MMTD, only the Ti–O–Ti vibration peak existed in the spectrum of synthesized MMTD calcined at 550 °C. It could be concluded that the hydroxyl, carbonyl and other extra groups were removed during calcination, which meant the synthesis was successful. In addition, based on the FT-IR spectra, it could be concluded that the organic matter in the as-synthesized MMTD was the chelate products of citric acid and the hydrolysates of tetrabutyl titanate.

Fig. 1 FT-IR spectra of (a) pure TiO₂, (b) as-prepared MMTD without calcination, and (c) synthesized MMTD calcined at 550 °C.

The N₂ adsorption–desorption experiments of synthesized MMTD and as-synthesized MMTD without calcination were conducted to investigate the textural properties of the materials. The N₂-sorption isotherms and corresponding pore size distribution curves are shown in Fig. 2. It is shown that the isotherms of the as-synthesized and calcined MMTD were both assigned to type IV, which commonly suggests the existence of a mesoporous framework. Moreover, the hysteresis loops were respectively type H4 and H3, associated with slit shape pores. The pore volume and surface area of the as-synthesized MMTD were found to be 0.35 cm³ g⁻¹ and 368 m² g⁻¹, while for calcined MMTD they were 0.26 cm³ g⁻¹ and 63 m² g⁻¹. Compared with that of the as-synthesized MMTD, the average pore diameter of calcined MMTD was broadened and mainly centered at 11.7 nm (inset of Fig. 2b). The decrease in the surface area and broadening of the pore size distribution suggest the presence of a partial collapse during the calcination. The main reason was probably that the mesostructured system was not very regular and was mostly generated from the stacking of self-assembly nanoparticles. A careful observation revealed that the hysteresis loop of the synthesized MMTD appeared at a high relative pressure ranging from 0.5 to 1.0, indicating that the quantity of secondary porosity of large pores (macropores) was considerable, which could be demonstrated using SEM. It should be noted that the macro-mesoporous structure could transport objects to the framework binding sites efficiently, which would enhance the binding capacity of MMTD to PPPs with molecules in the range of 1000 Da to 3000 Da.

Fig. 2 N₂ adsorption–desorption isotherms and pore size distribution profiles (inset) of (a) as-prepared MMTD without calcination and (b) synthesized MMTD after calcination at 550 °C for 4 h.

The morphological properties of synthesized MMTD were detected using SEM. As shown in Fig. 3, the synthesized MMTD exhibited a relatively uniform macroporous structure. Fig. 3a (the front view of MMTD at the magnification of 6000×) shows that the MMTD contained regular arrays of macropores with pore sizes ranging from 0.80 μm to 2.10 μm. Simultaneously the thicknesses of the walls were 0.75–1.80 μm. In addition, the macrochannels were mainly of a one-dimensional orientation, arranged parallel to each other, perpendicular to the tangent of the material surface, which could be explicitly seen from the profile view of the prepared MMTD (Fig. 3c and d). Moreover, from the close observation (Fig. 3b), at the magnification of 35 000×, it could be deduced that the macroporous walls with irregular wormlike mesopores consist of interconnected self-assembly nanoparticles, which was consistent with the results of the N₂-sorption measurements. Besides, the macro-mesoporous structure was preserved integrally after calcination at 550 °C, which demonstrated good thermal stability of the prepared MMTD. It was noticeable that the diameter of the prepared MMTD was nearly 22 μm which is efficient in isolation. At the same time, MMTD was formed spontaneously in the absence of surfactant through the combined effects of microphase separation and organic acid chelation. Therefore the simplicity of the synthesis process would make MMTD feasible for large-scale preparation.

Fig. 3 SEM images at different magnification and views of the synthesized MMTD calcined at 550 °C. (a) Front view of MMTD at the magnification of 6000×. (b) Close observation of MMTD at the magnification of 35 000×. (c) and (d) are the side view of MMTD at the magnification of 4000×.

3.2 Effect of pH on the adsorption

MMTD is an amphoteric compound and can act as Lewis acids or bases by adjusting the solution pH, hence pH was considered to have an important influence which would affect the affinity of PPPs in the adsorption process. It is known that the N/P is considered as a criterion for the purity of PPPs, which could identify the length of the peptide and the density of the phosphoric group (the lower the N/P, the higher the purity of the PPPs). Therefore, the N/P was used as a crucial index to assess the effect of the pH on the adsorption. Fig. 4 shows that the lowest value of the N/P appeared at pH 3.00. Once the pH value was changed, the N/P would increase. Therefore, the optimal pH of the medium for the enrichment of PPPs on MMTD was 3.00, at which PPPs were firmly adsorbed by MMTD with the highest purity.

Fig. 4 Effect of pH on the N/P of obtained PPPs.

The zeta potential of synthesized MMTD was studied to clearly account for the influence of the pH on the adsorption. Fig. 5 shows that the zero point of charge (pzc) of this amphoteric oxide was around 5.23. According to Zhang,²³ the pzc of purified PPPs was around 2.70. Hence there would be electrostatic repulsion between MMTD and PPPs when the pH value was lower than 2.70 or higher than 5.23, and the negatively charged phosphopeptides would not be adsorbed onto MMTD. When the pH value was between the isoelectric points of PPPs and MMTD, it was supposed that the titanium surface with positive charges (Ti⁴⁺) would adsorb the anionic phosphate groups of PPPs through electrostatic binding. It could be concluded that at pH 3.00 the electrostatic interaction between PPPs and MMTD was the strongest, and MMTD possessed the highest binding affinity for phosphate groups. Besides, according to Jenkins,³³ the pK_a values of aspartic acids in different isomers were 3.55, 3.04 and 3.50, hence foreign peptides with aspartic acid would not be adsorbed onto MMTD at pH 3.00. As a result, PPPs with phosphate groups could be effectively captured from crude polypeptides by MMTD at pH 3.00.

Fig. 5 Zeta potential of the synthesized MMTD.

3.3 Adsorption kinetics of MMTD

To further explore the capture efficiency of synthesized MMTD, the adsorption kinetic curves for PPPs on MMTD were studied at 25 °C (16 mg mL⁻¹ of crude polypeptides). As shown in Fig. 6, the adsorption capacity of synthesized MMTD increased rapidly within 10 min and the N/P (molar ratio) decreased from 60.49 (the N/P of crude peptides) to 8.29, mainly due to the amount of active binding sites in the first 10 min. After 20 min, the adsorption level showed no significant changes and reached an equilibrium with a binding capacity of 36.47 mg g⁻¹, which was four times higher than the value previously obtained by Zhang.²³ The higher adsorption capacity of MMTD was ascribed to the superior interconnected pore system and active surface sites of the TiO₂/diatomite composite.

Fig. 6 (a) Adsorption curve for PPPs on MMTD, and (b) the N/P of obtained PPPs.

In order to better describe the adsorption behavior, kinetics models of pseudo-first-order and pseudo-second-order were chosen to evaluate the adsorption process.

The pseudo-first-order equation:

The pseudo-second-order equation:

In the above equations, Q_e (mg g⁻¹) stands for the adsorption capacity at the equilibrium and Q_t (mg g⁻¹) is the adsorption capacity at time t. K₁ and K₂ refer to the rate constants of the pseudo-first-order and pseudo-second-order kinetics models of the adsorption process, respectively.

By plotting 1/Q_t was versus 1/t according to the pseudo-first-order kinetics model, a straight line was obtained in Fig. 7a. Fig. 7b was obtained by plotting t/Q_t versus t. At the same time, the parameters of the adsorption kinetics for PPPs on synthesized MMTD were listed in Table 1. As shown in Table 1, the correlation coefficient (R²) of pseudo-second-order kinetic model was higher than the pseudo-first-order kinetic model. Simultaneously the pseudo-second-order equation matched well with the experimental data as the dynamic parameter of Q_e (38.91 mg g⁻¹) was consistent with the experimental value of Q_e (36.47 mg g⁻¹). So the adsorption process was more suitably described by the pseudo-second-order kinetics model. From the above results, it could be concluded that the intraparticle diffusion and boundary layer diffusion were the main rate-controlling steps for the enrichment of PPPs onto MMTD.³⁴

Fig. 7 Adsorption kinetics for PPPs on synthesized MMTD. (a) Pseudo first-order kinetics, the linear plot of 1/Q_t vs. 1/t. (b) Pseudo second-order kinetics, the linear plot of t/Q_t vs. t.

Table 1 Adsorption kinetics parameters for PPPs on MMTD

Kinetics models	Equations	Qe (mg g^−1)	K1 (min^−1)	R^2
Pseudo-first-order	y = 0.1181x + 0.0231	43.29	5.1125	0.8843
Pseudo-second-order	y = 0.0257x + 0.0664	38.91	9.95 × 10^−3	0.9964

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3.4 Adsorption isotherms of PPPs on MMTD

To clearly understand the adsorption properties and mechanisms, adsorption isotherms were successfully constructed with a series of initial concentrations of crude polypeptides (6.0, 10.0, 14.0, 16.0, 18.0, and 22.0 mg mL⁻¹) at 25 °C. Simultaneously, the N/P was also measured to detect the purity of the obtained PPPs. As shown in Fig. 8, the adsorption capacity of MMTD for PPPs increased with the increasing crude polypeptide concentration. When the initial crude polypeptide concentration reached 16.0 mg mL⁻¹, the value of the N/P decreased to 5.4 and remained relatively stable, whereas the adsorption capacity still increased. It was reported that the molecular sizes and quantities of the phosphate groups among PPPs were different.⁷ A study by Kweon showed that TiO₂ preferentially enriched multiple phosphorylated peptides.³⁵ When the initial concentration of peptides was low, PPPs with single or multiply phosphate groups were all adsorbed on the abundant active sites of MMTD, as well as the non-phosphorylated peptides. Therefore, the N/P of the obtained PPPs was relatively high. With the increase of polypeptide concentration, PPPs with multiple phosphate groups were specifically enriched by MMTD and the value of the N/P decreased rapidly until the crude polypeptide concentration reached 16.0 mg mL⁻¹. On enhancing the concentration, the adsorption capacity of the PPPs still increased and on the whole the N/P remained stable. It could be deduced that multiply and singly phosphorylated peptides were adsorbed at the same time to form a multilayer adsorption. Besides, more singly phosphorylated peptides were adsorbed since there were no more redundant active sites on MMTD for multiple phosphate groups. In order to better describe the adsorption isotherms and reveal the accumulation pattern of PPPs onto MMTD, Langmuir and Freundlich models were adopted.³⁶ The equations were expressed as follows.where Q_e (mg g⁻¹) and C_e (mg mL⁻¹) are the adsorption capacity and initial concentration at equilibrium, respectively. Q_m (mg g⁻¹) is the maximum adsorption capacity, and K_L (mL mg⁻¹) is the Langmuir constant referred to as the adsorption energy. K_F is the Freundlich constant, and is an indicator of adsorption intensity.

Fig. 8 (a) Adsorption isotherm of MMTD for PPPs, and (b) the N/P of obtained PPPs.

The equations and coefficient correlations (R²) for the Freundlich and Langmuir models are listed in Table 2. It was found that the adsorption properties of MMTD for PPPs were better fitted to the Freundlich model rather than the Langmuir model (evidenced by the higher correlation coefficient for the Freundlich model). It was noticeable that the Freundlich model was generally used for non-ideal adsorption systems and mainly described the formation of multilayers on adsorbents with infinitely available sites. Besides, the hierarchical porous structured MMTD possessed different types of active sites, therefore the adsorption behavior of MMTD for PPPs belongs to a multi-molecular layer.

Table 2 Langmuir and Freundlich adsorption parameters of PPPs on MMTD

Langmuir equation	R^2	Freundlich equation	R^2
y = 0.0093x + 0.3395	0.9854	y = 5.6335x^0.6661	0.9983

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3.5 Reusability of the synthesized MMTD

Considering the practical applications, repeated adsorption–desorption experiments were successfully carried out twenty times using the same batch of MMTD. The performance of the synthesized MMTD was evaluated by the N/P of obtained PPPs, along with the adsorption capacity and recovery of MMTD. As shown in Fig. 9a, the value of the N/P was maintained steadily around 5.66 in the repeated experiments, indicating the high purity of the obtained PPPs. Furthermore, it also illustrated the strong affinity between PPPs and MMTD. Simultaneously, it could be observed that the adsorption capacity of MMTD for PPPs was still above 35 mg g⁻¹ after being repeated twenty times. It meant that the synthesized MMTD was very stable and had good resistance to acid and alkali solutions. Additionally, the recovery of MMTD was also investigated by measuring the weight of MMTD every few repetitions, which could evaluate the loss of MMTD in the repeated experiment. According to Fig. 9b, there was a slight weight loss of MMTD after the adsorption–desorption experiments, which was probably induced by the process of transferring MMTD from the sand core funnel to the centrifuge tube. The process should be carefully controlled to maintain a higher recovery of MMTD in repeated adsorption–desorption processes. In conclusion, the synthesized MMTD was very stable and could be used in large-scale affinity separation due to its good binding capacity and satisfactory reusability.

Fig. 9 Reusability of the synthesized MMTD. (a) The N/P of obtained PPPs and adsorption capacity of MMTD in the repeated experiments. (b) Recovery of MMTD in the repeated adsorption–desorption experiments.

4. Conclusions

Hierarchical MMTD with good stability, strong binding capacity and satisfactory repeatability was prepared using a feasible template-free method. Simultaneously, the synthesized MMTD was successfully applied as an adsorbent in the purification of PPPs for the first time. The specially interconnected porous structure made a great contribution to the binding capacity of PPPs onto MMTD. In addition, the purity of the obtained PPPs was relatively high with a N/P of 5.4, and the performance of the material was excellent in the repeated experiments. Hence it is believed that this work is the starting point for investigating applications of MMTD in the purification of PPPs from hydrolysates of EYP, and more complex biomacromolecules which are well worth exploring in further studies.

Acknowledgements

This work was financially supported by the National High Technology Research and Development Program of China (863 Program) (No. 2013AA102207).

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