Phenylboronic Acid Functionalized Adsorbents for Selective and

Aug 15, 2018 - (1−3) Currently, lactulose can be produced by chemical-based ... Before functionalization, AR-0 was first swelled thoroughly with DMF...
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Food and Beverage Chemistry/Biochemistry

Phenylboronic Acid Functionalized Adsorbents for Selective and Reversible Adsorption of Lactulose from Syrup Mixtures Mingming Wang, Fayin Ye, He Wang, Habtamu Admassu, Yinghui Feng, Xiao Hua, and Ruijin Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02152 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Journal of Agricultural and Food Chemistry

Phenylboronic Acid Functionalized Adsorbents for Selective and Reversible Adsorption of Lactulose from Syrup Mixtures

Mingming Wang1, 2, Fayin Ye3, He Wang4, Habtamu Admassu1, 2, Yinghui Feng1,2, Xiao Hua1, 2*, Ruijin Yang1, 2*

1. State Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, China 2. School of Food Science and Technology, Jiangnan University, 214122 Wuxi, China 3. College of Food Science, Southwest University, 400715 Chongqing, China 4. Jiyang College, Zhejiang Agriculture and Forestry University, Zhuji, Zhejiang 311800, China

*Correspond to: Dr. Xiao Hua and Dr. Ruijin Yang Xiao Hua: [email protected] (X. Hua) Ruijin Yang: [email protected] (R. Yang)

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ABSTRACT: :Boronate affinity materials have been widely used for enrichment of

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cis-diol molecules. In this work, phenylboronic acid functionalized adsorbents were

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prepared via a simple and efficient procedure by grafting phenylboronic acid groups

4

onto amino macroporous resins. Elemental analysis has confirmed the successful

5

functionalization of AR-1M and AR-2M with approximately 2.17% and 0.73% weight

6

percentage of boron. Comparatively, AR-1M possessed higher lactulose adsorption

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capacity ( , 84.78±0.95 mg/g dry resin) under neutral condition (pH=7), while

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the introduced glutaraldehyde spacer arms on AR-2M resulted in excellent adsorption

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selectivity (α~23), high adsorption efficiency (π~22%), and fast adsorption/desorption

10

rate. The purity of lactulose ( ) through pH-driven adsorption (pH 7-8) and

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desorption (pH 1.5) can be effectively improved depending on the ratio of lactulose to

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lactose. When lactulose: lactose ≥1:1,  ~95% was achieved. No significant drop

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in  (>90%) was observed after ten-consecutive repeats. Results demonstrated

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that the newly developed method may achieve satisfactory performance in lactulose

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purification.

16 17

KEYWORDS: Lactulose, phenylboronic acid functionalized adsorbent, adsorption

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selectivity, pH-responsive, recyclability

19 20 21 22 23 24 25 2

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INTRODUCTION

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As a valuable lactose-originated non-digestive disaccharide, lactulose has been

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commonly used in pharmaceuticals, nutraceuticals and food industries due to its

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well-documented prebiotic properties.1-3 Currently, lactulose can be produced by

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chemical-based isomerization of lactose (industrial scale) or through enzymatic-based

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synthesis (lab-scale) using β-galactosidase, Caldicellulosiruptor saccharolyticus

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cellobiose 2-epimerase (CsCE), etc.4-8 However, undesirable by-products of

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monosaccharides and residual lactose remained in the isomerization syrup mixture

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will most often lead to rising the costs associated with downstream lactulose

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purification, since high-purity lactulose is mandatory for its medical uses and food

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applications, especially for those who are lactose intolerance.1, 4, 9

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Purification processing is a key operation for high-purity lactulose production.

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Although monosaccharide can be efficiently removed from raw lactulose syrup by

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using membrane separation, the fractionation of lactulose and lactose from

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isomerization mixture is a challenging task due to the complexity of the syrup and the

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highly structural similarity among them. Recently, several separation technologies,

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such as methanolic crystallisation procedure10,

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(SC-CO2) with alcohol-type cosolvent technique12, pressurized liquid extraction

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(PLE)13 and room temperature ionic liquids (ILs)14, 15 approach have been applied to

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obtain high-purity lactulose. However, those methods, efficient as they are, may not

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appropriate for industrial process due to their sophisticated and tedious procedures,

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considerable energy and organic solvent consumption. Very recently, a new innovative

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strategy for lactulose and oligosaccharide purification has been developed through

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selective fermentation of monosaccharides and lactose with Saccharomyces cerevisiae

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and Kluyveromyces marxianus.9, 16 However, the final product was still a mixture of

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, supercritical carbon dioxide

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lactulose and oligosaccharides.

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Boronate affinity based technique has been extensively used in the selective capture

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and enrichment of cis-diol molecules (carbohydrates, nucleosides, glycoconjugates

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and etc) from complex samples.17-23 The high selectivity inherits from the unique

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chemical properties of boronic acids, which can reversibly form complexation with

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cis-1,2- or cis-1,3-diols to generate 5- (stable) or 6-membered (less stable) cyclic

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esters.17, 24 Furthermore, lactulose with typical cis-1,2-diol units shows higher binding

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affinity than lactose with boronic acid groups (Supplementary Figure 1), which would

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facilitate the separation of lactulose and lactose.25 To promote solid phase separation

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and enhance the reusability of boronic acid groups, it generally requires immobilizing

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this functional group on insoluble solid supports.26 Macroporous adsorption resins

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(MARs) with high specific surface areas, large pore volumes (adsorption capacity),

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regular and tunable pore sizes, and low cost, have been studied extensively and used

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widely as excellent supports for industrial adsorption.26-28 Moreover, MARs generally

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possess stable and interconnected frameworks with active pore surfaces, which make

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them possible for further modification or functionalization with functional groups.26

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The grafting of functional boronic acids onto MARs may somewhat favors the

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selective capture of target lactulose from isomerization mixtures.

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In the present work, a simple and effective modification procedure for the synthesis of

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phenylboronic acid functionalized adsorbents (AR-1M and AR-2M) was established

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by grafting phenylboronic acid groups onto amino macroporous resins (AR-0).

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Physical characterization and adsorption/desorption properties (adsorption/desorption

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kinetics and adsorption selectivity) of phenylboronic acid functionalized adsorbents

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towards lactulose from isomerisates were investigated. The feasibility of adsorbent

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recyclability and stability in ten consecutive capture/release cycles was also explored. 4

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MATERIALS AND METHODS

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Materials. Aminated poly (styrene-co-divinylbenzene) resin (AR-0, ~6.4 mmol/g dry

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resin of -NH2 functional groups) was kindly donated by Tianjin Nankai Hecheng S&T

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Co., Ltd. (Tianjin, China). 3-Aminophenylboronic acid (APBA, purity 98%),

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p-formylphenylboronic acid (p-FPBA, purity 95%), sodium cyanoborohydride

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(NaBH3CN, purity 95%), N,N-dimethylformamide (DMF, purity 99.8%) and HPLC

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grade (purity>98%) of lactulose, lactose and epilactose were purchased from

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Sigma-Aldrich (Shanghai, China). All other chemicals were of analytical grade and

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obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

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Preparation of Phenylboronic Acid Functionalized Adsorbents. Two synthetic

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protocols were used to functionalize poly(styrene-co-divinylbenzene) resins with

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phenylboronic acid groups as depicted in Fig.1. Before functionalization, AR-0 was

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first swelled thoroughly with DMF for 24 h. Then for route one, p-FPBA was

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covalently bound to AR-0 via the formation of Schiff’s base between the formyl

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groups of p-FPBA and the amino groups on the surface of AR-0.22, 23, 26 As to route

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two, AR-0 was activated first by grafting of glutaraldehyde, and then the activated

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AR-0 was chemically modified with APBA. Finally, sodium cyanoborohydride power

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was added portionwise to reduce the Schiff’s base and the phenyboronic acid

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functionalized adsorbents AR-1M (route 1) and AR-2M (route 2) were obtained after

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washed with DMF, ethanol and deionized water and air-dried.

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Characterization of Phenylboronic Acid Functionalized Adsorbents. Fourier

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transform infrared spectroscopy (FT-IR) measurements of AR-0, AR-1M and AR-2M

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were carried out on a Thermo Nicolet IS10 spectrometer (Nicolet, USA). X-ray

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photoelectron spectroscopy (XPS) measurements for elemental analysis (C, N and B)

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were performed using an AXIS Ultra DLD spectrometer (Shimadzu/Kratos, Japan). 5

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The surface morphology of AR-0, AR-1M and AR-2M was observed with scanning

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electron microscopy (SEM, 5.0 kV, SU1510; Hitachi Co., Japan).

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Selective Adsorption of Lactulose and Lactose. The densities of phenylboronic acid

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groups (µmol/g dry resin) on AR-1M and AR-2M was determined by calculating the

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content of p-FPBA (λmax 256 nm) and APBA (λmax 293 nm) on a UV-3600

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spectrometer (Techcomp, Shanghai, China) before and after the formation of boronic

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ester27, 28, respectively. Unless otherwise stated, the adsorption studies were conducted

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as follows: 1.0 g (dry mass) of the phenyboronic acid functionalized adsorbents was

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added to 25 mL of lactulose and lactose binary solution (Lu:La=1:1, ×5 mg/mL) in a

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100 mL Erlenmeyer flask. The initial pH values of the syrup ranged from 6.0 to 10.0

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were adjusted by 0.1 M HCl/NaOH solution. The mixture was then shaken (150 r/min)

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at 10-45oC for 12 h and samples were withdrawn at varying predetermined times

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intervals for further analysis.

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The equilibrium adsorption capacity in adsorption experiment,  (mg/ g dry resin),

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was calculated according to Eq. (1):  = −   ×

 1 

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where and  are the initial and equilibrium substrate concentrations (mg/mL),

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respectively.  refers the volume of syrup solution (mL), and  is the dry mass (mg)

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of the adsorbents.

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The adsorption capacity at contact time (t) in adsorption experiment,  (mg/ g dry

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resin), was calculated as:  = −   ×

 2 

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where  represents the substrate concentrations (mg/mL) at contact time (min/h).

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Adsorption selectivity (α) and adsorption efficiency (π, %) of adsorbents towards 6

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lactulose from Lu-La binary solution were quantitatively defined as Eqs. (3) and (4): α=

 ⁄ 3  ⁄ 

π % = 100 ×

1000 ×  4  × 342.30

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where  and  (mg/g dry resin) denote respectively the equilibrium adsorption

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capacities of lactulose and lactose.  and  are the equilibrium concentrations

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(mg/mL) of lactulose and lactose, respectively.  represents the content of

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phenylboronic acid groups (µmol/g dry resin) on adsorbents.

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pH-driven Desorption of Lactulose and Lactose from Adsorbents. After the

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adsorption equilibrium was reached, pH-driven desorption of lactulose and lactose

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from adsorbents was carried out as follows: the adsorbate-loaded AR-1M/AR-2M was

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filtered and then desorbed with 25 mL pre-treated deionized water (pH 1-5, adjusted

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by 1M HCl/NaOH) in 100 mL Erlenmeyer flask. Subsequently, the flask was

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incubated at 25oC on a rotary shaker (150 r/min) for 2 h. Samples were withdrawn at

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certain time intervals for further HPLC analysis.

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The desorption capacity of lactulose and lactose ( , mg/g dry resin) and desorption

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ratio (D, %) were defined to assess the desorption efficiency:  =  ×

 5 

% =  ×

 × 100% 6

−  

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where  is the concentrations (mg/mL) of lactulose and lactose in the desorption

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solution.  is the volume (mL) of the desorption solution.

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Selective Capture of Sugars by Phenylboronic Acid Functionalized Adsorbents

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from Syrup Mixtures. AR-1M and AR-2M were tested for selective adsorption of

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lactulose, lactose, fructose, glucose and epilactose from four syrup solutions: (1) 7

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lactulose-lactose binary mixture (Lu:La=1:5, 1:2.5, 1:1, 2:1, 3:1, 4:1 and 5:1, ×5

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mg/mL); (2) fructose-glucose binary mixture (Fru:Glu=1:1, ×5 mg/mL); (3)

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fructose-glucose-lactulose-lactose mixture (Fru:Glu:Lu:La=1:1:1:1, ×5 mg/mL); and

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(4) enzymatic reaction mixture (Lu:Epi:La=6.35:1.58:4.0, mg/mL) by using CsCE.6, 25

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The pH values of these four different syrup solutions were adjusted to 8.0 by adding

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0.1 M NaOH/HCl. Adsorption and desorption experiments were performed in an

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identical manner as described above. The aliquots were sampled at different time

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intervals and sugar concentrations were determined with HPLC analysis.

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Initial purity ( " , %) and desorption purity (" , %) were defined as Eqs. (7) and (8)

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in order to quantitatively evaluate the adsorption and desorption selectivity of

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AR-1M/AR-2M towards different sugars from syrup solutions.  " % =

" ×  × 100% 7 

" % =

" ×  × 100% 8 

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" and " refer the initial and desorption concentration (mg/mL) of certain sugar,

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respectively.  and  are, respectively, the total amount (mg) of sugars in the

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initial and desorption solutions.

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Recyclability of Phenylboronic Acid Functionalized Adsorbents towards

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Lactulose. The reusability of AR-1M/AR-2M in sugar capture was investigated in

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ten-consecutive adsorption/desorption cycles. Briefly, 1.0 g of AR-1M/AR-2M (dry

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mass) was added to 25 mL of Lu-La binary solution (Lu:La=1:1, ×5 mg/mL, pH 7.0

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for AR-1M and pH 8.0 for AR-2M) in a 100 mL Erlenmeyer flask. The mixture was

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shaken (150 r/min) for 2 h at 25oC, and then the adsorbents were filtered. The

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supernatant was collected for further HPLC analysis. Next, desorption process was

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performed as described above (pH 1.5, 25oC, for 2 h). The separated adsorbent was 8

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washed three times by deionized water. After air-dried overnight, the adsorbent was

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used for sugar adsorption once again. The above adsorption/desorption cycles were

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repeated for ten consecutively times.

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HPLC Method for Sugar Analysis. Quantification of sugars concentration was

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performed on a Hitachi L-2000 HPLC system (Hitachi Co., Japan) equipped with an

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RI detector, using Asahipak NH2P-50 4E chromatographic column (4.6 mm×250 mm;

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Showa Denko K.K, Japan).29 CsCE reaction mixture (lactulose, epilactose and lactose)

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was analyzed using a Waters Alliance e2695 HPLC system (Waters Co., USA)

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equipped with an RI detector (Waters 2414; Waters Co., USA) and Shodex VG-50 4E

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chromatographic column (4.6 mm×250 mm; Shodex, Tokyo, Japan).27, 28

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Statistical Analysis. All data expressed in this study were reported as mean±SD

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(standard deviation). Each value represents the mean for three independent

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experiments performed in duplicate with average standard deviations not exceeding

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5%. SPSS statistical software 22.0 (SPSS Inc., Chicago, IL, USA) was used to

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perform the statistical analysis.

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RESULTS AND DISCUSSION

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Characterization of Phenylboronic Acid Functionalized Adsorbents. FT-IR

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spectra of AR-0, AR-1M, p-FPBA (Fig.2a) and AR-2M, APBA (Fig.2b) were used to

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verify the successful grafting of phenylboronic groups. N-H stretching vibration at

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3440 cm-1, 1501 cm-1, 903 cm-1 and C-N stretching vibration at 1108 cm-1 in both

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AR-0, AR-1M and AR-2M indicated the presence of amino groups.24, 25 In Fig.2a and

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b, it was found that both p-FPBA and APBA showed a characteristic stretching

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vibration at 1342 cm-1, which is corresponded to B-O stretching of the phenylboronic

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groups.18, 30, 31 When compared with AR-0, the adsorption band around 1342 cm-1 was

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emerged for both AR-1M and AR-2M. The obtained results could indirectly prove that 9

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the phenylboronic groups have been successfully introduced to AR-1M and AR-2M

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through the amidation reaction.21-23

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Chemical bonding of phenylboronic groups onto AR-1M and AR-2M surface was

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further confirmed by XPS analysis. As shown in Fig.3, N 1s signal appeared at ~400

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eV was observed for both AR-0 (Fig.3a), AR-1M (Fig.3c) and AR-2M (Fig.3e), but

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only AR-1M (Fig.3d) and AR-2M (Fig.3f) exhibited the typical B 1s peak observed at

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184-194 eV. Specifically, the peak emerged at 190.04 eV was corresponded to the

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typical trigonal sp2-hybridized neutral B-species of the phenylboronic acid.19, 31 These

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data indeed confirmed the success coupling of phenylboronic groups onto adsorbents

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through boronic ester formation method.30, 31

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The morphologies of the adsorbents were characterized by SEM. It was observed that

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AR-0 showed a compact structure with a smooth surface (Fig.4a), whereas the pure

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resin sample exhibited a rough surface, which occurs due to solvent swelling effect

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(Fig.4b). The rougher surface also stated that the inner amino groups were much more

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exposed, thus enabling the subsequent amidation reactions. In addition to AR-0,

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AR-1M (Fig.4c) and AR-2M (Fig.4d) also had a rough surface accompanying by

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pores with well distributed tiny holes, while all the skeletons retained their porous

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structure and showed nearly uniform particle size (~300 µm). The changes on the

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surface morphology would be beneficial for grafting more phenylboronic acid groups

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which could obviously promote sugars capture.

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Adsorption Properties of Phenylboronic Acid Functionalized Adsorbents. The

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adsorption capacities of AR-1M (Fig.5a) and AR-2M (Fig.5b) on lactulose ( )

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and lactose ( ) were gradually increased with the increasing of phenylboronic

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acid groups due to the reversible covalent interaction between phenylboronic acid

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groups and cis-diol compounds. More importantly, as depicted in Fig.S1, the binding 10

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affinity of phenylboronic acid for lactulose (4-O-β-D-galactosyl-D-fructose) and

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fructose was much higher than that of lactose (4-O-β-D-galactopyranosyl-D-glucose)

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and glucose because of the typical cis-1,2-diol unit supplied by lactulose and

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fructose,25 which can be easily linked with boronic acid group and to form a stable

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5-membered boronic ester ring.17, 24 This is evidenced in Fig.5 that the increasing in

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 was much higher compared with that of  . The discordance in adsorption

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performance for lactulose and lactose could obviously increase the adsorption

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selectivity (α) according to Eq.(3) and can be a principle of separation lactulose from

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complex syrup. Furthermore, as depicted in Fig.1, the residual amino groups (-NH2,

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~3,800 µmol/g dry resin) of AR-1M could cause nonspecific adsorption21, besides, the

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residual amino groups might also participate in the adsorption of cis-dilos (lactulose

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and lactose) through the B-N coordination.21, 32, 33 Those obviously exert influence on

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 and α. Whereas for AR-2M, the adsorption performance was well maintained

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because of the presence of the interaction between amino groups and glutaraldehyde.

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Moreover, the introduced flexible chains of glutaraldehyde (spacer arms) on AR-2M

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were obviously favorable for boronate esterification with cis-diol compounds.

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Therefore, it can be seen from Fig.5 that AR-2M not only has a superior adsorption

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selectivity but also displays a higher adsorption efficiency (α=7.64, π~15.4% at

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phenylboronic acid content of ~750 µmol/g dry resin) than AR-1M (α=2.27, π~11.2%

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at phenylboronic acid content of ~2,600 µmol/g dry resin).

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Accordingly, the weight percentages of the major elements in the phenylboronic acid

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functionalized adsorbents were listed in Table 1. The mass concentrations of boron in

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AR-1M (~2600 µmol of phenylboronic acid groups) and AR-2M (~750 µmol of

237

phenylboronic acid groups) were ~2.17% and ~0.73%, respectively. Comparatively,

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the weight percentage of boron in AR-1M was almost 3 times higher than that of 11

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AR-2M. Such a drastic decline in the content of boron could be reasonably attributed

240

to the successful grafting of bifunctional glutaraldehyde (spacer arms) on AR-2M

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(Fig.1, route 2), which may exist in two types: resin-NH2-glutaraldehyde-NH2-resin

242

and resin-NH2-glutaraldehyde-phenylboronic acid group. These observations and

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explanations provide insight that phenylboronic acid was presumably the sole

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functional group of AR-2M, which might result in high lactulose selectivity, but failed

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to increase the lactulose adsorption capacity.

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Adsorption pH is a critical factor for boronate affinity. As stated in previous reports,

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the binding of boronic acid groups (usually phenylboronic acid groups) to cis-1,2-diol

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compounds occurred in two steps: phenylboronic acid group was first transformed to

249

tetrahedral anionic form [-B(OH)4-] (sp3) at alkaline conditions, and [-B(OH)4-] (sp3)

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subsequently bonded with cis-1,2-diol units to form a stable 5-membered cyclic ester

251

(Fig.1).17, 30, 34 Given that most boronic acids were generally weak acids having a pKa

252

value of 8-9 (pKa of 8.7-8.9 for phenylboronic acid), it means that at pH≤ the pKa

253

value, most of the boronic acids still existed in the form of trigonal [-B(OH)2] (sp2),

254

which cannot react with cis-diol groups.21, 24 Based on this principle, phenylboronic

255

acid functionalized adsorbents required a basic pH (usually the pH should be> the pKa

256

value of the phenylboronic acid ligand) for binding.35 This is in good agreement with

257

the results obtained from AR-2M. As described above, phenylboronic acid was the

258

sole functional group on AR-2M, thus  of AR-2M was definitely pH-dependent

259

and dramatically increased from ~5 to ~85 (mg/g dry resin) when the pH increased

260

from 6.0 to 8.0 and then kept almost unchanged (Fig.6b). Interesting,  of

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AR-2M was almost constant regardless the pH applied, this further confirmed that the

262

phenylboronic acid was the sole functional group of AR-2M. However, for AR-1M

263

(Fig.6a),  was significantly decrease from approximately 60 mg/g dry resin at 12

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pH 6.0 to about 30-40 mg/g dry resin at pH 7.0-10.0. The reason for this decline may

265

due to the influence of the pH affecting residual amino groups (-NH2), which is

266

stronger for lactose (through nonspecific adsorption) than for lactulose (through

267

reversible covalent binding with phenylboronic acid groups). Moreover, no obviously

268

change on  was observed as the pH increased from 6.0 to 10.0, this might be

269

reasonably ascribed to the influence of residual amino groups, which could form an

270

alkaline microenvironment around the surface of AR-1M. Besides, as illustrated in

271

Fig.1, the residual amino groups of AR-1M may also react with the trigonal [-B(OH)2]

272

(sp2) and to form a B-N coordination complex (regard as Wulff-type boronic acids).32

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B-N coordination complex with a low pKa value was obviously favors the adsorption

274

of cis-diols under neutral pH conditions.21, 32, 33 This interesting phenomenon is a great

275

advantage compared with traditional boronate affinity materials (such as AR-2M),

276

because it allows the selective adsorption of lactulose under neutral condition, which

277

is more favorable for industrial applications. Further studies are needed to understand

278

the positive adsorption of cis-diols through the B-N coordination.

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Fig.6c and 6d provides the effect of adsorption temperature on sugars capture. It was

280

shown that  of AR-1M was significantly (p96%, respectively. The same trend was also observed in AR-2M. Such a rapid 16

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reduction in  could be largely attributed to the inhibitory effect of the load of

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more lactulose molecules at higher ratio of lactulose to lactose, since the binding

366

affinity of phenylboronic acid for lactulose (cis-1,2-diol unit) is much higher than that

367

of lactose.25 However, Fig.S2 shows that both  and  increased with the

368

concentration of lactulose and lactose at fixed Lu-La ratio of 1:1, which further

369

confirming the decisive effect of the ratio of lactulose to lactose on lactulose selective

370

capture. Moreover, it can be seen from Table 2A that AR-2M exhibited a lower 

371

but a much higher  at fixed Lu-La ratio compared with AR-1M, demonstrating

372

again that the introduced glutaraldehyde spacer arms on AR-2M can enhance the

373

adsorption selectivity towards lactulose but failed to improve the adsorption capacity

374

due to the reduction in phenylboronic acid groups.

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Structure difference between lactulose (4-O-β-D-galactosyl-D-fructose) and lactose

376

(4-O-β-D-galactopyranosyl-D-glucose), namely the fructose moieties of lactulose

377

with typical cis-1,2-diol unit is responsible for their different binding affinity with

378

boronic groups.17, 22, 24 This explanation can be further supported by using fructose

379

and glucose as the model substrates. Table 2B clearly shows that a higher binding

380

affinity towards fructose was observed as expected at 1:1 ratio of fructose to glucose,

381

which ultimately resulted in a much higher &' for AR-1M (79.17±3.25%) and

382

AR-2M (90.45±5.47%).

383

Considering the fact that commercial lactulose syrup produced from chemical based

384

isomerization is a mixture of monosaccharides (i.e. fructose and glucose) and

385

disaccharides (e.g. lactulose and lactose).1,

386

mixtures of fructose-glucose-lactulose-lactose at a ratio of 1:1:1:1 was formulated to

387

investigate the applicability of AR-1M and AR-2M to selective adsorption of target

388

lactulose from the chemical isomerisates. Apparently, selective enrichment of

4

Herein,a model syrup consisting in

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389

lactulose and fructose from isomerisates by using AR-1M and AR-2M are presented

390

in Table 2C. As a result, &' and  were remarkably increased from 25% to

391

43.47±1.25%, 38.83±2.09% for AR-1M and 45.35±2.97%, 45.44±2.02% for AR-2M,

392

respectively. More importantly, small molecules such as monosaccharides can be

393

efficiently separated from lactulose syrup by using nanofiltration (NF) due to different

394

molecular weight cut-offs,37, 38 thus the affect of fructose on the selective adsorption

395

of AR-1M and AR-2M towards lactulose can be remarkably diminished through prior

396

NF process, implying that both AR-1M and AR-2M are still satisfactory in purifying

397

real isomerisates.

398

Table 2D claims the selective enrichment effect of AR-1M and AR-2M towards

399

lactulose from real CsCE-catalyzed reaction mixture, which contains lactulose,

400

epilactose and residual lactose at the ratio of 6.35:1.58:4.00 (×g/L).  was

401

considerably increased from ~53% to 75.75±1.43% for AR-1M and 85.95±0.59% for

402

AR-2M, respectively. More importantly, both of the loaded lactose and epilactose

403

expect for lactulose could be released from adsorbents by using deionized water

404

(pH~5.8) because of their weak binding strength with boronic groups and/or the

405

nonspecific adsorption with amino groups (-NH2).21 Therefore, a higher  * could

406

be obtained for both AR-1M (91.58±1.50%) and AR-2M (>98%) after first desorbing

407

with deionized water. The above results exhibited a good practicability of AR-1M and

408

AR-2M for real lactulose-enriched syrup.

409

Recyclability of Phenylboronic Acid Functionalized Adsorbents towards

410

Lactulose. From the viewpoint of practical applications, regeneration and

411

recyclability of phenylboronic acid adsorbents are other important features. Thus the

412

reutilization potential of AR-1M and AR-2M was evaluated in ten-consecutive

413

pH-driven adsorption/desorption cycles. As illustrated in Fig.8, no dramatic loss of 18

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414

lactulose adsorption capacity was observed and the recovery was still up to ~90%

415

after 10-repeats. The slight reduction in adsorption capacity was probably attributed to

416

the cleavage of some unreduced Schiff’s base (-CH=N-) (Fig.1) under desorption

417

acidic condition (pH96

>96

50.80±0.76

53.18±1.77

80.68±3.10

97.33±2.07

102.33±2.86

116.21±2.23

1.36±0.23

0.61±0.41

ND

ND

ND

ND

92.75±3.70

95.67±1.92

>98

>98

>98

>98

(B) fructose-glucose mixture (Fru:Glu=1:1, ×5 mg/mL). Fru:Glu  &' (%) AR-1M &' (mg/g) ,- (mg/g) ()./) (%) * AR-2M &' (mg/g) ,- (mg/g) ()./) (%) *

1:1 50 68.50±2.49 56.49±3.54 79.17±3.25 37.25±3.01 8.91±0.76 90.45±5.47

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(C) fructose-glucose-lactulose-lactose mixtures (Fru:Glu:Lu:La=1:1:1:1, ×5 mg/mL). Fru:Glu:Lu:La

1:1:1:1 25 46.79±1.95 34.18±2.27 37.15±2.57 4.42±0.96 43.47±1.25 9.19± ±1.76 38.83± ±2.09 8.51± ±0.87 27.30±3.40 7.01±0.59 32.84±2.47 0.67±0.28 45.35±2.97 5.18± ±0.39 45.44± ±2.02 4.04± ±0.24

 &' ,  ,- ,   ,   AR-1M

AR-2M

(%) &' (mg/g) ,- (mg/g)  (mg/g)  (mg/g) ()./) ) * (%) 12) ()* (%) ) ()+) ) * (%) ()+3 ) * (%) &' (mg/g) ,- (mg/g) 45 (mg/g) 46 (mg/g)

()./) ) * (%) 12) ()* (%) ) ()+) ) * (%) ()+3 ) * (%)

(D) CsCE reaction mixture (Lu:Epi:La=6.35:1.58:4.00, ×mg/mL). Lu: Epi: La 6.35:1.58:4.00 (g/L) 53.23   (%) 97.74±3.95 AR-1M  (mg/g) 14.78±1.13 789 (mg/g)  (mg/g) 40.20±2.07 75.75± ±1.43 ()+) ( %) ) * * 91.58±1.50 ()+) ( %) ) * 98.68±2.88 AR-2M  (mg/g) 10.17±1.34 789 (mg/g) 15.14±3.25 46 (mg/g) +) 85.95± ±0.59 ()* (%) ) * >98 ()+) ( %) ) *  *: desorption purity of lactulose from AR-1M and AR-2M after first washed with deionized water (pH 5.8~6.0).

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Figures Fig. 1.

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Fig.2.

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Fig.3.

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Fig. 4. (a)

(b)

(c)

(d)

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Fig. 5.

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Fig.6.

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Fig.7.

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Fig.8.

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Graphic for table of contents

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