Synergistic Effect of a Thermal Reaction and Vacuum Dehydration on Improving Xylose-Phenylalanine Conversion to N-(1-Deoxy- d -xylulos-1-yl)-phenylalanine during an Aqueous Maillard Reaction

Article abstract of DOI:10.1021/acs.jafc.8b04448

The synergistic effect of a thermal reaction and vacuum dehydration on the conversion of xylose (Xyl) and phenylalanine (Phe) to a Maillard-reaction intermediate (MRI) was researched. The yield of N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine was successfully improved and increased from 13.62 to 47.23% through the method combining a thermal reaction and vacuum dehydration. A dynamic process was involved in the transformation of Xyl and Phe (Xyl-Phe) to N-substituted d-xylosamine and in the transformation of N-substituted d-xylosamine to N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine during the initial stage of dehydration; then, only the transformation of N-substituted d-xylosamine to N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine occurred during the final stage. Furthermore, the MRI was prepared under optimized conditions (90 °C and pH 7.4), and the obtained MRI was characterized and confirmed by ESI mass spectrometry and NMR.

Full text of DOI:10.1021/acs.jafc.8b04448

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Food and Beverage Chemistry/Biochemistry
Synergistic Effect of Thermal Reaction and Vacuum Dehydration
for Improving Xylose-Phenylalanine Conversion to N-(1-deoxy-D-
xylulos-1-yl)-phenylalanine during an Aqueous Maillard Reaction
Heping Cui, Khizar Hayat, Chengsheng Jia, Emmanuel
DUHORANIMANA, Qingrong Huang, Xiaoming Zhang, and Chi-Tang Ho
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04448 • Publication Date (Web): 06 Sep 2018
Downloaded from http://pubs.acs.org on September 8, 2018
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Page 1 of 36
Journal of Agricultural and Food Chemistry
Synergistic Effect of Thermal Reaction and Vacuum Dehydration for Improving
XyloseꢀPhenylalanine Conversion to Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine
during an Aqueous Maillard Reaction
§
†
†
Heping Cui †,‡, Khizar Hayat , Chengsheng Jia , Emmanuel Duhoranimana ,
Qingrong Huang ‡, Xiaoming Zhang †*, ChiꢀTang Ho ‡*
†
State Key Laboratory of Food Science and Technology, School of Food Science and
Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, P. R.
China.
‡
Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick
08901, NJ, USA.
§ Department of Food Science and Nutrition, College of Food and Agricultural Sciences,
King Saud University, P. O. Box 2460, Riyadh 11451, Saudi Arabia.
Author information
* Corresponding Author: Xiaoming Zhang & ChiꢀTang Ho
(1) Xiaoming Zhang, Ph.D., Professor
Postal address: State Key Laboratory of Food Science and Technology, School of
Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, Jiangsu
214122, People’s Republic of China. Eꢀmail: xmzhang@jiangnan.edu.cn (X. Zhang).
Tel.: +86 510 85197217. Fax: +86 510 85884496.
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(2) ChiꢀTang Ho, Ph.D., Professor
Postal address: Department of Food Science, Rutgers University, 65 Dudley Road,
New Brunswick 08901, NJ, USA. Eꢀmail: ho@aesop.rutgers.edu
Heping Cui, Ph.D.
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4
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Postal address: School of Food Science and Technology, Jiangnan University, Lihu
Road 1800, Wuxi, Jiangsu 214122, People’s Republic of China. Eꢀmail:
cuihepingdavid@126.com
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Khizar Hayat, Ph.D.
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Postal address: Department of Food Science and Nutrition, College of Food and
Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi
Arabia. Eꢀmail: khayat@ksu.edu.sa
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Chengsheng Jia, Ph.D.
Postal address: School of Food Science and Technology, Jiangnan University, Lihu
Road 1800, Wuxi, Jiangsu 214122, People’s Republic of China. Eꢀmail:
chshjia@126.com
Emmanuel Duhoranimana, Ph.D.
Postal address: School of Food Science and Technology, Jiangnan University, Lihu
Road 1800, Wuxi, Jiangsu 214122, People’s Republic of China. Eꢀmail:
emmadu2020@yahoo.fr
Qingrong Huang, Ph.D.
Postal address: Department of Food Science, Rutgers University, 65 Dudley Road,
New Brunswick 08901, NJ, USA. Eꢀmail: qhuang@aesop.rutgers.edu
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ABSTRACT
The synergistic effect of thermal reaction and vacuum dehydration on the
conversion of xylose (Xyl) and phenylalanine (Phe) to Maillard reaction intermediate
(MRI) was researched. The yield of the Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine
was successfully improved and increased from 13.62% to 47.23% through the method
combining thermal reaction and vacuum dehydration. A dynamic process was involved
in the transformation of Xyl and Phe (XylꢀPhe) to Nꢀsubstituted Dꢀxylosamine and
Nꢀsubstituted Dꢀxylosamine to Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine at the
initial stage of dehydration; then only the transformation of Nꢀsubstituted Dꢀxylosamine
to Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine at the final stage. Furthermore, the MRI
was prepared under the optimized conditions (temperature 90 °C and pH 7.4), and the
obtained MRI was characterized and confirmed by ESIꢀmass spectra and NMR.
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KEY
WORDS:
Synergistic
effect;
Maillard
reaction
intermediate;
Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine; Transformation
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Chemical compounds studied in this article
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dꢀxylose (PubChem CID: 135191); lꢀphenylalanine (PubChem CID: 6140); Ethanol
(PubChem CID: 702); Deuteroxide (PubChem CID: 24602).
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INTRODUCTION
Maillard reaction is a cascade of complex and competitive reactions involving
amino compounds and reducing sugar during food processing leading to structural
changes and flavor formation of food. 1ꢀ3 The important Maillard reaction intermediates
(MRIs) known as Amadori or Heyns rearrangement products (ARPs or HRPs) occur in
the early stage of Maillard reaction, condensation of aldose with amine occurs, leading
to the formation of a labile Nꢀsubstituted amino sugar such as Nꢀsubstituted
4, 5
glycosylamine and Nꢀsubstituted fructosamine.
Specifically, after the nucleophilic
addition, the Schiff base of glycosylamine (or fructosamine) rearranges via
6
1,2ꢀeneaminol which leads to the glycation products of ARPs or HRPs . These MRIs
7, 8
widely exist in both wholefood and processed foods,
they are consequently
9ꢀ11
considered as potential natural food ingredients;
and their extensive application
11ꢀ14
prospect was proposed.
Especially, for the trend that using traditional cooking and
minimal processing conditions has become an attractive approach to generate authentic
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flavor profiles resonating with consumers’ demand for more naturalness,
a good
control of reaction steps to generate the stable MRIs adapted to the thermal processes is
required. 16 Flowerꢀlike flavor compounds were formed during the heat treatment of the
ARP derived from xylose and phenylalanine (XylꢀPhe), and this ARP could be used for
16
fresh process flavors of bakery foods . Thus, it is significant to prepare the XylꢀPhe
derived ARP and there is a need for its characterization at a molecular level. 12, 13
Water is a common solvent for Maillard reaction and Maillard flavorings production.
17ꢀ19
However, previous researchers have reported that the ARP hydrolysis could occur
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in aqueous solution during heating, generating free sugar and amino acid.
In order
to increase the ARP yield, in this study, vacuum dehydration combined with thermal
reaction was employed to improve the transformation from Nꢀsubstituted Dꢀaldosamine
to the ARP derived from XyleꢀPhe (Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine). The
ARP derived from Xyl and Phe was synthesized through two stages: (i) refluxing at a
constant temperature in aqueous solution; (ii) and continued thermal reaction with
vacuum dehydration treatment. When the vacuum dehydration was conducted after the
time when the ARP optimally formed, the dehydration process would result in the
secondary reaction during the excessive reaction. Thus, the reaction time of stage (i)
was shortened from the optimized time, to maintain the total preparation time equal to
the optimized time. The change in concentration of Nꢀsubstituted Dꢀaldosamine and the
conversion of XylꢀPhe to ARP during the thermal reaction combined with vacuum
dehydration was researched. Synergistic effect of thermal reaction and vacuum
dehydration
for
improving
xyloseꢀphenylalanine
conversion
to
Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine was proposed. Additionally, the relationship
between the ARP yield and the water content during dehydration was evaluated, and the
mechanism of Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine yield improvement was
discussed. Furthermore, the molecular structure of the ARP derived from Xyl and Phe
was characterized to support the effectiveness of ARP preparation through the
synergistic effect of thermal reaction and vacuum dehydration.
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MATERIALAND METHODS
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Chemicals. lꢀphenylalanine, dꢀxylose, anhydrous ethanol and deuteroxide were
purchased from SigmaꢀAldrich Chemical Co. Ltd (Shanghai, China). Formic acid,
acetonitrile, 2ꢀ(2,3,4ꢀtrihydroxybutyl)ꢀquinoxaline (97%) and oꢀphenylenediamine were
obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Standard ARP
(Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine) (98%) was prepared in our lab.
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Preparation of ARP. Model system (5 g) of Xyl and Phe mixture in a 2:1 relative molar
ratio of Xyl to Phe, was solubilized in 80 mL deionized water. The solution pH was then
adjusted to different values (5.9ꢀ7.9) with the NaOH solution at the concentration of 3
mol/L. The reaction was divided into two stages. Firstly, the solution was heated in a
water bath for refluxing at a constant temperature of 90 °C, and then the atmospheric
pressure inside the bottle was decreased to 25 mbar in 5 min using rotary evaporator
under vacuum (Rꢀ215, Büchi, Flawil, Switzerland). After dehydration at 90 °C for 15
min, the mixture was immediately cooled in ice water. The obtained solid product was
dispersed in anhydrous ethanol (40 mL), and rotary evaporated under vacuum at 30 °C
for 30 min (the vacuum pressure was above 0.07 MPa). The objective of sample
dispersion in anhydrous ethanol was to form azeotropic system of waterꢀethanol, so that
the residual water could be removed at low boiling point using rotary evaporation, to
stabilize the prepared Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine.
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Moisture measurement. All the ingredients with known moisture content were mixed
in a certain ratio, thus, initial moisture of the system was accordingly calculated. The
weight loss of the system after rotary evaporation was roughly regarded as the decrease
of the water weight. The moisture content of the system was expressed as the weight
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percentage of remained water to the solution.
Purification of the ARP. The preliminary purification of ARP was performed using the
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method of Cui et al. . The ARP solution was dried by rotary evaporator at 45 °C, and
the obtained solid was mixed with 500 mL anhydrous ethanol. The undissolved
components were filtered off and the solution was dried using a rotary evaporator again,
then the remaining solid of ARP was dissolved in water. The obtained ARP was further
purified using the method of Davidek et al. 22 with slight modifications. A column filled
with Dowex 50WX4 ion exchange resin in H+ꢀform was selected for purification of the
ARP. The column was firstly eluted with water till the eluent showed negative to
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2,3,5ꢀtriphenyltetrazoliumchloride (TTC) test.
Then the ARP was eluted with
ammonium hydroxide (0.2 mol/L). The fractions containing the ARP were further
purified using semiꢀpreparative RPꢀHPLC. The sample was eluted at 1.0 mL/min by
linear gradient from 2 to 100% acetonitrile/0.1% formic acid over 18 min; and C18
RPꢀHPLC (10 ꢁm, 22 × 200 mm) column was used. The recovery percentage of the
purified ARP was 65%.
Analysis of ARP by HPLC. The method of Cui et al. 16 for ARP analysis was employed
for the analysis of ARP. The ARP was dissolved in water (80 mL). The resulted aqueous
solution was filtered using polyvinylidene fluoride filters (PVDF, 0.22 Millipore) before
injection into RPꢀHPLC system. UV detection was performed at 215 nm with a photo
diodeꢀarray detector 2996 (Waters, Milford, MA, USA). The elution conditions were
similar to those of semiꢀpreparative RPꢀHPLC. The XꢀSelect C18 RPꢀHPLC (3.5 ꢁm, 4.6
× 150 mm) column was used for the analysis.
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The ARP in the sample was quantified based on calibration curve of purified
product (y = 1.1039 x﹣2.8301, R2 = 0.9971). The yield of ARP was calculated as the
percentage of the measured molar concentration of ARP to the initial molar
concentration of phenylalanine.
Mass Spectrometry analysis of Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine,
Nꢀsubstituted
Dꢀxylosamine
and
and
deoxyosone.
When
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine
Nꢀsubstituted
Dꢀxylosamine
simultaneously existed in the sample, they were hardly separated through HPLC. Thus,
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Maillard reaction intermediates were often analyzed using mass spectrometry. In this
research, purified ARP, Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine and Nꢀsubstituted
Dꢀxylosamine in the reaction solution were analyzed using QꢀTOF MS spectrometry
(Waters Synapt QꢀTOF MS, USA). Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine and
Nꢀsubstituted Dꢀxylosamine in the samples were quantified based on calibration curve
of purified ARP, as the ARP was stable to be prepared and purified, additionally
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine and Nꢀsubstituted Dꢀxylosamine appeared
in the same peak in the chromatogram. The UPLCꢀESIꢀMS system with positive ESI
mode mass spectra was used to obtain the spectra and a Waters Acquity PDA detector
was used. The ionization conditions were as follows: capillary voltage 3.5 kV, cone
voltage 20 V, and extractor voltage 7 V. The source block temperature and the
desolvation temperature were 100 °C and 400 °C, respectively. The cone gas flow was
adjusted to 50 L/h. For the analysis of Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine and
Nꢀsubstituted Dꢀxylosamine in the reaction solution, MS detection was in multiple
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reaction monitoring (MRM) mode performed with the m/z 316, 298 for Nꢀsubstituted
Dꢀxylosamine, and the m/z 298, 280 for Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine.
For the analysis of purified ARP, MS detection was in full scan mode over the range of
m/z 20ꢀ1000 with 1 s scanning time and an interꢀscanning delay of 0.1 s. The
quantification was based on the identification of the peaks of Phe (m/z 166 and 120) and
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine (m/z 298 and 280). Furthermore, to
facilitate protonation of Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine required for the
detection by ESI+ꢀMS, water containing 0.1% formic acid was used as an eluent. The
CSH C18 (1.7 ꢁm, 2.1 × 100 mm) column was used for Ultra Performance Liquid
Chromatography (UPLC) analysis. Analytical UPLC conditions on LCꢀMS were as
follows: flow rate was 0.3 mL/min, and UV detector was set at 200ꢀ600 nm. The
samples were analyzed by linear gradient from 2 to 100% methanol /0.1% formic acid
over 20 min by directly injecting the sample. The samples were analyzed using an
autosampler, and the injection volume was 1 ꢁL. Data were acquired using Mass Lynx
software (version 4.1, Milford, MA, USA).
Additionally, the analysis of deoxyosone was performed using the method of
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Kocadaǧlı et al. with some modification.
Briefly, the sample of Maillard reaction
solution (100 ꢁL) was mixed with 0.05 mol/L oꢀphenylenediamine solution (200 ꢁL).
The mixture was placed in dark at 25 °C for 2 h before being filtered using
polyvinylidene fluoride filters (PVDF, 0.22 Millipore). Then the resulted solution was
injected into HPLC system. The HPLC column was LiChrospher C18 (250 mm × 4.6
mm, 5 ꢁm, and the UV detection was performed at 320 nm. The samples were analyzed
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using an autosampler, and the injection volume was 10 ꢁL. Mobile phase A was 0.1%
formic acid aqueous solution, while mobile phase B was methanol. The flow rate of the
mobile phase was 1 mL/min, and the samples were analyzed by linear gradient: 0ꢀ35
min, B 35%ꢀ70%; 35ꢀ40 min, B 70%ꢀ35%, 40ꢀ50 min, B 35%. The ionization
conditions were as follows: capillary voltage 3.5 kV, cone voltage 20 V, and extractor
voltage 7 V. The source block temperature and the desolvation temperature were 100 °C
and 400 °C, respectively; the cone gas flow was adjusted to 50 L/h. MS detection was in
full scan mode over the range of m/z 20ꢀ1000 with 1 s scanning time and an
interꢀscanning delay of 0.1 s. The quantification was based on the identification of the
peaks of deoxyosone derivative with oꢀphenylenediamine (m/z 205 and 187) and the
calibration curve of 2ꢀ(2,3,4ꢀtrihydroxybutyl)ꢀquinoxaline. Data were acquired using
Mass Lynx software (version 4.1, Milford, MA, USA).
NMR analysis of ARP. The purified ARP powder was dissolved in deuteroxide (500
ꢁL), and the mixture was then transferred to a 5 mm NMR tube for NMR analysis. The
analysis was performed on a Bruker DRX 400 MHz spectrometer (Bruker Bio Spin,
Ettlingen, Germany) equipped with a 5 mm PABBO probe and operated at 25 ºC (298
K). Data were obtained using MestReNova software (version 9.0.1, Mestrelab Research,
Escondido, CA, USA).
Statistical Analysis. The results were presented as mean values ± standard deviation.
Each reaction was done for three times and each sample was analyzed in triplicate.
SPSS version 19.0 (IBM, Armonk, NY, USA) was used for all statistical analyses.
Bivariate Pearson correlation analysis among the parameters tested was also conducted
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using SPSS. Duncan’s multiple range test was used, and p < 0.05 was considered as
significant.
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RESULTS AND DISCUSSION
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Effect of dehydration on ARP yield. According to Nursten (Chapter 2, Paragraph 8, 9)
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degradation of ARP. Thus, in this study, a temperature (80 °C) below the normal
Maillard reaction procedure (usually 100ꢀ180 °C) was used for ARP preparation, with
or without dehydration. The yields of Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine at
different dehydration temperature are shown in Fig. 1.
Without dehydration, the yield of ARP increased up to 13.62% at 80 °C with
extended reaction time (Fig. 1). The optimal yield of ARP prepared by dehydration at
50 °C was 13.78%, which was similar to that prepared at 80 °C without dehydration
(13.62%) (Fig. 1). However, the yield of ARP prepared by only dehydration at 50 °C
without the first reaction stage (reaction time t = 0 min) reached 7.45% and was higher
than that obtained after reacting at 80 °C for 20 min of reaction without dehydration
(Fig. 1). These results indicated that dehydration for 15 min increased the yield of ARP.
Moreover, dehydration at 80 °C increased the optimal yield to 20.21%, which was much
higher than the optimal yield obtained with dehydration at 50 °C (13.78%) (Fig. 1).
These results demonstrated that dehydration was important for yielding the ARP from
XylꢀPhe during the Maillard reaction in aqueous medium, and the increase of
temperature could further improve the yield. Thus, in order to improve the ARP yield,
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an increased temperature was needed for efficient dehydration in the following research.
When the ARP was prepared at 80 °C without dehydration, the yield of
Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine was only 13.62%, and Nꢀsubstituted
Dꢀaldosamine, the precursor of Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine, was
observed
in
heated
XylꢀPhe
aqueous
solution
during
Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine preparation. It was probably because that
Nꢀsubstituted Dꢀaldosamine was labile in aqueous medium that it could either
disintegrate to regenerate the original sugar and amino acid or dehydrate to complete
Amadori rearrangement. 3, 21
Improvement of ARP yield by vacuum dehydration synergistically with thermal
reaction. The optimal yield of ARP during the reaction at 80 °C without dehydration
was 13.62%, and the optimal yield during the reaction at 80 °C and dehydration at
50 °C was 13.77% (Fig. 1). These results indicated that the yield increased by only
1.10% through the dehydration at low temperature. Results, showed that the optimal
yield of XylꢀPhe to ARP during the reaction at 90 °C without dehydration was 15.15%
(Fig. 2a), which has increased by 11.23% compared to the optimal yield during the
reaction at 80 °C without dehydration. However, the optimal yield during the reaction at
80 °C and dehydration at 80 °C was 20.21% (increased by 48.38% compared to the
optimal yield during the reaction at 80 °C without dehydration), and the optimal yield
during the reaction at 90 °C and dehydration at 90 °C was 47.23% (increased by
211.75% compared to the optimal yield during the reaction at 90 °C without
dehydration) (Fig. 1 and 2a). Both of the yield growth rates were much higher than the
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improvement by either increasing the temperature or combining with dehydration at a
low temperature. These results revealed that synergistic effect of thermal reaction and
vacuum dehydration for improving the ARP yield existed during the reaction. Hence,
the ARP yield could be improved through the adjustment of temperature and vacuum
during the Maillard reaction in aqueous medium.
According to previous studies, the activation energy of Amadori arrangement (33.5
kJ·molꢀ1) was found to be slightly greater than that of Schiff base formation (27.3
kJ·molꢀ1) and dehydration (31.3 kJ·molꢀ1) in Maillard reaction of Phe, which indicated
that the ARP generation was more sensitive to temperature and favored by high
21
temperature. Particularly under low moisture conditions, dehydration was favored by
rising temperature, which would probably improve Amadori arrangement 20. This
theory might account for the synergistic effect of thermal reaction and vacuum
dehydration for improving XylꢀPhe conversion to the ARP.
With dehydration at 80 and 90 °C, the yield of ARP increased firstly and then
decreased with increasing the reaction time (Fig. 1 and 2a). When the reaction
temperatures were 80, 90 and 100 °C, the optimal reaction time was 60, 40 and 30 min,
respectively (Fig. 1 and 2a). This result showed that the optimal reaction time for ARP
preparation was decreased when the reaction temperature was increased, which would
be an advantage in the industrial production of ARP. However, the optimal yield of
ARP was 47.23% when the reaction was at 90 °C, which was higher than that obtained
after the reaction at 100 °C (45.28%). It suggested that higher temperature expedited the
formation of ARP, meanwhile overreaction led to the disintegration of accumulated
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ARP. These results are in accordance with some previous researches,
which
reported that the reaction rate at the intermediate stage of Maillard reaction was
enhanced when reaction temperature increased above 90 °C, which resulted in the
formation of 1ꢀdeoxyglucosone, furfural, methylglyoxal, and others. Additionally,
increasing temperature could also promote the degradation of original sugar or amino
acid (the sugar or amino acid in reactants), and their reaction with the products at
7, 18, 20, 26
intermediate stage of Maillard reaction.
Accordingly, 90 °C was selected for
the reaction and dehydration when the effect of initial pH on the XylꢀPhe conversion to
ARP was evaluated.
pH 7.4 showed the optimal improvement in ARP yield, compared with the other
pHs in the pH range 5.9ꢀ7.9 (Fig. 2b). This result was similar to previously reported
27, 28
findings.
According to Mossine et al, 29 protonation of reducing sugar is necessary
for generating ARP. The Maillard reaction was initiated by the nucleophilic attack of the
amino group on the sugar to form a Schiff base, which is a precursor of ARP. 30 Besides,
reverse Amadori rearrangement was accelerated in aqueous solution heated during
29, 31
alkalization,
while the reverse rearrangement could be inhibited under neutral
29
condition. In addition, the rate of intermediate and final stages could be increased
32
under alkaline conditions, facilitating the secondary reactions.
On the other hand,
excessive protonation of amine group led to a decrease in glycation of amino acids at
low pH levels, which was not favorable to ARP generation. 3, 9, 33 Furthermore, the yield
30
of original sugar formed from ARP degradation was found to decrease at pH > 7,
whereas, a relatively neutral pH was favorable to ARP generation.
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Therefore, overtime reaction, excessively high temperature and pH were adverse to
improve the yield of ARP, and the optimal reaction conditions based on synergistic
effect of thermal reaction and vacuum dehydration were evaluated as follows: reaction
time of 40 min, reaction and dehydration temperature of 90 °C and the initial pH 7.4.
Relationship between the water content and XylꢀPhe conversion to intermediates
during dehydration with thermal reaction. Since the synergistic effect of thermal
reaction and vacuum dehydration was crucial on improving the ARP yield, the Maillard
reaction with vacuum dehydration was proposed as an effective method to prepare ARP
in aqueous medium, and the yield was increased from 13.62% to 47.23% (Fig. 1 and 2).
The dehydration with thermal treatment was critical during the intermediates (both of
Nꢀsubstituted
generation, and great influence of dehydration extent on the total intermediate
(including Nꢀsubstituted Dꢀxylosamine and
Dꢀxylosamine
and
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine)
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine) yield was observed (Fig. 1). The decrease
of water content resulted in the change of water activity (aW), which had a great
influence on the kinetics of ARP formation and degradation, especially change of aW
34
from 0.86 to 0.96. Additionally, higher aW had an unfavorable influence on the ARP
34
stability, and the reducing sugar degradation probably occurred at a high aW. Thus,
the effect of moisture change on intermediates generation during the dehydration with
thermal reaction was evaluated.
During the dehydration, the moisture content of the reaction system sharply
decreased with dehydration time and after 20 min it reached zero percent (Fig. 3).
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313
314
315
Meanwhile, the yield of ARP also increased up to the climax at also 20 min dehydration
time (Fig. 3). The Pearson’s correlation was applied to see the relationship between the
water content and the yield of ARP, and strong negative correlation was observed in the
results (r = ꢀ0.902, p < 0.01). These results revealed that dehydration facilitated the ARP
yield, and the optimal dehydration time was 20 min. Additionally, in aqueous medium,
an
equilibrium
among
XylꢀPhe,
Nꢀsubstituted
Dꢀxylosamine
and
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine occurred during the Maillard reaction (eq.
1).
k2
k1
Phe + Xyl
Nꢀsubstituted Dꢀxylosamine
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine
+ H2O
kꢀ1
316
317
(eq. 1)
318
319
320
321
322
323
324
325
326
327
328
329
Based on the observed concentration of the intermediates at equilibrium, k1, k2 and kꢀ1
were calculated as 0.018, 20.48 and 55.87, respectively, according to the method
35
reported in previous study. The kꢀ1 and k2 were much greater than k1, suggesting that
Nꢀsubstituted
Dꢀxylosamine
formation,
but
not
the
21
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine formation, was rateꢀlimiting;
and this
rateꢀlimiting effect of Nꢀsubstituted Dꢀxylosamine formation was more remarkable with
25
the changes in reaction temperature.
These results were similar to the previous
reported research on the kinetic significance of the Schiff base reversion in the aqueous
35
Maillard reaction model of a phenylalanineꢀglucose. Therefore, increasing XylꢀPhe
conversion to Nꢀsubstituted Dꢀxylosamine was important for the improvement of
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine formation; and the formation of
Nꢀsubstituted Dꢀxylosamine was improved at the reaction and dehydration temperature
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334
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348
349
350
351
of 90 °C, which probably further accelerated Amadori arrangement.
Change of the intermediates concentration during the dehydration with thermal
reaction. Before the dehydration, the yield of ARP was only 10.03% (Fig. 3). By using
UPLCꢀMS/MS, the concentrations of Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine and
Nꢀsubstituted Dꢀxylosamine were measured as 58.92 mmol/mol and 39.28 mmol/mol,
respectively (Fig. 4). However, the concentration of Nꢀsubstituted Dꢀxylosamine
dropped to zero after 20 min dehydration (Fig. 4), and the concentration of
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine increased by 425%. According to the
Pearson’s correlation analysis, the Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine
concentration showed strong positive correlation with dehydration time (r = 0.913, p <
0.01). Moreover, the concentration of Nꢀsubstituted Dꢀxylosamine showed strong
negative correlations with dehydration time (r = ꢀ0.841, p < 0.01). On the other hand,
the Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine concentration (r = ꢀ0.885, p < 0.01)
and ARP yield (r = ꢀ0.902, p < 0.01) showed strong correlations with the moisture
content. These results suggested that the decrease of moisture at a thermally intensified
condition facilitated the generation of Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine.
Before
dehydration,
the
concentrations
of
both
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine and Nꢀsubstituted Dꢀxylosamine were low
(Fig. 4). At that time, the equilibrium (eq. 1) was favorable to the left side; Xyl and Phe
were the major components in the reaction system. During the dehydration, the
transformation of Xyl and Phe to Nꢀsubstituted Dꢀxylosamine and then to
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine was gradually improved. In the first 5 min
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dehydration, the concentration of Nꢀsubstituted Dꢀxylosamine was maintained steadily,
and the concentration of Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine was slightly
increased (Fig. 4). During dehydration (between 5ꢀ10 min dehydration time), significant
and rapid increase of Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine concentration was
observed, while the concentration of Nꢀsubstituted Dꢀxylosamine still remained steady
(Fig. 4). These results indicated that a plenty of formed Nꢀsubstituted Dꢀxylosamine was
transformed to Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine during this period of
dehydration. After 10 minutes dehydration, the concentration of Nꢀsubstituted
Dꢀxylosamine gradually decreased and reached 0 mg/mL at the 20th minute; meanwhile,
the improvement of Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine concentration receded
and its concentration reached to the apex (Fig. 4). Afterward,
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine concentration decreased slightly (Fig. 4),
due to the ended transformation of Nꢀsubstituted Dꢀxylosamine to
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine, and the subsequent degradation of
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine. Hence, twenty minutes was the most
optimal dehydration time for the transformation of Nꢀsubstituted Dꢀxylosamine to
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine. Due to the ARP concentration increased
during the dehydration, the generated deoxyosone also has increased (Fig. 4). However,
the concentration of deoxyosone increased by only 116.5%, while the concentration of
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine increased by 706.1%. These results
indicated that the ARP was accumulated during the dehydration.
Xyl was transformed into Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine through
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protonation, combination with Phe and dehydration (Fig. 6). Then, dehydration of
Nꢀsubstituted Dꢀxylosamine led to the formation of Schiff base which is the origin of
Amadori arrangement (Fig. 6). During the vacuum dehydration with thermal reaction,
Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine accumulation was improved through the
synergistic effect of thermal reaction and vacuum dehydration on a gradually dynamic
transformation promotion. At initial stage of dehydration, both the transformation of
XylꢀPhe to Nꢀsubstituted Dꢀxylosamine and Nꢀsubstituted Dꢀxylosamine to
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine were improved. Moreover, at the final
stage of dehydration, the dehydration rate was expectantly increased when the amount
of water became limited, 21 and the improvement of the transformation of Nꢀsubstituted
Dꢀxylosamine to Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine became dominant.
Additionally, during the vacuum dehydration with thermal reaction, the
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine could be hydrated to form Nꢀsubstituted
Dꢀxylosamine
in
aqueous
medium;
and
the
degradation
of
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine may also occur via hydrolysis, leading to
36
1ꢀdeoxyxylosone generation via retroꢀMichael reaction.
The hydrolysis could
probably be inhibited during dehydration, thus, the conversion of XylꢀPhe to
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine was improved and the yield of
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine increased.
Identification of prepared ARP. Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine was
prepared at the optimal conditions (temperature and pH) and then purified. In order to
confirm the obtained compound, the ARP samples before and after purification were
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417
analyzed using UPLCꢀMS/MS. Two peaks (at 3.89 and 4.30 min) were identified in
total ions chromatogram of the sample before purification (Fig. 5a) as Phe and ARP
derived from Xyl and Phe respectively. Whereas, after purification, the sample showed a
single peak at 4.28 min on the chromatogram (Fig. 5b). As shown in Fig. 5c and Fig. 7,
the fragmentation pattern observed was typical to the pattern reported in other previous
22, 37ꢀ39
researches.
The molecular mass and proposed molecular formula, were 297 Da
and C14H19O6N, respectively, calculated by Mass Lynx software. Then, the ARP
structure was further identified by NMR. The NMR analysis results showed the signals
in
the
expected
regions,
and
five
major
isomers
of
Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine were observed (1, 2ꢀα, 2ꢀβ, 3ꢀα, 3ꢀβ in Fig.
6). These isomers existed simultaneously in aqueous medium and could not be
separated through the procedure provided in the section of Purification of the ARP due
1
to their similar structure and chemical properties. The chemical shifts (δ values) of H
NMR and 13C NMR spectra are shown in the supplementary data, which are in good
agreement with what had been reported in previous studies. 40, 41
In the present work, the synergistic effect of thermal reaction and vacuum
dehydration was found to be crucial to improve the formation of ARP during aqueous
Maillard reaction. The reaction temperature and pH were optimized as initial pH 7.4 and
temperature 90 °C for reaction and dehydration, and the yield of MRI was improved
from 13.62% to 47.23% under these conditions. Furthermore, thermal reaction and
vacuum
dehydration
synergistically
improved
the
Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine yield through
a gradual dynamic
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431
432
433
434
435
436
437
438
439
transformation promotion: involving the transformation of XylꢀPhe to Nꢀsubstituted
Dꢀxylosamine
and
Nꢀsubstituted
Dꢀxylosamine
to
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine at first stage; and only the transformation
of Nꢀsubstituted Dꢀxylosamine to Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine at the
final stage. The obtained compound was characterized and confirmed by ESIꢀmass
spectra and NMR. The molecular mass and proposed molecular formula of the MRI
were 297 Da and C14H19O6N, respectively.
ACKNOWLEDGEMENTS
The research was supported by the National Natural Science Foundation of China
31671826, the National Key R&D Program of China 2017YFD0400105, National
Firstꢀclass Discipline Program of Food Science and Technology JUFSTR20180204, and
“Collaborative innovation center of food safety and quality control in Jiangsu
Province”.
SUPPORTING INFORMATION
Statement on the manuscript’s significance
COMPLIANCE WITH ETHICAL STANDARDS
Conflict of Interest
The authors declare no conflict of interest.
Ethical Approval
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441
442
443
444
445
446
This article does not contain any study with human participants or animals performed
by any of the authors.
Informed Consent
Not applicable.
Figure Color
No figures need to be colored in print.
447
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FIGURE CAPTIONS
Fig. 1. Effect of dehydration degree on the yield of ARP with initial pH 7.4,
reaction temperature 80 °C
Fig. 2. Effect of reaction temperature (a) and pH (b) on the yield of ARP on the
basis of increasing dehydration degree
(a: Initial pH 7.4, dehydration temperature 90 °C; b: reaction temperature 90 °C,
dehydration temperature 90 °C)
Fig. 3. Correlation between the water content and the yield of Nꢀsubstituted
Dꢀxylosamine and Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine during dehydration
under optimum reaction conditions (Initial pH 7.4, reaction temperature 90 °C, and
dehydration temperature 90 °C)
Fig. 4. Change in concentration of Nꢀsubstituted Dꢀxylosamine and
Nꢀ(1ꢀdeoxyꢀαꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine during dehydration
(Initial pH 7.4, reaction temperature 90 °C, and dehydration temperature 90 °C)
Fig. 5. Total ions chromatogram (a) and LCꢀMS/MS spectra (b) of the prepared
ARP
Fig. 6. Generation and isomerization of Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine
(Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀlꢀphenylalanine
Nꢀ(αꢀDꢀXylofuranosꢀ1ꢀyl)ꢀlꢀphenylalanine
Nꢀ(βꢀDꢀXylofuranosꢀ1ꢀyl)ꢀlꢀphenylalanine
Nꢀ(αꢀDꢀXylofuranosꢀ1ꢀyl)ꢀlꢀphenylalanine
Nꢀ(βꢀDꢀXylofuranosꢀ1ꢀyl)ꢀlꢀphenylalanine 3'ꢀβ).
1,
3ꢀα,
3ꢀβ,
3'ꢀα,
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Journal of Agricultural and Food Chemistry
Fig. 7. Fragmentation of Nꢀ(1ꢀdeoxyꢀDꢀxylulosꢀ1ꢀyl)ꢀphenylalanine ([M+H]+ 298,
280, 262, 252, 234) in the mass spectra
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