Carbohydrate Polymers 218 (2019) 126–135

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Enzymatic synthesis and characterization of maltoheptaose-based sugar T

esters
Phu Cuong Nguyena, My Tuyen Thi Nguyenb, Chang-Kyu Leec, Il-Nam Oha, Jae-Han Kimb,
⁎
Soon-Taek Honga, Jong-Tae Parka,c,
a
Department of Food Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea
b
Department of Food Nutrition, Chungnam National University, Daejeon 34134, Republic of Korea
c
CARBOEXPERT Inc., Daejeon 34134, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, maltoheptaose (G7)-based sugar esters were synthesized from maltoheptaose and fatty acids
Maltoheptaose (C10–C16) using a commercial lipase. With the exception of dimethyl sulfoxide (DMSO; 76.4%, w/v), G7
Sugar esters showed only limited solubility in organic solvents. Among the fatty acids, palmitic acid (PA) was the best
Esterification substrate for G7-based ester formation. G7-PA ester was successfully synthesized as the monoester structure
Emulsifier
exclusively in 10% DMSO of t-butanol with a 22% conversion yield. NMR and enzymatic analyses of the purified
Lipase
monoester product revealed that the ester bond in the G7 was located at C-6 of the glucose at the reducing end.
The G7-PA monoester showed the melting temperature at 56.3 °C that was 6.5 °C lower than that of the free PA
and exhibited a different endothermic pattern from the free G7. The G7-PA monoester exhibited excellent
emulsifier potential with more even droplet size distribution compared with the commercial sucrose esters for an
oil-in-water emulsion system.

1. Introduction and the ratio between the hydrophilic head (mono-, di-, oligo- or
polysaccharides) and the lipophilic tail (the chain length and amount of
Sugar fatty acid esters (SFAE) have been commonly used as drug saturated or unsaturated fatty acid) (Pedersen, Wimmer, Emmersen,
delivery agents in pharmaceuticals (Csoka, Marton, Zelko, Otomo, & Degn, & Pedersen, 2002; Zhang et al., 2015). The interest in the
Antal, 2007), and as emulsifiers, emollients, surfactants, and anti- synthesis of SFAE has increased significantly in recent years. In parti-
bacterial and antioxidant agents in foods and cosmetics (Khan & cular, numerous studies on the synthesis of mono- and disaccharides
Virendra, 2015; Lucarini et al., 2016; Meng, Sun, Fang, Chen, & Li, fatty acid esters have been reported (Inprakhon et al., 2017; Lee,
2014; Neta, Teixeira, & Rodrigues, 2015; Perinelli et al., 2018; Szuts & Guneev, & Walsh, 2017; Lin et al., 2016; Pappalardo, Boeriu, Zaccheria,
Szabó-Révész, 2012). They can be synthesized from renewable raw & Ravasio, 2017; Perinelli et al., 2018; Ren & Lamsal, 2017; Sebatini,
materials such as carbohydrates and fatty acids (FA) by both chemical Jain, Radha, Kiruthika, & Tamilarasan, 2016; Staroń, Dąbrowski,
and enzymatic esterification (Hidayat, Fitria, Supriyanto, & Hastuti, Cichoń, & Guzik, 2017). Some SFAE such as sucrose esters have become
2016; Inprakhon et al., 2017; Šabeder, Habulin, & Knez, 2006; Wan, popular on the surfactant market (Szuts & Szabó-Révész, 2012), while
Lim, & Hameed, 2017). The enzymatic pathway is much more attractive oligosaccharides are rarely used for the synthesis because of their
and eco-friendlier than the chemical method. It is carried out at a mild limitations such as low solubility in organic solvents. Nevertheless,
temperature, avoiding the degradation of the substrates and products oligosaccharide-based esters showed distinct properties such as high
during the reaction. Enzymes catalyze the reaction with a high speci- interfacial tension values (Chauhan, Sharma, & Sharma, 2013; van
ficity and region-selectivity, generating ester products with homo- Kempen et al., 2013), and stronger inhibitory effects on tumor cell lines
geneous structure and functionality (Gumel, Heidelberg, Heidelbergc, & compared to their mono- and disaccharides counterparts (Ferrer, Perez,
Chisti, 2011; Jiang et al., 2015; Plou et al., 2002; van Kempen et al., Plou, Castell, & Ballesteros, 2005). To the best of our knowledge, the
2013). feasibility of using oligosaccharides in a specific length greater than DP
The specific properties of SFAE depend on the degree of substitution 3 for the synthesis of sugar ester has not been evaluated to date. In a

⁎
Corresponding author at: Department of Food Science & Technology, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of
Korea.
E-mail address: jtpark@cnu.ac.kr (J.-T. Park).

https://doi.org/10.1016/j.carbpol.2019.04.079
Received 28 January 2019; Received in revised form 25 April 2019; Accepted 25 April 2019
Available online 29 April 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
P.C. Nguyen, et al. Carbohydrate Polymers 218 (2019) 126–135

previous study, using a packed-bed reactor, we have successfully pro- separation was conducted using the Dionex CarboPac PA1 column
duced high-purity maltoheptaose (G7) (Cuong et al., 2016) that can be (4 mm × 250 mm, Thermo Scientific) with a hyperbolic gradient of
a great source of oligosaccharides for new sugar esters synthesis. 0–600 mM sodium acetate containing 150 mM sodium hydroxide, and
G7 are linear α-1,4-glucans composed of 7 glucose units that can be then extrapolating to quantify the concentration of G7 from the stan-
employed in food, cosmetics, and pharmaceutical processing. G7 and dard curve that was determined using the G7 standards in the range of
their derivatives (ethylidene-4-nitrophenyl-α-D-maltoheptaoside) have 0.3–70 μg/mL.
been used as substrates in the α-amylase activity test kit (Badenoch &
Bais, 1989). It was reported that G7 exhibited encapsulation capacity 2.3. Enzymatic synthesis of maltoheptaose fatty acid esters and purification
for flavor/aroma in foods and cosmetics (Min et al., 2010). In addition,
G7 was used as a building block for the copolymer that was further To produce maltoheptaose-based sugar esters, the fatty acid with
applied in nanoencapsulation of gold (Otsuka et al., 2013; Zepon et al., various lengths, including capric acid (C10), lauric acid (C12), myristic
2015). It was also reported that G7 was used for the development of acid (C14), and palmitic acid (C16) were esterified with G7 (molar ratio
oligosaccharide biosensors (Bouchet-Spinelli et al., 2013). Overall, G7 sugar/FA: 1:5) by the lipase reaction. First, 0.1 mmol substrates were
is attracting increased attention from food, cosmetics industry, and prepared in 100-mL glass bottles with screw-caps and were dissolved in
nanotechnology sectors. 5 mL DMSO at 60 °C for 30 min using a magnetic stirrer (400 rpm).
For biocatalyst selection among the enzymes and proteins that Then, 45 mL t-butanol and 2 g molecular sieve (4 Å) were added to each
catalyze ester bond formation between acyl acceptor (carbohydrate) bottle, and the mixture was kept stirring for a further 15 min. Next,
and acyl donors (fatty acids), lipases are the most widely utilized for 0.5 mL enzyme solution (CALA) was added to the mixture and the re-
catalyzing this type of reaction (Gumel et al., 2011; Hidayat et al., action was conducted at 60 °C for 72 h with stirring at 400 rpm
2016; Jiang et al., 2015; Kobayashi, 2011; Yang & Ze-Lin, 2012). For throughout the incubation. At the end of the reaction, 400 μL of the
the enzymatic reaction, many factors contribute to the reaction yield solution was withdrawn to measure the concentration of unreacted FA
including the solubility of the substrates, carbohydrate to fatty acid and to identify the G7-FA ester products by using the HPLC-evaporative
molar ratio, the solvent type, the amount and source of the enzyme, light scattering detector (ELSD) and Matrix-assisted laser desorption/
temperature and reaction time (Gumel et al., 2011; Kobayashi, 2011; ionization time-of-flight (MALDI-TOF) measurements. All experiments
Pappalardo et al., 2017; Ren & Lamsal, 2017). Lipases are efficient were performed in triplicate.
catalysts for the hydrolysis of ester in aqueous media; however, in or- To purify esterification products, the mixture was filtered to remove
ganic solvents where water molecules are completely limited, they can molecular sieves after the completion of the reaction, and the filtrate
catalyze the reverse reaction (esterification). In addition, the yield of was concentrated under vacuum using a rotary evaporator (Eyela,
ester products is inversely proportional to the amount of water either Tokyo, Japan). Then, ester products and unreacted G7 were pre-
available in the reaction system or formed during the reaction. cipitated by adding n-hexane. The supernatant containing n-hexane and
In this work, we aimed to synthesize a novel oligosaccharide-based unreacted PA were eliminated after centrifugation at 20,000×g for
fatty acid ester from high-purity G7 and fatty acid by a lipase-catalyzed 5 min. The solid was suspended in acetone, and only G7-FA esters could
reaction. In addition, we attempted to elucidate the structure of the be dissolved in the supernatant. Finally, the supernatant was collected
oligosaccharide-based sugar ester along with physical properties char- and dried overnight in a hot-air oven at 60 °C.
acterization. Finally, the emulsion properties of the new sugar ester To investigate the effect of the concentration on the rate of the es-
were compared to those of commercial sucrose palmitate esters, which terification, G7 (0.1 mmol) and palmitic acid (PA) in different molar
is a common emulsifier in the industry. ratios sugar/FA (1:1; 1:5; 1:10; 1:20) were prepared using the steps
described above. In the purification steps, G7-PA ester was further
2. Materials and methods purified using a Sep-Pak C18 6 cc cartridge (Waters, Milford, MA, USA)
with the following protocol (1 mL of sample, 4 mL of distilled water,
2.1. Materials 4 mL of 50% methanol, 8 mL of 90% methanol) in order to obtain high
purity of the product. The fraction of 90% methanol was collected for
Candida antarctica lipase A (CALA), molecular sieves (4 Å beads) characterization experiments.
were purchased from Sigma. Maltoheptaose (G7) (> 90%) and mal-
todextrin standards were purchased from CARBOEXPERT Inc. 2.4. Analysis and characterizations
(Daejeon, South Korea). Capric acid (> 98%), lauric acid (> 98%),
myristic acid (> 99%), palmitic acid (> 95%), PS750 (Sucrose 2.4.1. HPLC- evaporative light scattering detector (ELSD)
Palmitate, HLB 16, monoester 75%), and β-amylase were purchased The reaction mixtures were analyzed using a Waters 1525 Binary
from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). DMSO-d6 HPLC system (Waters Corp.) equipped with an evaporative light scat-
(99.8%) used for NMR analyses was obtained from Eurisotop (Saint tering detector (Alltech 2000, Deerfield, IL, USA) and YMC-triart C18
Aubin, France). All the other chemicals used were of analytical grade. column (4.6 mm × 250 mm, YMCKOREA, Seongnam, Korea). The mo-
bile phase was water and acetonitrile with the following gradient
2.2. Determination of maltoheptaose solubilities system: water (A), acetonitrile (B) at a flow rate of 1 mL/min: 0 min
(50% B); 5 min (90% B);10 min (90% B); 20 min (100% B); 20.1 min
To determine the solubility of G7 in various organic solvents, G7 (50% B); 25 min (50% B). The injection volume was 20 μL, and the
powder (10–140 mg) was suspended in 300–500 μL of different solvents eluate was monitored by ELSD at a drift-tube temperature of 90 °C, with
(t-butanol, n-hexane, acetone, 2-butanone, dimethyl sulfoxide (DMSO), a carrier gas flow rate of 2.0 L/min, and the gain of the detector was set
and acetonitrile). Subsequently, the mixture was heated to 60 °C and at 1. The calibration curves for the G7-PA monoester were obtained
vortexed for 2 h in order to obtain the saturated solutions. Then, the using the purified products as described above (Section 2.3). The con-
mixture was centrifuged at 20,000×g for 5 min. The supernatant was version was defined as the molar ratio of the amount of G7-esters to that
collected, filtered through a 0.2-μm membrane filter from ADVANTEC of G7 at the beginning of the reaction.
(Tokyo, Japan) and diluted with distilled water prior to the analysis.
The initial concentration of G7 and the concentration of G7 in the su- 2.4.2. MALDI- TOF MS
pernatant were quantified using high-performance anion-exchange Mass spectrometry of G7-FA ester product was performed on an
chromatography (HPAEC) with pulsed amperometric detection (PAD) ultrafleXtreme instrument (Bruker Daltonics, Bremen, Germany)
(Thermo Scientific Dionex ICS-5000, Sunnyvale, CA, USA). G7 equipped with a smart beam laser (355-nm Nd: YAG), and 2,5-

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dihydroxybenzoic acid (DHB) was used as a matrix. To prepare the DHB water (O/W) food emulsions was evaluated and compared to the
matrix solution, 10 mg of DHB was dissolved in 0.2 mL of a solvent commercial sucrose palmitate (PS750). The emulsion was prepared
mixture of acetonitrile and water (1:1). Then, 1 μL aliquot of the sample with 0.25% of ester, and 10% of canola oil in water. The mixture
was applied to a stainless-steel target plate, then mixed with 0.5 μL of (50 mL) was premixed at an ambient temperature and 10,000 rpm for
DHB matrix and 0.3 μL of 0.1 M NaCl solution in order to increase ion 2 min using a mixer (Polytron PT-2100, Kinematica AG, Switzerland),
formation. Subsequently, the MALDI plate was dried under a stream of then this premix was homogenized three times with increasing pres-
air until forming a microcrystal layer. The machine was externally ca- sures at 300, 600, and 1000 bars using a PandaPLUS 2000 homogenizer
librated using a mixture of maltodextrins with known molecular (GEA Niro Soavi, Italy) in order to prepare the final O/W emulsions.
masses. For TOF/TOF analysis, the ion fragments of target mass were The emulsion droplet size distribution of the G7-PA monoester and
formed under laser-induced dissociation by using LIFT mode. The sucrose palmitate were measured using a laser light-scattering particle
measurements were performed in a mass range from 500 to 2000 m/z size analyzer (Mastersizer Hydro 2000, Malvern Instruments Ltd.,
and the total shots were accumulated in reflectron positive ion mode. Malvern, Worcestershire, UK) during storage at the ambient tempera-
All spectra were acquired by flexControl and then evaluated by ture (0, 3, 7, 10, 14 days). Control emulsions were prepared without the
flexAnalysis™ software (Bruker Daltonics). emulsifier.

2.4.3. Nuclear magnetic resonance (NMR) 2.5. Statistical analysis

For NMR analysis, each of G7-PA monoester (5 mg), PA (5 mg), and
G7 (10 mg) was dissolved in 600 μL of d6-DMSO, then transferred to The data were analyzed by independent samples one-way ANOVA
NMR tubes (Eurisotop, Saint Aubin, France). ¹H NMR and ¹³C NMR test with Bonferroni analysis (Version 12.0, StataCorp, College Station,
spectra were obtained by a Bruker Avance-III-600 (Bruker, Seongnam, TX, USA). Differences were considered to be significant when p < 0.05.
South Korea) instrument at the frequencies of 600.23 MHz and
150.93 MHz, respectively. The chemical shift (δ) data are expressed in
parts per million (ppm) and the d6-DMSO solvent signal (δ (1 H) 3. Results and discussion
2.5 ppm, δ (13C) 39.52 ppm) were used as the chemical shift reference.
2D heteronuclear multiple bond correlation (HMBC) and heteronuclear 3.1. Solubility of maltoheptaose in organic solvents
single quantum correlation (HSQC) spectroscopy were used in order to
confirm the formation of ester bond and chemical structures of G7-PA Organic solvents have been used for enzymatic reactions in a large
monoester. Correlation spectroscopy (COSY) and total correlation number of studies, particularly for the lipase-catalyzed esterification of
spectroscopy (TOCSY) were applied to identify the maltoheptaose SFAE (Abdulmalek, Hamidon, & Abdul Rahman, 2016; Habulin, Saša, &
backbone. The 2D COSY, TOCSY, HMBC, HSQC NMR spectra were Knez, 2008). However, one of the main disadvantages of the enzymatic
acquired using (8 scans, 1 h), (64 scans, 12 h), (32 scans, 13 h) and (16 synthesis of SFAE is the low solubility of the carbohydrate substrates in
scans, 4 h), respectively. All measurements were conducted at the am- organic solvents that strongly affects the reaction yield. To understand
bient temperature and the data were processed using the academic the solubility of G7 and identify a solvent system that is appropriate for
version of the TopSpin™ 4.0 software (Bruker). dissolving both G7 and PA substrates, various types of solvents (cov-
ering a wide range of log P values) were tested. It was found that only
2.4.4. β-Amylase treatment of maltoheptaose-palmitate esters DMSO could dissolve the G7 substrate with high concentration, and it
To identify ester bond position β-amylase which is non-reducing had the lowest log P value among these solvents (Table 1).
end exo-active enzyme was treated on the purified G7-PA. 1 mg/mL of By contrast, other solvents with higher log P values such as t-bu-
β-amylase (Tokyo Chemical Industry Inc., Tokyo, Japan) was dissolved tanol, 2-butanone, and n-hexane hardly dissolve G7. G7 presented
in 50 mM sodium acetate buffer (pH 5.5), then centrifuged at 20.000 x g limited but apparently better solubility in t-butanol compared with
for 5 min. The supernatant was used directly for the hydrolysis reaction. other solvents in this group. Nevertheless, the solubility of G7 was re-
The reaction mixture was composed of 490 μL of 1% (wt/vol) G7- PA latively lower than that of glucose for the organic solvents that have log
ester in 50 mM sodium acetate buffer (pH 5.5) and 10 μL of enzyme P ranging 0.5–1.0, but the difference was not significant (Lin et al.,
solution. The mixture was incubated at 60 °C during 4 h. The reaction 2016). Notably, although DMSO can easily dissolve both G7 and PA, it
mixture was withdrawn at 1 h 30 min and 4 h for MALDI-TOF analysis. should be utilized in controlled amounts in the enzymatic reaction. A
high concentration of DMSO can disrupt the tertiary structure of the
2.4.5. Differential scanning calorimetry (DSC) enzymes by removing the water molecules from the enzyme surface. As
Thermal analysis of maltose (G2), maltohexaose (G6), G7, mal- a result, the enzyme begins to unfold and rapidly loses its activity
tooctaose (G8), PA, G7-PA mixture, and G7-PA monoester were carried (Kumar, Dhar, Kanwar, & Arora, 2016). In previous studies, 5–20% of
out on a DSC-1 instrument (Mettler Toledo, Greifensee, Switzerland). DMSO was utilized in combination with the other solvent for the
G7-PA mixture was prepared by mixing G7 and PA (molar ratio of 1:1)
in distilled water (85 °C, 10 min) before drying to powder. Table 1
Solubility of maltoheptaose in various solvents at 60 °C.
Approximately 2 mg of each dried sample was placed in a sealed alu-
minium pan. Then, the pan was heated from 25 to 400 °C with the rate Solvent Log P* Solubility of G7 (%)i
of 10 °C/min under a nitrogen atmosphere. An empty pan was used as a
t-Butanol 0.6 0.19 ± 0.12a
reference. Acetone −0.23 0.01 ± 0.01a
2-Butanone 0.81 0.03 ± 0.02a
2.4.6. Hydrophilic-lipophilic balance (HLB) and emulsion droplet size n-Hexane 3.5 0.01 ± 0.01a
distributions Acetonitrile −0.36 0.05 ± 0.01a
DMSO −1.3 76.39 ± 2.64b
The HLB value of the maltoheptaose-palmitate monoester was cal-
culated according to Griffin (Griffin, 1949; Pasquali et al., 2008) as * the log P values which are the logarithms of the partition coefficient ex-
follows: press the hydrophobicity of solvents and were taken from previous studies (Jia,
mass of hydrophilic groups Zhao, Feng, Zhang, & Xia, 2010; Vaisali, Belur, & Regupathi, 2017).
HLB = 20 × i
Each value was expressed as the mean ± SD and was the mean of two
total mass of surfactant (1)
replicates. Values followed by different letters within a column were sig-
The ability of maltoheptaose-palmitate monoester to stabilize oil-in- nificantly different (p < 0.05) according to Bonferroni’s multiple range test.

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successful synthesis of sugar ester, and it was found that these condi- G7-PA monoester for the structural and physicochemical analyses
tions not only improve the solubility of both carbohydrate and fatty (Fig. 1).
acid but also avoid the significant decrease of the enzyme activity (Plou It was reported that the conversion of phytosterols linolenate in the
et al., 2002; Salihu & Zahangir, 2015). lipase-catalyzed reaction increasing steadily from 67% to 84% when the
molar ratio of phytosterols: linolenic acid changed from 1:1 to 1:3,
respectively (Zheng et al., 2012). A higher concentrations of an acyl
3.2. Synthesis of maltoheptaose-fatty acid esters
donor appears to be appropriate for the esterification. This may be due
to the increase in the substrate concentration that gave rise to an in-
Based on the solubility data of G7 (Table 1), DMSO and t-butanol
crease in the interaction with the active site of the enzyme and there-
were chosen as the cosolvents for the synthesis of G7-FA esters. After
fore led to improved conversion yield. In addition, it could also be
72 h reaction, the G7 esters with different chain length of FA were
described by the equilibrium constant k as follows:
obtained. Intriguingly, only G7 monoesters were detected in the SFAE
products. The binding site of CALA has a tunnel-like structure that
[G7­PA ester] [water]
hardly allows the enzyme to accept many bulky substrates (Widmann, k=
[PA] [ G7] (2)
Juhl, & Pleiss, 2010). The low concentration of G7 monoesters in the
reactant may be another reason for these results. The relative abun-
dance of monoesters was compared based on the intensity of their peaks k × [PA] [ G7]
[G7­PA ester] =
in the mass spectrum. The highest intensity was obtained at m/z [water] (3)
1414.024 (G7-palmitate), followed by m/z 1329.567 (G7-caprate), m/z
1385.648 (G7-myristate), and m/z 1357.657 (G7-laurate), demon- The reaction with various PA concentrations was conducted in the
strating that the palmitic acid was the best substrate for the G7 esters same conditions. Here, the k value and G7 concentration were kept
synthesis. This result is similar to that in a previous study where various constant. Meanwhile, the water produced during the reaction was ef-
FAs were used for SFAE production (van Kempen et al., 2013). In ad- ficiently absorbed with a molecular sieve to maintain an unchanged
dition, the effect of the molar ratio G7: PA on the conversion yield of concentration. Hence, according to Eq. (3), higher concentrations of PA
the reaction was briefly evaluated. In this experiment, G7 concentration or high molar ratio of G7:PA will result in the increase in the G7-PA
was limited due to its low solubility in the organic solvent system. The ester amount. Notably, a too high increase in the concentrations of an
amount of G7 was kept constant and PA concentration was gradually acyl donor may give rise to a strong decrease in the FA-based conver-
increased. As the molar ratio of G7: PA changed from 1:1 to 1:20, the sion yield due to the inhibitory effects on the lipase activity (Zheng
conversion yield of G7-PA ester increased gradually from 10% to 22% et al., 2012).
(Supplementary data 3). While the production yield should be im-
proved and optimized further, we obtained a sufficient amount of the

Fig. 1. Maltoheptaose-fatty acid esters with different chain lengths detected by MALDI-TOF MS.

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Fig. 2. (A) Confirmation of the purified G7-PA monoester (m/z value of 1413.9) using MALDI-TOF MS. (B) Fragmentation analysis of the pure G7-PA using MALDI-
TOF/ TOF MS. The parent peak was detected at m/z 1413.9, and the possible fragmentation sites (B/Y and C/Z) are shown in the G7-PA structure (for more
information, refer to (Lavanant and Loutelier-Bourhis, 2012; Mechref et al., 1998)). As shown in the chromatogram, the predicted m/z values of the smaller
monoesters fragment were detected. All the MS peaks were detected as [M + Na]+ species. Other abbreviations: maltose (G2); maltotriose (G3); maltotetraose (G4);
maltopentaose (G5); maltohexaose (G6); maltoheptaose (G7); palmitic acid (PA).

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3.3. Characterizations of the maltoheptaose-palmitate monoester The amount of G7-PA monoester was decreased after 1 h 30 min, and it
was completely hydrolyzed after 4 h reaction (Fig. 3). Consequently, the
3.3.1. MALDI- TOF/MS and NMR position of the ester bond formed was clearly identified as the linkage
To identify the structure of the G7-PA ester, the purified G7-PA ester between the PA and the reducing end of the G7 moiety.
was first analyzed using positive ion MALDI-TOF MS (Fig. 2). The major To resolve the structure of the G7-PA ester molecule in more detail,
peak at m/z 1413.96 was attributed to the G7-PA monoester that con- 1D and 2D (13C and 1H) NMR measurements were conducted. The
tained a single PA and one G7 moiety (Fig. 2A). Subsequently, this m/z HMBC spectrum showed the sixth protons of glucose residue at
value was further analyzed by tandem mass spectrometry. The MALDI- 4.02 ppm (α-H) and 3.89 ppm (β-H) interacted with the ester carbonyl
TOF/TOF MS spectrum (Fig. 2B) showed that fragment ions from the carbon (173.32 ppm), demonstrating the formation of ester bonds be-
parent peak at m/z 1413.96 were produced under laser-induced dis- tween G7 and the PA molecule at C6 of the glucosyl residue (Fig. 4).
sociation. The relative intensities of various monoester fragment ions The interaction of the protons of C2 and C3 of PA with the ester car-
from Y6 to Y2 decreased gradually, indicating that the glycosidic bonyl carbon was also observed in this figure. Additional NMR data are
cleavages were found mainly at either end of the G7 residue. This provided in the supplementary material. Using the MALDI-TOF and
fragmentation pattern was similar to the pattern of a linear oligo- NMR techniques, we successfully identified the fine structure of the new
saccharide and their derivatives found in previous studies (Lang, Zhao, sugar ester molecule: 6-O-palmitoyl-α-D-maltoheptaose.
Liu, & Yu, 2014; Lavanant & Loutelier-Bourhis, 2012; Mechref, Milos, & Intriguingly, we obtained G7-FAs as monoesters only. As discussed
Novotny, 1998; Sandra, Devreese, Van Beeumen, Stals, & Claeyssens, below (Section 3.2), the characteristics of the lipase used in this study
2004). Moreover, the cross-ring cleavages (0.2A) occurred pre- and the low concentration of the monoester that must be used as the
dominantly at the reducing end of the molecule under high energy laser substrate for additional esterification in reaction mixture may affect
or collisional activation (Lavanant & Loutelier-Bourhis, 2012; Mechref this result. Moreover, we found that the G7-PA monoester had no iso-
et al., 1998). Taken these results together, we deduced that the ester mers that have different ester bond positions and its ester bond was
bond may be located at the reducing end of the G7 moiety. To confirm found only at the C6 of the reducing-end glucose. To date, very few
this, the product was treated with β-amylase. We examined two hy- studies that used pure maltooligosaccharides for sugar ester synthesis
potheses: first, if the free PA is attached to the reducing end of the have been reported. G3 was used for this purpose but the position of
molecule, β-amylase can hydrolyze the G7 backbone from the non- ester bond was not identified (Ferrer et al., 2005). G2 was acylated with
reducing end. Alternatively, if the ester bond was formed at the non- linoleic acid using various lipases and yielded G2-linoleate mono or
reducing end of the G7 moiety, no reaction of β-amylase could occur. diester (Fischer, Happe, Emery, Fornage, & Schütz, 2013). In this

Fig. 3. Enzymatic hydrolysis of the G7-PA monoester carried out to confirm the position of the ester bond in the G7 moiety. Exo-acting β-amylase that hydrolyzes
starch from the nonreducing end was used exclusively. It is clearly shown in the MS chromatogram that the G7-PA monoester was hydrolyzed and decreased as the
reaction progressed.

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Fig. 4. 2D 1H-13C HMBC spectrum of G7-PA monoester. The chemical shifts (ppm) of 1H proton and 13C are indicated on the horizontal and vertical axes, re-
spectively. The interaction of the proton of C6 of the glucosyl residue with the ester carbonyl group demonstrated the ester bond formation.

Scheme 1. CALA catalyzed esterification of maltoheptaose with palmitic acid.

previous study, both 6 and 6ˊ-O-linoleyl-α-D-maltose monoesters were maltodextrins, including G2, G6, and G8. The G2 thermogram pre-
produced as a mixture. When isomaltotriose (IG3) was used as the sugar sented a typical crystal melting peak at 125 °C and a relatively broad
moiety, only the C6ˊˊ position of the nonreducing end anhydroglucose degradation peak at 240 °C, whereas G7 showed no notable peaks
participated in the ester bond (Liu, 2017). However, for that study, around the crystal melting peak of G2 (Fig. 5A). Thermograms of G6
metalloprotease was used as the catalyst, and IG3 has only one free C6 and G8 were similar to that of G7, and these three maltodextrins ex-
position that was the C6ˊˊ. Notably, the C6 of the reducing-end glucose hibited higher degradation temperatures (260 °C) than G2. The melting
in G7 is a primary target for the esterification conducted by CALA point of the G7-PA monoester was 56 °C which is lower than that of free
(Scheme 1). The CALA catalytic selectivity on this position is pro- PA (62.81 °C), and no additional thermal events were detected up to
nounced, resulting in a homogenous G7-PA monoester structure. It is 250 °C (Fig. 5B). Meanwhile, the free G7/PA mixture showed a thermal
plausible that structural homogeneity can provide some benefits to G7- pattern similar to the sum of the free G7 and PA individual thermogram
PA as a new emulsifier. (Fig. 5C). G7-PA monoester formation was evident in the downshift of
the melting temperature observed in Fig. 5B. In addition, no apparent
3.4. Thermal properties interaction was detected between free G7 and PA, even though the
mixture were prepared by a process similar to that for G7-PA monoester
The thermal properties of sugar esters are of particular interest due synthesis. Based on this result, an inclusion complex or any kind of
to their feasibility for use in hot-melt technology (Szuts, Pallagi, strong interaction between free G7 and PA was excluded. Overall, it is
Regdon, Aigner, & Szabó-Révész, 2007; Szuts & Szabó-Révész, 2012). likely that G7-PA monoester will be a good candidate for use in hot-
Additionally, the melting temperature affects the G7-PA application melting technology in food, pharmaceuticals and cosmetics fields due to
range and could be another evidence for the ester bond formation its superior thermal properties compared to its mono or disaccharides
(Pappalardo et al., 2017). Therefore, the thermal properties of the counterparts.
product were evaluated by DSC. First, free G7 was compared with other

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Fig. 5. Thermal behavior analysis of G7-PA and relevant substrates. DSC thermograms of maltose (G2), maltohexaose (G6), maltoheptaose (G7), maltooctaose (G8)
(A), G7-PA monoester and PA (B), G7 and free G7/PA mixture (C) are shown.

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Fig. 6. Droplet size distribution of emulsions prepared with no emulsifier (negative control) (A), G7-PA monoester (B), and a commercial sucrose-PA ester for various
storage times.

3.5. Emulsion stability result indicated that the droplet size of the sucrose-PA emulsion was
slightly less homogeneous than that of the G7-PA emulsion. This may
The sugar esters with different HLB values have different emulsion due to the lower emulsion capacity of the sucrose-PA. Compared to
properties. The calculated HLB value of the G7-PA monoester was 16, molar concentration, the sucrose-ester used twice higher concentration
which is suitable for use in the O/W emulsions (Griffin, 1949; Pasquali, than G7-PA. It is notable that maltoheptaosyl moiety is 3 times larger
Taurozzi, & Bregni, 2008). The emulsion properties of the G7-PA than sucrose. It may provide a thicker hydrophilic layer than the mono-
monoester were investigated and compared with those of commercial or disaccharide-FA esters. On the other hand, G7 is still a small mole-
sucrose-PA (HLB value = 16) by measuring the droplet size distribution cule compared to proteins and modified starches that are often used as
during storage. The emulsion with G7-PA showed a mono-modal dis- emulsion stabilizers. To conclude, the advantages of this G7-FA ester
tribution that was similar to that of sucrose-PA. The distributions of suggest that further research should be performed for its industrial
both emulsions had a very small shoulder on the right (particle size applications (Fig. 6).
1 μm). The emulsion droplet distribution was very stable for the both
samples during 14-day storage and was narrow with D values [4,3] of
0.87 ± 0.142 μm and 0.73 ± 0.03 μm for the sucrose-PA and G7-PA 4. Conclusion
monoester emulsions, respectively. However, large particles with sizes
greater than 10 μm were detected in the emulsion prepared with the To the best of our knowledge, the present work is the first report on
commercial sucrose-PA, although the amount was not significant. The the direct esterification of G7 and fatty acids for the synthesis of sugar
esters. Due to the low solubility of G7 in organic solvents, a relatively

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low yield of G7-PA monoester was obtained. However, the product desorption/ionization time-of-flight mass spectrometry. Rapid Communications in
shows a homogeneous structure that has C-6 of the glucose at the re- Mass Spectrometry, 26, 1311–1319.
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PA ester shows that the G7-PA monoester exhibits emulsion properties Lin, X.-S., Zhao, K.-H., Zhou, Q.-L., Xie, K.-Q., Halling, P. J., & Yang, Z. (2016). Aspergillus
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sion capacity. To take advantage of this new sugar ester for industry, emulsifying property. Enzyme and Microbial Technology, 101, 51–56.
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Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Maltoheptaose and maltooctaose as the superior aroma encapsulating agents. Food
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