Enzymatic Preparation of a Homologous Series of Long-Chain 6- O -Acylglucose Esters and Their Evaluation as Emulsifiers

Article abstract of DOI:10.1021/acs.jafc.8b00913

Sugar fatty acid esters are nonionic surfactants that are widely exploited in the food and cosmetics industries, as well as in the oral care and medical supply fields. Accordingly, new methods for their selective synthesis and the tuning of their emulsifying properties are of considerable interest. Herein we report simple and irreversible enzymatic esterifications of d-glucose with seven fatty acid vinyl esters. The foaming and emulsifying effects of the resulting 6-O-acylglucose esters were then evaluated. In accord with expectations, when the length of the alkyl side chain associated with the 6-O-acylglucose esters increases, then their hydrophilic-lipophilic balance (HLB) values decrease, while the stabilities of the derived emulsions improve. In order to maintain good foaming properties, alkyl side chains of at least 9 to 11 carbons in length are required. In the first such assays on 6-O-acylglucose esters, most of those described herein are shown to be nontoxic to the HepG2, MCF-7, LNacp, SW549, and LO-2 cell lines.

Full text of DOI:10.1021/acs.jafc.8b00913

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Food and Beverage Chemistry/Biochemistry
Enzymatic Preparation of an Homologous Series of Long-chain
6-O-Acylglucose Esters and their Evaluation as Emulsifiers
Min-Yi Liang, Yongsheng Chen, Martin G Banwell, Yong Wang, and Ping Lan
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00913 • Publication Date (Web): 30 Mar 2018
Downloaded from http://pubs.acs.org on April 1, 2018
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Enzymatic Preparation of an Homologous Series of
Long-chain 6-O-Acylglucose Esters and their Evaluation as
Emulsifiers
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MinꢀYi Liang†,‡, Yongsheng Chen†, Martin G. Banwell§,#, Yong Wang*†,‡,
and Ping Lan*†
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†Guangdong Saskatchewan Oil Seed Joint Laboratory, Department of Food Science
and Engineering, Jinan University, Guangzhou, 510632, China
‡Guangdong Engineering Technology Research Center for Oils and Fats Biorefinery,
Guangzhou 510632, China
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§College of Pharmacy, Jinan University, Guangzhou, 510632, China
#Research School of Chemistry, Institute of Advanced Studies, The Australian
National University, Canberra, ACT 2601, Australia
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*Corresponding author (Telephone: +86ꢀ20ꢀ85221367. Fax: +86ꢀ20ꢀ85224766.
Eꢀmail: ping.lan@jnu.edu.cn)
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ABSTRACT
Sugar fatty acid esters are nonꢀionic surfactants that are widely exploited in the food
and cosmetics industries, as well as in the oral care and medical supply fields.
Accordingly, new methods for their selective synthesis and the “tuning” of their
emulsifying properties are of considerable interest. Herein we report simple and
irreversible enzymatic esterifications of Dꢀglucose with seven fatty acid vinyl esters.
The foaming and emulsifying effects of the resulting 6ꢀOꢀacylglucose esters were then
evaluated. In accord with expectations, when the length of alkyl side chain associated
with the 6ꢀOꢀacylglucose esters increases then their HLB values decrease while the
stabilities of the derived emulsions improve. In order to maintain good foaming
properties alkyl side chains of at least 9 to 11 carbons in length are required. In the
first such assays on 6ꢀOꢀacylglucose esters, most of those described herein are shown
to be nonꢀtoxic to the HepG2, MCFꢀ7, LNacp, SW549, and LOꢀ2 cell lines.
KEYWORDS: 6ꢀOꢀacylglucose esters, foaming properties, emulsion stability,
structureꢀfunction profiles
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INTRODUCTION
Those sugar fatty acid esters used in industry are normally obtained by
combining sustainably produced and inexpensive carbohydrates such as sucrose or
glucose with lauric, palmitic or stearic acids or their corresponding low molecular
weight esters via esterification or transꢀesterification reactions, respectively.
Longꢀchain fatty acid esters of sucrose, in particular, are high volume commodity
chemicals and act as nonꢀionic surfactants with the sucrose moiety serving as a
hydrophilic head group and the longꢀchain fatty acid residue as the lipophilic tail. The
ensuing amphipathic properties mean such esters are especially important in the food
and cosmetics industries as well as in the oral care and medical supply fields, not least
because they exhibit good emulsifying properties while also being readily
biodegradable and nonꢀtoxic.1ꢀ3 Moreover, their functional characteristics can be
“dialed up” over a wide range through the attachment of varying numbers and types
of fatty acyl moieties to the sucrose core.4
Most of the sucrose esters on the market are currently manufactured by very
traditional chemical techniques.5 Given that there are eight nonꢀequivalent hydroxyl
groups associated with the sucrose molecule, the fundamental challenge associated
with the required derivatization process arises from the often concurrent formation of
monoꢀ, diꢀ, triꢀ and even higherꢀorder esters.6,7 None of the current industrial
processes is particularly selective for the formation of any given sucrose ester, thus
dramatically reducing the yield of the target compound(s) as well requiring the
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application of purification regimes. Of course, the formation of byꢀproducts also
creates waste steams and so further increasing the costꢀofꢀgoods.8
The deficiencies associated with the current methods for manufacturing sucrose
esters have attracted considerable attention, particularly from academic laboratories.
Manifold efforts have been made to improve the current methods of manufacture by
focusing on, for example, alternate acyl donors7 and reaction solvents,9,10 new modes
of promoting reaction (including through ultrasonic irradiation),11 and the use of
biological catalysts (enzymes).12ꢀ16 The main difficulties encountered in attempts to
improve the yields of enzymatic couplings arises from the low activity of the
biological catalysts in polar solvents such as DMSO and DMF that are required to
dissolve the carbohydrates.13 To prevent enzyme deactivation it has been suggested
that these highly polar solvents are doped with alcoholic ones such as 2ꢀbutanol12 or
tꢀamyl alcohol1,14 that serve as adjuvants. Increasing the concentration of sucrose in
the reaction medium so as to improve throughput whilst retaining the activity of the
enzyme represents another challenge. The highest sucrose loading successfully
deployed to date is 0.7 M (in DMSO) although a “preꢀtreated” lipase was required to
establish an effective process but thereby delivering sucrose monoꢀcaprate at useful
scale and rate.13 The reaction times required for the enzymatic generation of sucrose
esters has reportedly ranged from 6 h to several days, with the longer periods
representing a major drawback for industrial development.15,16
While there have been extensive studies on the enzymeꢀmediated synthesis of
sucrose monoesters, the corresponding glucoseꢀderived systems are much less studied.
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Compared to sucrose, glucose displays higher regioselectivity in the
transꢀesterification process since there is only one primary hydroxyl group present,
whereas there are three in sucrose.17 Most efforts to prepare glucose fatty acid esters
have focused on enzymatic approaches, since these offer the possibility of employing
mild reaction conditions and could, in principle at least, proceed at high volume and
with excellent regioselectivities. A range of commercially available and immobilized
lipases, most notably Novozym435, Lipozyme RMIM and Lipozyme LIM, has been
used to catalyze the regioselective acylation of glucose with various series of
longꢀchain fatty acids.15ꢀ20 Most of these delivered the 6ꢀOꢀacylglucose ester as the
major product. For instance, Ren et al reported17 enzymatic syntheses of the
6ꢀOꢀpalmitoylꢀ, 6ꢀOꢀlauroylꢀ and 6ꢀOꢀhexanoylꢀglucose esters in DMSO and tꢀamyl
alcohol media using the corresponding fatty acids as the acyl donor. Significantly, a
standꢀalone but highly efficient Novozym435ꢀcatalyzed synthesis of
6ꢀOꢀmyristoylglucopyranose was achieved by Davis et al20 using the relevant enol
ester as the acyl donor and a mixture of THF/pyridine as the solvent system.
Surprisingly, this attractive process does not appear to have been deployed more
extensively even though it should be capable of delivering a series of such glucose
monoꢀesters and so allowing for the systematic study of the impacts of varying the
fatty acid chain on the emulsification properties of this monosaccharide. Accordingly,
the aims of the current study were to undertake an inꢀdepth investigation of the
structureꢀfunction profiles of an homologous series 6ꢀOꢀacylglucose esters and
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thereby gain insights into their potential utility in the food industry as, for example,
emulsifiers.
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MATERIALS AND METHODS
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Materials. Dꢀglucose (anhydrous, 99.0%), pyridine, tetrahydrofuran (THF),
N,Nꢀdimethylformamide (DMF), benzene, tꢀamyl alcohol, tꢀBuOH, Span 20, Span 40,
Span 60, Span 80, Tween 20 and Tween 80 were purchased from the Energy
Chemical Co., Ltd (Shanghai, China). Vinyl hexanoate (>99.0%), vinyl nꢀoctanoate
(>99.0%), vinyl decanoate (>99.0%), vinyl laurate (>99.0%), vinyl myristate
(>99.0%), vinyl palmitate (>96.0%) and vinyl stearate (>95.0%) were purchased from
the Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan) while Novozym435 was
supplied by SigmaꢀAldrich (St. Louis, MO). Pure canola oil was purchased from a
local grocery store while commercial sucrose stearic esters Sꢀ570 and Sꢀ770 were
obtained from the MitsubishiꢀChemical Foods Corporation (Tokyo, Japan). Cell
culture media were purchased from the Gibco U.S. Biotechnology Co. (Waltham, MA)
while foetal bovine serum (FBS) was obtained from the Tianhang Biotech Co. Inc.
(Zhejiang, China).
General Protocols. Proton (1H) and carbon (13C) NMR spectra were recorded at
25 °C on a Bruker spectrometer (Billerica, MA) operating at 500 MHz for proton and
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125 MHz for carbon nuclei. For H NMR spectra, the residual protioꢀsolvent signals
were used as internal standards. Lowꢀresolution ESI mass spectra were recorded on an
Agilent singleꢀquadrupole liquid chromatographꢀmass spectrometer (Santa Clara,
CA).
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Synthesis of the 6-O-Acylglucose Esters. Enzymatic syntheses of the seven
6ꢀOꢀacylglucose esters were conducted (Figure 1) using modifications of a previously
reported method.20 In brief, commercially available Novozym435 was used as the
catalyst while seven long alkyl chain (5 to 17 carbons) fatty acid vinyl esters, namely
vinyl hexanoate, vinyl octanoate, vinyl decanoate, vinyl laurate, vinyl myristate, vinyl
palmitate and vinyl stearate, were selected as the acyl donors. The use of vinyl esters
has proven advantageous since, in each instance, the transꢀesterification reactions are
irreversible and the byꢀproduct is acetaldehyde that acts as an internal water scavenger,
and so thus promoting the desired transformation.20 THF/pyridine (4:1 v/v) served as
the reaction solvent and allowed for effective solubilization of the free glucose
without adversely impacting on the activity of the enzyme. Under such conditions,
complete reaction was achieved within 48 h on multiꢀgram scales and delivered each
6ꢀOꢀacylglucose ester in a completely regioselective manner. The products were
isolated by trituration with diethyl ether, affording the target compounds in high purity,
as determined by NMR spectroscopic analysis. A typical protocol was as follows:
A mixture of anhydrous Dꢀglucose (1.00 g, 5.6 mmol) and THF/pyridine (40 mL,
4:1 v/v) contained in a 100 mL roundꢀbottom flask was subject to sonication and once
dissolution had taken place (ca. 5 min), Novozym435 (50 to 200 mg) was added along
with the relevant fatty acid vinyl ester (3.0 equiv., 16.8 mmol). The ensuing mixture
was stirred magnetically at 40 °C until all the glucose had been consumed (normally
24 to 48 h) as determined by TLC analysis. The ensuing solution was decanted from
the bulk of the solid residue and the residual enzyme removed by filtration and the
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solid thus retained washed with methanol (2 x 10 mL) then dichloromethane (1 x 10
mL). The combined filtrates were dried (Na2SO4), filtered then concentrated under
reduced pressure to afford a yellow solid, trituration of which (with 100 mL of diethyl
ether) followed by suction filtration gave the corresponding 6ꢀOꢀacylglucose ester 2ꢀ8
as an amorphous white solid in high yield. The optimization of the reaction through
varying the enzyme loading, the solvent(s) and/or reaction times is detailed in Table 1.
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6-O-Hexanoylglucopyranose, 2. Amorphous white solid. H NMR (500 MHz,
DMSOꢀd6) δ 6.34 (d, J = 4.7 Hz, 1H, 1ꢀOH), 5.03 (d, J = 5.7 Hz, 1H, 4ꢀOH), 4.90
(app. t, J = 4.5 Hz, 1H, H1), 4.75 (d, J = 4.8 Hz, 1H, 3ꢀOH), 4.53 (d, J = 6.7 Hz, 1H,
2ꢀOH), 4.27 (dd, J = 11.6 and 1.6 Hz, 1H, H6ʹ), 3.99 (dd, J = 11.6 and 6.3 Hz, 1H,
H6), 3.76 (ddd, J = 9.9, 6.1 and 2.0 Hz, 1H, H5), 3.43 (td, J = 9.1 and 4.8 Hz, 1H, H3),
3.12 (m, 1H, H2), 3.03 (m, 1H, H4), 2.27 (td, J = 7.3 and 2.2 Hz, 2H), 1.52 (m, 2H),
1.26 (m, 4H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, DMSOꢀd6) δ 173.4, 92.8
(C1), 73.3 (C3), 72.6 (C2), 71.0 (C4), 69.6 (C5), 64.3 (C6), 33.9, 31.1, 24.6, 22.3,
14.3. MS (ESI, +ve): m/z 279 ([M+H]+, 20%), 296 ([M+H2O]+, 70), 301 ([M+Na]+,
100).
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6-O-Octanoylglucopyranose, 3. Amorphous white solid. H NMR (500 MHz,
DMSOꢀd6) δ 6.33 (d, J = 4.7 Hz, 1H, 1ꢀOH), 5.03 (d, J = 5.7 Hz, 1H, 4ꢀOH), 4.90
(app. t, J = 4.5 Hz, 1H, H1), 4.74 (d, J = 4.8 Hz, 1H, 3ꢀOH), 4.52 (d, J = 6.7 Hz, 1H,
2ꢀOH), 4.26 (dd, J = 11.7 and 2.0 Hz, 1H, H6ʹ), 3.99 (dd, J = 11.7 and 6.2 Hz, 1H,
H6), 3.76 (ddd, J = 10.1, 6.2 and 2.0 Hz, 1H, H5), 3.42 (td, J = 9.1 and 4.9 Hz, 1H,
H3), 3.12 (m, 1H, H2), 3.03 (m, 1H, H4), 2.27 (td, J = 7.3 and 2.3 Hz, 2H), 1.52 (m,
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2H), 1.26 (m, 8H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, DMSOꢀd6) δ 173.4,
92.8 (C1), 73.3 (C3), 72.7 (C2), 71.0 (C4), 69.6 (C5), 64.3 (C6), 33.9, 31.6, 28.9, 28.8,
24.9, 22.5, 14.4. MS (ESI, +ve): m/z 307 ([M+H]+, 10%), 324 ([M+H2O]+, 100), 329
([M+Na]+, 50).
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6-O-Decanoylglucopyranose, 4. Amorphous white solid. H NMR (500 MHz,
DMSOꢀd6) δ 6.34 (d, J = 5.2 Hz, 1H, 1ꢀOH), 5.03 (d, J = 5.7 Hz, 1H, 4ꢀOH), 4.90 (m,
1H, H1), 4.75 (d, J = 4.8 Hz, 1H, 3ꢀOH), 4.52 (d, J = 6.7 Hz, 1H, 2ꢀOH), 4.26 (dd, J =
11.7 and 2.0 Hz, 1H, H6ʹ), 3.99 (dd, J = 11.7 and 6.3 Hz, 1H, H6), 3.76 (ddd, J = 10.1,
6.3 and 2.0 Hz, 1H, H5), 3.43 (td, J = 9.2 and 4.8 Hz, 1H, H3), 3.12 (m, 1H, H2), 3.04
(m, 1H, H4), 2.27 (m, 2H), 1.52 (m, 2H), 1.26 (m, 12H), 0.86 (t, J = 7.0 Hz, 3H). 13C
NMR (125 MHz, DMSOꢀd6) δ 173.4, 92.8 (C1), 73.3 (C3), 72.7 (C2), 71.0 (C4), 69.6
(C5), 64.3 (C6), 33.9, 31.7, 29.3, 29.2, 29.1, 28.9, 24.9, 22.6, 14.4. MS (ESI, +ve):
m/z 335 ([M+H]+, 10%), 352 ([M+H2O]+, 100), 357 ([M+Na]+, 50).
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6-O-Lauroylglucopyranose, 5. Amorphous white solid. H NMR (500 MHz,
DMSOꢀd6) δ 6.35 (d, J = 4.3 Hz, 1H, 1ꢀOH), 5.05 (d, J = 5.7 Hz, 1H, 4ꢀOH), 4.95 (m,
J = 4.7 Hz, 1H, H1), 4.76 (d, J = 4.8 Hz, 1H, 3ꢀOH), 4.54 (d, J = 6.7 Hz, 1H, 2ꢀOH),
4.26 (m, 1H, H6ʹ), 3.99 (m, 1H, H6), 3.76 (m, 1H, H5), 3.43 (m, 1H, H3), 3.16 (m, 1H,
H2), 3.04 (m, 1H, H4), 2.27 (m, 2H), 1.52 (m, 2H), 1.26 (m, 16H), 0.86 (t, J = 7.0 Hz,
3H). 13C NMR (125 MHz, DMSOꢀd6) δ 173.4, 92.8 (C1), 73.9 (C3), 72.7 (C2), 71.0
(C4), 69.6 (C5), 64.4 (C6), 33.9, 31.8, 29.5, 29.4, 29.2, 29.1, 28.9, 24.9(4), 24.9(2),
22.6, 14.4. MS (ESI, +ve): m/z 363 ([M+H]+, 40%), 380 ([M+H2O]+, 50), 385
([M+Na]+, 100).
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6-O-Myristoylglucopyranose, 6. Amorphous white solid. H NMR (500 MHz,
DMSOꢀd6) δ. 6.34 (broad s, 1H, 1ꢀOH), 5.03 (broad s, 1H, 4ꢀOH), 4.89 (m, 1H, H1),
4.30 (d, J = 8.8 Hz, 1H, 2ꢀOH), 4.27 (d, J = 11.6 Hz, 1H, H6ʹ), 3.99 (dd, J = 11.6 and
6.3 Hz, 1H, H6), 3.76 (m, 1H, H5), 3.43 (t, J = 9.1, 1H, H3), 3.12 (m, 1H, H2), 3.04
(m, 1H, H4), 2.27 (m, 2H), 1.52 (m, 2H), 1.26 (m, 2ꢀOH), 0.86 (t, J = 7.0 Hz, 3H). 13C
NMR (125 MHz, DMSOꢀd6) δ 173.4, 92.8 (C1), 75.2 (C3), 73.3 (C2), 71.0 (C4), 69.6
(C5), 64.3 (C6), 33.9, 31.8, 29.5(3), 29.5(0), 29.4(8), 29.4(7), 29.3(7), 29.2, 29.1, 28.9,
24.9, 22.6, 14.4. MS (ESI, +ve): m/z 391 ([M+H]+, 30%), 408 ([M+H2O]+, 50), 413
([M+Na]+, 70), 647 (100).
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6-O-Palmitoylglucopyranose, 7. Amorphous white solid. H NMR (500 MHz,
DMSOꢀd6) δ 6.34 (d, J = 4.3 Hz, 1H, 1ꢀOH), 5.03 (d, J = 5.7 Hz, 1H, 4ꢀOH), 4.90 (t, J
= 4.3 Hz, 1H, H1), 4.75 (d, J = 4.8 Hz, 1H, 3ꢀOH), 4.53 (d, J = 6.7 Hz, 1H, 2ꢀOH),
4.27 (dd, J = 11.7 and 2.0 Hz, 1H, H6ʹ), 3.99 (dd, J = 11.7 and 6.2 Hz, 1H, H6), 3.76
(ddd, J = 10.0, 6.2 and 2.0 Hz, 1H, H5), 3.43 (td, J = 9.2 and 4.6 Hz, 1H, H3), 3.13
(ddd, J = 9.9, 6.5 and 3.5 Hz, 1H, H2), 3.04 (m, 1H, H4), 2.27 (m, 2H), 1.52 (m, 2H),
1.26 (m, 24H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, DMSOꢀd6) δ 173.4, 92.8
(C1), 73.3 (C3), 72.7 (C2), 71.0 (C4), 69.6 (C5), 64.3 (C6), 33.9, 31.8, 29.5 (x5), 29.4,
29.3, 29.2, 29.1, 28.9, 24.9, 22.6, 14.4. MS (ESI, +ve): m/z 419 ([M+H]+, 70%), 436
([M+H2O]+, 30), 441 ([M+Na]+, 100).
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6-O-Stearoylglucopyranose, 8. Amorphous white solid. H NMR (500 MHz,
DMSOꢀd6) δ 6.34 (d, J = 5.3 Hz, 1H, 1ꢀOH), 5.03 (d, J = 5.7 Hz, 1H, 4ꢀOH), 4.90 (dd,
J = 5.3 and 3.4 Hz, 1H, H1), 4.76 (d, J = 4.8 Hz, 1H, 3ꢀOH), 4.53 (d, J = 6.7 Hz, 1H,
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2ꢀOH), 4.27 (dd, J = 11.7 and 2.0 Hz, 1H, H6ʹ), 3.99 (dd, J = 11.7 and 6.2 Hz, 1H,
H6), 3.76 (ddd, J = 10.0, 6.2 and 2.0 Hz, 1H, H5), 3.43 (td, J = 9.2 and 4.7 Hz, 1H,
H3), 3.13 (ddd, J = 9.9, 7.0 and 3.5 Hz, 1H, H2), 3.04 (ddd, J = 10.1, 8.7 and 5.5 Hz,
1H, H4), 2.27 (m, 2H), 1.52 (m, 2H), 1.26 (m, 28H), 0.86 (t, J = 7.0 Hz, 3H). 13C
NMR (125 MHz, DMSOꢀd6) δ 173.3, 92.7 (C1), 73.3 (C3), 72.7 (C2), 71.0 (C4), 69.6
(C5), 64.4 (C6), 33.9, 31.8, 29.5(3) (x7), 29.5(0), 29.4, 29.2, 29.1, 28.9, 24.9, 22.6,
14.4. MS (ESI, +ve): m/z 447 ([M+H]+, 10%), 469 ([M+Na]+, 70), 758 (100).
Determination of Hydrophilic-Lipophilic Balance (HLB) Values. The HLB
values of the seven products obtained as detailed above, namely esters 2ꢀ8, were
determined experimentally using the water number method.21 Thus, 100 mg of each
compound was dissolved in DMF/benzene (40 mL, 100:5 v/v). The resulting solution
was titrated with distilled water while being maintained at 20 ±1 °C until a permanent
turbidity was obtained. All results were reproducible to within 0.5 units of HLB. A
plot of consumed water number vs HLB was then established using emulsifiers with
known HLB values, including Span 80 (HLB = 4.3), Span 60 (HLB = 4.8), Span 40
(HLB = 6.7), Span 20 (HLB = 8.6), Tween 80 (HLB = 15.6) and Tween 20 (HLB =
16.7), for calibration purposes. The HLB values of 6ꢀOꢀacylglucose esters 2ꢀ8, as well
as the commercial sucrose stearic esters Sꢀ570 and Sꢀ770, were then read from this
plot. All the experiments were performed in triplicate.
Evaluation of Foamability and Foam Stability. Evaluation of the foamability
and foam stability of esters 2ꢀ8 together with controls Sꢀ570, Sꢀ770, Span20 and
Span80 were undertaken using a previously described method.22,23 Specifically, 10 mL
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of 0.1%, 0.2% and 0.5% w/w aqueous dispersions of each sample were placed in a 50
mL, flatꢀbottomed measuring cylinder and the height of solution (H1) was measured
before being homogenized, using a blender, at 3000 rpm for 2 min. The initial foam
height (H2) was measured immediately. Following resting periods of 10, 20, and 30
min, the foam heights (H3) were then recorded. All the experiments were performed in
triplicate. The foamability and foaming stabilities of these compounds were calculated
using the following equations:
Foamability (%) = H2/ H1 × 100%
Foam stability (%) = H3/ H2 × 100%
Determination of Emulsion Stability Index. The emulsifying stability indices
of esters 2ꢀ8 as well as the four controls were determined using minor modifications
of literature protocols.17,24 Specifically, 10.0 g of 0.1%, 0.2% and 0.5% w/w aqueous
solutions of the test compounds were each mixed with 1.0 g of canola oil while a
solution lacking a test material was used as the control. The ensuing mixtures were
homogenized for 2 min at 20,000 rpm using a blender and a 20 µL sample of the
resulting emulsion was diluted with 2 mL deionized water before being drawn into a
clean spectrometric cuvette. The absorbance of the emulsion at 500 nm was measured
at 0 min and 20 min. All the experiments were performed in triplicate. The emulsion
stability indices (ESI) was calculated using the following equation:
ESI (%) = A0 × 20/[A0 – A20] × 100%
where A0 and A20 are the absorbances obtained at 0 min and 20 min.
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Evaluation of Cytotoxicities. In order to determine the biosafety of the
6ꢀOꢀacylglucose esters 2ꢀ8 at the cellular level, their cytotoxicities were measured.
The methylene blue method of analysis was employed with some modifications.
Specifically, cells were seeded at 4 × 104/well on a 96ꢀwell plate in 100 ꢁL of
complete medium (DMEM+ 5% FBS, 50 units/mL penicillin, 50 ꢁg /mL streptomycin,
100 ꢁg /mL gentamicin). After being incubated for 24 h at 37 °C, the medium was
removed and the cells were washed once with 100 ꢁL of sterile and cold phosphate
buffer solution (PBS, 0.01M, pH7.4). Free FBS medium containing the test material
was then added to the well while the same medium lacking any test compound served
as the control. After an additional 24 h of incubation, the medium was removed and
the wells were washed with PBS. 50 ꢁL of methylene blue (Hank's balanced salt
solution + 1.25% glutaraldehyde + 0.6% methylene blue) was added to each well,
then the cells were incubated for 1 h at 37 °C in 5% CO2. After incubation, the
staining agent was removed and the cells were washed six times with deionized water
then elution buffer (100 ꢁL) was added to each well. The plates were placed on a table
oscillator for 20 min, and absorbance was measured at 570 nm using a microplate
reader. Concentrations of samples that decrease the absorbance by >10% when
compared to the blank control were considered to be cytotoxic. All the experiments
were performed in triplicate.
Statistical Analysis. Each evaluation experiment was performed three times. The
results are presented as the mean values ± standard deviation (SD). Statistical analyses
were performed using SPSS software (SPSS, Inc., Chicago, IL) to identify any
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significant differences. All values were assessed by oneꢀway analysis of variance
(ANOVA) in conjunction with Duncan's new multipleꢀrange test (MRT).
RESULTS AND DISCUSSION
Syntheses and Characterization of Products. The optimization of the
enzymatic acylation of glucose at Cꢀ6 was explored by varying the enzyme loading,
the solvent(s) and/or reaction times. In each instance, three equivalents of the relevant
fatty acid vinyl ester were used as the acyl donor. As shown in Table 1, when 5% w/w
of the enzyme was used in anhydrous THF/pyridine then good to excellent yields of
the target compound were obtained within 24 h. When DMSO/tꢀBuOH, was used as
the solvent system then yields ranging from 47ꢀ57% were observed and 48 h was
required for reactions to go to completion. Similar trends were observed in the
DMSO/tꢀamyl alcohol solvent system (entry 4). Higher enzyme loadings (entry 3)
only resulted in marginally higher yields. The reaction seemed to be particularly water
sensitive, as lower yields (entry 5) were obtained when undried/reagentꢀgrade
THF/pyridine was used. Among the seven vinyl esters, the laurate and
myristateꢀbased systems exhibited the best reactivity and productivity.
The structures of all products 2ꢀ8 were confirmed by undertaking MS
spectrometric and NMR spectroscopic analyses, including those arising from the
1
outcomes of Hꢀ1H COSY and HMBC experiments. In all instances, two sets of
chemical shifts were observed in the NMR spectra, reflecting the presence of both the
αꢀ and βꢀanomeric forms of the product monosaccharide.17 With the exception of
1
6ꢀOꢀlauroylglucose, the former anomer predominated. Key elements of the Hꢀ1H
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COSY and HMBC correlation spectra derived from 6ꢀOꢀpalmitoylglucopyranose are
shown in Figure 2. These are representative of the series as a whole. As revealed
through HMBC experiments, the protons at Cꢀ6 of the pyranose ring interact with the
carbonyl carbon of the ester moiety and thus establishing the site of esterification. The
1Hꢀ1H COSY data allowed for the assignment of the resonances due to the
oxymethine protons as well as those arising from the sideꢀchain protons.
The protocols defined here are robust and offer the capacity to generate a
significant number of variants of the title 6ꢀOꢀacylglucose esters in high yields.
Furthermore, they are capable of producing such esters in a completely regioselective
fashion and at multiꢀgram scale. Additionally, product purification is simple and
efficient.
Hydrophile-Lipophile Balance (HLB) Values. The hydrophilicꢀlipophilic
balance (HLB) represents one of the standard means for emulsifier classification and
selection.25 This value is a measure of the degree to which a potential emulsifier is
hydrophilic (high HLB values are above 11) or lipophilic (low HLB values are below
9). Those in the range 9–11 are considered intermediate.26 The HLB values of each
product derived by the water number method are shown in Figure 3 and reveal that as
the length of the hydrophobic sideꢀchain increases from esters 2ꢀ8, the HLB values
decrease from 9.0 to 5.7. Since the potential utility of emulsifiers is often defined
using the HLB system, these values suggest that esters 2ꢀ8 could have practical
applications. Based on the Griffin method26,27 of predicting surfactant properties,
esters 2 and 3 have the potential to act as oilꢀinꢀwater emulsifiers (HLB >8), while
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esters 4ꢀ8 might be used as waterꢀinꢀoil emulsifiers (HLB <8). Compounds 2ꢀ5 could
also be expected to serve as wetting and spreading agents.
Foaming Properties. Surfactants stabilize the foams by preventing bubble
coalescence.28ꢀ30 Given their significance, foams are generally described in terms of
their “foamability”, which is defined as the capacity of the surfactants to form foams.
The stability of a foam, which tracks variations in foam height or volume over time, is
first monitored immediately after foam generation.22 The issues of foamability and
foam stability are vital for food emulsifiers since certain foods such as ice cream,
Chantilly cream, mousses and marshmallow need to possess good shelfꢀlives and
maintain their appearance even when subject to variations in storage conditions.31,32
The foaming properties of each product, as well as the four controls, were evaluated
in terms of their “foamability” at different concentrations and the outcomes of such
studies are shown in Figure 4. Unsurprisingly, foamability is positively correlated
with sample concentration. At all three concentrations, ester 2 displayed the weakest
foamability even though it had the highest HLB value. Compound 3 showed superior
properties but these were no better than those of the four controls. When the length of
alkyl side chain increased to 9 carbons (as seen in 4), the highest foamability was
observed (39.1% at 0.5% w/w). Indeed, ester 4 had remarkably better foamability than
the controls at the same concentrations. Nevertheless, it is clear that foamability
begins to decrease when products with longer (>9 carbons) alkyl side chains are
involved. Although ester 5 exhibited a slightly higher foamability than those of
controls under all concentrations tested, ester 6 was only comparable to such controls
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at high concentrations (29.4% at 0.5%). When the alkyl side chains were increased to
15 carbons (as in 7) or 17 carbons (as in 8), their foamabilities were lower than most
of the other samples. These results are rather counterintuitive since there appears to be
no correlation between the HLB values and foamability. The inverted Vꢀtype profile
revealed in Figure 4 is also intriguing in that while esters 2, 3, Sꢀ570, Sꢀ770 and
Span20 are regarded as more hydrophilic than compounds 4 and 5, the last pair
exhibited the best foamability. Presumably these outcomes are due to the differing
surface tensions of the compounds. Those with higher surface tensions, such as
compounds 2, 3, 7, 8, are less able to increase surface area to facilitate foam
generation and, thus, display poorer foamability.33
Foaming Stability. The foaming stability (FS) of esters 2ꢀ8 as well as the four
controls were measured over 10 min at 0.1%, 0.2%, and 0.5% w/w concentrations and
the outcomes of the relevant experiments are shown in Figure 5A. While ester 2 failed
to give any meaningful data, the FS of all the remaining esters increased with
concentration although the differences were very modest, if detectable, at the 0.2%
and 0.5% concentration levels. As was the case with the studies described above, an
inverted Vꢀshaped profile was observed in moving through the full series of glucose
esters. For esters 3ꢀ5, their FSs increased steadily from 30% through to 97.5% and
thus indicating the benefits of longer side chains in promoting foaming stability. In
contrast, with esters 6ꢀ8 the increasing length of the respective sideꢀchains
significantly reduced the capacity to maintain foams. Since foaming stability is
influenced by standing time, these were determined for all samples at 0.2% w/w
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concentration over 10, 20, 30 min. The outcomes of the relevant set of experiments
are shown in Figure 5B and reveal that the FSs for all esters decreased significantly
between the standing times of 10 and 30 min. At the 30 min mark the foams generated
using both esters 3 and 8 disappeared completely, indicating the poor stability of the
derived foaming systems. In contrast, the four commercial controls exhibited
excellent stability over time.
Emulsion Stability. Emulsion stability refers to the ability of an emulsion to
resist change in its properties over time.34 Such stability is highly dependent upon
liquid droplet size and distribution, emulsion processing conditions, the emulsifiers'
physical characteristics, solvents, the phase volume ratio and continuous phase
viscosity.35 The ability to form stable emulsions is a classic characteristic of good
surfactants.33 The emulsifying stability index of an emulsifier provides a measure of
stability, over a defined period of time, for the emulsion. Accordingly, the emulsion
stability indices (ESIs) of each product, as well as the four controls, were determined
at 0.1%, 0.2%, and 0.5% w/w concentrations in an oilꢀinꢀwater system. The data so
obtained are shown in Figure 6 and indicate that in each instance the test samples
displayed higher ESIs than the blank control and reveal that there is a good correlation
between the length of side chain and the ESI. In all instances, the ESIs were
proportional to concentration. Among the seven glucose esters, congener 2 showed the
lowest ESIs. A relatively high ESI (421.1 ± 12.9) was seen when ester 8 was tested at
low concentration (0.1% w/w) and this rose to 919.3 ± 34.7 at 0.5% w/w, thus
representing the highest ESI value observed. This profile could be attributed to the
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stronger hydrophobic interactions between the longer alkyl side chains in independent
molecules of this form and with the oil droplets thus assuming a more compact
structure and so resulting in higher emulsion stability.17 The ESIs of esters 2ꢀ8 are
significantly lower than those of the four controls at every concentration. This is
consistent with a previous study17 that revealed, after measuring the diameters of the oil
droplets in emulsions derived from two glucose esters, that unlike the sucrose esters,
the former delayed but did not ultimately prevent the coalescence of oilꢀinꢀwater
emulsions.
Cytotoxicity. Food emulsifiers must not only possess suitable functional
properties but, above all else, must be nonꢀtoxic.36 Sugar fatty acid esters are
generally accepted as safe emulsifiers in the food and cosmetics industries because
they are converted into harmless carbohydrates and fatty acids in the human stomach.
However, the cellular toxicities (cytotoxicities) of the glucose fatty acid esters have
not yet been established. In the present study, the cytotoxicities of the title esters were
evaluated against four cancer cell lines, specifically HepG2 (a liver cancer cell line),
MCFꢀ7 (a breast cancer cell line), LNacp (a prostate cancer cell line) and SW549 (a
thyroid cancer cell line) as well as LOꢀ2 (a normal liver cell line). As shown in Table
2, all seven 6ꢀOꢀacylglucose esters were devoid of significant cytotoxic effects.
Specifically, ester 4 showed weak cytotoxic activity towards all the cell lines
employed, the concentration required to inhibit cell proliferation at the 10% level
ranging from 210 to 600 ꢁmol/L. Ester 8 displayed weak inhibitory activity toward
SW549 but was inactive against the other four cell lines. The remaining esters had no
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notable cytotoxicity towards any of the five cell lines. Therefore, esters 2, 3, 5, 6, 7
can be regarded as nonꢀtoxic toward all of the cell lines screened in this study.
The structureꢀfunction profile of the 6ꢀOꢀacylglucose esters, as established
through this study, can be summarized as follows: when the length of alkyl side chain
of the 6ꢀOꢀacylglucose esters increases, their HLB values decrease, while, contrarily,
the stabilities of the derived emulsions increase. To have good foaming properties, a 9
or 11 carbon alkyl side chain is required. The presence of a side chain with less or
more carbons than these decreases the foaming properties. The protocols and data
presented here should provide an important reference point for further studies on the
preparation and evaluation of fatty acid esters derived from diꢀ and higher
orderꢀsaccharides.
ASSOCIATED CONTENT
Supporting Information
1
1H and 13C NMR Spectra of compounds 2ꢀ8, HMBC, Hꢀ1H COSY spectra of
compound 7. This material is available free of charge via the internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +86ꢀ20ꢀ85221367. Fax: +86ꢀ20ꢀ85224766.
Eꢀmail: twyong@jnu.edu.cn (Y. W.)
Eꢀmail: ping.lan@jnu.edu.cn (P. L.)
Funding
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We gratefully acknowledge financial support from the National Key Research and
Development Program of China under grant 2017YFD0400200, and the Department
of Science and Technology of Guangdong Province under grants 2017B090907018
and 2014A010107014 as well as the Fundamental Research Funds for the Central
Universities, China.
Notes
The authors declare no competing financial interest.
REFERENCES
(1) Ferrer, M.; Cruces, M. A.; Bernabe, M.; Ballesteros, A.; Plou, F. J.
Lipaseꢀcatalyzed regioselective acylation of sucrose in twoꢀsolvent mixture.
Biotechnol. Bioeng. 1999, 65, 10–16.
(2) Yang, Z.; Huang, Z. L. Enzymatic synthesis of sugar fatty acid esters in ionic
liquids. Catal. Sci. Technol. 2012, 2, 1767–1775.
(3) Polat, T.; Linhardt, R. J. Syntheses and applications of sucroseꢀbased esters. J.
Surfactants Deterg. 2001, 4, 415-421.
(4) Kobayashi, T. Lipaseꢀcatalyzed syntheses of sugar esters in nonꢀaqueous media.
Biotechnol. Lett. 2011, 33, 1911–1919.
(5) Zhao, R.; Chang, Z.; Jin, Q.; Li, W.; Dong, B.; Miao, X. Heterogeneous base
catalytic transesterification synthesis of sucrose ester and parallel reaction control.
Int. J. Food Sci. Technol. 2014, 49, 854–860.
(6) Yan, Y.; Bornscheuer, U. T.; Stadler, G.; LutzꢀWahl, S.; Reuss, M.; Schmid, R. D.
Production of sugar fatty acid esters by enzymatic esterification in a stirredꢀtank
21
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Page 22 of 35
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
membrane reactor: optimization of parameters by response surface methology. J.
Am. Oil. Chem. Soc. 2001, 78, 147–152.
(7) Cruces, M. A.; Plou, F. J.; Ferrer, M.; Bernabé, M.; Ballesteros, A. Improved
synthesis of sucrose fatty acid monoesters. J. Am. Oil Chem. Soc. 2001, 78, 541–
546.
(8) Khan, R. Chemistry and new uses of sucrose: how important? Pure Appl. Chem.
1984, 56, 833–844.
(9) Park, S.; Kazlauskas, R. J. Improved preparation and use of roomꢀtemperature
ionic liquids in lipaseꢀcatalyzed enantioꢀ and regioꢀselective acylations. J. Org.
Chem. 2001, 66, 8395–8401.
(10)Liu, Q.; Janssen, M. H. A.; van Rantwijk, F.; Sheldon, R. A. Roomꢀtemperature
ionic liquids that dissolve carbohydrates in high concentrations. Green Chem.
2005, 7, 39–42.
(11)Huang, D.; Jiang, X.; Zhu, H.; Fu, X.; Zhong, K.; Gao, W. Improved synthesis of
sucrose fatty acid monoesters under ultrasonic irradiation. Ultrason. Sonochem.
2010, 17, 352–355.
(12)Zhong, X.; Qian, J.; Liu, M.; Ma, L. Candida rugosa lipase catalyzed synthesis of
sucroseꢀ6ꢀacetate in a 2ꢀbutanol/buffer two phase system. Eng. Life Sci. 2013, 13,
563–571.
(13)Inprakhon, P.; Wongthongdee, N.; Amornsakchai, T.; Pongtharankul, T.;
Sunintaboon, P.; Wiemann, L. O.; Durand, A.; Sieber, V. Lipaseꢀcatalyzed
synthesis of sucrose monoester: increased productivity by combining enzyme
22
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Journal of Agricultural and Food Chemistry
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
pretreatment and nonꢀaqueous biphasic medium. J. Biotechnol. 2017, 259, 182–
190.
(14)Plou, F. J.; Cruces, M. A.; Ferrer, M.; Fuentes, G.; Pastor, E.; Bernabé, M.;
Christensen, M.; Comelles, F.; Parra, J. L.; Ballesteros, A. Enzymatic acylation of
diꢀ and triꢀsaccharides with fatty acids: choosing the appropriate enzyme, support
and solvent. J. Biotechnol. 2002, 96,55ꢀ66.
(15)Habulin, M.; Šabeder, S.; Knez, Ž. Enzymatic synthesis of sugar fatty acid esters
in organic solvents and in supercritical carbon dioxide and their antimicrobial
activity. J. Supercrit. Fluids 2008, 45, 338–345.
(16)Shi, Y. G.; Li, J. R.; Chu, Y. H. Enzymeꢀcatalyzed regioselective synthesis of
sucroseꢀbased esters. J. Chem. Technol. Biotechnol. 2011, 86, 1457–1468.
(17)Ren, K.; Lamsal, B. P. Synthesis of some glucoseꢀfatty acid esters by lipase from
Candida antarctica and their emulsion functions. Food Chem. 2017, 214, 556–
563.
(18)Ganske, F.; Bornscheuer, U. T. Optimization of lipaseꢀcatalyzed glucose fatty
acid ester synthesis in a twoꢀphase system containing ionic liquids and tꢀBuOH. J.
Mol. Catal. B: Enzym. 2005, 36, 40–42.
(19)Poehnlein, M.; Ulrich, J.; Kirschhoefer, F.; Nusser, M.; MuhleꢀGoll, C.;
Kannengiesser, B.; BrennerꢀWeiss, G.; Luy, B.; Liese, A.; Syldatk, C.; Hausmann,
R. Lipaseꢀcatalyzed synthesis of glucoseꢀ6ꢀOꢀhexanoate in deep eutectic solvents.
Eur. J. Lipid Sci. Technol. 2015, 117, 161–166.
(20)Davis, R. A.; Lin, C. H.; GervayꢀHague, J. Chemoenzymatic synthesis of
23
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Page 24 of 35
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
cholesterylꢀ6ꢀOꢀtetradecanoylꢀαꢀDꢀglucopyranoside: a product of host cholesterol
efflux promoted by Helicobacter pylori. Chem. Commun. 2012, 48, 9083–9085.
(21)Gupta, R. K.; James, K.; Smith, F. J. Sucrose esters and sucrose ester/glyceride
blends as emulsifiers. J. Am. Oil Chem. Soc. 1983, 60, 862–869.
(22)Zhang, X.; Song, F.; Taxipalati, M.; Wei, W.; Feng, F. Comparative study of
surfaceꢀactive properties and antimicrobial activities of disaccharide monoesters.
PLoS One 2014, 9, 1–19.
(23)Lajnaf, R.; PicartꢀPalmade, L.; Cases, E.; Attia, H.; Marchesseau, S.; Ayadi, M. A.
The foaming properties of camel and bovine whey: the impact of pH and heat
treatment. Food Chem. 2018, 240, 295–303.
(24)Pearce, K. N.; Kinsella, J. E. Emulsifying properties of proteins: evaluation of a
turbidimetric technique. J. Agric. Food Chem. 1978, 26, 716–723.
(25)Chun, A. H.; Martin, A. N. Measurement of hydrophileꢀlipophile balance of
surfaceꢀactive agents. J. Pharm. Sci. 1961, 50, 732–736.
(26)Griffin, W. C. Classification of surface active agents by HLB. J. Soc. Cosmet.
Chem. 1949, 1, 311–326.
(27)Griffin, W. C. Calculation of HLB values of nonionic surfactants. Am. Perfum.
and Essent. Oil Rev. 1995, 65, 26–29.
(28)Wege, H. A.; Kim, S.; Paunov, V. N.; Zhong, Q.; Velev, O. D. Longꢀterm
stabilization of foams and emulsions with inꢀsitu formed microparticles from
hydrophobic cellulose. Langmuir 2008, 24, 9245–9253.
(29)Belhaij, A.; AlꢀMahdy, O. Foamability and foam stability of several surfactants
24
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Journal of Agricultural and Food Chemistry
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
solutions: the role of screening and flooding. J. Pet. Environ. Biotechnol. 2015, 6,
1–6.
(30)Malysa, K.; Lunkenheimer, K. Foams under dynamic conditions. Curr. Opin.
Colloid Interface Sci. 2008, 13, 150–162.
(31)Hutzler, S.; Lösch, D.; Carey, E.; Weaire, D.; Hloucha, M.; Stubenrauch, C.
Evaluation of a steadyꢀstate test of foam stability. Philos. Mag. 2011, 91, 537–
552.
(32)Foegeding, E. A.; Luck, P. J.; Davis, J. P. Factors determining the physical
properties of protein foams. Food Hydrocolloids 2006, 20, 284–292.
(33)Kothekar, S. C.; Ware, A. M.; Waghmare, J. T.; Momin, S. A. Comparative
analysis of the properties of tweenꢀ20, tweenꢀ60, tweenꢀ80, arlacelꢀ60, and
arlacelꢀ80. J. Dispersion Sci. Technol. 2007, 28, 477–484.
(34)Silvestre, M. P. C.; Decker, E. A.; McClements, D. J. Influence of copper on the
stability of whey protein stabilized emulsions. Food Hydrocolloids 1999, 13,
419–424.
(35)Avramenko, N. A.; Low, N. H.; Nickerson, M. T. The effects of limited
enzymatic hydrolysis on the physicochemical and emulsifying properties of a
lentil protein isolate. Food Res. Int. 2013, 51, 162–169.
(36)Kaur, K.; Kaur, J.; Kumar, R.; Mehta, S. K. Formulation and physiochemical
study of αꢀtocopherol based oil in water nanoemulsion stabilized with non toxic,
biodegradable surfactant: sodium stearoyl lactate. Ultrason. Sonochem. 2017, 38,
570–578.
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FIGURE CAPTIONS
Figure 1. Enzymatic syntheses of the 6ꢀOꢀacylglucose esters 2ꢀ8.
Figure 2. Key HMBC and 1Hꢀ1H COSY correlations for 6ꢀOꢀpalmitoylglucopyranose
7.
Figure 3. The HLB values of compounds 2ꢀ8 and the commercial sucrose stearic
esters Sꢀ570 and Sꢀ770 (mean ± SD, n = 3).
Figure 4. The foamability of compounds 2ꢀ8 and the commercial sucrose stearic
esters Sꢀ570 and Sꢀ770 as well as span20 and span80 at 0.1%, 0.2%, and 0.5% w/w
concentrations (mean ± SD, n = 3).
Figure 5. (A) The foaming stability of compounds 2ꢀ8 and the commercial sucrose
stearic esters Sꢀ570 and Sꢀ770 as well as span20 and span80 over 10 min at 0.1%,
0.2%, and 0.5% w/w concentrations; and (B) over 10, 20, 30 min at 0.2% w/w
concentration (mean ± SD, n = 3).
Figure 6. The emulsion stability index of compounds 2ꢀ8 and the commercial sucrose
stearic esters Sꢀ570 and Sꢀ770 as well as span20 and span80 over 20 min at 0.1%,
0.2%, and 0.5% w/w concentrations (mean ± SD, n = 3).
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Journal of Agricultural and Food Chemistry
Table 1. Investigations of Acylation Conditionsa
Enzyme
Entry Loading
(w/w %)
Yields (%)
Solvents
(v/v ratio)
THF/pyridineb
(4:1)
Time
(h)
2
3
4
5
6
7
8
1
2
3
4
5
24
78
84 82 88 88 80 72
54 56 57 57 51 52
89 87 90 92 90 75
48 56 55 52 47 43
5
DMSO/tꢀBuOHb
(1:1)
48
24
48
47
82
41
10
10
THF/pyridineb
(4:1)
DMSO/tꢀamyl
alcoholb
(4:1)
5
6
20
20
THF/pyridinec
(4:1)
48
48
44
89
51 57 60 58 52 29
94 95 95 95 87 82
THF/pyridineb
(4:1)
aThe reactions were carried out using glucose (1 mmol) and the relevant fatty acid
vinyl ester (3 mmol) under the conditions shown above. The products were normally
purified via trituration but in the cases involving incomplete reaction, column
b
chromatographic separations were performed. Strictly anhydrous solvents used.
cReagentꢀgrade solvents used.
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Journal of Agricultural and Food Chemistry
Page 28 of 35
Table 2. The Cytotoxic (> 10% Inhibition) Concentrations of Glucose Esters 2ꢀ8
Cell Line
HepG2
(ꢁM)
LNacp
(ꢁM)
MCFꢀ7
(ꢁM)
SW549
(ꢁM)
LO2
Sample
(ꢁM)
2260±9
2
＞2300±3
＞2300±70
＞2120±20
＞2300±20
＞2120±2
＞2300±10
1800±30
1240±50
3
＞2120±6
360±2
600±30
510±2
480±9
880±3
210±30
4
5
1240±10
1740±20
830±10
＞1760±60
1640±10
6
7
8
＞1650±20
＞1650±10
＞1220±6
＞1130±20
＞1650±8
＞1220±10
＞1130±6
＞1650±50
＞1220±90
＞1130±30
＞1220±10
＞1220±20
1120±20
560±3
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Journal of Agricultural and Food Chemistry
Figure 1.
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