Effect of Variations in the Fatty Acid Residue of Lactose Monoesters on Their Emulsifying Properties and Biological Activities

Article abstract of DOI:10.1021/acs.jafc.8b05794

Lactose fatty acid esters are high-value-added derivatives of lactose and represent a class of biodegradable, non-ionic, low-molecular-weight surfactants (emulsifiers) that have considerable potential in the food, cosmetic, and pharmaceutical industries. Certain lactose esters have also garnered attention for their biological activities. In this work, we detail syntheses of a homologous series of 6′-O-acyllactose esters of varying alkyl chain length (from 6 to 18 carbons) and report on their activities as surfactants as well as their antimicrobial and cytotoxic properties. The structure-property profiles established in this work revealed that while the medium-chain esters displayed excellent emulsifying properties and moderate antimicrobial activities, their longer chain congeners exhibited the highest cytotoxicities. As such, we have established that certain 6′-O-acyllactose esters are superior to their sucrose-derived and commercially exploited counterparts. These results will serve as a useful guide for the development of lactose esters as, inter alia, emulsifiers in the food industry.

Full text of DOI:10.1021/acs.jafc.8b05794

Article
pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Effect of Variations in the Fatty Acid Residue of Lactose Monoesters
on Their Emulsifying Properties and Biological Activities
Min-Yi Liang,† Martin G. Banwell,‡,§ Yong Wang,*,†,‡ and Ping Lan*,†,‡
†Department of Food Science and Engineering, Jinan University, Guangzhou, Guangdong 510632, People’s Republic of China
‡Institute for Advanced and Applied Chemical Synthesis, Jinan University, Zhuhai, Guangdong 519070, People’s Republic of China
§Research School of Chemistry, Institute of Advanced Studies, The Australian National University, Canberra, Australian Capital
Territory 2601, Australia
S
* Supporting Information
ABSTRACT: Lactose fatty acid esters are high-value-added derivatives of lactose and represent a class of biodegradable, non-
ionic, low-molecular-weight surfactants (emulsifiers) that have considerable potential in the food, cosmetic, and pharmaceutical
industries. Certain lactose esters have also garnered attention for their biological activities. In this work, we detail syntheses of a
homologous series of 6′-O-acyllactose esters of varying alkyl chain length (from 6 to 18 carbons) and report on their activities as
surfactants as well as their antimicrobial and cytotoxic properties. The structure−property profiles established in this work
revealed that while the medium-chain esters displayed excellent emulsifying properties and moderate antimicrobial activities,
their longer chain congeners exhibited the highest cytotoxicities. As such, we have established that certain 6′-O-acyllactose
esters are superior to their sucrose-derived and commercially exploited counterparts. These results will serve as a useful guide
for the development of lactose esters as, inter alia, emulsifiers in the food industry.
KEYWORDS: 6′-O-acyllactose esters, surfactant properties, practical applications, antimicrobial activity, cytotoxicity
microorganisms.18−23 These lipases usually affect esterification
at the non-reducing end of lactose, viz., at the C6′ hydroxyl, thus
INTRODUCTION
■
Lactose is a disaccharide comprised of a glucose residue linked in
affording a variety of 6′-O-acyllactose esters.
a 4,1′ manner to a galactose unit. As the most abundant
Although the lipase-catalyzed synthesis of lactose fatty acid
saccharide component in the milk of most mammals, lactose
esters has been studied extensively, along with their properties
makes up around 80% of the dry weight of whey, a byproduct
and associated applications,4,24,25 their structure−property
from the cheese and casein manufacturing industries.1,2
profiles, especially those concerned with the influence of the
Unprocessed lactose has been used extensively in the food and
alkyl chain length on the surface-active properties of 6′-O-
pharmaceutical sectors, including, for instance, as a key
acyllactose esters, remain to be clearly defined. As such, the
supplement in baby formulae, cream stouts, and confectionary
influence of the alkyl chain length on their surfactant properties
products and as an excipient for many tableted drugs.2 Beyond
and their bioactivities remains to be studied in any detail.
those direct applications, lactose is also a versatile raw material
Herein, we report on the synthesis, purification, rigorous
for “upgrading”, by fermentation or chemical transformation,
spectroscopic characterization, and systematic evaluation of
and thus providing a variety of value-added derivatives,
the surface-active properties and bioactivities of a homologous
including lactitol, lactobionic acid, lactulose, lactosucrose,
series of 6′-O-acyllactose esters bearing varying alkyl chains
lactosyl urea, galacto-oligosaccharides, tagatose, and lactose
(from 6 to 18 carbons in length). These studies reveal clear
esters.2−4 Most of the commercially available derivatives of
relationships between the nature of the fatty acid residue of the
lactose are either used as ingredients in functional foods5−7 (as
esters and both their emulsifying functionalities and their
encountered in prebiotoics, low-calorie sweeteners, preserva-
bioactivities. The structure−property profile established as a
tives, and food acidulants) or employed as laxatives8 or chelating
result of the current work, along with efforts to identify practical
agents3 in pharmaceuticals. In recent years, lactose esters have
gained considerable attention from the food, cosmetic,
applications for these esters, provides a useful guide for the
development of lactose esters as emulsifiers in the food industry.
detergent, and pharmaceutical industries, not least because
they possess excellent emulsifying properties9−11 that are
accompanied, in various instances, by antimicrobial,9,12−15
MATERIALS AND METHODS
■
antiproliferative,14 permeability-enhancing,15 and/or other bio-
Materials. D-Lactose (anhydrous, 99%), sucrose (99%), sodium
benzoate (99%), and vancomycin were purchased from the Energy
logical activities.16,17
Lactose-ester-based surfactants are normally prepared using
enzyme-catalyzed esterification or transesterification reactions.
Various enzymes have been employed for such purposes, most
notably lipases, such as Novozym 43511,18−20 and Lipozyme
TLIM,9 along with certain others derived from a range of
Received: October 21, 2018
Revised: November 1, 2018
Accepted: November 5, 2018
Published: November 5, 2018
© XXXX American Chemical Society
A
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Figure 1. Enzymatic syntheses of 6′-O-acyllactose esters 2−8.
Chemical Co. Ltd. (Shanghai, China). Fatty acid vinyl esters (>99%)
were purchased from the Tokyo Chemical Industry Co., Ltd. (Tokyo,
Japan), while Lipozyme TLIM was supplied by Novo Nordisk
(Bagsværd, Denmark). Pure canola oil, full cream milk, and coconut
were purchased from a local grocery store, while commercial sucrose
esters P-1570 and P-1670 were obtained from the Mitsubishi-Chemical
Foods Corporation (Tokyo, Japan).
Synthesis of the 6′-O-Acyllactose Esters and 6-O-Lauroylsu-
crose Ester (LSE). A solution of D-lactose (3.42 g, 10.0 mmol) in
anhydrous tetrahydrofuran (THF)/pyridine (50 mL, 1:1, v/v) was
treated with Lipozyme TLIM (3.42 g, 100%, w/w loading) and the
relevant fatty acid vinyl ester (3.0 equiv, 30.0 mmol). The ensuing
mixture was stirred magnetically at 55 °C for 48 h and then cooled; the
residual enzyme was removed by filtration, and the solid thus-retained
was washed with dichloromethane (2 × 30 mL) and then methanol (2
× 30 mL). The combined filtrates were dried (Na2SO4), filtered, and
then concentrated under reduced pressure to afford a yellow oil that was
subjected to flash chromatography on silica and eluting with methanol/
dichloromethane (1:9, v/v). Concentration of the relevant fractions (Rf
= 0.2) then afforded the corresponding 6′-O-acyllactose esters 2−8
(Figure 1) as amorphous, white solids. LSE that served as a positive
control in the following evaluations was prepared via the same protocol
using sucrose and vinyl laurate.
Determination of Hydrophilic−Lipophilic Balance (HLB)
Values. The water number method26 was used to determine the
HLB values of each sample. Thus, 1.0 g of each product was dissolved in
a dimethylformamide (DMF)/benzene mixture (25 mL of a 100:5, v/v
mixture), and the ensuing solution was titrated with distilled water
while being maintained at 25 1 °C until a permanent turbidity was
observed. All results were reproducible to within 0.5 units of HLB. A
plot of the consumed water number versus HLB was then established
using surfactants (Span 20, Span 40, Span 60, Span 80, Tween 20, and
Tween 80) with known HLB values for calibration purposes. The
experimental HLB value of each sample was then read from this plot.
The theoretical HLB values were calculated using Griffin’s method.9
Evaluation of Water Solubility. A saturated aqueous solution of
each sample in distilled water maintained at 25 1 °C was filtered; 1
mL of each filtrate was then transferred into a preweighed round-
bottom flask, the water was removed by evaporation under reduced
pressure, and the amount of sugar ester remaining was then determined.
The derived water solubilities are presented in milligrams per milliliter.
Measurement of the Critical Micelle Concentration (CMC).
The CMCs of each sample were measured using the Wilhelmy plate
method.27 Specifically, a series of variably concentrated aqueous
solutions of the samples was prepared and then maintained at 28 °C.
The equilibrium surface tension of each solution was then measured
solutions of each sample at concentrations above their CMCs was
mixed with equal volumes of canola oil. The oil−water interfacial
tension was measured with a horizontal hanging platinum ring of
known geometry (platinum circle radius of 9.55 mm and platinum wire
radius of 0.3 mm). The ring was dipped into the solutions maintained at
28 °C and then pulled out again. The maximum force, in milliNewtons
per meter, required to pull the ring through the interface is defined as
the oil−water interfacial tension (γO/W).
Determination of the Contact Angle. Pellets of each test sample
were prepared using 0.08 g of powdered compound that was placed on a
13 mm pellet die and pressed under a force of 5000 kg for 10 s in a HY-
12 hydraulic compressor (Tianguang, Tianjin, China). An approx-
imately 4 μL drop of deionized water was applied to the surface of the
pellet, and the contact angle was then measured using a CAM-PLUS
goniometer (Tantec, Lunderskov, Denmark). Values of θ were
determined at 0 s.29
Evaluation of Foamability and Foaming Stability. A total of 10
mL of 0.1, 0.2, and 0.5% (w/w) aqueous dispersions of each sample
were placed in a 50 mL centrifuge tube, and the height of the solution
(H0) was measured. Each solution was then homogenized using a XW-
80A rotary mixer worm (Shanghai Jiapeng Technology Co., Ltd.,
China) operating at a frequency of 2800/min for 2 min. The resulting
foam height (H2) and the total height (H1) were measured
immediately. Following resting periods of 10, 20, and 30 min, the
new foam heights (H3) were then recorded. The foamabilities and the
foaming stabilities of each test sample were calculated using the
following equations:30
foamability (%) = (H − H0)/H0 × 100%;
1
foaming stability (%) = H3/H2 × 100%
Determination of the Emulsion Stability Index (ESI). A total of
10.0 g of 0.1, 0.2, and 0.5% (w/w) aqueous solutions of each sample
were each mixed with 1.0 g of canola oil. The ensuing mixtures were
homogenized for 2 min at 10 000 rpm using a blender, and a 20 μL
sample of the resulting emulsion was diluted with 2 mL of deionized
water before being transferred to a clean spectrometric cuvette. The
absorbance of the emulsion at 500 nm was recorded at 0 and 20 min.
The ESIs were then calculated using the following equation:31
ESI (%) = A0 × 20/[A0 − A20] × 100%
where A0 and A20 are the absorbances taken at 0 and 20 min,
respectively.
Measurement of Particle Size Distributions of Coconut Milk
Emulsions. Fresh coconut pulp (100 g) was mixed with water (33 mL)
in a juicer and after being processed for 5 min, the resulting blend was
filtered through cheesecloth, thereby providing fresh coconut
milk.11,32,33 A total of 10.0 g of 0.3% (w/w) solutions of lactose ester
5, Tween 20, and P-1670 in fresh coconut milk were homogenized for 2
min at 10 000 rpm using a blender. One drop of each emulsion was then
transferred to a microscope slide and a coverslip placed over the sample.
The samples were then analyzed using a model BX61 optical
microscope (Olympus, Tokyo, Japan) fitted with a camera and
objective lens of 200× and capable of 1024 × 720 resolution. Image
using a Kruss tensiometer (Hamburg, Germany). The CMC of each
̈
sample was then calculated from the break in the surface tension values
versus log10 concentration and is expressed in micromolars (γCMC is the
surface tension corresponding to the CMC).
Evaluation of the Oil−Water Interfacial Tension. The Du
Nou
̈
y ring method28 was employed to evaluate the oil−water interfacial
tension using a JYW-200B automatic interfacial tensiometer (Chengde
Jin He Instrument Manufacturing Co., Ltd., China). A series of aqueous
B
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Figure 2. Key HMBC correlations for 6′-O-hexanoyllactose 2.
Products 6.0 software (Olympus, JP, Ltd.) was then used to determine
the distribution and size of the particles in each emulsion.
for 4 h at 37 °C in 5% CO2. After incubation, the staining agent was
removed and 100 μL of DMSO was added to each well. The plates were
placed on a table oscillator for 20 min, and absorbance was measured at
570 nm using an Epoch2 microplate reader (Biotek Instruments,
Winooski, VT, U.S.A.). Results are expressed as half maximal inhibitory
concentration (IC50), which is the dose of sample resulting in a 50% loss
of cell viability.
Statistical Analysis. Experiments was performed in triplicate, and
the results are presented as mean values standard deviation (SD).
Statistical analyses were performed using SPSS software (SPSS, Inc.,
Chicago, IL, U.S.A.) to identify any significant deviations. All values
were assessed by one-way analysis of variance (ANOVA) in
conjunction with Duncan’s new multiple-range test (MRT). A
difference was considered significant when p < 0.05.
Analysis of Thermal Stability. The thermal stabilities of each
sample were evaluated by thermogravimetric analysis. Specifically, 10
mg of each sample was placed in a TG209F3-ASC thermogravimetric
analyzer (Netzsch, Germany); the testing temperature range was set
from 50 to 350 °C and a heating rate of 7 °C/min was employed. The
samples were maintained under a nitrogen atmosphere in an Al2O3
crucible. The reductions in mass of each sample as a function of the
temperature during this heating process were recorded.
Evaluation of Antimicrobial Activity. The minimum inhibitory
concentrations (MICs) of lactose esters 2−8 against six bacterial strains
were determined by microdilution methods.15 Thus, a 1.28 mg sample
of each ester was dissolved in dimethyl sulfoxide (DMSO, 1 mL), while
several colonies of each bacterial strain were used to inoculate 10 mL of
Mueller-Hinton broth (Oxoid, Waltham, MA, U.S.A.) before being
incubated at 37 °C for 24 h. The ensuing bacterial suspensions were
then adjusted to a turbidity of 5 × 105 colony-forming units (CFU)/
mL. Aliquots (100 μL) of each such standardized bacterial suspension
were then added to a 96-well plate, along with a solution of each ester, to
yield final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2, and 1 μg/
mL. The optical density (OD) at 595 nm in each well was measured at t
= 0 and 24 h using a Multiskan MK3 microplate reader (Thermo
Labsystems, Helsinki, Finland). MICs were defined as the lowest
concentration at which the bacterial growth was completely inhibited
(OD < 0.05) after 24 h.
Evaluation of Cytotoxicity. The 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method, with some modifica-
tions, was employed to evaluate the cytotoxicities of lactose esters 2−8.
Specifically, cells were seeded at densities of around 3000 cells/well on a
96-well plate in 100 μL of complete medium [Dulbecco’s modified
Eagle’s medium (DMEM) + 5% fetal bovine serum (FBS), 50 units/mL
penicillin, and 50 μg/mL streptomycin]. After incubation for 12 h at 37
°C in 5% CO2, the medium was then removed. Fresh, complete
medium containing the test sample 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, 20 μL of the 5 mg/mL MTT
stock solution was added to each well. Then, the cells were incubated
RESULTS AND DISCUSSION
■
Synthesis of 6′-O-Acyllactose Esters 2−8. Esters 2−8
and LSE were prepared through the enzyme-mediated trans-
esterification of lactose (sucrose for LSE) using 3 mol equiv of
the relevant and commercially available fatty acid vinyl ester.
Screening a range of solvents established that THF/pyridine
(1:1, v/v) provided the best medium, while a survey of various
commercially available enzymes indicated that Lipozyme TLIM
was the most effective in the present setting. Reactions were run
at 55 °C for 48 h, and after cooling and conventional workup, the
crude reaction mixture was subjected to flash chromatography.
By such means, pure samples [>99% as judged by high-
performance liquid chromatographic analysis using an evapo-
rative light scattering detector (HPLC−ELSD)] were obtained
in yields ranging from 58 to 78% based on recovered starting
material (b.r.s.m.), viz., the lactose. Each lactose ester was
1
subjected to detailed H and 13C nuclear magnetic resonance
(NMR) spectroscopic as well as mass spectrometric analysis,
and the derived data were in complete accord with the illustrated
monoester structures. Key elements of the hetereonuclear
multiple bond correlation (HMBC) spectra derived from 6′-O-
C
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Table 1. HLB, Water Solubility, Contact Angle, CMC, γCMC, and γO/W of 6′-O-Lactose Esters 2−8, LSE, and Commercial Sucrose
Esters P-1570 and P-1670
a
b
sample
HLB measured
HLB calculated
water solubility (mg/mL)
contact angle, θ (deg)
CMC (μM)
γCMC (mN/m)
γO/W (mN/m)
2
3
4
5
6
7
8
17.1
16.7
16.4
15.9
13.5
10.2
10.1
14.0
15.0
16.0
15.5
14.6
13.8
13.0
12.4
11.8
11.2
13.0
715
650
604
312
154
74
27
1284
215
18.0
29.4
30.0
30.7
33.0
34.5
42.5
9.4
1258
850
684
308
63
15
12
437
34
11
35.1
30.6
32.7
34.5
33.7
31.4
31.8
33.9
36.7
38.2
8.1
7.6
4.8
4.2
3.7
2.8
2.6
3.8
1.4
1.8
LSE
P-1570
P-1670
258
a
b
HLB values were determined using the water number method. HLB values were calculated by Griffin’s method.
Figure 3. (A) Foamability of compounds 2−8, LSE, and the commercial sucrose esters P-1570, P-1670, Span 20, and Tween 20 at 0.1, 0.2, and 0.5%
(w/w) concentrations (mean SD; n = 3). (B) Foaming stability of compounds 2−8 and the five controls over 10, 20, and 30 min at 0.2% (w/w)
concentration (mean SD; n = 3). Values in each group with different letters denote a significant difference (p < 0.05). Capital letters indicate a
comparison of different concentrations of the same surfactant. Lowercase letters in the same column indicate a comparison of different surfactants at
the same concentration.
hexanoyllactose 2 are shown in Figure 2, and this is
representative of the series as a whole. The methylene protons
at C6′ interact with the carbonyl carbon of the ester moiety, thus
establishing the site of esterification. Other key interactions
allowed for the assignment of the resonances arising from the
non-hydroxylic protons associated with the lactose and side-
chain substructures.
Surface-Active Properties. The HLB is a measure of the
degree to which a surfactant is hydrophilic or lipophilic and is
one of the standard means for classifying surfactants, thus
allowing for consideration of them for particular applications.34
HLBs can be predicted by assigning values, as described by
Griffin,35 to the various substructures associated with a given
compound or by experimental measurement using, for example,
the water number method. As shown in Table 1, the
D
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Figure 4. ESI of compounds 2−8, LSE, and the commercial surfactants P-1570, P-1670, Span 20, and Tween 20 over 20 min at 0.1, 0.2, and 0.5% (w/
w) concentrations (mean SD; n = 3). Values in each group with different letters denote a significant difference (p < 0.05). Capital letters indicate a
comparison of different concentrations of the same surfactant. Lowercase letters in the same column indicate a comparison of different surfactants at
the same concentration.
experimentally determined HLBs for lactose esters 2−8 range
from 17.1 to 10.1, a trend in broad agreement with the calculated
values, although there was some notable variation. In particular,
while the molecular formulae of ester 5 and LSE are the same, as
are their calculated HLB values (13.0), their experimentally
derived HLB values were 15.9 and 14.0, respectively. Broadly
speaking, then, esters 2−8 can all be regarded as hydrophilic,
with their measured HLB values indicating that they could serve
as oil-in-water emulsifiers, while esters 2−6 could also act as
detergents.35 The water solubilities of esters 2−8 match their
HLB profiles, although compound 5 shows a lower water
solubility than LSE, a feature that is attributed to the inferior
solubility of the associated lactose residue.
The contact angle (θ) is a commonly used measure of the
wetting behavior of surfactants, species that have broad practical
applications, including in the food, agriculture, soil, and
pharmaceutical industries.29,36 Small contact angles (<90°)
correspond to high wettability, while larger contact angles
(>90°) suggest low wettability. Upon initial deposition of a
droplet of deionized water on the surface of compressed powder
discs of lactose esters 2−8, values of θ between 18° and 42.5°
were measured (Table 1). In keeping with expectations, the
trend in contact angles for these esters ran counter to the
corresponding HLB values and led to the conclusion that these
compounds have high wettability.
The minimum surfactant concentration required to form
micelles (the so-called critical micelle concentration or CMC) of
each test sample is listed in Table 1 and parallels the
corresponding HLB value. In essence, the CMC values of esters
2−8 are correlated with the length of the hydrophobic side
chain, with, for instance, compound 2 displaying the highest
CMC (1258 μM). Congeners 3−8, with their increasingly long
side chains, have CMC values that decrease from 850 to 12 μM.
This “steady” trend varies from those seen in the sucrose,
maltose, and fructose monoester series, where the stearoyl (C18)
esters show higher CMCs than their palmitoyl (C16) counter-
parts.27,28 Lactose esters 6−8 displayed similar CMC values (63,
15, and 12 μM, respectively) to the commercially deployed, non-
ionic sucrose esters P-1570 (34 μM) and P-1670 (11 μM),
indicating that those with the appropriate hydrophobic tail could
be useful in industrial settings. The γCMC values determined for
esters 6−8 are shown in Table 1, but these do not show any clear
correlation with the length of the associated side chains.
Because compounds 2−8 have potential as oil-in-water
emulsifiers, the interfacial tensions of their aqueous solutions
with canola oil were measured. As anticipated and shown in
Table 1, these esters displayed low interfacial tensions within the
oil−water system, further encouraging their deployment as
emulsifiers in such settings.37 Clearly, the length of the
associated hydrophobic chain plays an important role in
determining the interfacial tension values of compounds 2−8
because the γO/W values decreased from 8.1 to 2.6 mN/m as the
size of this moiety increased. This is entirely consistent with the
outcomes of earlier studies28 that have shown that interfacial
tension is directly related to surfactant hydrophobicity. This
effect is attributed to the adsorption of the surfactants at the
phase boundary and an accompanying transfer of the surfactant
molecules across this.38 In the present study then, those lactose
esters with more hydrophobic moieties are more soluble in the
oil, thus creating a lower interfacial tension with the water.
Significantly, in comparison to their commercially deployed
sucrose counterparts, the lactose esters, in general, displayed
slightly higher interfacial tension values, even though the former
series of compounds are contaminated with di- or higher order
esters. This remains the case when lactose ester 5 is compared to
the pure sucrose monoester LSE embodying the same lauroyl
side chain. Such results clearly emphasize the likely utility of
esters 5−8 as emulsifiers because of their ability to effectively
reduce the interfacial energy barrier.
Foaming Properties. The foamabilities of lactose esters 2−
8 together with five controls were determined at varying
concentrations, and the results of such studies (Figure 3A)
revealed a positive correlation between foamability and sample
concentration. Specifically, compound 2 exhibited the poorest
foamability at all three concentrations. Although ester 3 showed
superior properties to lower homologue 2, it was no better than
those of the five controls. When the length of the alkyl side chain
increased to 9 carbons (as seen in ester 4), then foamability
matching those of the commercial controls was observed, at least
at high concentrations. The highest foamability was realized
E
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Figure 5. Micrographs (100× magnification) and particle size distribution of (A) fresh coconut milk and fresh coconut milk with 0.3% (w/w) (B)
Tween 20, (C) P-1670, and (D) lactose ester 6.
with ester 5. Indeed, this sample had remarkably better
foamability than the four commercial controls and was slightly
superior to the LSE at the same concentrations. As just implied,
foamability began to decrease with lactose esters embodying
longer (>11 carbons) alkyl side chains. Thus, although ester 6
exhibited a higher foamability than those of the commercial
controls at all concentrations tested (but not LSE), esters 7 and
8 were only comparable to congeners 3 and 4. Because all lactose
ester concentrations being used in these experiments were
significantly higher than their CMCs, the inverted V-shaped
profile is rather puzzling. Presumably the poorer foamabilities of
esters 7 and 8, viz., those possessing the longest fatty acid side
chains, can be attributed to their increased molecular size and
relatively higher hydrophobicity that thus reduce both
adsorption speed and transport to the interface.39
After foam creation, the foam and liquid heights were
monitored as a function of time. Figure 3B shows the foaming
stability of all samples at 0.2% (w/w) concentrations over 10, 20,
and 30 min. As clearly indicated, it was not possible to maintain a
foam with ester 2 for even 10 min. Similarly, the foam derived
from ester 3 collapsed within 10 min. While compound 4
exhibited (modest) foaming stability values, ester 5 had the best
such effects (94.0, 92.0, and 82.2%) among all of the testing
samples. The foaming stability effects exerted by ester 6 were
found to be comparable to the controls, while higher
homologues 7 and 8 showed significantly poorer behaviors,
thus mirroring trends encountered in sucrose,40 fructose,28
glucose,26 and maltose esters.41 Such observations reinforce the
notion that the foamability and foaming stability of low-
molecular-weight surfactants depend upon a variety of factors,
including the adsorption capacity of the surfactants at the air/
water interface, interface rheological properties, and diffusion
rate of the gas captured in the foam.42
quantitating such properties.31,43 Accordingly, the ESI of each
lactose ester and the four controls in an oil-in-water emulsion
was determined and the outcomes of the relevant experiments
are shown in Figure 4. In all cases, the ESIs correlate positively
with the concentration, while an inverted V-type profile is, once
again, observed in moving through the homologous series of
esters. Thus, for compounds 2−6, the ESIs increased from 167.2
to 1897.1 at 0.5% (w/w), with the latter value being the highest.
This trend correlates with the corresponding γO/W values (from
8.1 to 3.7 mN/m) and suggests an increasing capacity to lower
the tension at the oil−water interface. However, the ESIs for
esters 7 and 8 at the same concentration are significantly lower
(797.1 and 308.6), a feature contrary to the trends observed for
their γO/W values. It is clear from the derived ESI data that lactose
monoesters bearing medium-length side chains, such as
compounds 5 and 6, display significantly better ESIs than the
four commercial controls as well as the LSE, thus indicating that
they could have greater efficacy in stabilizing emulsions.
Particle Size Distributions. To further evaluate its
emulsifying properties and potential applications, a particle
size distribution analysis of ester 6 in a coconut milk emulsion
system was undertaken. Figure 5 illustrates the particle size
distributions of fresh coconut milk (A) and fresh coconut milk
processed with Tween 20 (B), P-1670 (C), and lactose
monoester 6 (D). Not surprisingly, the fresh coconut milk
emulsions contain variably sized droplets up to 3000 μm in
diameter and are non-uniformly dispersed, with some
aggregation being evident.11 The fresh coconut milk emulsion
containing ester 6 had droplet sizes ranging from 0 and 500 μm
in diameter. While some larger droplets were present, the
obviously reduced overall particle size distribution contributes
to the lower probability of coalescence and, therefore, results in
higher emulsion stability.11 The analogous coconut milk system
incorporating Tween 20 and P-1670 had the particle (droplet)
sizes and distributions shown in panels B and C of Figure 5.
Overall, then, the effective droplet diameter within coconut milk
Emulsion Stability. How long an emulsified state can be
maintained is a key issue associated with the development of
new food emulsifiers.43 The ESI is commonly used for
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Journal of Agricultural and Food Chemistry
Article
emulsions is reduced by the addition of either lactose monoester
6 or Tween 20 and P-1670. Both additives enhance emulsion
stabilities to similar degrees. Such results are accordance with
the ESI measurements reported here and those deriving from
previous studies.11,33,41
Thermal Stability. Many of the most common food-
processing operations, such as canning, baking, and pasteuriza-
tion, rely on heating. Accordingly, good thermal stability is an
important characteristic in a food emulsifier. The outcomes of
the thermogravimetric analysis of each sample are shown in
Figure 6 and reveal that, in general, lactose esters 2−8 showed
for the lactose esters within the same temperature range.
Interestingly, pure LSE was also found to be more stable than P-
1570 and P-1670 at high (>250 °C) temperatures.
Antimicrobial Activity. Numerous studies have reported
the microbial growth inhibitory effects of a range of sugar esters,
although the mechanism by which they exert these remain
unclear. In a recent study,44 mannose esters were found to
promote bacterial membrane pore formation, suggesting a
possible mode of action. Of the sugar esters previously
investigated, sucrose esters have been studied the most
thoroughly,45 and while the antimicrobial activity of certain
lactose esters has been reported,9,12−15,46 any relationship
between such activity and the nature of the fatty acid side chain
remains unclear. Accordingly, in the current study, the microbial
growth inhibitory effects of lactose monoesters 2−8 against six
microorganisms were measured by determining their MICs. As
shown in Table 2, like the emulsifying properties, their
antimicrobial activities are impacted by the length of the fatty
acid chain with esters 2, 7, and 8, showing no inhibitory
potencies against all six microorganisms. While ester 3 showed
very weak inhibitory effects against Salmonella paratyphi B at
concentrations above 512 μg/mL, ester 4 displayed slightly
better inhibition toward Enterococcus faecalis (MIC = 512 μg/
mL), Staphylococcus aureus, and Pseudomonas aeruginosa.
Compounds 5 and 6 were found to be active against E. faecalis,
with MIC values of 256 and 64 μg/mL, respectively. The latter
value represents the lowest MIC obtained for all of the lactose
esters. Ester 5 also showed broad inhibitory effects against all of
the other test microorganisms, although the MICs in these cases
were in excess of 512 μg/mL, a profile observed previously.12,46
The positive control used in the present study was sodium
benzoate, a commonly used food preservative, and this showed
better inhibitory effects against the three Gram-positive bacteria
than esters 5 and 6 but was also inactive against the three Gram-
negative bacteria. Vancomycin exhibited significantly better
potencies for the three Gram-positive bacteria, with a MIC down
to 1 μg/mL, but it was less effective against Gram-negative
strains. Overall, then, lactose esters 5 and 6 (viz., the esters
bearing medium-length side chains) displayed moderate
antimicrobial activity against certain Gram-positive bacteria,
while the other esters were either inactive or only weakly active.
Cytotoxicity. In previous studies, fatty acid esters of
maltose,47 sucrose,48,49 trehalose,49 and lactose11,14,15,20 were
shown to be cytotoxic against various tumor cell lines, but no
Figure 6. Thermogravimetric analysis of compounds 2−8, LSE, and the
commercial sucrose esters P-1570 and P-1670.
better thermal stabilities than the pure sucrose ester LSE,
although they are more heat-sensitive than commercial sucrose
esters P-1570 and P-1670 [because the temperature range at
which a 5% loss of mass for lactose esters 2−8 was 156−176 °C,
while that for P-1570 and P-1670 was significantly higher (210
°C)]. It seems, therefore, that the length of the alkyl side chain of
esters 2−8 has no obvious influence on their thermal stabilities.
Surprisingly, however, the thermal stabilities of the lactose esters
were superior to those of their sucrose counterparts above 250
°C. In overall terms, a significant loss of mass was observed for P-
1570 and P-1670 between 220 and 270 °C, while this was less so
Table 2. MICs of 6′-O-Lactose Esters 2−8 and the Controls Sodium Benzoate and Vancomycin Against Six Bacteria as
Determined by Optical Density Measurements
microorganism
Staphylococcus aureus
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Salmonella enterica
Enterococcus faecalis
ATCC 25923
(MRSA) ATCC 43300
ATCC 25922
ATCC 27853
CMCC 50094
ATCC 29212
a
a
b
b
b
c
sample
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
d
2
3
4
5
6
7
8
na
na
na
na
>512
>512
na
na
na
na
>512
na
na
na
na
>512
>512
>512
na
na
>512
na
>512
>512
na
na
na
512
256
64
na
>512
>512
na
na
na
na
na
na
na
na
na
sodium
benzoate
64
32
>512
>512
>512
64
vancomycin
2
1
512
>512
>512
2
a
b
c
d
Gram-positive pathogens. Gram-negative pathogens. Gram-positive commensal bacterium. na = not active.
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Table 3. Cytotoxicity (IC50) of Lactose Monoesters 2−8 Against Five Cell Lines
cell line
sample
MCF-7 (μM)
A549 (μM)
H1299 (μM)
HEPG2-DOX (μM)
AC16 (μM)
2
3
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
4
>256
>256
>256
>256
>256
5
>256
>256
>256
>256
>256
6
7
8
161.35 8.70
94.12 6.11
80.86 5.77
5.11 1.27
231.57 18.62
133.88 15.45
69.25 6.34
9.20 1.42
184.65 24.83
124.86 16.87
82.55 5.59
18.16 1.67
173.85 27.22
142.65 30.05
79.65 25.19
>25.6
146.90 1.56
103.76 22.27
74.33 2.54
3.1 1.04
doxorubicin
Spectroscopic data, NMR spectra (1H, 13C, and HMBC),
and purity analysis reports derived from the HPLC−
ELSD-based evaluation of compounds 2−8 and LSE
(PDF)
definitive IC50 values appear to have been reported. Given the
lack of relevant previous studies that establish the effect of the
variation in the fatty acid side chain, the cytotoxicities of the 6′-
O-lactose esters 2−8 were determined. As revealed in Table 3,
the observed cytotoxicities showed some (positive) correlation
with the length of the alkyl side chain. Esters 2 and 3, embodying
the shortest side chains, displayed no inhibitory properties
against any of the five cell lines tested (IC50 > 250 μM) nor did
the congeners bearing medium-length side chains, viz.,
compounds 4 and 5. However, the longer chain esters, including
compound 6, displayed some inhibitory effects on the normal
human cell line AC-16 as well as the human cancer cell lines
MCF-7, A549, and H1229. Intriguingly, this ester (6) also
exerted a weak antiproliferative effect on the drug-resistant
HEPG2-DOX cell line. Higher homologue 7 displayed an IC50
value of 103.76 μM against AC16 and superior effects, relative to
congener 6, against MCF-7, A549, H1229, and HEPG2-DOX.
Ester 8, possessing the longest side chain, displayed the best
cytotoxicities for every cell line, with IC50 values of 80.86, 69.25,
82.55, 79.65, and 74.33 μM. Clearly, then, and as was the case
with the corresponding maltose esters,47 the longer the alkyl side
chain the greater the cytotoxicities of the present series of lactose
esters. It is worth noting that the IC50 values of compounds 6−8
are significantly higher than their CMCs, thus implying that they
would serve as relatively safe surfactants. Indeed, sugar esters are
generally considered perfectly safe for use in the food and
cosmetics industries50 because they are readily converted into
harmless carbohydrates and fatty acids in the human gut. That
said, it has been established that the cytotoxicities of sugar esters
arise from the compounds themselves and not their metabol-
ically derived and constituent fatty acids and sugars.48 Further
studies will be necessary to establish the pathway by which esters
6−8 inhibit cell proliferation.
AUTHOR INFORMATION
Corresponding Authors
*Telephone: +86-20-85226326. E-mail: twyong@jnu.edu.cn.
*Telephone: +86-20-85221367. E-mail: ping.lan@jnu.edu.cn.
ORCID
Min-Yi Liang: 0000-0003-3651-257X
Martin G. Banwell: 0000-0002-0582-475X
Yong Wang: 0000-0001-7547-1542
Ping Lan: 0000-0002-9285-3259
■
Funding
The authors thank the National Key Research and Development
Program of China under grant 2017YFD040020002, Program
for Guangdong YangFan Introducing Innovative and Entrepre-
neurial Teams (grant 2016YT03H132), the Department of
Science and Technology of Guangdong Province under Grants
2016A010105010, 2017B090907018, and 2014A010107014,
the Guangzhou Science and Technology Project under Grant
201508020007, and the Famous Foreign Supervisor Program
(Grant 2018-HWMS001) of the Ministry of Education, People’s
Republic of China, for financial support.
Notes
The authors declare no competing financial interest.
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