CYTA - JOURNAL OF FOOD

2020, VOL. 18, NO. 1, 688–697

https://doi.org/10.1080/19476337.2020.1839566

Modification of lecithin-based emulsions with phospholipases

Dely Rubí Chávez-Garay a, Néstor Gutiérrez-Méndez a
, Blanca Estela Sanchez-Ramirez a, Iván Salmeron a
,
León Raúl Hernández Ochoa a, David Chávez-Flores a
and Sergio Martínez-Monteagudo b
a
The Graduate School, Graduate Program in Chemistry, Chemistry School, Autonomous University of Chihuahua, Mexico; bDairy Science
Department, South Dakota State University, Brookings, SD, USA

ABSTRACT ARTICLE HISTORY

Phospholipases have been used in different food processes, but mainly on degumming vegetable Received 13 August 2020
oils. More recently, the use of these enzymes has been extended to the manufacture of bread and Accepted 15 October 2020
dairy products. However, little is known on phospholipases effect on lecithin-based emulsions and KEYWORDS
how the emulsion size contributes to such effect. This work aimed to explore the effect of Lecithin-based emulsions;
phospholipase type A1 (PLA1) on lecithin-based emulsions with different droplet size distribution. phospholipase A1;
The PLA1 was able to hydrolyze lecithin phospholipids aggregated in (oil-in-water) emulsions, nanoemulsion; emulsion
generating different lysophospholipids. The larger the particle size in the emulsion, the higher the stability; lysophospholipids;
enzymatic activity of PLA1. According to theoretical calculations, the lysophospholipids had higher hydrophile-lipophile balance
hydrophile-lipophile balance (HLB) and had a lower critical packing parameter (p) than phospholi­
PALABRAS CLAVE
pids. In consequence, the emulsions having more lysophospholipids were more prone to flocculate emulsions a base de lecitina;
and to coalescence. fosfolipasa A1;
nanoemulsión; estabilidad
de las emulsiones;
Modificación de emulsions a base de lecitina con fosfolipasas lisofosfolípidos; balance
hidrófilo lipófilo
RESUMEN
Las fosfolipasas se utilizan en diferentes procesos alimenticios, principalmente en el desgomado de
aceites vegetales. Recientemente, se ha extendido el uso de estas enzimas en la manufactura de
pan y productos lácteos. Sin embargo, se conoce poco sobre el efecto que tienen las fosfolipasas
sobre emulsiones que usan lecitina, y como el tamaño de las emulsiones contribuye en tal efecto.
En este trabajo se exploró el efecto de la fosfolipasa tipo A1 (PLA1) sobre emulsiones a base de
lecitina con diferente distribución de tamaño de partícula. La PLA1 fue capaz de hidrolizar los
fosfolípidos de la lecitina que se agregaban en forma de emulsión (aceite en agua), generando
diferentes lisofosfolípidos. Entre mayor era el tamaño de partícula en las emulsiones, mayor fue la
actividad enzimática de la PLA1. Según los cálculos teóricos obtenidos, los lisofosfolípidos tuvieron
un mayor balance hidrófilo-lipófilo (HLB) y un menor parámetro de empacado (p) en comparación
que los fosfolípidos. En consecuencia, las emulsiones con lisofosfolípidos más propensas a flocular
y a presentar coalescencia.

1. Introduction and phosphatidic acid (PA). Lecithin phospholipids have an

Phospholipases have been used in different food processes,
but mainly on degumming vegetable oils or modifying egg
yolk’s emulsion properties. More recently, the use of these
enzymes has been extended to the manufacture of bread
and dairy products. In bread making, it has been reported
that phospholipases elevate the loaf volume and bread soft­
ness. In the dairy industry, phospholipases have been used
to raise the cheese yield (Clausen, 2001; Guerrand, 2017; De
Maria et al., 2007). According to different authors, the phos­
pholipase type 1 (PLA1) promotes water retention in the
curd and decreases fat loss in the whey (Lilbæk et al., 2006;
Trancoso-Reyes et al., 2014).
In the food industry, the most widely used emulsifier
ingredient is lecithin. Lecithins are extracted from different
natural sources like plant seeds (soybeans and rapeseeds),
and egg yolk. However, lecithin extracted from soybean oil is
the most common used for food emulsions. Lecithins con­
tain a complex mixture of glycerophospholipids (Table 1),
including phosphatidylcholine (PC), phosphatidylethanola­
mine (PE), phosphatidylinositol (PI), phosphatidylserine (PS),
amphiphilic structure that confers emulsifying, wetting, and
self-assembly characteristics (Friberg et al., 2003; J. Li et al.,
2015; David Julian McClements, 2015).
Phospholipases are a ubiquitous group of hydrolases which
have the property of hydrolyzing phospholipids (Wilton, 2008).
These enzymes are classified based on their site of hydrolysis.
The phospholipase type A1 (PLA1) attack the Sn1-acyl ester
(3.1.1.32), the phospholipase type A2 (PLA2) acts on the Sn2-
acyl ester (3.1.1.4), and the type B act on both the Sn1 and Sn2
position (PLB). Accordingly, phospholipid hydrolysis by PLA1
and PLA2 yields lysophospholipids and free fatty acids
(Table 1). The biological role of these enzymes is to hydrolyze
phospholipids to prevent membrane degradation and control
the formation of phospholipid-derived signaling molecules. In
nature, phospholipases share another particularity, being
more active on aggregated phospholipids than on monomeric
substrate. Unlike other esterases, phospholipases (and most
lipases) show an enhanced activity at the lipid-water interface
due to conformational changes in the enzyme resulting from
the interfacial binding (Dowhan et al., 2008; Sandoval, 2012;

CONTACT Néstor Gutiérrez-Méndez ngutierrez@uach.mx Circuito Universitario, Chihuahua City, Mexico

© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Table 1. Theoretical calculation on the hydrophile-lipophile balance (HLB), and the surfactant critical packing parameter (p) of phospholipids and their corresponding lysophospholipids.
Tabla 1. Cálculos teóricos del balance hidrófilo lipófilo (HLB), y de el valor crítico de empaque (p) de fosfolípidos y sus correspondientes lisofosfolípidos.
v l a0 p tail
HLB (3) () (2) (Sn1/Sn2) Molecular structure
PC 4.655 620.4 15.4 156.5 0.257 18:2/18:2

LPC 7.201 327.6 14.3 156.5 0.146 -/18:2

PE 3.912 626.4 18.7 116.7 0.287 16:0/18:2

LPE 6.046 325.7 18.7 116.7 0.149 -/18:2

PA 2.95 618.3 21.4 80.7 0.357 16:0/16:0

LPA 4.89 299.5 20.9 80.7 0.177 -/16:0

CYTA - JOURNAL OF FOOD

PC = phospatidylcholine, LPC = lysophosphatidylcholine, PE = phosphatidylethanolamine, LPE = lysophosphatidylethanolamine, PA = phosphatidic acid, LPA = lysophosphatidic acid, v = volume of the lipophilic group, l = length of the
lipophilic group, a0 = cross-sectional area of the hydrophilic group.
PC = fosfatidilcolina, LPC = lisofosfatidilcolina, PE = fosfatidiletanolamina, LPE = lisofosfatidiletanolamina, PA = ácido fosfatídico, LPA = ácido lisofosfatídico, v = volúmen del grupo lipofílico, l = longitude del grupo lipofílico, a0 = area de la
sección transversal del grupo hidrophílico.
689
690 D. R. CHÁVEZ-GARAY ET AL.
Wilton, 2008). It has been suggested that the property
described above is related with the low critical micelle con­
centration (CMC) of phospholipids in an aqueous milieu. Since
a low CMC indicates that at very low concentrations (<10−9 M),
phospholipids will self-assemble into liposomes, micelles, or
hexagonal structures in water (Wilton, 2008).
Despite phospholipases are currently used in different
industrial processes, it is not clear the effect these enzymes
have on food emulsions, chiefly on lecithin-based emulsions.
However, it can be hypothesized that phospholipases are
able to hydrolyze phospholipids arranged in oil-in-water
emulsions, modifying consequently the stability of emulsion.
This work aimed to study phospholipase type A1 (PLA1)
capability to hydrolyze phospholipids arranged in oil-in-
water emulsions with different droplet size distribution.
2. Materials and methods
2.1. Materials
Extra virgin olive oil (Gallo, España), soybean lecithin (CTR
Scientific, México), and deionized water (>18 MΩcm−1)
obtained from ultra-pure water purification system (Thermo
Scientific, D8611, Dubuque, IO) were employed for the pre­
paration of all solutions. Emulsions were modified using the
phospholipase A1 (3.1.1.32) from Thermomyces lanuginosus/
Fusarium oxysporum and expressed in Aspergillus oryzae
(LecitaseUltra ≥ 10 KLU/g, Sigma-Aldrich, Germany).
2.2. Preparation of emulsions
Emulsions were formed using olive oil, lecithin, and deio­
nized water. First, a surfactant solution of water-lecithin was
prepared in a glass baker by dissolving 2.5 g of soybean
lecithin in 45 g of warm distilled water (40°C). Then, 2.5 g of
oil was added in the water-lecithin solution formerly pre­
pared to obtain a 5% O/W emulsion. The pH was adjusted (if
necessary) to 6.8 with NaOH (1 N). Emulsions with different
size distribution were induced by mechanical stirring or
applying high power ultrasound. For the mechanical
induced emulsions (MI), the mixture of water-lecithin-oil
was stirred for two minutes at 40°C with a high-shear
mixer (IKA T18 basic Ultra-turrax with S18N-10 G dispersing
tool, Japan) at 7200, and 15500 rpm (MI-7200 and MI-15500).
For the ultrasound-induced emulsions (USI), 15 milliliters of
the water-lecithin-oil mixture were poured in a stainless-
steel chamber. Water was pumped with a peristaltic bomb
(EP1- Econo pump, Biorad, Hercules, CA) through the cham­
ber’s jacket to prevent an increase in temperature during
sonication. Sonication was applied for five minutes with an
ultrasound processor (Branson Ultrasonic Corporation,
Danbury, CT; model Sonifier 450 W) with a 12.7 mm dia­
meter probe placed at 1.57 cm from the chamber bottom.
Two ultrasound powers were used 8 W and 10 W, obtained
by varying the vibrational amplitude of the probe at 40 and
70%, respectively. The ultrasound energy densities (Q) deliv­
ered in the water-lecithin-oil mixture were estimated with
the calorimetric method described elsewhere (Sánchez-
García et al., 2018). Sonication with an ultrasound power of
8 W generated a Q = 167 JmL−1 (USI-167), and the ultra­
sound power of 10 W had a Q = 200 JmL−1 (USI-200).
2.3. Enzymatic treatment of emulsions
Fifty milliliters of freshly prepared emulsions were poured in
a glass baker and kept under gentle agitation at 40°C with
a water bath. Then, 50 μL of phospholipase A1 (PLA1) was
added in each emulsion and incubated at 40°C. The enzy­
matic activity of PLA1 was followed by measuring the release
of free fatty acids with a pH-meter (Hanna Instruments
HI4522, Rumania). The pH values were transformed into
hydrogen ion (μmolar) concentration [H+], graphed, and
fitted to the logistic model (Eq. 1).
� �
d½Hþ � ½Hþ �
¼ k½Hþ � 1 (Eq:1)
dt ½Hþ �s
Where d[H+]/dt represents the change in hydrogen ion con­
centration (H+) with time (t), k represents the rate of hydro­
lysis (μmoles per minute) and ½Hþ �s the maximum
concentration of H+. The enzymatic activity of PLA1 in the
emulsions was expressed as lecitace units (LU), where one LU
represented the release of one μmole of free fatty acids
per minute. The protein concentration was measured using
the Bradford reagent (Bradford, 1976).
Additionally, one and two dimensions thin-layer chromato­
graphies (1D-TLC and 2D-TLC), were used to monitor the hydro­
lysis of phospholipids in the emulsions. During the enzymatic
incubation, aliquots of 0.5 mL from each treatment were col­
lected at different time intervals (i.e., 0, 5, 15, and 30 min). The
aliquots were taken up in glass tubes with three milliliters of
chloroform/methanol (1:1). Then, 10 µL from these glass tubes
were applied to TLC plates of silica gel (60 F254, E. Merck Co.,
Darmstadt, Germany). The TLC was run using chloroform/
methanol/acetic acid/water (65:40:8:3). The 2D-TLC used chloro­
form/methanol (2:1) as a mobile phase. Eluted compounds
were detected by spraying 5% phosphomolybdic acid in etha­
nol, followed by heating at 100°C (Hossen & Hernandez, 2005).
2.4. Changes in viscosity during the enzymatic
treatment of emulsions
Fifty milliliters of freshly prepared emulsions (see section 2.2)
at 40°C were added with 50 μL of PLA1 (see section 2.3).
Then, a sample of 1.3 mL was promptly placed in the rhe­
ometer (AR2000, TA Instruments, New Castle, DE) to follow
the changes in viscosity during the enzymatic reaction. The
rheometer conditions were as follows: temperature before
and during the test = 40°C, geometry = aluminum plate of
40 mm diameter with a solvent trap (to prevent sample
evaporation), fixed shear rate = 100−s, sample points 180,
test duration = 60 minutes. Data were analyzed and graphed
with the Rheology Advantage Data Analysis software (TA
Instruments, New Castle, DE). Considering the time required
to setting up the rheological experiments (1–2 min), the
initial viscosity (ηinitial) was designed as the viscosity in the
emulsions during the first 2 minutes of reaction. The final
viscosity (ηfinal) was established as the viscosity in the emul­
sions after one hour of reaction.
2.5. Oil droplet size distribution
All emulsions treated or not with PLA1 were analyzed by
optical microscopy. Briefly, a sample of 10 µL from each
treatment (MI-700, MI-7200 MI-15500, USI-167, and USI-200)
was placed in the microscope glass slide immediately after

the dispersion took place. Samples were analyzed with an
optical microscope (Bx41 Olympus Optical Co. Ltd., Tokyo)
with a digital camera (KP-D50, Hitachi Kokusai Electronic Inc.,
Tokyo). Multiple images were taken from each sample and
were analyzed with the Software Image Pro Plus v.7.0 (Media
Cybernetics, Inc., USA) to estimate the size of oil droplets.
Data collected were analyzed and graphed in boxplots to
describe the oil droplet size distribution.
2.6. Theoretical calculations
The hydrophile-lipophile balance (HLB) for PC (18:2/18:2), PE
(16:0/18:2), and PA (16:0/16:0), as well as their corresponding
lysophospholipids (LPC, LPE, LPA), were calculated with
Griffin’s equation (Eq. 2):
Mh
HLB ¼ 20 � (Eq:2)
M tion (Figure 1). The emulsions induced by high-intensity
whereby M is the molecular mass of the entire molecule, and
Mh is the molecular mass of the hydrophilic portion of the
molecule (Lange et al., 2014). On the other hand, the surfac­
tant critical packing parameter (p) for the PC, PE, LPC, LPE,
and LPA were calculated using equation 3:
v droplets into the continuous phase. During the stable cavita­
p¼
a0 l
where a0 is the cross-sectional area of the hydrophilic group
(Å2), v is the volume of the lipophilic group (Å3), and l is the
length of the lipophilic group (Å) (Nagarajan, 2002). These
parameters were calculated with the computational method
described by Khalil and Zarari (2014) but using the software
UCSF-Chimera (v 1.13.1, National Institutes of Health, USA),
and PyMOL (v 2.2.2, Schrodinger LLC, USA). Molecular struc­
tures were obtained from PubChem (PC = CID 5288075,
PE = CID 46891780) and edited with ChemDraw (Version
3.1, Cambridge Scientific Computing, MA).
2.7. Statistical analysis
A factorial design (2 x 4) was used to assess the effect of
adding phospholipase A1 (PLA1) in different oil-in-water
emulsions. The first categorical factor (x1 = addition of
PLA1) had two levels: with PLA1 and without PLA1 (controls).
The second categorical factor (x2 = type of emulsion)
involved 4 levels: MI-7200, MI-15500, USI-167, and USI-200.
In total, eight treatments were performed, and each treat­
ment was carried out in triplicate. Data collected were
Figure 1. Oil droplet size distribution of emulsions induced by mechanical stirring (MI) or by applying high ultrasound energy (USI) after 10 minutes of
incubation with a phospholipase A1 (B), or without the addition of phospholipase (A).
Figura 1. Distribución de tamaño de las gotas de aceite en emulsiones inducidas por agitación mecánica (MI) o por la aplicación de ultrasonido de alta energía
(USI) después de 10 minutos de incubación con un fosfolipasa A1 (B) o sin la adición de la fosfolipasa (A).
CYTA - JOURNAL OF FOOD 691
subject to analysis of variance (ANOVA) and multiple mean
comparisons (Tukey-Kramer analysis) with the Minitab soft­
ware 17 (Minitab Inc. State College, PA).
3. Results and discussion
3.1. Characterization of emulsions
All emulsion showed droplets with a spheroid-like structure,
although their sizes varied with the method of emulsification
(Figure 1). Mechanical induced (MI) emulsions had a larger
median (Q2) diameter (MI-7200 = 0.882 μm, MI-
15000 = 0.636 μm) than ultrasound-induced (USI) emulsions
(USI-167 = 91 nm, USI-200 = 67 nm). The droplet size dis­
tribution was also broader for MI emulsions than USI emul­
sions (Figure 1). The USI emulsions had a diameter of <
100 nm and were monodisperse with a narrow size distribu­
ultrasound have been reported to be less polydisperse and
more stable than emulsions prepared with mechanical
devices (Ghosh et al., 2013; P.-H. Li & Chiang, 2012).
Ultrasound irradiation first generates local perturbations of
the interface leading to the eruption of dispersed phase
(Eq:3) tion, a water-in-oil or oil-in water-in-oil emulsion is formed
inside the bulk oil phase. Then, the collapse of micro gas
bubbles (transient cavitation) near the interface breaks up
the emulsion, resulting in tiny oil-in-water droplets with
a diameter < 100 nm (Canselier et al., 2002; Mahdi Jafari
et al., 2006; Stepišnik Perdih et al., 2019).
The MI emulsions had yellow-opaque color, whereas the
USI emulsions exhibited a milky-opaque color (Figure 2).
The viscosity of emulsions during the first two minutes
(ηinitial) was significantly different between emulsions with
and without PLA1 (Table 2). These results pointed to the
immediate effect of the PLA1 on lecithin-based emulsions,
as was further confirmed with the enzyme activity curves
(Figure 3). The viscosity discrepancy between emulsions
with and without PLA1 was more evident after one hour
of incubation (ηfinal), mainly in the mechanical-induced
emulsions. On the other hand, the viscosity of USI emul­
sions was significantly lower than observed in MI emulsions
(Table 2), which makes sense since USI emulsions had
smaller emulsion droplets than MI emulsions (Figure 1). It
has been reported that emulsions with a small particle size
(10 to 100 nm) had lower viscosity than macro emulsions (1
to 20 mm) (Kale & Deore, 2017). Referring to the stability of
692 D. R. CHÁVEZ-GARAY ET AL.

emulsions with time, the rheological analysis showed
that MI and USI emulsions (without enzyme) were reason­
ably stable within the first 60 minutes; the changes in
viscosity (Δη) in both types of emulsions were in the
order of 1–2 mPa.s (Table 2). Nevertheless, the stability of
MI emulsions decreased after the first 24 hours (Figure 2).
The MI-7200 showed flocculation of micelles that eventually
turned into coalescence at seven days, and into partial
phase separation at 30 days of storage. The MI-15500
emulsion showed flocculation on the seventh day and pro­
gress to coalescence after 30 days but not turned into
phase separation. The USI emulsions (USI-167 and USI-
200) were stable during the 30 days of storage at 40°C
(Figure 2).

Figure 2. Stability of mechanical- and ultrasound-induced emulsions (MI and USI) treated or not with a phospholipase A1 (PLA1) and incubated at 40°C.
Figura 2. Estabilidad de las emulsiones inducidas mecánicamente o por ultrasonido (MI y USI) tratadas o no con una fosfolipasa A1 (PLA1) e incubada a 40°C.

Table 2. Changes in viscosity (η) with time in mechanical- and ultrasound-induced emulsions (MI and USI) treated or not
(control) with a phospholipase A1 (PLA1).
Tabla 2. Cambios en la viscosidad (η) con respecto al tiempo de las emulsiones inducidas mecánicamente y por
ultrasonido (MI y USI) tratadas o no (control) con una fosfolipasa A1 (PLA1).
Initial viscosity Viscosity after 1 h Δη
(ηinitial) (ηfinal) (ηinitial- ηfinal)
Treatment mPa.s mPa.s mPa.s
Control MI-7200 8.315 ± 0.31a 6.481 ± 0.43a 1.833 ± 0.27bc
MI-15500 6.837 ± 0.33b 4.823 ± 0.24b 2.014 ± 0.14bc
USI-167 2.909 ± 0.67 c 1.649 ± 0.83de 1.260 ± 0.21 c
USI-200 2.710 ± 0.14 c 1.083 ± 0.03e 1.627 ± 0.16bc
Treated with PLA1 MI-7200 6.962 ± 0.42b 3.714 ± 0.73bc 3.247 ± 0.60a
MI-15500 5.976 ± 0.68b 2.810 ± 0.26 cd 3.165 ± 0.42a
USI-167 2.486 ± 0.04 c 1.064 ± 0.18e 1.422 ± 0.18bc
USI-200 3.296 ± 0.46 c 1.084 ± 0.14e 2.211 ± 0.32b
ηinitial = viscosity observed during the first two minutes of incubation at 40°C. ηfinal = viscosity after 60 minutes of
constant shear rate at 100−s and 40ºC. Means followed by different letters in the same column are statistically different
(ANOVA, Tukey-Kramer test, p > 0.05). Data represent the average of three replicates (n).
ηinitial = viscosidad observada durante los primeros dos minutos de incubación a 40°C. ηfinal = viscosidad después de 60
minutos bajo un esfuerzo de sizalla constante de 100−s a 40ºC. Medias seguidas de letras diferentes en una misma
columna son estadísticamente diferentes (ANDEVA, prueba de Tukey-Kramer p > 0.05). Los datos representan el
promedio de tres repeticiones (n).

Figure 3. Enzymatic activity observed in mechanical- and ultrasound-induced
emulsions (MI and USI) added with a phospholipase A1 (PLA1). The upper
graphic shows the rate of free fatty acids released in each emulsion. The
bottom graphic describes the enzymatic activity of PLA1 in each emulsion.
Bars sharing the same letter had no significant difference in their mean values
(p > 0.05).
Figura 3. Actividad enzimática observada en emulsions inducidas mecánicamente
o por ultrasonido (MI y USI) tratadas o no con una fosfolipasa A1 (PLA1) e incubada
a 40°C. La gráfica superior muestra la velocidad de liberación de ácidos grasos en
cada tipo de emulsión. La gráfica del fondo describe la actividad enzimática de la
PLA1 en cada emulsión. Las barras que comparten la misma letra no presentaron
diferencia significativa en los valores de sus medias (p > 0.05).
According to the size, color, stability, and viscosity, the
mechanical-induced (MI) emulsions can be considered con­
ventional emulsions, whereas the ultrasound-induced (USI)
emulsions were classified as nano-emulsions. A conventional
emulsion is thermodynamically unstable and has droplet
sizes ranging from 0.1 to 100 μm. Additionally, the conven­
tional emulsions show a white and opaque color that comes
from the light scatter. A nano-emulsion is considered
a conventional emulsion containing very small droplets
and is also a thermodynamically unfavorable system. The
diameter of nano-emulsions varies between 10 to 100 nm.
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The relatively small droplet size compared to the wavelength
of light means that they tend to be transparent or only
slightly turbid when the particle diameter falls below about
60 nm. However, if the droplet diameter approaches 80 nm,
the emulsion becomes turbid and milky. The nano-
emulsions have in general lower viscosity than conventional
emulsions (Kale & Deore, 2017; Khan et al., 2014; David Julian
McClements, 2012; David Julian McClements & Rao, 2011).
3.2. Phospholipase activity on emulsions
The PLA1 showed activity in all the lecithin-based emulsions
tested (Figure 3), confirming the capability of this enzyme to
hydrolyze aggregated phospholipids, as suggested by other
authors (Dowhan et al., 2008; Sandoval, 2012; Wilton, 2008).
The specific activity of PLA1 varied from 0.20 to 0.25 LU/mg
of protein (Figure 3). Other authors have reported activities
of 0.8 and 1.6 LU/mg for lipases from P. fluorescens and
M. miehei hydrolyzing phospholipid dispersions (Bourlieu
et al., 2012). The kinetic behavior of PLA1 was sigmoidal in
all the treatments, but the rates of hydrolysis (k) varied
depending on the type of emulsion (Figure 2). The conven­
tional emulsions had higher rates of hydrolysis (kMI7200
= 0.254 μM min−1, and kMI15000 = 0.245 μM min−1) than the
nano-emulsions (kUSI167 = 0.202 μM min−1, and kUSI200
= 0.227 μM min−1). These results suggest that nano-
emulsions’ small surface may limit the amount of enzyme
in the lipid-water interface, and therefore the hydrolysis of
phospholipids.
According to the one-dimensional and two-dimensional
TLC, the soybean lecithin presented PA, PE, PS, PI, and PC.
The most abundant phospholipid was PC, followed by PE,
PA, and PS (Figure 4). These results were in agreement with
those reported in the literature for soybean lecithin
(Gunstone, 2011). The TLC analysis also confirmed the hydro­
lysis of lecithin phospholipids by incorporation of PLA1 into
the emulsions (Figure 5). Overall, the PA and the PE were the
phospholipids more attacked by the PLA1 (Figure 5). The PC
was barely hydrolyzed in all the emulsions, and the PS was
hydrolyzed only in the USI emulsions (Figure 5). As the
enzymatic reaction proceeded, the accumulation of lysopho­
spholipids was evident, particularly those derived from the
hydrolysis of PE (LPE). The extensive hydrolysis of PE could
be related to its structure (e.g., the unsaturated fatty acid at
the Sn1 position) or its arrangement at the emulsions’ inter­
face, which facilitated the interaction with the PLA1.
3.3. Theoretical calculations of phospholipids and
lysophospholipids
The hydrophile-lipophile balance (HLB) of lecithin phospho­
lipids fell from 2.95 to 4.65 (Table 1). For instance, the PC had
a calculated HLB value of 4.65, which agrees with the litera­
ture data (HLB = 4.5) for this phospholipid (Celli et al., 2020).
The removal of one fatty acid at position Sn1 almost doubled
the phospholipids’ HLB values (4.89 to 7.20), making the
lysophospholipids more hydrophilic. A surfactant with low
HLB value (3–5) is predominantly hydrophobic and partition
into the oil phase. Additionally, when the surfactant has
a low HLB value, this is surface-active but does not decrease
the interfacial tension so much, which makes the emulsion
stable. In contrast, a surfactant with an intermediate HLB
value (7–9) partition into both the water and the oil phase
694 D. R. CHÁVEZ-GARAY ET AL.

Figure 4. Thin layer chromatography (TLC) of emulsion constituents (olive oil and lecithin) and phosphatidylcholine (PC) standard using one-dimensional TLC
(1D-TLC), or two-dimensional TLC (2D-TLC). In 1D-TLC (a), lane 1 = phosphatidilcholine standard, lane 2 = soybean lecithin, lane 3 = extra virgin olive oil. In 2D-
TLCs, the (b) shows an ultrasound induced emulsion (USI 200) without enzymatic treatment, the (c) shows the extra virgin olive oil, d) = soybean lecithin. NL:
neutral lipids, FFA: free fatty acids, PA: phosphatidic acid, PE: phosphatidylethanolamine, PS: phosphatidylserine, PI: phosphatidylinositol, PC: phosphatidylcho­
line, LysoPLs: Lysophospholipids.
Figura 4. Cromatografía de capa fina (TLC) de los constituyentes de la emulsión (aceite de oliva y lecitina) y de un estándar de fosfatidilcolina (PC) utilizando
TLC de una dimensión (1D-TLC), o TLC de dos dimensiones (2D-TLC). En el 1D-TLC (a) el carril 1 = estandar de fosfatidilcolina, carril 2 = lecitina de soya, carril 3
aceite de oliva extra virgen. En los 2D-TLC la (b) muestra una emulsión inducida por ultrasonido (USI200) sin ser tratada por fosfolipasas, el (c) muestra al aceite
oliva extravirgen, y el (d) muestra a la lecitina de soya. NL: lípidos neutrales, FFA: ácidos grasos libres, PA: ácido fosfatídico, PE: fosfatidiletanolamina, PS:
fosfatidilserina, PI: fosfatidilinositol, PC: fosfatidilcolina, LysoPLs: lisofosfolípidos.

with no particular preference. Therefore, this type of surfac­
tant is prone to accumulate at the emulsion interface,
decreasing the interfacial tension markedly and facilitating
the coalescence among emulsion droplets (Claesson et al.,
2001; Hasenhuettl & Hartel, 2008; David Julian McClements,
2015).
The ratio of lipophilic-group volume to lipophilic-group
length (v/l) ranged between 28–40 Å2 for phospholipids, and
14–22 Å2 lysophospholipids. In agreement with these results,
Nagarajan (2002) described that surfactants with double tail
usually show a ratio v/l of 42 Å2, and 21 Å2 for single tail
surfactants. The critical packing parameter (p) oscillated
between 0.35 to 0.25 for phospholipids and 0.14 to 0.17
for lysophospholipids (Table 1). The packing parameter
gives an insight into the shape of amphiphilic molecules
and their self-assembly structures. If p-values are greater
than 0.333 (p > 1/3), the surfactants will form a cylinder or
rod-like micelles. However, if p-values are smaller than 0.333
(p < 1/3), the surfactants will assemble spherical micelles
(Khalil & Zarari, 2014; Nagarajan, 2002). Accordingly, and
based on the results of Table 1, all the phospholipids and
lysophospholipids (excepting the PA) might form spherical
micelles. On the other hand, a total phase inversion would
not be expected in the lecithin-based emulsions modified
with the PLA1, since a packing parameter greater than one
(p < 1) will be required to form reverse micelles (David Julian
McClements, 2015).
3.4. Properties of emulsions treated with the
phospholipase A1
All the emulsions treated with PLA1 showed a narrower oil
droplet size distribution and a slightly lower viscosity than
the emulsions non-added with the enzyme (Figure 1, Table
2). Both effects were more evident in the conventional emul­
sions than in the nano-emulsions. The stability of conven­
tional emulsions and nano-emulsions was also affected by
the addition of phospholipase A1. For instance, the emul­
sions added with PLA1 exhibited more prominent changes in
viscosity (Δη) during the first hour of incubation than the
emulsions non-treated with the enzyme (Table 2). The
instability of emulsions with PLA1 turned even more evident
at longer times (e.g., 1, 7, and 30 days). The conventional
emulsions (MI-7200 and MI-15500) treated with PLA1 had
partial phase separation on the seventh day, and the nano-
emulsions (USI-167 and USI-200) presented an oiling ring or
creaming on the thirtieth day. Such instability was not
observed in the emulsions non-treated with PLA1 (Figure 2).
As previously described, phospholipase A1 was very
active on lecithin-based emulsions (Figure 3). The hydrolysis
of lecithin phospholipids generated different lysophospho­
lipids but mainly lysophosphatidylethanolamine or LPE
(Figure 5). According to the theoretical calculations, the
LPE had an HLB value of 6 (Table 1). Surfactants with
intermediate HLB value (7) are prone to accumulate at
the interface and to reduce the interfacial tension drasti­
cally. The much lower interfacial tension created by lyso­
phospholipids also derives from their small molecular size
(critical packing parameter) that speeds the diffusion and
adsorption of these molecules at the emulsion interface
(Dowhan et al., 2008; David Julian McClements, 2015). In
general, lowering the interfacial tension can improve the
emulsifying properties of a surfactant. When the interfacial
tension is too low (0 mN/m), a thermodynamically stable
emulsion can be formed. In this type of emulsion (referred
to as microemulsion), the interfacial tension is almost zero
(1x10−2 – 1 × 10−3 mN/m), and the coalescence of droplets
does not occur (Paul & Moulik, 2001; Salager et al., 2005).
However, when the interfacial tension is low but not too
low to form a thermodynamically stable emulsion, the
emulsions droplets are prone to Ostwalt ripening or droplet
coalescence, since little free energy is required to disrupt
the interfacial layer (Hasenhuettl & Hartel, 2008; David
Julian McClements, 2015). It has been reported that lyso­
phospholipids decrease the interfacial tension to
32–49 mN/m(Stafford et al., 1989); this interfacial tension
is lower than produced by phospholipids (51.8 mN/m) but
is not too low to induce the formation of thermodynami­
cally stable emulsions (Kabalnov et al., 1995). Consequently,
the emulsions containing lysophospholipids are more
 CYTA - JOURNAL OF FOOD 695

Figure 5. Hydrolysis of phospholipids and lipids during the incubation of mechanical- and ultrasound-induced emulsions (MI and USI) added with
phospholipase A1. a) MI-7200; b) MI-15500; c) USI-167; d) USI-200. NL: neutral lipids, PA: phosphatidicacid, PE: phosphatidylethanolamine, PS: phosphatidylser­
ine, PC: phosphatidylcholine, PI: phosphatidylinositol, LPE: lysophosphatidylethanolamine, LPS: lysophosphatidylserine, LPC: lysophosphatidylcholine, LPI:
lysophosphatidylinositol.
Figura 5. Hidrólisis de fosfolípidos y lípidos durante la incubación de emulsions inducidas por acción mecánica o por ultrasonido (MI y USI) adicionadas con una
fosfolipasa A1. a) MI-7200; b) MI-15500; c) USI-167; d) USI-200. NL: lípidos neutrales, FFA: ácidos grasos libres, PA: ácido fosfatídico, PE: fosfatidiletanolamina, PS:
fosfatidilserina, PI: fosfatidilinositol, PC: fosfatidilcolina, LPE: lisofosfatidiletanolamina LPS: lisofosfatidilserina, LPC: lisofosfatidilcolina, LPI: lisofosfatidilinositol.

unstable and susceptible to coalescence than emulsions changes in interfacial tension and to the increased polarity
with phospholipids. Correspondingly, it has been reported and hydration of lysophospholipids (Stafford et al., 1989).
that lysophospholipids can disrupt the structure or perturb The larger the particle size in the emulsion, the higher the
the stability of natural phospholipid membranes due to enzymatic activity of PLA1 (Figure 3), and the higher the
696 D. R. CHÁVEZ-GARAY ET AL.
emulsion instability (Figure 2). The largest emulsion droplets
might accommodate more enzyme molecules at the interface
than the smallest droplets. Accordingly, the droplets with the
largest sizes inside each emulsion treatment were probably
the most affected by the PLA1. Consequently, these large and
unstable droplets could be the first and more abundant to
flocculate and coalescence. Such effect is illustrated in Figure
1, where it is observed that emulsions treated with the PLA1
narrowing their droplet size distribution, particularly the upper
quartiles. The decrease in viscosity over time (Table 2) also
exhibited the aggregation and separation of large emulsion
droplets destabilized by the PLA1.
4. Conclusion
It was evidenced that phospholipase A1 (PLA1) can hydrolyze
lecithin phospholipids aggregated in (oil-in-water) emul­
sions. However, the hydrolysis of phospholipids by the
PLA1 depended on the size of the emulsion droplets. The
bigger the emulsion, the higher the enzymatic activity of
PLA1. The hydrolysis of phospholipids generated lysopho­
spholipids, which had higher HLB values and smaller mole­
cular sizes. In consequence, the emulsions having
lysophospholipids were more prone to flocculate and coa­
lescence than phospholipid-based emulsions.
Acknowledgments
The Mexican Nacional Council of Science and Technology (CONACYT) sup­
ported this research through the grant conceded to Dely R. Chávez-Garay.
Funding
This work was supported by the Consejo Nacional de Ciencia
y Tecnología [Doctoral grant].
Author contributions
Dely Chávez collected test data and interpreted the results. Nestor
Gutiérrez designed the study and drafted the manuscript. Blanca
Sanchez helped to acquire and to interpret data from the microscope.
Ivan Salmerón and León Hernández helped acquire and interpret data
from the enzymatic reactions and thin layer chromatography analysis.
David Chavez participated with the theoretical calculations. Sergio
Martínez aided in drafting the manuscript.
Conflicts of Interest
The authors have declared that there is no conflict of interest.
ORCID
Dely Rubí Chávez-Garay http://orcid.org/0000-0001-6649-3758
Néstor Gutiérrez-Méndez http://orcid.org/0000-0002-0791-3211
Blanca Estela Sanchez-Ramirez http://orcid.org/0000-0002-7284-7855
Iván Salmeron http://orcid.org/0000-0003-1182-2571
León Raúl Hernández Ochoa http://orcid.org/0000-0002-5886-8617
David Chávez-Flores http://orcid.org/0000-0001-6852-0406
Sergio Martínez-Monteagudo http://orcid.org/0000-0002-9717-807X
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