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1. Introduction
The worldwide prevalence of obesity is rising at an alarming rate due
to an increasingly sedentary lifestyle and an increased preference for
unhealthy food (Ferretti, Mariani, & Sarti, 2021). Additionally, there is a
burgeoning problem of childhood obesity that requires mandatory
intervention to help reduce this phenomenon (Klingelhofer et al., 2021).
The growing obese population has become a serious cause of concern
due to its association with chronic diseases such as cardiovascular dis
eases (CVD), diabetes mellitus (type-2 diabetes) and cancers (Stefan,
Birkenfeld, & Schulze, 2021). To alleviate this phenomenon, numerous
anti-obesity treatments were developed to improve obesity complica
tions while limiting the side effects (Saad et al., 2017). One such method
was through the use of functional food containing bioactive compounds
to prevent and manage obesity. For instance, saponins and polyphenols
were reported to have potent lipase inhibitory activities and could serve
as promising anti-obesity treatments (Marrelli et al., 2016; Boccellino &
D’Angelo, 2020). However, studies showed that polyphenols could
interact with protein, thereby reducing its effectiveness (Singh et al.,
2020). Commercially, a synthetic anti-obesity drug such as Orlistat is
commonly consumed as it reduces the absorption of dietary fats in the
body by up to 30% by inhibiting gastrointestinal lipase (Aabideen et al.,
2020). Unfortunately, the consumption of Orlistat was accompanied by
undesirable side effects such as abdominal bloating, liquid stools, and
pain. Therefore, there have been continuous investigations into the antiobesity potential of edible fruits and vegetables due to their natural
origin, cost-effectiveness, and potentially limited adverse effects
(Aabideen et al., 2020).
Ipomoea aquatica Forssk. (Family: Convolvulaceae) is commonly
consumed globally, particularly in Southeast Asia. These plants were
traditionally used as an effective remedy to ease the symptoms of
numerous diseases (diabetes, hypertension, and fever) (Abu Bakar Sajak
et al., 2016). The presence of rich phytochemicals (flavonoids, alkaloids,
saponins, and steroids) might be responsible for the numerous health
benefits (Igwenyi et al., 2011; Miean & Mohamed, 2001). Apart from the
above-mentioned metabolites, Ipomoea aquatica also contains resin
glycosides (RG). Aquaterins I-XI were isolated from the leaves of Ipo
moea aquatica and these RG showed potent anti-cancerous activity
against human liver cancer (HepG2) cells (Fan et al. 2014). While ample
research was reported on the anti-cancerous properties of this plant, no
research had been carried out to look at the anti-obesity potential of
Ipomoea aquatica. Therefore, the present work aimed to evaluate the
pancreatic lipase inhibitory potential of RG in Ipomoea aquatica in
simulated in vitro digestion systems.
2.4. Characterisation of resin glycosides (RG) in Ipomoea aquatica
adjusting pH to 7.0 using potassium carbonate (1.0 M). Simulated in
testinal fluid (SIF) was prepared with the following concentration of
salts: 6.8 mM KCl, 0.8 mM KH2PO4, 85 mM NaHCO3, 38.4 mM NaCl,
0.33 mM MgCl2(H2O)6 and 0.3 mM CaCl2(H2O). The pH of the solution
was adjusted to 7.0 with 1 M NaOH. Intestinal digestion was initiated by
adding 100 U/mL of pancreatin to 12 mL of SIF and porcine bile (10
mM). The control consists of just the food sample and enzymes without
any RG extract.
The digestion during the intestinal phase was monitored using a pHstat automatic titration unit (Metrohm Ltd, Herisau, Switzerland) at pH
7.0 with 0.025 M NaOH solution. The degree of hydrolysis was calcu
lated using Eq. (3),
( )
VNaOH (L) x mNaOH mol
L(
/
) x100
(3)
Degree of hydrolysis(%)
g
(Wlipid (g) x Flipid (%) Mlipid mol
)x2
The extract treated with activated charcoal was subjected to LC-MS2
analysis using the Bruker Amazon ion trap mass spectrometer equipped
with a Dionex ultimate 3000 RS Diode array detector (Billerica, MA,
USA). The heated capillary and spray voltage were maintained at 250 ◦ C
and 4.5 kV, respectively. Additionally, nitrogen was operated at 80 psi
for the sheath gas flow rate and 20 psi for the auxiliary gas flow rate. The
MS2 collision gas used was helium with a collision energy of 30% of the
5 V end-cap maximum tickling voltage. The mass spectra were scanned
from m/z 100–2000 in both the positive and negative ion modes with a
scan speed of one scan per second. During the high-performance liquid
chromatography (HPLC) run, the fraction extracted with activated
charcoal was carried out on an analytical reversed-phase C18 column
(Phenomenex, 5 μm, 4.6 × 250 mm), a flow rate of 1.0 mL/min with a
sample injection of 20 μL (20 mg/mL in methanol) at 30 ◦ C column
temperature. The PDA detector was set to scan from 190 to 800 nm. The
solvent gradient of methanol (mobile phase B) and water (mobile phase
A) was used to elute the samples. The gradient elution was programmed
as follows: 0–3 min, 90% B; 3–10 min, 90% B to 96% B; 10–35 min, 96%
B; 35–37 min, 96% B to 100% B; 37–75 min, 100% B.
where VNaOH is the consumed volume (L) of NaOH solution to maintain
pH 7, mNaOH is the molarity (mol/L) of the NaOH solution, Wlipid is the
weight of the food in the digestion system (3 g), Flipid is the fat per
centage of the food (%) and Mlipid is the molecular weight (g/mol) of
lipids in food.
Apart from the creamer, both butter and salad dressing containing
different concentrations of RG (3.2, 6.5, 8.7, 10.8% (w/w of fat)) and
Orlistat (0.005, 0.011, 0.013, 0.016% (w/w of fat)) were tested using the
in vitro digestion model.
High-temperature, short-time (HTST) pasteurisation (72 ◦ C, 15 s)
and low-temperature, long time (LTLT) (63 ◦ C, 30 min) pasteurisation
conditions were also performed to look at its effects on the RG extract. At
a constant temperature of 60 ◦ C, the effect of different time points, 5
min, 15 min and 30 min were analysed. The RG extract (30 mg) was first
added to 3 mL of deionised water. This mixture was then subjected to
HTST or LTLT treatment conditions in a water bath and then cooled
down to room temperature (27 ◦ C) immediately using an ice water bath.
6 mL of dichloromethane was added to the heated RG mixture to extract
the resin glycoside. The control treatment consisted of keeping the RG at
room temperature (27 ◦ C) and not subjecting the RG to any heat treat
ments. After which, the dichloromethane fraction was dried and dis
solved into methanol (2 mg/mL) and injected into the HPLC with a
sample injection of 20 μL. The HPLC analysis was performed on a Waters
2695 HPLC system that is equipped with a Waters 2996 photodiode
array (PDA) detector (Milford, MA) and this system was installed with
an Empower program. An analytical reversed-phase C18 column (Phe
nomenex, 5 μm, 4.6 × 250 mm) was used with a flow rate of 1.0 mL/min
at 30 ◦ C, column temperature. The PDA detector was set to scan from
190 to 800 nm. The solvent gradient of methanol (mobile phase B) and
water (mobile phase A) was used to elute the samples. The gradient
elution was programmed as follows: 0–3 min, 90% B; 3–10 min, 90% B
to 96% B; 10–35 min, 96% B; 35–37 min, 96% B to 100% B; 37–75 min,
100% B.
2.5. Measurement of free fatty acids released during in vitro digestion of
high-fat food with RG extract
The pancreatic lipase inhibition activity of the Ipomoea aquatica
extract that was treated with activated charcoal was evaluated after
incubating with α-amylase or pepsin. The RG extract (50 mg) was
incubated with 75 U/mL of α-amylase in 3 mL of simulated salivary fluid
(SSF) and CaCl2 (0.75 mM) for 2 min at pH 7.0. Simulated salivary fluid
(SSF) was prepared with the following concentration of salts: 15.1 mM
KCl, 3.7 mM KH2PO4, 13.6 mM NaHCO3, 0.15 mM MgCl2(H2O)6, 0.06
mM (NH4)2CO3. The pH of the solution was adjusted to 7.0 using a
hydrochloric acid solution (6.0 M). After the digestion treatment, RG
was extracted with dichloromethane and the pancreatic lipase inhibition
activity was tested. RG extract (50 mg) was also incubated with 2000 U/
mL of pepsin in 5 mL of simulated gastric fluid (SGF) and 0.075 mM
CaCl2. The mixture was subsequently placed in a water bath (SW22,
Julabo, Germany) (37 ◦ C, 150 rpm, 2 h). Simulated gastric fluid (SGF)
was prepared freshly before use with the following concentration of
salts: 6.9 mM KCl, 0.9 mM KH2PO4, 25 mM NaHCO3, 47.2 mM NaCl, 0.1
mM MgCl2(H2O)6 and 0.5 mM (NH4)2CO3. The pH of the solution was
subsequently adjusted to 3.0 with hydrochloric acid (6.0 M). After the
digestion process, RG was extracted with dichloromethane and the
pancreatic lipase inhibition activity was determined.
The in vitro digestibility of creamer was conducted using the stand
ardised static in vitro digestion of food with minor modifications from
Minekus et al., (2014). The powdered creamer (1.5 g) was first weighed
and dissolved in 1.5 mL of boiling water. The mixture was mixed thor
oughly and cooled to 40 ◦ C. Two dosages of RG (6.5 and 10.8% (w/w of
fat)) were prepared to test its effect on lipolysis in creamer and were
added in a different sequence. Firstly, the RG was added to the solid
creamer before the addition of hot water (90 ◦ C). Secondly, the RG was
added to the solid creamer only after the solution was cooled to 40 ◦ C.
Thirdly, RG extract at different dosages was incubated with intestinal
enzymes (Pancreatic lipase and bile) for 30 min first at 37 ◦ C with
continuous shaking in the water bath at 150 rpm before adding it into
the creamer at the intestinal phase. This would mimic the gastrointes
tinal conditions of consuming RG extracts before the consumption of
high-fat food.
In order to stimulate oral digestion, 75 U/mL of α-amylase in 3 mL of
SSF and CaCl2 (0.75 mM) were added to the mixture before vortexing
the mixture for 2 min. After the oral phase, gastric digestion was
simulated by adding 2000 U/mL of pepsin in 5 mL of SGF and 0.075 mM
CaCl2 into the mixture and subsequently placed in a water bath (SW22,
Julabo, Germany) (37 ◦ C, 150 rpm, 2 h). Pepsin was then inactivated by
2.6. Statistical analysis
All experiments were conducted in triplicate and the mean and
standard deviation (SD) were reported. Statistical analysis was per
formed using IBM SPSS version 22 computer software (IBM Corp.,
Armonk, NY, USA). A one-way analysis of variance (ANOVA) with
Tukey’s test (p < 0.05) were used where three or more groups of data
were compared to determine the differences within the groups while an
independent T-test was performed to compare two samples containing
the same RG concentration but with different treatment conditions (p <
0.05).
3
lipase inhibition effect of Ipomoea aquatica was comparable or even
higher than some fruits and vegetables. However, in comparison to some
edible plants like hot pepper, caffeic acid was found to have a higher OE
value of 9.96 × 10-3 (IC50 401.5 ± 32.1 μM) (Martinez-Gonzalez et al.,
2017). This might be due to the presence of impurities in the extract
treated with activated charcoal, thereby reducing the PL inhibition
effect.
combination of both the pH-stat method and the INFOGEST protocol
was advantageous as it enabled continuous monitoring of lipolysis
without requiring samplings at specific time points. During the diges
tion, a pH stat titrator was utilised to neutralise the released free fatty
acids (FFA) to maintain the pH at pH 7.0. During the experiment, two
treatments were performed. For the first treatment, the RG was added
when hot water was added to the creamer at 90 ◦ C. As shown in Fig. 4a,
the RG extract did not exhibit any pancreatic lipase inhibition effect
after the heat treatment. On the other hand, a dose-dependent rela
tionship was observed when the RG was incubated at a lower temper
ature (40 ◦ C) instead. When RG was added to the creamer at 40 ◦ C with a
concentration of 0.0, 6.5, and 10.8% (w/w), the fat digested were 95.3
± 2.6, 73.9 ± 4.2, and 58.1 ± 3.2% respectively. This showed that the
extract was temperature sensitive and would degrade at higher tem
peratures, thereby losing its pancreatic lipase inhibition effect. In gen
eral, a variety of binding interactions could occur in the gastrointestinal
tract, which would affect lipid digestibility. This was because creamer
contains a wide variety of different components, which include sugars,
protein, polysaccharides and lipids. In fact, these components could
interact with each other and RG, thereby altering the rate and extent of
lipid digestion (McClements & Li, 2010). Moreover, lipid droplets in
creamer were simply dispersed in a low viscosity aqueous liquid hence,
digestive enzymes like PL are readily accessible to triglycerides
(McClements & Li, 2010). As such, fat hydrolysis occurs quickly (20–30
min) in the intestinal phase.
High-temperature short-time (HTST) pasteurisation (72 ◦ C, 15 s) and
low-temperature long-long time (LTLT) (63 ◦ C, 30 min) pasteurisation
conditions were also performed to look at its effects on the RG extract.
Various high-fat food products (dairy produce) undergo pasteurisation
to eliminate pathogens and prolong the shelf life. If RG were to be uti
lised as an active ingredient in food products, it would have to undergo
thermal processing. For this experiment, HPLC was employed to quan
tify the total resin glycoside composition. Based on the results, HTST
(72 ◦ C, 15 s) led to a 36.3 ± 3.9% decrease and LTLT (63 ◦ C, 30 min) led
to a 49.8 ± 5.1% reduction in the total RG composition. This further
enforces the idea that RG was thermally unstable and could degrade if
the food were to undergo pasteurisation. Moreover, the duration of the
heating process affected the extent of degradation as well. When the RG
was heated at 63 ◦ C for 5 and 15 min, the reduction in the total RG
content was 23.2 ± 3.5% and 29.3 ± 2.2%, respectively. When sub
jected to longer heating, the RG content decreased significantly, which
showed that more RG was required to be fortified into the functional
food product to exert its effects. In this context, it was favorable to
consume RG as a whole (supplement form) instead of fortifying it into
functional food products that were subjected to thermal processing.
Further studies could also be carried out to look at the thermal stability
of RG at different temperatures and times.
3.2. Characterisation of resin glycoside in Ipomoea aquatica extract
Identification of the active compounds responsible for the pancreatic
lipase inhibition activity was performed through HPLC-MS2. Fig. 2a
shows the HPLC chromatogram of the activated charcoal extract with
the tentatively assigned resin glycosides based on the MS data (shown in
the supporting information). However, the use of mass spectrometry was
limited as it was unable to determine the position of the side chains that
were linked to the sugar backbone (R1 to R5). Moreover, the mass
fragmentation was unable to indicate where the bonding takes place
between the first sugar moiety and jalapinolic acid, hence there were
two proposed bonding positions (Type A and Type B) that could be
present in all the resin glycosides. In general, the main structure RG
contains either a pentasaccharide or tetrasaccharide, which consists of
one fucose/glucose and three or four rhamnose moieties. Based on the
fragmentation pattern, a series of fatty acid side chains (C8 to C12),
arylalkyl acid (cinnamic acid), and short-chain aliphatic acids (2methylbutyric acid, isobutyric acid) were determined.
A wide range of RG were found in the extract, with the same
saccharide backbone but with different alkyl chains. Based on the
negative-ion mass spectra, Ipomoea aquatica extract at 42.7 min dis
played fragment ion peaks at m/z 1361 [M H]-, 1215 [1361–146
(rhamnose)]-, 1033 [1215–182 (dodecanoyl unit)]-, 963 [1033–70
(isobutanoyl unit)]-, 837 [963–126 (octanoyl unit)]-, 691 [837–146
(rhamnose)]-, 545 [691–146 (rhamnose)]-, 417[545–128]- (Figs. 2 & 3).
Apart from rhamnose moiety, glucose moiety may bind to the tetra
saccharide backbone as well. For instance, fragment ion peaks at 22.1
min displayed peaks at m/z 1209 [M - H]-, 1047 [1209–162 (glucose)]-,
921 [1047–126 (octanoyl unit)]-, 837 [921–84 (2-methylbutanoyl
unit)]-, 691 [837–146 (rhamnose)]-, 545 [691–146 (rhamnose)]-, 417
[545–128]- (Fig. 2). m/z 417 [271 (jalapinolic acid) + 146 (hexosyl
unit)]- was present in all compounds which indicated the presence of an
ester linkage between jalapinolic acid unit and a fucose or rhamnose
moiety. In fact, a macrocyclic bond was formed due to the intracellular
bond between the carboxyl group of jalapinolic acid and a hydroxyl
group from the sugar moiety. Since RG contained different isomeric
forms and were highly complex, it was impractical to isolate them
individually and to investigate their activity for application purposes. In
view of this, the activated charcoal extract with the highest pancreatic
lipase inhibition activity was utilised during the in vitro lipolysis to
investigate its stability in gastrointestinal conditions.
3.4. In vitro digestion of salad dressing and butter with RG extract from
Ipomoea aquatica
3.3. In vitro digestion of creamer with RG extract from Ipomoea aquatica
During the simulated digestion experiment, the amount of released
FFA was measured when RG was added to the salad dressing. From
Fig. 5, the addition of RG significantly decreases the rate and extent of
lipolysis in the intestinal phase. Moreover, the lipid digestion rate
decreased in a dose–response manner with increased RG dosage. This
was evident as the amount of fatty acid released was slower and lower at
higher concentrations of RG. This illustrated the effectiveness of RG in
inhibiting pancreatic lipase despite being in the gastrointestinal tract.
Orlistat at various dosages was also added to the salad dressing during
the simulated digestion experiment. Based on Fig. 5, 10.8% RG had
similar total fat digested results as compared to the addition of 0.005%
Orlistat. Interestingly, the degree of hydrolysis did not reach 100% for
both the butter and salad dressing control and this discrepancy was due
to several reasons. Firstly, FFA could bind to β-lactoglobulin, a globular
protein that was present in the butter (Le Maux et al., 2013). Since these
To determine whether the various enzymes would degrade the RG
and thereby reduce the inhibition activity, the pancreatic lipase inhi
bition activity of the activated RG extract was determined before and
after incubating it with individual enzymes, α-amylase or pepsin. The
RG extract before alpha-amylase treatment had an IC50 of 45.4 ± 3.0 μg/
mL and did not change (IC50 of 50.4 ± 5.5 μg/mL) much after treatment.
Likewise, when RG was incubated with pepsin at pH 3 for 2 h, the RG
extract had an IC50 of 51.3 ± 7.1 μg/mL, which was not significantly
different from the initial inhibition activity as well. Therefore, this
indicated that the RG extract was relatively stable when exposed to
different enzymes and pH.
In order to verify the effectiveness of RG during lipolysis, Ipomoea
aquatica extract was added to the creamer and a simulated digestion
process was performed using an in vitro digestion model. The
6
Fig. 4. In vitro digestibility of fats in creamer whereby (a) RG extract is incubated with creamer at 90 ◦ C; (b) RG extract is added to the creamer solution at 40 ◦ C; (c)
Fat digested at different RG concentrations or treated at different temperatures. *Different capital letter represents significant differences in the fat digested within
each heat treatment as evaluated by one-way ANOVA Tukey test (p < 0.05) while different lowercase letter represents significant differences in the fat digested
between each concentrations as evaluated by independent T-test (p < 0.05).
proteins are hydrophobic, they could easily bind to FFA, hence reducing
the amount of FFA being liberated. Another possible reason was that the
addition of calcium-binding agents like ethylenediaminetetraacetic acid
(EDTA) in salad dressing would affect the rate and extent of lipid hy
drolysis by binding to calcium ions. In general, lipid digestion could be
inhibited when long-chain free fatty acids were accumulated at the lipid
droplet surface, thereby preventing lipase from reacting with tri
glycerides (Hu et al., 2010). The presence of calcium helped to precip
itate these accumulated FFA from the lipid surface, which would
increase the accessibility of the emulsified lipids to lipase (Hwang, Lee,
Ahn, & Jung, 2009). Moreover, calcium acts as a cofactor for pancreatic
lipase to work (Kimura et al., 1982). Therefore, lipid digestion would be
significantly reduced when there was a reduction of calcium ions in the
presence of chelating agents like EDTA.
In addition, factors such as particle size, macronutrient composition,
and food structure (semi-solid, solid or liquid) would also affect the
digestive behavior (Dias et al., 2019). Therefore, to better understand
the effects of RG on various determinants, other high-fat food matrices
such as butter were also explored. In general, the fat hydrolysis for salad
dressing (30–40 min) was much faster than that of butter (50–60 min).
Since salad dressing has a liquid-like consistency whereby the lipids are
less bounded to the structure, lipolysis was generally faster as shown in
Fig. 5 since the fats were more accessible to enzymatic action as
compared to butter (Fig. 6) (Calvo-Lerma et al., 2018). As a result, the
substrate was less accessible to the pancreatic lipase, which led to lower
digestion kinetics. Moreover, the slower rate of titration for butter could
be attributed to the larger oil droplet size that is dispersed in it. Since
hydrolysis is an interfacial enzymatic reaction, the larger the oil droplet,
the smaller the oil/water interfacial area, which will contribute to a
slower reaction rate (Mat et al., 2016). When RG was added to the butter
at the oral phase with a concentration of 0.0, 3.2, 6.5 and 10.8% (w/w),
the fat digested were 71.9 ± 2.2, 58.2 ± 2.1, 46.5 ± 2.7, 39.2 ± 0.6%
respectively (Fig. 5). This showed that the RG extract was effective and
stable in the gastrointestinal tract despite being incubated at both pH 3
(gastric phase) and 7 (oral and intestinal phase).
In order to investigate the effect of the sequence of addition on the
inhibitory activity of RG, RG was incubated with PL first before the
addition of the salad dressing. The changes in sequence resulted in 1.7 to
5.2 times higher percentage inhibition as compared to incubating RG
with lipid substrate (salad dressing) first. Additionally, the addition of
10.8% RG was sufficient to achieve a 55.2 ± 4.1% reduction in fat hy
drolysis. This implies that the inhibitor requires sufficient time to bind
and effectively inhibit PL before high-fat food enters the small intestine.
Hence, RG might be more effective when consumed before a high-fat
meal.
4. Conclusions
In conclusion, our result revealed a series of RG content in Ipomoea
aquatica and these compounds were responsible for the PL inhibitory
effect. In general, the consumption of RG and high-fat food shows
promise for the development of novel anti-obesity supplements. Based
7
Fig. 5. In vitro digestibility of fats in salad dressing containing (a) RG extract (RG added to salad first), (b) RG extract (RG reacts with intestinal enzymes first), (c) fat
digested when RG was added, (d) Orlistat (Orlistat added to salad first), (e) Orlistat (Orlistat reacts with intestinal enzymes first) and (f) fat digested when Orlistat
was added. *Different capital letters represent significant differences in the fat digested within each treatment conditions as evaluated by one-way ANOVA Tukey test
(p < 0.05) while different lowercase letter represents significant differences in the fat digested between different treatment conditions as evaluated by independent Ttest (p < 0.05).
8
Fig. 6. In vitro digestibility of fats in butter with different dosages of RG extract. *Different letters represent significant differences in the fat digested as evaluated by
one-way ANOVA Tukey test (p < 0.05).
on the in vitro digestion result, a dose–response relationship was
observed in high-fat food products (Creamer, butter and salad) in the
presence of RG. This inhibitory effect was also significantly dependent
on the sequence of addition of the inhibitor to the enzyme. In fact, preincubating RG with PL prior to the addition of substrate resulted in a
strong inhibition effect. These results showed that RG extract could be
consumed alone as a supplement before the ingestion of high-fat food,
which helped to retard fat digestion. However, the present data still
warrants further in vivo studies to evaluate the effects of RG on managing
fat absorption in obese and weight-conscious consumers.
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Marrelli, M., Conforti, F., Araniti, F., & Statti, G. A. (2016). Effects of saponins on lipid
metabolism: A review of potential health benefits in the treatment of obesity.
Molecules, 21(10), 1404. https://doi.org/10.3390/molecules21101404
Martinez-Gonzalez, A. I., Alvarez-Parrilla, E., Díaz-Sánchez, Á. G., de la Rosa, L. A.,
Núnez-Gastélum, J. A., Vazquez-Flores, A. A., & Gonzalez-Aguilar, G. A. (2017). In
vitro inhibition of pancreatic lipase by polyphenols: A kinetic, fluorescence
spectroscopy and molecular docking study. Food Technology and Biotechnology, 55(4),
519–530. https://doi.org/10.17113/ftb.55.04.17.5138
Mat, D. J. L., Le Feunteun, S., Michon, C., & Souchon, I. (2016). In vitro digestion of foods
using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This study was funded by a Singapore Ministry of Education Aca
demic Research Fund Tier 1 grant R160-000-B04-114.
References
Aabideen, Z. U., Mumtaz, M. W., Akhtar, M. T., Mukhtar, H., Raza, S. A., Touqeer, T., &
Saari, N. (2020). Anti-obesity attributes; UHPLC-QTOF-MS/MS-based metabolite
profiling and molecular docking insights of Taraxacum officinale. Molecules, 25(21),
4935. https://doi.org/10.3390/molecules25214935
Abu Bakar Sajak, A., Abas, F., Ismail, A., & Khatib, A. (2016). Effect of different drying
treatments and solvent ratios on phytochemical constituents of Ipomoea aquatica and
correlation with α-glucosidase inhibitory activity. International Journal of Food
Properties, 19(12), 2817–2831. https://doi.org/10.1080/10942912.2016.1141295.
Ado, M. A., Abas, F., Mohammed, A. S., & Ghazali, H. M. (2013). Anti- and pro-lipase
activity of selected medicinal, herbal and aquatic plants, and structure elucidation of
an anti-lipase compound. Molecules, 18(12), 14651–14669. https://doi.org/
10.3390/molecules181214651
Boccellino, M., & D’Angelo, S. (2020). Anti-obesity effects of polyphenol intake: Current
status and future possibilities. International Journal of Molecular Sciences, 21(16),
1–24. https://doi.org/10.3390/IJMS21165642
9
kinetics of macronutrients, proteins and lipids. Food Research International, 88 (Part
B), 226–233. https://doi.org/10.1016/j.foodres.2015.12.002.
McClements, D. J., & Li, Y. (2010). Review of in vitro digestion models for rapid screening
of emulsion-based systems. Food and Function, 1(1), 32–59. https://doi.org/
10.1039/c0fo00111b
Miean, K. H., & Mohamed, S. (2001). Flavonoid (myricetin, quercetin, kaempferol,
luteolin, and apigenin) content of edible tropical plants. Journal of Agricultural and
Food Chemistry, 49(6), 3106–3112. https://doi.org/10.1021/jf000892m
Minekus, M., Alminger, M., Alvito, P., Ballance, S., Bohn, T., Bourlieu, C., … Brodkorb, A.
(2014). A standardised static in vitro digestion method suitable for food - an
international consensus. Food & Function, 5(6), 1113–1124. https://doi.org/
10.1039/c3fo60702j
Saad, B., Zaid, H., Shanak, S., & Kadan, S. (2017). Anti-obesity medicinal plants. AntiDiabetes and Anti-Obesity Medicinal Plants and Phytochemicals, 59–93. https://doi.org/
10.1007/978-3-319-54102-0_3
Singh, M., Thrimawithana, T., Shukla, R., & Adhikari, B. (2020). Managing obesity
through natural polyphenols: A review. Future Foods, 1–2, Article 100002. https://
doi.org/10.1016/j.fufo.2020.100002
Stefan, N., Birkenfeld, A. L., & Schulze, M. B. (2021). Global pandemics interconnected
— obesity, impaired metabolic health and COVID-19. Nature Reviews Endocrinology,
17(3), 135–149. https://doi.org/10.1038/s41574-020-00462-1
10
The Effect of Resin Glycoside Extracts from Ipomoea
Aquatica on Fat Digestion: An in vitro study
Introduction
Objective
Rising incidence of obesity has sparked interest in effective anti-obesity treatments using vegetables and fruits.
The synthetic drug Orlistat reduces fat absorption but has undesirable side effects (Aabideen et al., 2020).
Ipomoea aquatica Forssk. is commonly consumed globally and used traditionally for various health benefits; It contains resin glycosides
(RG), with anti-cancer activity against liver cancer cells (Fan et al. 2014).
Evaluate inhibitory effects of resin glycosides
(RG) from Ipomoea aquatica on pancreatic
lipase (PL) in an in vitro digesting model.
Previous research focused on its anti-cancer properties, with no exploration of its anti-obesity potential.
Methodology
Extraction and enrichment of RG
In vitro digestion
Result and Discussion
Conclusion
◼ Ipomoea aquatica extract exhibits comparable or stronger PL inhibitory effects than some vegetables
and fruits.
◼ Range of RG contents with different alkyl chains in Ipomoea aquatica found to be responsible for PL
inhibition.
◼ Dose-response relationship observed between RG consumption and high-fat food digestion,
suggesting potential for innovative anti-obesity supplements.
◼ The sequence of substance consumption significantly impacts the inhibitory effect.
◼ RG extract is temperature-sensitive and loses its PL inhibitory effect at high temperatures.
◼ Further in vivo research required to assess the regulation of fat absorption by RG based on available
data.
References
(a)
(b)
(c)
(d)
Figure S1: Dose-response curve of pancreatic lipase inhibition against different
concentrations of (a) DCM extract, (b) MeOH extract, (c) extract treated with activated
charcoal and (d) Orlistat.
Intens.
x104
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 13.6min #531
1335.72
1381.68
2
618.76
269.14
0
3000
793.48
1069.08
1183.41
1610.221693.73
1928.97
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1335.72), 13.6min #532
1336.68
2000
1000
417.17
543.26
0
673.35743.30 819.31
963.47
1065.43
1191.60
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1381.68), 13.6min #533
4000
1335.62
2000
1080.49
1382.73
0
200
400
600
800
1000
1200
1400
1600
1800
m/z
Figure S2: LC-MS2 spectral data for peak shown in Figure 2 at 13.6 min.
Intens.
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1423.80), 16.2min #624
1377.86
4000
2000
1233.67
0
800
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1377.80), 16.3min #625
1379.74
600
400
417.01
200
946.31
543.16
1089.49
1251.67
0
200
400
2
600
800
1000
Figure S3: LC-MS spectral data for peak shown in Figure 2 at 16.3 min.
1200
1400
1600
1800
m/z
Intens.
x104
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 17.7min #676
1281.63
0.5
1235.67
403.16
793.46
1093.55
1435.76
932.63
0.0
1635.66
1767.78
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1281.63), 17.7min #677
1500
1235.65
1000
981.47
500
417.17
1091.50
1283.60
837.35
0
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1235.67), 17.8min #678
200
1237.58
100
0
200
400
600
800
1000
1200
1400
1600
1800
m/z
Figure S4: LC-MS2 spectral data for peak shown in Figure 2 at 17.7 min.
Intens.
x105
1.0
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 20.3min #769
1239.66
1193.68
0.5
387.22
775.41
1395.76
0.0
x104
1505.90
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1239.66), 20.3min #770
4
1193.69
2
417.19
0
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939.47
543.19
1049.52
1241.59
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1193.68), 20.3min #771
1195.55
1
417.18
0
200
400
2
543.23
647.28
600
757.37 837.28
800
939.39
1049.45
1000
Figure S5: LC-MS spectral data for peak shown in Figure 2 at 20.3 min.
1200
1400
1600
1800
m/z
Intens.
x105
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 22.1min #841
1209.64
1255.64
2
745.47
833.48
1381.79
987.71 1065.66
0
1519.81
1930.11
1753.00
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1209.64), 22.1min #842
4000
417.17
981.41
2000
545.17
1107.51
1211.52
775.45
1835.09
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1255.64), 22.1min #843
0
x104
1209.61
1
417.18
0
200
400
653.37
600
1107.52
953.45
800
1000
1257.70
1200
1400
1600
1800
m/z
Figure S6: LC-MS2 spectral data for peak shown in Figure 2 at 22.1 min.
Intens.
x105
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 29.4min #1133
1688.00
1251.67
1
764.45
485.26
1036.77
605.96
0
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1409.87
1540.93
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1688.00), 29.4min #1134
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0.5
0.0
4000
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1251.67), 29.5min #1135
981.44
2000
417.19
563.25
1107.54
1253.59
855.35
0
200
400
2
600
800
1000
Figure S7: LC-MS spectral data for peak shown in Figure 2 at 29.5 min.
1200
1400
1600
1800
m/z
Intens.
x105
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 34.3min #1337
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1319.75
815.45
514.27
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1165.38
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(721.44), 34.4min #1338
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255.05
4000
2000
391.06
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698.96
0
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1319.75), 34.4min #1339
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2000
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837.35
939.37
1065.57
1175.57
1447.84
0
200
400
600
800
1000
1200
1400
1600
1800
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Figure S8: LC-MS2 spectral data for peak shown in Figure 2 at 34.4 min.
Intens.
x106
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 38.9min #1523
1345.99
0.5
1279.75
578.44
766.48
0.0
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20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1345.99), 38.9min #1524
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1347.91
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20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1279.75), 38.9min #1525
1281.74
2
981.45
417.21
563.29
1125.56
837.40
0
200
400
2
600
800
1000
Figure S9: LC-MS spectral data for peak shown in Figure 2 at 38.9 min.
1200
1400
1600
1800
m/z
Intens.
x105
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 40.4min #1583
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1381.85
2
1
766.51
1004.77
0
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1521.94
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1335.81), 40.4min #1584
1337.72
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417.19
545.26
689.35
0.0
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4
819.38
963.40
1107.56
1209.63
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1381.85), 40.5min #1585
1335.81
2
1191.64
543.37
0
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400
600
800
1000
1200
1400
1600
1800
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Figure S10: LC-MS2 spectral data for peak shown in Figure 2 at 40.5 min.
Intens.
x105
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 42.7min #1673
1407.83
1361.82
2
742.50
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1523.05
825.54
0
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20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1407.83), 42.7min #1674
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543.31
0
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837.37
963.48
1107.64
1217.72
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1361.82), 42.7min #1675
1363.77
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417.14
543.29
0.0
200
400
2
600
1107.67
669.41 742.43
837.34
800
981.43
1000
Figure S11: LC-MS spectral data for peak shown in Figure 2 at 42.7 min.
1217.61
1200
1400
1600
1800
m/z
Intens.
x105
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 43.6min #1709
1423.86
1377.85
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606.52
742.52
0
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20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1423.86), 43.6min #1710
1377.97
0.5
417.17
0.0
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543.32
1123.64
1233.71
1425.76
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1377.85), 43.6min #1711
768.56
1.0
1378.85
0.5
963.36
417.23
0.0
200
400
600
800
1107.58
1000
1233.62
1200
1400
1600
1800
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Figure S12: LC-MS2 spectral data for peak shown in Figure 2 at 43.6 min.
Intens.
x105
20201118 Kang kong meoh_BB1_01_6586.d: -MS, 47.8min #1877
1405.88
744.53
1
339.74
0
x104
889.60
592.21
1004.77
1662.05
1879.15
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1405.88), 47.8min #1878
1406.85
4
2
417.20
0
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697.45
963.42
1107.55
1261.66
20201118 Kang kong meoh_BB1_01_6586.d: -MS2(744.53), 47.8min #1879
6
281.08
4
509.12
2
711.38
0
200
400
2
600
800
1000
Figure S13: LC-MS spectral data for peak shown in Figure 2 at 47.6 min.
1200
1400
1600
1800
m/z
(a)
(b)
(c)
Figure S14: Dose-response curve of pancreatic lipase inhibition of extract treated with
activated charcoal (a) before enzymatic treatment, (b) after incubating with -amylase at pH
7 and (c) extract incubated with pepsin at pH 3.
F ST 5 198
PO ST ER DESIG N
Week 6
Credit to Dr. Foo Maw Lin
https://pollev.com/joannetoy493
Scan here!
WHAT DO YOU
INCLUDE IN YOUR
POSTER?
Acid adaptation increases resistance of
Escherichia coli O157:H7 in bok choy (Brassica rapa
subsp. chinensis) juice to high pressure processing
ANDREA KOO1 ,2 , VINAYAK GHATE 1, WEIBIAO
ZHOU
Title, authors, and affiliations
1,2
1Integrative Sciences and Engineering Programme, NUS Graduate School, National University of Singapore, University Hall,
Tan Chin Tuan Wing Level 5, #05-03, 21 Lower Kent Ridge Road, Singapore 119077
2Department of Food Science and Technology, National University of Singapore, 2 Science Drive 2, Singapore 117542
Methodology
Introduction
Introduction/Background
High pressure processing (HPP) is a pasteurisation technique commonly
used to produce 'fresh-like' juices
Bok choy is a leafy vegetable commonly cultivated in Asia and ugly bok choy
can be upcycled via juicing
Acid stress is a major stressor in juice processing environments1
The adaptive response of E. coli O157:H7 has been shown to confer crossresistance against pasteurisation technologies e.g. heat 2 , X-ray3
Objectives/Aims
37˚C, 24 h
Process lethality &
survival during storage
Total viable counts on TSA
Cellular damage
Plating on TSA + metabolic inhibitor
DNA - nalidixic acid (1.5 ppm)
Proteins - chloramphenicol (6 ppm)
Acid adaptation
TSB - dextrose + HCl
(pH 5.0)
Methodology
Staining & flow cytometry
Cell membrane - SYTO9/PI
Enzymes - NBDG
37˚C, 24 h
No acid
adaptation TSB dextrose
(pH 7.3)
Does acid adaptation affect the resistance of E.
coli O157:H7 to HPP in bok choy juice? If so, why?
Inoculation
~10 log CFU/mL in
bok choy juice
Statistical analysis
HPP treatment
400 MPa and 5˚C
Student's t-test between acid
adapted and non adapted cells
(n = 3, p < 0.05)
Results & Discussion
Process lethality
Survival during storage @ 4˚C
Survival curves at 400 MPa and 5˚C
fitted with a first-order kinetic model
Total viable counts decreased
during storage post-HPP
No change in control cells
D-value = time required to achieve a 1-log
reduction
Acid adapted = 1.2 ± 0.1 min
Non adapted = 0.8 ± 0.1 min*
Depending on requirements,
• Abstract
• Acknowledgements
Slower rate of decrease observed
in acid adapted cells
Could acid adapted cells be less
injured?
Longer processing time required to
achieve 5D reduction for pasteurisation
Cellular
damage
Acid adaptation may protect
against DNA damage
A
DN
Acid adapted cells
better retained
membrane integrity
Cell m
em
br
Adaptive changes in
membrane composition
may protect against
pressure disruption
↑cyclopropane FA
↑saturated FA
e
an
N.S. = Counts on TSA &
selective media did not differ (p
> 0.05)
dps is known to protect DNA
from oxidative damage and
correlates with acid and
pressure resistance in E. coli
Glucose pump activity
was better preserved
in acid adapted cells
Protein damage may be
attenuated by acid adaptive
response
Upregulation of chaperones
HdeA and DegP in acid tolerance
response may prevent protein
aggregation during HPP
Conclusions
against cellular
damage
Kindly shared by Andrea Koo
Adaptation also
protected cells against
the juice matrix, with
lower levels of damage
even in untreated
samples (p < 0.05)
Pro
teins & Enzymes
References
Conclusion
Acid adaptation
protects
Results
Increased
Processing parameters
developed based on non
pressure
resistance
adapted cells may not be
sufficient!
The effect of acid adaptation should be considered in the
selection of HPP parameters for E. coli O157:H7
inactivation.
1.Kang, J.-W., & Kang, D.-H. (2019). Increased resistance of Salmonella enterica serovar
Typhimurium and Escherichia coli O157:H7 to 222-nanometer krypton-chlorine excilamp
treatment by acid adaptation. Applied and Environmental Microbiology, 85(6), e02221-02218.
2.Usaga, J., Worobo, R. W., & Padilla-Zakour, O. I. (2014). Effect of acid adaptation and acid
shock on thermal tolerance and survival of Escherichia coli O157: H7 and O111 in apple
juice. Journal of Food Protection, 77(10), 1656-1663.
3.Lim, J.-S., & Ha, J.-W. (2021). Effect of acid adaptation on the resistance of Escherichia coli
O157: H7 and Salmonella enterica serovar Typhimurium to X-ray irradiation in apple juice.
Food Control, 120, 107489.
References
Acid adaptation increases resistance of
Escherichia coli O157:H7 in bok choy (Brassica rapa
subsp. chinensis) juice to high pressure processing
ANDREA KOO1 ,2 , VINAYAK GHATE 1, WEIBIAO
ZHOU
SIZE
A0 (84.1 X 118.9 CM) OR
A1 (59.4 X 84.1 CM)
1,2
1Integrative Sciences and Engineering Programme, NUS Graduate School, National University of Singapore, University Hall,
Tan Chin Tuan Wing Level 5, #05-03, 21 Lower Kent Ridge Road, Singapore 119077
2Department of Food Science and Technology, National University of Singapore, 2 Science Drive 2, Singapore 117542
Introduction
Methodology
High pressure processing (HPP) is a pasteurisation technique commonly
used to produce 'fresh-like' juices
Bok choy is a leafy vegetable commonly cultivated in Asia and ugly bok choy
can be upcycled via juicing
Acid stress is a major stressor in juice processing environments1
The adaptive response of E. coli O157:H7 has been shown to confer crossresistance against pasteurisation technologies e.g. heat 2 , X-ray3
37˚C, 24 h
Process lethality &
survival during storage
Total viable counts on TSA
Cellular damage
Plating on TSA + metabolic inhibitor
DNA - nalidixic acid (1.5 ppm)
Proteins - chloramphenicol (6 ppm)
Acid adaptation
TSB - dextrose + HCl
(pH 5.0)
Staining & flow cytometry
Cell membrane - SYTO9/PI
Enzymes - NBDG
37˚C, 24 h
No acid
adaptation TSB dextrose
(pH 7.3)
Does acid adaptation affect the resistance of E.
coli O157:H7 to HPP in bok choy juice? If so, why?
Inoculation
~10 log CFU/mL in
bok choy juice
Statistical analysis
HPP treatment
400 MPa and 5˚C
Student's t-test between acid
adapted and non adapted cells
(n = 3, p < 0.05)
Results & Discussion
Process lethality
Survival during storage @ 4˚C
Survival curves at 400 MPa and 5˚C
fitted with a first-order kinetic model
Total viable counts decreased
during storage post-HPP
No change in control cells
D-value = time required to achieve a 1-log
reduction
Acid adapted = 1.2 ± 0.1 min
Non adapted = 0.8 ± 0.1 min*
Slower rate of decrease observed
in acid adapted cells
Could acid adapted cells be less
injured?
Longer processing time required to
achieve 5D reduction for pasteurisation
Acid adaptation may protect
against DNA damage
N.S. = Counts on TSA &
selective media did not differ (p
> 0.05)
A
DN
Cell m
em
br
Adaptive changes in
membrane composition
may protect against
pressure disruption
↑cyclopropane FA
↑saturated FA
dps is known to protect DNA
from oxidative damage and
correlates with acid and
pressure resistance in E. coli
Typically, portrait but
could be landscape too
Glucose pump activity
was better preserved
in acid adapted cells
Protein damage may be
attenuated by acid adaptive
response
Upregulation of chaperones
HdeA and DegP in acid tolerance
response may prevent protein
aggregation during HPP
Acid adaptation
protects
against cellular
damage
Adaptation also
protected cells against
the juice matrix, with
lower levels of damage
even in untreated
samples (p < 0.05)
Pro
teins & Enzymes
References
Conclusion
Kindly shared by Andrea Koo
Acid adapted cells
better retained
membrane integrity
e
an
ORIENTATION
Cellular
damage
Increased
Processing parameters
developed based on non
pressure
resistance
adapted cells may not be
sufficient!
The effect of acid adaptation should be considered in the
selection of HPP parameters for E. coli O157:H7
inactivation.
1.Kang, J.-W., & Kang, D.-H. (2019). Increased resistance of Salmonella enterica serovar
Typhimurium and Escherichia coli O157:H7 to 222-nanometer krypton-chlorine excilamp
treatment by acid adaptation. Applied and Environmental Microbiology, 85(6), e02221-02218.
2.Usaga, J., Worobo, R. W., & Padilla-Zakour, O. I. (2014). Effect of acid adaptation and acid
shock on thermal tolerance and survival of Escherichia coli O157: H7 and O111 in apple
juice. Journal of Food Protection, 77(10), 1656-1663.
3.Lim, J.-S., & Ha, J.-W. (2021). Effect of acid adaptation on the resistance of Escherichia coli
O157: H7 and Salmonella enterica serovar Typhimurium to X-ray irradiation in apple juice.
Food Control, 120, 107489.
• Not too wordy
• Include figures and an
explanation
• Clear to readers
LAYO U T & F O RM AT ARE CRI T I CAL
Easy to the eye
1. Easy to tell where to read next
2. Appropriate text/figure ratio
3. Appropriate colour scheme and font size
LAYO U T & F O RM AT ARE CRI T I CAL
1. Easy to tell where to read next
Top to bottom
LAYO U T & F O RM AT ARE CRI T I CAL
2. Appropriate text/figure ratio
Which poster is better?
LAYO U T & F O RM AT ARE CRI T I CAL
3. Appropriate colour scheme and font size
Colour
Colour
Colour
Colour
Colour
Colour
It is better to use a light-coloured background with dark-coloured fonts.
LAYO U T & F O RM AT ARE CRI T I CAL
3. Appropriate colour scheme and font size
Never use more than 3 fonts,
typically 2
Font size
LAYO U T & F O RM AT ARE CRI T I CAL
Attention grabbing
1. Interesting title
2. Impactful photographs, figures and tables at the appropriate positions.
3. Conclusions that are easy to understand
LAYO U T & F O RM AT ARE CRI T I CAL
1. Interesting title
Orientate audience to
purpose of the poster
• Make sure the title is left or center justified
• Do not use all capital letters
• No jargons or acronyms
LAYO U T & F O RM AT ARE CRI T I CAL
2. Impactful photographs, figures and tables at the appropriate positions.
• Give the figure a title
• The simpler the figure, the better
• For graphs, always label the axis and make sure the lines are thick
enough
• Do not use screen capture images with low resolution (~300 dpi)
LAYO U T & F O RM AT ARE CRI T I CAL
3. Conclusions that are easy to understand
• Clearly state what is new, original, exciting and different from published
results
• Must fit into the objectives/aims
• Future work can be included
Least valuable space
Bottom of the poster
References
&
Funding
Optional
Optional
QR code
to link
to publication
Photograph of
yourself and
contact details
LET ' S TAKE A LOOK AT SOME
PO ST ER
TO O W O R DY
HA R D TO R EA D
• Easy to read(nice
colour)
• Methods are in
pictorial form
Kindly shared by Ricco Tindjau.
• Attractive colours
• Nice summary of
materials and methods
• Conclusion -> in a
pictorial form
Kindly shared by Sze Hui Yong.
ASSIGNMENT 2 (20%)
Prepare one poster based on your research project/any research topic. (A1 poster)
• Title, authors and affiliations
• Introduction
• Objectives
• Methods
• Results and discussion
• Reference
*For those with no research projects/results yet, you may choose a research article of your choice,
extrapolate information to create/design a poster. The journal article should be different from your
oral presentation.
Deadline: 14 April 2023 (Week 13) 11.59 pm
Submit in Powerpoint/pdf format on Canvas-> assignment 2.
