Introduction The worldwide prevalence of obesity

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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. Buchholz, T., & Melzig, M. F. (2016). 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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 x104 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 x104 1409.87 1540.93 20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1688.00), 29.4min #1134 1688.12 1.0 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 721.44 2 1319.75 815.45 514.27 0 978.73 1473.86 1165.38 20201118 Kang kong meoh_BB1_01_6586.d: -MS2(721.44), 34.4min #1338 6000 255.05 4000 2000 391.06 483.15 698.96 0 20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1319.75), 34.4min #1339 4000 1321.70 417.17 2000 543.30 837.35 939.37 1065.57 1175.57 1447.84 0 200 400 600 800 1000 1200 1400 1600 1800 m/z 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 x105 20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1345.99), 38.9min #1524 766.50 1.0 1347.91 0.5 0.0 x104 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 1335.81 1381.85 2 1 766.51 1004.77 0 x105 1.0 1521.94 20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1335.81), 40.4min #1584 1337.72 0.5 417.19 545.26 689.35 0.0 x105 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 0 200 400 600 800 1000 1200 1400 1600 1800 m/z 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 1 1523.05 825.54 0 x104 4 20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1407.83), 42.7min #1674 1361.89 2 417.21 543.31 0 x104 1.0 653.35 837.37 963.48 1107.64 1217.72 20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1361.82), 42.7min #1675 1363.77 0.5 417.14 543.29 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 2 606.52 742.52 0 x105 20201118 Kang kong meoh_BB1_01_6586.d: -MS2(1423.86), 43.6min #1710 1377.97 0.5 417.17 0.0 x105 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 m/z 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 x104 543.35 697.45 963.42 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.

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