Digestion fates of different edible oils vary with their composition specificities and interactions with bile salts

Accepted Manuscript Digestion fates of different edible oils vary with their composition specificities and interactions with bile salts

Zhan Ye, Chen Cao, Yuanfa Liu, Peirang Cao, Qiu Li PII: DOI: Reference:

S0963-9969(18)30408-3 doi:10.1016/j.foodres.2018.05.040 FRIN 7630

To appear in:

Food Research International

Received date: Revised date: Accepted date:

3 March 2018 10 May 2018 18 May 2018

Please cite this article as: Zhan Ye, Chen Cao, Yuanfa Liu, Peirang Cao, Qiu Li , Digestion fates of different edible oils vary with their composition specificities and interactions with bile salts. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi:10.1016/j.foodres.2018.05.040

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ACCEPTED MANUSCRIPT Title Page

Full Title Digestion fates of different edible oils vary with their composition specificities and interactions with bile salts Names of Authors a, b

*, Peirang Cao a, b, Qiu Li c]

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[Zhan Ye a, Chen Cao a, b, Yuanfa Liu

Author Affiliation(s)

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[a. School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, People’s Republic of China; b. National Engineering Laboratory for Cereal

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Fermentation Technology, State Key Laboratory of Food Science and Technology, 1800 Lihu Road, Wuxi 214122, Jiangsu, People’s Republic of China; c. Shandong LuHua group co., LTD,

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Laiyang 265200, Shandong, People’s Republic of China] Contact information for Corresponding Author

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Corresponding Author: Yuanfa Liu; Telephone: (+086)510-85876799; Fax: (+086)510-85876799 Complete mailing address: Room 108, Jiangnan University Synergetic Innovation Center, 1800

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Lihu Road, Wuxi 214122, Jiangsu, P.R. China E-mail address: [email protected]

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Contact information for First Author E-mail address: [email protected]

Complete mailing address: Room 104, Jiangnan University Synergetic Innovation Center, 1800

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Lihu Road, Wuxi 214122, Jiangsu, People’s Republic of China

END PAGE 1

ACCEPTED MANUSCRIPT Abstract: The digestion fates of different edible oils are different. The objective of this study was to understand the influences of lipid composition on their digestion fates, and investigate the roles of bile salts (BS) played in emulsified lipid system (whey protein isolate as emulsifier) in the

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in- vitro small intestine digestion stage. Three typical oils (palm oil (PO), rapeseed oil (RO) and linseed oil (LINO)) were chosen. Results showed that with the BS addition increased from 0.0 to

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2.0 mg/mL, the increasing magnitude of the different fatty acid (FA) apparent release rate

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constants were: PO >RO ≈ LINO. Although the maximum FA release extent changed with BS

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addition, the order were: PO> RO> LINO. These may probably be attributed to palmitic acids, the most abundant FA in PO, was mostly located on the Sn-1, 3 positions of triacylglycerol

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(TAG) molecules, which contributed to the pancreatic lipase hydrolysis action. The relatively short chain length and the lower hydrophobicity also favored this process. However, Sn-1, 3

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positions of TAGs in RO and LINO were mainly long chain mono- or poly-unsaturated FAs,

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which restricted the continuous lipid hydrolysis. Furthermore, the lipid composition may also affect the BS behavior on the O/W emulsion droplet surface, thus modulating lipase hydrolysis

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reaction. These findings can provide some basic understandings of the digestion differences of different oils.

Key words: Lipid digestion; Edible oil; Digestion fate; Bile salts; In-vitro digestion; Emulsion

ACCEPTED MANUSCRIPT 1. Introduction Lipids are important macronutrients and play an important role in the human diet. They store energy, deliver essential FAs and lipid soluble nutrients, and considered as a hedonic component to many foods (Golding & Wooster, 2010). Digestible lipids derived from different sources have

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different FA and TAG compositions (e.g. FA type, saturation and isomerism). FAs vary in their chain length and unsaturation can lead to appreciable differences in their physical and chemical

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properties (R. Zhang, Z. Zhang, H. Zhang, E. A. Decker, & D. J. Mcclements, 2015), thus

numerous physicochemical and biochemical events,

for example,

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process involving

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causing their different bioaccessibilities and nutritional values. Lipid digestion is a complex

emulsification, adsorption, desorption, enzymatic hydrolysis (Mu & Høy, 2004; Singh & Sarkar,

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2011; Zhu, Ye, Verrier, & Singh, 2013).

The digestion of lipids takes place primarily in the small intestine, although about 10-30%

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dietary lipids are hydrolyzed in mouth and stomach phase by lingual or gastric lipase (Armand,

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2007). In the small intestine, emulsified lipids are mixed with intestinal fluids that contain pancreatic lipase, co- lipase, proteases, BS and phospholipids. The BS may adsorb to the surfaces

adsorption

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of the lipid droplets before displacing other surface active materials through competitive mechanism

(Golding

&

Wooster,

2010;

Groff

&

Gropper,

2013;

Maldonado-Valderrama, Wilde, Macierzanka, & Mackie, 2011). Then in the assistance of BS, the pancreatic lipase/co- lipase complex adsorb to the lipid–water interface and hydrolyze the emulsified TAGs into free fatty acids (FFAs) and monoacylglycerols (MAGs). Finally, the lipid digestion products mix with BS and phospholipids to form the mixed micelle phase to be absorbed in intestine (Groff & Gropper, 2013; Sarkar, Ye, & Singh, 2016). During the digestion process, BS plays a vital role in keeping the lipid continuous hydrolysis reaction upon pancreatic

ACCEPTED MANUSCRIPT lipase as indicated in Fig. 1. The main roles of BS played on the lipid-emulsion droplet surface during lipid intestine digestion were to keep the lipid continuous hydrolysis reaction upon pancreatic lipase (Golding & Wooster, 2010). This particular actions have also been elaborated in many previous studies (Guo, Ye, Bellissimo, Singh, & Rousseau, 2017; Maldonadovalderrama et

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al., 2008; Sarkar et al., 2016; Wilde & Chu, 2011). Normally pancreatic lipase needs to be bound to co- lipase, so as to access the interface of

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emulsified lipids. The interfacial structure of some surfactants can sometimes prevent the

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adsorption of the co- lipase/pancreatic lipase complex from reaching the lipid emulsion surface

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(Fig. 1 i). ii). and iii)). While in the presence of BS, such inhibitory surfactants can effectively be removed through an orogenic displacement mechanism (Fig. 1 iv)). As lipolysis reaction

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proceeds, the hydrolysis products of the TAGs (2- monoglycerides (2-MAGs) and FFAs) accumulate on the surface of the lipid emulsion droplets, which can limit lipase adsorption, and

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further down regulate the hydrolysis rate (Engelking, 2015; Zou et al., 2016). However, these

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products are probably removed from the interface and transported across the intestinal wall by mixed BS and phospholipid micelles, although some argued that the highly surfactant nature of

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BS might restrict lipase adsorption at the interface, limiting lipid digestion by sterical hindrance (Nieva-Echevarría, Goicoechea, Manzanos, & Guillén, 2016; Reis, Holmberg, Watzke, Leser, & Miller, 2009; Sarkar et al., 2016) (Fig. 1 v)). Thus, it can be concluded that BS can either participate in the mediation and activation of pancreatic lipase, and/or facilitate continuous lipase hydrolysis reaction by removing the hydrolysis products from the droplet surface. Different FA and TAG composition of oils can affect their digestion and absorption. Previous studies reported the oil types affected the gastrointestinal fate of lipids in the form of O/W emulsions (Hur, Joo, Lim, Decker, & McClements, 2011; R. Zhang et al., 2015; Zhu et al., 2013).

ACCEPTED MANUSCRIPT Rong Liang, et al. (Liang et al., 2016) showed that lipid digestion rate increased in the following order: long-chain triacylglycerol (LCT) < medium-chain triacylglycerol (MCT) < short-chain triacylglycerol (SCT) when using tributyrin, caprylic/capric triglycerides and canola oil which represented as SCT, MCT and LCT, respectively. Liqiang Zou, et al (Zou et al., 2016) also

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reported that lipid composed of MCT was digested more faster than that composed of LCT, which might be attributed to the different FAs composition within oils (long chain, medium

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chain or short chain FFAs) in the small intestine environment. However, all these studies just

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provided some rough conclusions, rather than analyzing the influences of lipid FA compositions

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and TAG profiles on the digestion fates, or the connections between the lipid compositions and their interaction with BS during small intestine digestion process. Some researchers suggested

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that the digestion products of LCT (long chain FFAs) tended to accumulate at the oil–water interface, thereby restricting the access of lipase to the droplet surfaces. While the digestion

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products of MCT (medium chain FFAs) have a higher affinity for water and therefore rapidly

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move into the surrounding aqueous phase, thereby making it easier for lipase to access to the lipid droplet surface (Guo et al., 2017; R. Zhang et al., 2015). The degree of unsaturation seemed

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to have no significant effect on lipid digestion. However, little information is available on the different fatty acids and TAG composition, which affect oil digestion and absorption. Thus, many works remains to be proceeded to figure out the influences of lipid composition on the digestion fates. PO, RO and LINO are the three of the most widely consumed edible oils in China. The diversity of digestion fates of different oils might impose significant influence on their absorption efficiency and nutritional value to various people. So, in the present study, firstly, to investigate the influences of FA and TAG compositions on the lipid digestion behavior, and the roles of BS

ACCEPTED MANUSCRIPT during this process, PO, RO and LINO, which mainly contain palmitic acid (C16:0), oleic acid (C18:1) and linolenic acid (C18:3), respectively, were selected to conduct in vitro small intestine digestion experiment before analyzing their FA and TAG compositions by GC and UPLC-MS. Secondly, the FA release extent over a period of 120 min digestion time was further characterized

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by pH-Stat method and the fatty acid release dynamic behavior of different oils were detailedly analyzed. Thirdly, the potential connections between the lipid chemical composition and FA

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release rate and extent were briefly illustrated by analyzing the construction of BS and the

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underlying roles it played during the digestion process. The basic recognitions of the different

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digestion fates between PO, RO and LINO can give us a clue that digestion fates of different oils may vary with the FA and TAG composition specificities and interactions with BS. The findings

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from this work might supply information for the understanding of the digestion diversity of oils for their absorption and nutrition differences and would be useful for functional food design and

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dietary lipid intervention and regulation.

2. Materials and methods

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2.1 Materials

PO and RO were purchased from Wilmar International Ltd. (Shanghai, China). LINO was obtained from Mengcaotang Ecological Agriculture and Animal Husbandry Development Co., Ltd. (Inner Mongolia, China). Whey protein isolate (WPI) was obtained from Davisco Foods International Inc. (bilPro, Le Sueur, USA). Porcine bile salt (BS) (CAS 8008-63-7) was sodium salt of bile acid which was purchased from Sinopharm Chemical reagent Co., Ltd (Shanghai, China), as stated by the manufacturer, the total bile salt content ≥60% (calculated as bile acid). Lipase (CAS 9001-62-1) from porcine pancreas Type II (100-500 units/mg protein) was

ACCEPTED MANUSCRIPT purchased from Sigma-Aldrich (Sigma Chemical, Co., St. Louis, MO, USA). Other chemicals were of analytical grade purchased from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO), J&K Scientific Ltd (Beijing, China) or Sinopharm Chemical reagent Co., Ltd. (Shanghai, China). All solutions were freshly prepared using Milli-Q water (Milli-Q Direct 8, Millipore Corp.,

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Bedford, MA, USA) as solvent. 2.2 Fatty acid and triacylglycerol analysis

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FA composition was analyzed using a gas chromatography (GC) system (Shimadzu, GC-2010

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PLUS) equipped with a capillary gas chromatography column (TR-FAME 60m × 0.25mm i.d. ×

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0.25 μm) and a flame ionization detector (FID) according to AOCS Official Method Ce 2-66 (AOCS, 2009). Briefly, the initial oven temperature was maintained at 60 ºC for 3 min before it

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was increased to 175 ºC by 5 ºC/min and held for 15 min, then increased to 220 °C at 2 °C/min, and kept for 10 min. The temperatures of the injector and FID detector were set at 250 and

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280 °C, respectively. The constant carrier gas (N 2 ) flow was set as 1.2 mL/min, split ratio was

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100, and the injection volume was 1 μL. All the FAs were identified by comparing their retention time with those of the FAME standards.

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TAG profiles were analyzed by Ultra‑Performance Liquid Chromatography Mass Spectrometry (UPLC-MS). The UPLC system (Waters, Milford, USA) was equipped with a BEH C18 column (i.d. 2.1 mm × 50 mm, 1.9 μm). The formic acid /water (1: 99, v/v) was used as the mobile phase A. Acetonitrile as mobile phase B. The binary gradient started with 100% phase A for 5 min, then adjusted to 70% for 6 min, followed by 80 % phase A and 20% phase B for 7min, after that, 100% phase B for another 7.1 min, and then changed to 100% phase A until the UPLC analysis process ended. The flow rate was 300 μL/min. The temperature of the sample chamber was set at 20 ºC, the column temperature was set at 45 ºC and the injection volume was 1.0 μL for each analysis

ACCEPTED MANUSCRIPT with a concentration of 1 mg/mL (diluted by HPLC grade n-Hexane). Prior to analysis, the diluted samples were filtered using 0.25 μm filter membrane (Thermo Fis her Scientific, MA., USA) and collected in 2 mL injection bottle. The column was need to be flushed for 5 min with 100% of mobile phase A before the next analysis was started after each analysis. The quadrupole

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time of flight mass spectrometry (Q-TOF-MS) instrument (Waters, Milford, USA) with ESI probe was used to identify and quantify the TAG of the samples. Positive ion mode was used at

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the following optimized conditions: capillary voltage, 3.5 kV; cone voltage, 30 V; source block

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temperature, 100 ºC; desolvation temperature, 400 ºC collision gas, argon, desolvation gas (N 2 )

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flow rate, 700 L/h, cone gas flow 50 L/h, collision energy, 6/20 V, detector energy, 1800 V. The mass range was from 20 to 2000 m/z with a scan duration of 0.5 s. The corresponding adduct ion

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peaks from the different TAG classes were detected by positive ion full- scan ESI–MS analysis. Instrument control and data analysis were performed using the MassLynx 4.1 software (Waters).

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2.3 Preparation of O/W digestion emulsions

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Emulsifier stock solution (1 wt% WPI) was prepared by dispersing WPI in Milli-Q water, then stirring gently for at least 2 h at 20 ºC to ensure complete dissolution. The emulsifier solution

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was stored overnight under 4 ºC to ensure complete hydration. The pH of the solution was adjusted back to pH 7.0 using either 0.05 mol/L NaOH or HCl solution if required. Initially, pre-emulsions were prepared by blending 20.0 wt% oil with 80.0 wt% aqueous WPI solution using a conventional high speed mixer (T 18D S25, IKA, Germany) at 16 000 r/min for 3 min. These coarse emulsions were then passed three times through a two-stage valve ultra-high pressure homogenizer (AH 2010, ATS nano technology Co., Ltd., Suzhou, China) operating at 80 bar and 350 bar in the first and second stage respectively, to create a fine emulsion. 2.4 Droplet size determination

ACCEPTED MANUSCRIPT The particle size determined by static light scattering using a particle analyzer (Nano Brook Omni, Brookhaven Instruments Corporation) referred to Sarkar’s method with some modifications (Sarkar et al., 2016). The relative refractive indexes of the various emulsions were taken as 1.092 (PO), 1.101 (RO) and 1.111 (LINO), respectively, which were calculated by the

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ratio of the refractive index of different oils to that of the aqueous phase (1.333). The sizes of emulsion droplets were reported as the surface weighted mean diameter d3, 2 (μm) and were

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calculated using the equation d3, 2 = Σnidi3 /Σnidi2 , where ni is the number of particles and di is the

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diameter of emulsion droplet.

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2.5 Zeta-potential measurements

The Zeta-potential of emulsions was measured according to Salvia et al. and Sarkar et al. using a

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Zetasizer Nano instrument (Zetasizer nano ZS, Malvern Instruments Ltd) (Salvia-Trujillo et al., 2017; Sarkar et al., 2016). Prior to analysis, samples were diluted to approximately 0.005 wt%

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droplet concentration with either Milli-Q water or SIF for the initial emulsions and emulsions

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after intestine digestion, respectively. A proper volume of the diluted solution was placed in a folded capillary cell (DTS 1070, Malvern Instruments Ltd) to assess the electrophoretic mobility

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of the particles. An individual Zeta-potential measurement was calculated from the mean and the standard deviation of at least three readings from an individual sample. 2.6 Fluorescence microscope An upright fluorescence microscope (Leica DM2700 M, Germany) equipped with a Leica fluorescence generator (Leica EL6000, Germany) was used to capture images of the different emulsions during the intestine digestion process. Before analysis, 1 mL samples were mixed with 20 μL Nile Red solution (10 mg/mL in ethanol), and vortex for 3 min (Vortex 4 basic, IKA, Germany), and incubated for 15 min before taking images.

ACCEPTED MANUSCRIPT 2.7 Preparation of simulated intestinal fluid (SIF) and simulated small intestine digestion The SIF was prepared according to the US Pharmacopeia, which contained 39 mM K 2 HPO 4 and 150 mM NaCl, and the pH was maintained at 7.5 (Stippler, Kopp, & Dressman, 2004). For in vitro intestinal digestion with SIF, emulsions which contained 4 wt% oil phase was obtained by

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diluting the freshly prepared stock emulsions (contain 20 wt% oil phase) with SIF buffer (without added pancreatic lipase). The 0.2, 1, 2 or 5 mg/mL of BS (in powder form) were then

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added to every individual samples, respectively. After adding 1.6 mg/mL pancreatic lipase into

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each sample, the emulsion-SIF mixture was vortexed for 30 seconds (Vortex 4 basic, IKA,

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Germany). During the 120 min digestion process, small aliquots were withdrawn periodically for analysis.

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2.8 Free fatty acid release and hydrolysis kinetics analysis The emulsion-SIF mixture (4 wt% oil) was poured into a conical flask in a water bath at 37 ºC,

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then, the system was then adjusted to pH 7.5 using 0.05 M NaOH or HCl solution, followed by

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addition of pancreatic lipase powder (96 mg of powder to 60 mL diluted sample). After vortex mixing for 30 s, the conical flask was placed in a 37 ºC water bath and subjected to magnetic

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stirring to simulate the small intestine digestion processing. The pH of the system was maintained at 7.5 by the addition of 0.05 M NaOH during the 120 min digestion process, and the FFAs generated from emulsified oils during this process were monitored by pH-stat method using a pH-stat automatic titration system (ZDJ-4A, INESA Scientific Instrument Co., Ltd, Shanghai, China). The volume of 0.05 M NaOH (in mL) consumed was recorded and calculated as the amount of FFAs hydrolyzed from the lipid emulsions. The FFAs release rate with digestion time was obtained according to the previous study (Sarkar et al., 2016) with some modifications. Briefly, the volume of NaOH consumed in the pH-Stat

ACCEPTED MANUSCRIPT experiment was converted to the amount of FFAs (as μM FFAs/mL emulsion) based on the standard curve established by in- vitro small intestine digestion model using standard palmitic acid, oleic acid and linolenic acid, and was plotted as a function of the digestion time. Pancreatic lipase converted TAGs into a complex mixture of diacylglycerols (DAGs), MAGs and FFAs. The

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percentage of the FFAs released from the intestine digestion process was calculated from the number of moles of 0.05 NaOH required to neutralize the FFA by using the following equation

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(1) (assuming 2 FFAs was produced per TAG molecule by the action of lipase) (Ruojie Zhang,

VNaOH  mNaOH  M Lipid (1)

WLipid  2

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FFA(%)=100 

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Zipei Zhang, Hui Zhang, Eric Andrew Decker, & David Julian Mcclements, 2015):

mNaOH is the

molarity of the sodium

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Here VNaOH is the volume of sodium hydroxide (in mL),

hydroxide solution (0.05 M), WLipid is the total weight of lipid initially present in the reaction

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vessel, and MLipid is the average molecular weight of different oils. The relative percentage of

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each FA in the oil is multiplied by its relative molecular mass, and then be summed to gether as the average molecular weight of the corresponding oil. During the intestine digestion process, the FFA released gradually increased with digestion time,

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potentially attaining the total release (Φmax ). The kinetic parameters for the initial FFA release were calculated using Equation (2) (Ye, Cui, Zhu, & Singh, 2013):

ln (max  t ) / max   kt  b

(2)

In the Eq. (2), k is the first-order rate constant for FFA release (s-1 ) and t is the digestion time (s). The total FFA release level (Φmax , μM/mL) was obtained from the FFA release curve. 2.9 Statistic analysis All experiments were performed on at least three freshly prepared samples. Statistical ana lysis was performed using Spss 16.0 software (IBM SPSS software, USA). Figures were drawn using

ACCEPTED MANUSCRIPT Origin 8.5 software (Origin Lab Ltd., USA). One-way ANOVA was carried out and Tukey adjustment was used to determine the significant difference. Significant differences were at P<0.05.

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3. Results and discussion 3.1 Droplet characteristic of different oil emulsions

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The emulsion droplet behavior of different oils in the presence of BS without pancreatic lipase

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addition was first investigated in order to show the impact of BS on emulsions stabilized by WPI

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without any interference from lipolytic activity. The mean particle sizes and Zeta-potentials of

respectively) were shown in Fig. 2.

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WPI emulsions in the presence of different levels of BS (0.0, 0.2, 1.0, 2.0 and 5.0 mg/mL,

Initially, the droplet size of the three oil emulsions were 0.315, 0.331 and 0.316 μm, respectively

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(Fig. 2A), and their particle size distribution showed monomodal (data not shown). When the

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amount of BS added was under 0.2 mg/mL, the mean particle size was significant higher (P<0.05) than those with BS addition of above 1.0 mg/mL. However, there was no significant difference

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in the average droplet size with the BS addition from 1.0 to 5.0 mg/mL (P>0.05) between groups. The average droplet diameter of the three oil emulsions just slightly decreased from 0.313 to 0.285 μm. These results indicated that low level of BS addition might reduce the stability of the oil emulsions with a characteristic of the increase of mean particle diameter or Zeta-potential (Klinkesorn & Mcclements, 2010; Sarkar, Horne, & Singh, 2010). These result also suggested that the addition of BS could not induce droplet aggregation or coalescence in the emulsions, which were consistent with previous studies (Mun, Decker, & Mcclements, 2007; Sarkar et al., 2010). While the data within a group showed that no significant differences (P>0.05) were

ACCEPTED MANUSCRIPT observed in the mean particle size between different oils under the same amount of BS addition. Fig. 2B showed the Zeta-potential changes among different oil emulsions. As expected, the Zeta-potential of the three emulsions was negative at neutral pH, increased slightly from -29.29 to -33.36 mV as a function of an increased concentration of aqueous BS from 0.0 to 5.0 mg/mL.

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The overall changes in Zeta-potential with BS addition (Δ Zeta potential = 4 mV) might be attribute to the anionic components within the BS adsorbed to the droplet surfaces (Mun et al.,

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2007), which might induce the displacement of the WPI from the interface of droplets (Euston,

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Baird, Campbell, & Kuhns, 2013). No significant differences were observed between groups,

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except for the LINO emulsion with 0.0 mg/mL and 5.0 mg/mL BS addition (P<0.05). This indicated that, although the BS addition ranged from 0.0 to 5.0 mg/mL, the interface charge of

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WPI-Oil emulsions stayed unchanged. However, it was worth noting that, significant differences were observed within a group under same BS content. Zeta-potential of the three emulsions were

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PO >RO >LINO from high to low. It demonstrated that the influences of BS exerted on different

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WPI-Oil emulsions were different, which were supported by previous studies (Sun, Joo, Lim, Decker, & Mcclements, 2011; Zhu et al., 2013).

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Thus we make the following hypothesizes: The different digestion fates of PO, RO and LINO in small intestine digestion stage may probably be caused by their unique FA and TAG constitutions. The BS plays a vital role in the digestion process by interacting with oil emulsions on the lipid droplet surface. Some related works have also been reported (Bellesi & Pilosof, 2014; Maldonado-Valderrama et al., 2011; Reis et al., 2009). To address these intriguing problems, three different groups with BS addition was 0.0, 1.0 and 2.0 mg/mL were chosen to conduct in- vitro small intestine digestion experiment in terms of the FFA release extent and lipid digestion kinetic. These groups were chose in the aim of achieving the similar BS secretion state

ACCEPTED MANUSCRIPT in small intestine during lipid digestion (Maldonado-Valderrama et al., 2011). 3.2 FFA release analysis 3.2.1 Effect of BS concentration on FFA release extent Fig. 3 showed the total FFA release from different oil emulsions stabilized by WPI in the

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presence of 1.6 mg/mL pancreatic lipase with or without the addition of BS. In the absence of BS (Fig. 3A), the absorbed WPI on the droplet surface seemed to restrict hydrolysis of emulsified

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lipids by pancreatic lipase. The FFA released were all relatively low, i.e. 6.05 μM/mL for PO,

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5.52 μM/mL for RO and 4.66 μM/mL for LINO within 120 min digestion time (FFA released

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were all under 15%). It also could be concluded that the differences of the FFA release extent between groups were not significant, which may due to the limited lipid hydrolysis without BS

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assistance. During lipid digestion, the coated WPI on the emulsion droplet surface not only acted as an emulsifier, but also a barrier to protect the integrity and stability of emulsion droplets

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which could keep the outer substances from reaching the inner core, including pancreatic lipase

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and some other surfactants, such as DAGs (Farfán, Villalón, Ortíz, Nieto, & Bouchon, 2015). In the presence of BS (Fig. 3B and 3C), the lipid hydrolysis extent were significantly improved.

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Specifically, in the presence of 1.0 mg/mL BS, the FFA released reached 19.81 μM/mL for PO, and about 16.0 μM/mL for RO and LINO after 120 min digestion. The similar tendency was observed in the presence of 2.0 mg/mL BS, in which the maximum fatty acid release extent for the three oils were 28.36, 23.01 and 21.63 μM/mL, respectively. Different with many previous results, the addition of BS could not only affect the hydrolysis extent, but also the overall FFA release trends of different oils. It showed that the FFA release extent of PO was significantly higher than RO and LINO with the addition of both 1.0 mg/mL and 2.0 mg/mL BS (Fig. 3B and 3C). Moreover, in the first 30 minutes, the FFA release extent of PO increased much faster than

ACCEPTED MANUSCRIPT the RO and LINO, which could be further explained by the FFA release first-order kinetics. In the first 10 minutes, RO and LINO showed similar FFA release trends. However, from 10 min to 30 min, the FFA release rate and extent of LINO seemed to be higher than RO, and after 30 minutes, the FFA release trends just become reverse.

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The fluorescence microscope images (Fig. 4) displayed the microscopic changes of oil emulsion droplets upon pancreatic lipase hydrolysis at the time of 30 min with or without BS addition. In

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the absence of BS, there seemed no significant differences between the three lipid emulsions, nor

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did the initial lipid emulsions (Fig. 4A and 4B). However, in the presence of BS, the PO was

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obviously hydrolyzed by the pancreatic lipase as few fluorescence particles were observed after 30 min digestion. While no significant changes were observed for either RO or LINO emulsions.

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These were consistent with the FFA release results in Fig. 3. and supported by previous studies, which showed that lipase could adsorb to O/W interface with or without BS addition during

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intestine digestion, however, the rate and the extent of lipid digestion were highly dependent on

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the presence of BS (Maldonado-Valderrama et al., 2011; Torcellogómez et al., 2011). Actually, BS was a surfactant with high surface activity, and in many cases, the high surfactantcy of BS

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actually could aid digestion. Above their critical micelle concentration (CMC) (about 2 ~ 4 mM) during lipid digestion, on the one hand, the BS could displace surfactants (e.g. WPI in present study) from O/W interface. The lipase then could bind with the BS covered on the interface thereby facilitating lipolysis (Golding & Wooster, 2010); On the other hand, as lipolysis reaction progressed, the lipolysis products (FFAs, MAGs and DAGs) were continuously accumulated on the surface of oil droplets, and there was a buildup of FFAs and MAGs on the droplet surface (Fig. 1). These digested products may tend to exclude pancreatic lipase from the O/W interface, thus leading to the continual decline of lipase hydrolysis efficiency (Gargouri, Julien, Bois,

ACCEPTED MANUSCRIPT Verger, & Sarda, 1983; Pafumi et al., 2002). However, BS, and a small amount of phospholipids, could remove the digested products from the interface by solubilizing them in mixed micelles in the aqueous phase (Fig. 1) (Bauer, Jakob, & Mosenthin, 2005; Mu & Høy, 2004). Therefore, BS facilitated further lipid digestion by removing these inhibitory surfactants and driving the

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reaction equilibrium towards continuous lipolysis. As for the present results showed in Fig. 3B and 3C, sharp increase of the initial FFA release rate could be observed because of the easily

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accessibility of pancreatic lipase to the O/W interface with the assistant of BS. Moreover, it was

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reported that WPI adsorption layer was porous to BS adsorption, which could further facilitate

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the pancreatic lipase action (Yi, Li, Zhong, & Yokoyama, 2014). After 30 minutes digestion, the rate of lipolysis slowed down. This might be attributed to the deceasing accessibility of

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pancreatic lipase to the oil droplet surface due to the overload accumulation of lipolytic products on the oil-water interface (Gallier, Tate, & Singh, 2013; S & H, 2012).

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3.2.2 Effect of BS concentration on FFA release kinetics

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Various factors which may be responsible for the above mentioned multi- faceted results have been reported, such as the emulsifier types (Chang & Mcclements, 2016), the content of BS

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addition (Sarkar et al., 2016) and the lipid types (Zhu et al., 2013), etc. The effect of BS concentration on FFA release first-order kinetics was carried out to investigate whether the multiple effects of BS addition and oil type could lead to the different digestion fates. The linear relationships for FFA release with time were obtained by using Eq. (2) (Fig. 5). As shown in the Fig. 5A, without BS, the FFA release rate constants for the three oils were very low, which were 0.0265 s-1 , 0.0268 s-1 and 0.0272 s-1 for PO, RO and LINO, respectively. These results just reflected the low FFA release rate and extent showed in Fig. 3. Besides, there seemed no significant differences in the rate constants between different oils in the absence of BS, which

ACCEPTED MANUSCRIPT were also in line with the above total FFA release profiles when the BS addition was 0.0 mg/mL. With 1.0 and 2.0 mg/mL BS addition, the rate constants for FFA release all showed a significant increase (Fig. 5B and 5C) with different extent of different oils. In the presence of 1.0 mg/mL BS (Fig. 5B), the rate constants were 0.0336 s-1 , 0.0316 s-1 and 0.0311 s-1 for PO, RO and LINO,

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respectively. The FFA release rate constant of PO was the highest, which increased by nearly 1.4 times, while that of RO and LINO were only 17.91% and 14.34% increase by compared with the

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groups without BS addition. The FFA release rate constants were continually increase in the

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presence of 2.0 mg/mL BS (Fig. 5C). After being experienced 30 min intestine digestion, the

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FFA release rate constants of PO, RO and LINO were 0.0373 s-1 , 0.320 s-1 and 0.0315 s-1 , and the corresponding FFA released were 28.36, 23.01 and 21.63 μM/mL, which were equivalent to

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53.44%, 43.53% and 40.76% of FFA released (data not shown). These data were in line with a study on digestion of protein stabilized corn oil emulsion using similar pH-stat techniques (Li &

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Mcclements, 2010).

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In summary, it could be concluded that BS addition did have an obvious impact on the FFA release rate constant, and the differences were also varied with different lipids. As for the present

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study, the FFA released rate of PO was more significantly affected by BS addition than RO and LINO. However, the differences in rate constants between RO and LINO were not significant in the presence of 1.0 or 2.0 mg/mL BS addition, which were in consistent with the FFA release extent results showed in Fig. 3B and 3C. A quantitatively analysis of the roles of BS in the digestion of emulsified lipids by separating the unabsorbed BS during lipid digestion indicated that lipid digestion rate and kinetics of the total FAs released increased with the increase of BS addition. This probably could be attributed to the presence of considerable amount of unabsorbed BS in the aqueous phase, which could effectively remove the lipolysis products (FFAs, MAGs,

ACCEPTED MANUSCRIPT DAGs) in mixed micelles (Sarkar et al., 2016). These were consistent with the results showed in the present work. 3.3 Analysis of the connections between oil compositions, BS addition and digestion fates To illustrate the connections between the lipid compositions and their different digestion fates

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showed above, the FA compositions and the TAG profiles of the experimental oils were analyzed by GC-FID and UPLC-MS (Table 1, Fig. 6A and 6B).

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Lipid digestion was a complicated interfacial process dependent on the adsorption of lypolytic

competitive

adsorption,

surfactants

removal

and

lipid

droplet

disruption

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as

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enzymes to the oil droplets surface, which was involved in a series of interfacial reactions, such

(Maldonado-Valderrama et al., 2011; Wilde & Chu, 2011). In general, the human body had an

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excess capacity for oil digestion, and the rate of lipid digestion was controlled by the ability of lipase binding to the interfaces which were further influenced by the droplet size and interfacial

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lipids were also different.

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compositions, i.e. droplet structure or composition. While, the droplets composed of different

Significant differences could be observed in the saturated fatty acid (SFA) content between PO

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and the other two oils. The total SFA content of PO was 50.72 ± 2.01 %, 43.24 ± 1.81% of which was palmitic acid (C16:0) (Fig. 6A and 6B). The top three TAGs in the PO were PPP, OOP and POP, which composed 55.59 % of the total TAG content. Especially, the PPP content was over 35 % (Table 1). It was also worth noting that the palmitic acids were mostly located on the Sn-1 and Sn-3 positions of the TAG molecules in the PO (Table 1). Previous studies showed that the hydrolysis extent and rate of lipids composed of short chain FA were higher than lipids composed of long chain FA (Liang et al., 2016; Ozturk, Argin, Ozilgen, & Mcclements, 2015; Zou et al., 2016). Moreover, during intestine digestion, the pancreatic lipase was more prone to

ACCEPTED MANUSCRIPT hydrolyze the Sn-1 and Sn-3 ester bonds of TAG molecules due to its substrate specificity, and producing two FFAs and a 2-MAG (Mu & Høy, 2004). Thus, it could be easy to conclude that the FFA release rate constant and FFA release extent of PO increased significantly with the addition of 1.0 or 2.0 mg/mL. This may probably be attributed to that the saturated and

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short-chain palmitic acids (C16:0) were mainly located on the Sn-1 and Sn-3 positions of TAG molecules in PO.

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There was no significant difference in the total amount of unsaturated fatty acid (UFA) between

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RO and LINO (p>0.05, Fig. 6B), however, they had significant difference in the

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monounsaturated fatty acids (MUFA) and poly unsaturated fatty acids (PUFA) content (p<0.05, Fig. 6B). More specifically, RO contained more C18:1 (62.39 ± 2.36%), while, LINO was most

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abundant of C18:3 (53.06 ± 2.33%). Moreover, from the Table 1, it could be seen that the Sn-1 and Sn-3 positions of TAGs in RO and LINO were mainly long chain MUFAs or PUFAs, which

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were oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3), besides, these TAGs

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were mainly UUU (Unsaturated- Unsaturated- Unsaturated) types (Table 1). The influences of positional distribution and unsaturation of FAs on TAG molecules were observed on the lipid

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digestion rates in many previous studies, and it showed that positional distribution of FA within TAG molecules could affect lipid digestion, especially for the long-chain FA located on the Sn-1 or Sn-3 positions (Farfán, Villalón, Ortíz, Nieto, & Bouchon, 2013; Guo et al., 2017; Lien, 1994; Nagata, Kasai, Watanabe, Ikeda, & Saito, 2003). Since the effect of degree of unsaturation of FA (i.e., poly- or mono-unsaturation) seemed to be less obvious on lipid digestion (R. Zhang et al., 2015), the structural difference of PO, RO and LINO might be responsible for the different FFA release rate constants of the three lipids in the presence of different content of BS. However, from the above results, traces evidence could be seen that the degree of FA unsaturation and TAG

ACCEPTED MANUSCRIPT types (UUU and SSS (Saturated- Saturated- Saturated); OOO and LLL) could affect lipid digestion. So further work still need to be proceeded in these intriguing areas. The above results suggested that different digestion fates of different oils might be ascribe to the FA composition and positional distribution, short chain saturated FAs on the Sn-1 or Sn-3

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position favored lipase hydrolysis reaction as in the case of PO. While the relatively high content of UFA in the Sn-1, 3 positions in RO and LINO seemed to impose negative effect upon their

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FFA release extent and FFA release rate constants. In addition, the added BS played an important

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role in the emulsion properties during digestion, which could indicate from the Fig. 2B. The

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above results were probably caused by the different interactions between lipids and BS. When at a lower BS concentration (<5.0 mg/mL), the Zeta-potential values decreased with the increasing

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of BS amount. However, the Zeta-potential value tended to be constant when the BS concentration exceeds 5.0 mg/mL (Data not shown). This indicated that BS might compete with

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WPI to be adsorbed on the emulsion droplet surface, or inserted on the surface-active molecules

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to form mixed micelles by hydrophobic interaction, thus, leading to the high negative electrophoretic mobility, which might contribute to the higher emulsion stability (Euston et al.,

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2013; Liang et al., 2016).

In fact, the interactions between BS and emulsion droplet not only depend on BS structure itself (i.e., the stronger the hydrophobic action, the more obvious of the adsorption ability), but also be affected by hydrophobic groups in the emulsion droplets (i.e., the properties of lipids in the droplet core imposes different effects on these interactions), which have already been discussed above. In other words, the oil composition may influence the competitive adsorption ability of the BS with WPI on the emulsion droplet surface. The present study showed that BS seemed to be more prone to compete with WPI on PO emulsion droplet to displace it from the surface, then

ACCEPTED MANUSCRIPT facilitating the lipase hydrolysis process. Namely, the binding force of WPI to PO droplet may be weaker than that of RO and LINO. As shown in Fig. 7. BS was composed of two connecting units, a rigid steroid backbone with a hydrophobic and a hydrophilic face to which a flexible aliphatic tail was attached. The steroid nucleus of bile acids included three six- member rings and

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a five- member ring (Fig. 7). The hydrophobic surface lied on the convex side of the rigid steroid ring system. The concave side of the molecule contained one, two or three hydroxyl groups

glycine

(-NHCH2 COO−)

or

other

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(-NH(CH2 )2 SO3 −),

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(position R1 , R2 and R3 ) and an amino group that could be conjugated with taurine amino

acids

(position

R4 )

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(Maldonado-Valderrama et al., 2011). Although BS differed in the number, position and stereochemistry of the hydroxyl group as well as on the conjugated amino acid, they behaved in

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a qualitative similar manner.

When the BS was added to the O/W emulsion, the surface elasticity drastically reduced, causing

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the severely damage of the surfactant interface network structure. Then, the BS displaced the

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WPI from the emulsion oil-water interface (Euston et al., 2013; Nieva-Echevarría et al., 2016; Zangenberg, Müllertz, Kristensen, & Hovgaard, 2001 ). Many previous studies had reported this

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particular action, and defined as "orogenic displacement" substitution mechanism (Groff & Gropper, 2013; Maldonadovalderrama et al., 2008). This was the explanation for the BS competitive adsorption with WPI on the emulsion droplet surface. Due to the particular chemical structure, the arrangement of BS on the emulsion droplet surface was different from the other surfactants. The adsorption of BS on the droplet surface was characterized as the parallel arrangement of the chemical steroidal ring with the hydrophobic droplet surface, the nonpolar face of which interact with the inner part of the droplet (oil phase), while the polar face (carboxyl or hydroxyl parts) pointed to the aqueous phase (Fig. 1) (Tiss et al., 2001), forming a new BS-oil

ACCEPTED MANUSCRIPT emulsion system. The enrichment of palmitic acid in the PO led to the relatively lower hydrophobicity than RO and LINO. So the negative charge of RO and LINO emulsions was higher than PO, which indicated the surface stability of PO emulsion might be lower than the other two oils, thus, the

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PO emulsion film could be destroyed easily by the BS, which further competed with the WPI and replaced it to adsorb on the droplet surface. Furthermore, the palmitic acids were mainly

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located on the Sn-1, 3 positions of the TAG molecules, which was an optimal substrate for the

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effective hydrolysis of PO by pancreatic lipase with the assistance of BS.

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Many previous researchers did research to investigate the connection between the pancreatic lipase hydrolysis efficiency and the FA chain length and saturation, and gave a common

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conclusion that the TAGs composed with short or medium chain FAs were more prone to be hydrolyzed by lipase (Benito-Gallo et al., 2015; Liang et al., 2016). Also higher BS

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concentration could result in higher rate and extent of long chain triglyceride digestion (P et al.,

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2008), which were consistent with the present study. The effect of unsaturation on the FA polarity could be ignored due to the long carbon chain length. And studies also showed that the

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degree of unsaturation of TAGs (i.e., poly or mono- unsaturation) did not appear to significantly affect lipid digestion by doing research in fish oil (rich in C20:5 and C22:6) and corn oil (rich in C18:1 and C18:2), both of which mainly contained very long-chain polyunsaturated FAs (Engelking, 2015; R. Zhang et al., 2015).

4. Conclusions The present study had explored the influences of FA composition, TAG profiles and the BS addition on the digestion fates of the three common representative oils using in vitro small

ACCEPTED MANUSCRIPT intestine digestion model. Results showed that the addition of BS could significantly influence the digestion fates by changing the hydrolysis extent and FFA release rate. Compared with RO and LINO, PO was more prone to be influenced. The order of maximum FFA release level upon pancreatic lipase hydrolysis was: PO> RO> LINO, and the increase magnitude of different FFA

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apparent release rate constants were: PO >RO ≈ LINO. This could be explained by the different FA compositions and TAG profiles. The relatively short chain length and high saturation p almitic

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acids, which were the most abundant FA in PO, were mostly located on the Sn-1 and Sn-3

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positions of TAG molecules in PO, which contributed to the pancreatic lipase hydrolysis reaction

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due to the enzyme specificity. However, the Sn-1 and Sn-3 position of RO and LINO were mainly C18:1, C18:2 or C18:3 FAs, which restricts the continuous hydrolysis reaction upon

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pancreatic lipase. BS played a pivotal role during lipid digestion process, which could replace the WPI by competitive adsorption before acting as a lipase activator and a lipase-assisted

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adjuvant. These results could not only provide evidence for the potential differences of digestion,

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absorption and nutrition values of PO, RO and LINO, but also have important implications for the formation of functional foods or beverages that can control the rate and extent of lipid

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digestion.

Conflict of interest

No conflicts of interest are declared for any of the authors.

Acknowledgement This work was supported by the Natural Science Foundation of China (31701528 and 31671786), National Key R&D Program of China (2016YFD0401404), Northern Jiangsu province science

ACCEPTED MANUSCRIPT and technology projects (BN2016137), and the Fundamental Research Funds for the Central

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Universities (JUSRP51501).

ACCEPTED MANUSCRIPT References Armand, M. (2007). Lipases and lipolysis in the human digestive tract: where do we stand? Current Opinion in Clinical Nutrition & Metabolic Care, 10 (2), 156-164. Bauer, E., Jakob, S., & Mosenthin, R. (2005). Principles of physiology of lip id digestion. Asian Australasian Journal of Animal Sciences, 18 (18), 282-295. Bellesi, F. A., & Pilosof, A. M. R. (2014). Behavior of p rotein interfacial films upon bile salts addition. Food Hydrocolloids, 36 (5), 115-122. Benito-Gallo, P., Franceschetto, A., Wong, J. C., Marlow, M., Zann, V., Scholes, P., & Gershkovich, P. (2015). Chain length affects pancreatic lipase activity and the extent and pH-time profile o f triglyceride lipolysis.

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S, G., & H, S. (2012). Behavior o f almond o il bodies during in v itro gastric and intestinal digestion. Food & function, 3 (5), 547-555.

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digestion, micelle format ion and carotenoid bioaccessibility kinetics: Influence of emu lsion droplet size. Sarkar, A., Horne, D. S., & Singh, H. (2010). Interactions of milk protein -stabilized o il-in-water emu lsions with bile salts in a simulated upper intestinal model. Food Hydrocolloids, 24 (2–3), 142-151.

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Singh, H., & Sarkar, A. (2011). Behaviour of protein-stabilised emu lsions under various physiological conditions. Advances in Colloid & Interface Science, 165 (1), 47.

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Zhu, X., Ye, A., Verrier, T., & Singh, H. (2013). Free fatty acid profiles of emu lsified lip ids during in v itro digestion with pancreatic lipase. Food Chem, 139 (1-4), 398-404.

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Zou, L., Zheng, B., Zhang, R., Zhang, Z., Liu, W., Liu, C., Zhang, G., Xiao, H., & McClements, D. J. (2016). Influence of Lip id Phase Co mposition of Exc ipient Emu lsions on Curcu min So lubility, Stability, and

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Bioaccessibility. Food Biophysics, 11 (3), 213-225.

ACCEPTED MANUSCRIPT Figure captions and table legend Fig. 1. Interfacial processes occurring during pancreatic lipase lipolysis: The major site of lipid digestion is the small intestine, where pancreatic lipase acts in concert with various co- factors (co-lipase, BS and calcium) to ensure efficient oil digestion.

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Fig. 2. Mean particle size (d3, 2 ) (A) and Zeta-potential (B) of WPI-stabilized PO, RO and LINO emulsions in the presence of BS. Error bars represent the standard deviation calculated from

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three independent experiments (P= 0.05). Significant differences (P < 0.05) are indicated with

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different letters above the bars. Capital letters indicate significant difference exists between

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groups; while lowercase letters indicate significant difference within a group. Fig. 3. Levels of total FFAs released within 120 min from PO, RO and LINO WPI emulsions in

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the presence of 0.0, 1.0 and 2.0 mg/mL of BS (Fig. 3A, 3B and 3C respectively). Fig. 4. Fluorescence microscope images of different oil emulsions. (A) for the initial emulsions.

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(B), (C) and (D) are the real-time monitoring of PO, RO and LINO digestion at the point of 30

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min during the total 120 min digestion process at each level of BS addition, (B) for BS=0.0mg/mL; (C) for BS=1.0 mg/mL; (D) for BS=2.0 mg/mL.

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Fig. 5. Corresponding FFA release data plotted as a first-order kinetics reaction as a function of lipolysis time. The effect of different concentrations of BS (0.0, 1.0 and 2.0 mg/mL) on the first-order kinetics reactions were displayed in Fig. 5A, 5B and 5C, respectively. The inset table shows the respective apparent rate constants k (s-1 ) and the regression coefficient (R2 ) of each oil emulsion as calculated from Eq. (2). Fig. 6. (A) The FA compositions of the PO, RO and LINO. (B) The content of saturated and unsaturated fatty acids in the PO, RO and LINO. SFA, Saturated fatty acid; MUFA, Monounsaturated fatty acid; PUFA, Polyunsaturated fatty acid; UFA, Unsaturated fatty acid.

ACCEPTED MANUSCRIPT Different letters above the bars in the same group indicate significant difference exists between FFA compositions of different oils (P < 0.05). Fig. 7. Chemical structure of BS.

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Table 1. The TAG profiles of the PO, RO and LINO.

ACCEPTED MANUSCRIPT Table 1 Table 1 The TAG profiles of the PO, RO and LINO.

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LnOLn LnLnLn LnLLn LOLn OLnO LnPLn PLnO LnStLn LLnL StOLn

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PPP OOP POP PStP PPO PPM POS OPL PPSt POM

PO 35.42 ± 1.50 11.49 ± 1.08 8.68 ± 0.83 8.31 ± 0.52 6.09 ± 1.15 5.43 ± 1.23 4.46 ± 1.22 4.54 ± 1.00 2.91 ± 0.87 1.59 ± 0.85

* TAG Profiles (%) RO OOL 18.84 ± 1.37 OLSt 11.80 ± 1.56 LLO 10.04 ± 1.40 SOO 8.86 ± 0.70 OOO 8.15 ± 0.72 LLL 6.12 ± 1.11 OLO 4.25 ± 0.67 LLP 4.79 ± 0.53 LLPo 3.73 ± 0.58 LLLn 3.51 ± 0.88

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* P, palmitic; St, stearic; O, oleic; L, linoleic, M, Myristic; Ln, Linolenic; Po, Palm oleic.

LINO 18.61 ± 1.42 14.88 ± 2.60 12.57 ± 2.98 10.16 ± 0.33 8.02 ± 2.75 4.79 ± 0.51 4.25 ± 0.68 3.44 ± 0.39 3.41 ± 1.04 3.07 ± 0.70

ACCEPTED MANUSCRIPT Highlights: 1. Maximum FFA release level upon pancreatic lipase hydrolysis: PO> RO> LINO. 2. Apparent FFA release constant during in-vitro small intestine digestion: PO > RO ≈ LINO. 3. FA chain length and distribution within different oil modulate lipid hydrolysis reaction.

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SC

RI

PT

4. Bile salts impose significant effect on lipid digestion course.

Graphics Abstract

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