Food & Function

PAPER

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aUMB – University of Life Sciences, Departm

Science (IKBM), Norwegian University of Li

1432 As, Norway. E-mail: kristi.aarak@um

96 58 60bNoma – Norwegian Institute of Food, F

NorwaycIFR – Institute of Food Research, Norwich R

Cite this: Food Funct., 2013, 4, 1819

Received 20th August 2013Accepted 2nd October 2013

DOI: 10.1039/c3fo60346f

www.rsc.org/foodfunction

This journal is ª The Royal Society of Ch

The impact of meal composition on the release of fattyacids from salmon during in vitro gastrointestinaldigestion

Kristi Ekrann Aarak,*ab Neil Marcus Rigby,c Bente Kirkhus,b Louise Jane Salt,c

Stefan Sahlstrøm,b Gunnar Bengt Bengtsson,b Gerd Elisabeth Vegaruda

and Alan Robert Mackiec

We hypothesize that the rate of release of lipids from salmon muscle during in vitro digestion is altered by

additional meal components. In vitro digestion of salmon was performed using a mixture of porcine

gastrointestinal enzymes and bile salts. Broccoli and barley were also added to the digestion simulating

a meal. The extent of lipolysis was determined by measuring the release of fatty acids (FAs) during

sampling at the simulated gastric phase endpoint (60 minutes) and 20, 40, 60, 80, 110 and 140 minutes

simulated small intestinal phase, using solid phase extraction and GC-FID. Adding barley resulted in a

lower overall release of FA from salmon, whereas broccoli caused an initial delay followed by increased

release from 80–140 min when lipid digestion of salmon alone plateaued. The impact of broccoli and

barley on the release of peptides and digesta viscosity were also measured. The effect of different

components in the meal shown by this in vitro study suggests that it would be possible to make dietary

changes affecting the lipolysis, further triggering specific responses in the gastrointestinal tract.

However, these observations need to be validated in vivo, and the mechanisms need to be further

examined.

Introduction

Lipids have been shown to be linked to the development ofobesity, and suggested to be a risk factor in the development oflifestyle diseases like cardiovascular disease (CVD), diabetestype II and cancer.1–3 This correlation has led to the miscon-ception that lipids in general are a negative component of food,only affecting health in a bad way. Lipids are the most energydense macronutrient; hence over-consumption will lead toaccumulation of lipids in the body leading to obesity.3,4

However, due to their superior energy density, lipids also playan important role in ensuring that we meet our daily energyrequirement. Furthermore, lipids are also essential for theuptake and absorption of lipid soluble vitamins and caroten-oids.5 Lipids are also essential for building cell membranes andfor production of both signal molecules and formation of theirprecursors.6 Lipids are a complex group of macronutrients,mainly built up of fatty acids (FAs). FAs are dened by the

ent of Chemistry, Biotechnology and Food

fe Sciences, Chr. M. Falsens vei 1., BTB,

b.no; Fax: +47 64 96 59 00; Tel: +47 64

isheries and Aquaculture Research, As,

esearch Park, Norwich, UK

emistry 2013

numbers and placement of double bonds within the carbonchain. Briey; a FA without any double bonds is considered asaturated FA (SFA), a FA containing one double bond is amonounsaturated FA (MUFA), and a FA containing $2 doublebonds is a polyunsaturated FA (PUFA). Furthermore, the PUFAsare dened by which carbon the rst double bond is attached to(n � X), counted from the COOH– end of the FA, commonlycalled the omega end. The type of FA affects the physiologicalresponse.7 A risk assessment by WHO/FAO evaluated the asso-ciation between different types of FA and common lifestylediseases, showing conicting effects. For instance SFAincreased the risk of CVD and type II diabetes, while the n � 3PUFAs did the opposite; decreasing the risk.3 However, the linkbetween diet and various diseases is complex and the specicmechanisms are difficult to detect, thus, evidence is stillaccumulating.7

Previous studies have emphasized the need to look into therole of interfacial composition on the digestion of emulsiedlipids, as a route to getting better control of lipid bioavailability.Moreover, inhibition of lipid digestion and absorption,decreasing the total lipid bioavailability, has been suggested asa practical approach to tackling the growing problem of obesitythrough increasing satiety, reducing total energy intake andthereby decreasing the positive energy balance.8 During the lastdecade the health benets of dietary fats, especially MUFAs andPUFAs, especially the n � 3 PUFAs, have received increased

Food Funct., 2013, 4, 1819–1826 | 1819

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interest.7 Because of the limited conversion of the essential FAa-linolenic acid (ALA) into the active metabolites eicosa-pentaenoic acid (EPA) and docosahexaenoic acid (DHA)9 dietarysources are a major contributor of these FAs.7,10 Fish lipids tendto contain naturally high concentrations of long chain n � 3PUFAs, of which EPA and DHA are predominant, and are knownto be an important source of these FAs in the human diet.11 Thepositive health implications of EPA and DHA are well docu-mented in regard to protective effects of CVD, autoimmunityand mental disorders12,13 and several studies have emphasizedthe importance of introduction of these FAs through diet.12,14

Ruxton et al. concluded that the absence of EPA and DHA in thediet was unlikely to lead to deciency, but that there was apossibility that people with enhanced requirements could bedisadvantaged by this type of diet.15

Several studies have emphasized the crucial role of the foodmatrix with regard to lipid digestion16,17 and previous studieshave shown that the bioavailability of lipids is affected directlyby the lipolytic environment.18,19 This has been explained byboth indirect and direct mechanisms, e.g. changes in propertiesof the emulsion, physical obstruction and/or through directinuence on different enzymes and cells in the gastrointestinaltract. However, there is still a need for more knowledge oninuence of meal components on lipid digestion, and potentialbenecial effects on weight control.

The hypothesis of the work presented was therefore thatcomponents of a complex meal would affect the rate of lipolysisduring in vitro digestion. The present study aimed to observethe effect of broccoli and barley on the rate of lipolysis of lipidsfrom salmon muscle, furthermore studying the effect on therelease on the n� 3 PUFAs EPA and DHA which are known to beof nutritional importance to several population groups, assalmon provides a substantial amount of these FAs. The threeraw materials were chosen because they are likely to appeartogether in a typical “healthy meal”. They are all known to berich in components which have been proven to have positivebioactive properties, e.g. dietary bre from barley, polyphenolsand thylakoids from broccoli, and the polyunsaturated fattyacids from salmon. The release and activity of these compo-nents are hypothesized to be affected by matrix properties,which could further affect lipolysis.

Materials and methodsRaw materials

Salmon muscle (SM), Atlantic salmon (Salmo salar L.), wasobtained from Bremnes Seashore AS, Bremnes, Norway. GroundSM was defrosted overnight at 4 �C, before transferring into aplastic bag, making a 3–5 mm thick layer. Subsequently, air wasremoved, and the vacuum-packed SM was heated (70 �C, 2 min)in a water bath and cooled in an ice bath before freezing at�20 �C. The broccoli (Br) (Brassica oleracea, L. var. italica, cv.‘Ironman’) was obtained from a commercial grower on Jeløy,Moss, Norway. Broccoli orets (10–30 g) with 2 cm stalks wereheated (97 �C, 5 min) in a water bath, cooled for 3 min in waterwith ice, before vacuum packing in one layer and rapid freezingon aluminium plates (�40 �C). Commercially dehulled barley

1820 | Food Funct., 2013, 4, 1819–1826

(Hordeum vulgare L.), from Ottadalen Mølle, Lom, Norway, wasboiled for 45 min (150 g barley (Ba) in 450 ml water) cooled toroom temperature and vacuum packed before freezing at�20 �C. All samples were stored at �80 �C.

Experimental design

The meal was designed to simulate a main dinner course,consisting of 125 g cooked salmon, 125 g cooked broccoli and150 g cooked barley, resulting in a total of 2192 kJ which iswithin the denition of a main meal (1670–3150 kJ).20 Theenergy content and macronutrient composition are given inTable 1. To t into the in vitro digestion model the meal wasdownscaled, by grinding the food components and taking arepresentative sample. The ratio between the solid componentsand the simulated digestive uid was kept constant: 3.43 gsalmon muscle, 3.43 g broccoli and 4.13 g barley included in atotal of 32 ml.

In vitro digestion

This semi-dynamic model of adult human digestion wasdeveloped in-house at the Institute of Food Research (IFR),based on methods and biochemical conditions used for large-scale digestions by a dynamic gastric model.21 The lipolysisexperiments were performed using a pH-stat to regulate the pHin the small intestinal phase.22 All enzymes and reagents werepurchased from Sigma Aldrich (Poole, UK) (unless otherwisestated).

Oral phase. To mimic sheer and crush forces from chewing,meal components were passed through a domestic mincer(Lakeland, UK), using a coarse mincer attachment (holes:8.0 mm). The minced food was mixed for 2 minutes (37 �C) withsimulated salivary uid [0.15 M NaCl, pH 6.5; 2 units per mlhuman salivary a-amylase]23 making a food bolus.

Gastric phase. The food bolus was transferred to a pH-stat(Titrando, Metrohm, Germany) mixing vessel containingsimulated digestion uid 0.15 M NaCl, pH: 6.5; 0.17 mMphosphatidylcholine (Grade 1 egg lecithin, Lipid Products,USA). Pepsin (P7012, Sigma-Aldrich UK) was added (170 unitsper mg protein substrate) and the pH was titrated in four stepsfrom 6.5–6.0–5.0–2.5 for 30 minutes. Total incubation time was60 minutes (37 �C) with constant stirring. Pepsinolysis washalted by increasing the pH to 7.5 and holding at this pH for10 minutes.

Small intestinal phase. The pH of the digestion mixture wasadjusted to 6.5 and small intestinal chemicals were added[0.5 mM CaCl2; 0.1 M Bis–Tris; 1.8 mM phosphatidyl choline,11.8 mM bile salts (1 : 1 ratio of sodium taurocholate : sodiumglycodeoxycholate)] followed by addition of small intestinalenzymes trypsin (T0303, Sigma-Aldrich UK) (34.5 BAEE unitsper mg protein substrate), chymotrypsin (C4129, Sigma-AldrichUK) (0.4 BTEE units per mg protein substrate), porcinepancreatic lipase (L0382, Sigma-Aldrich UK) (60 units per mldigestion uid) and co-lipase (C3028, Sigma-Aldrich UK) (2 : 1molar ratio of co-lipase : lipase).24 Samples were withdrawn induplicate from 60min simulated gastric phase and 0, 20, 40, 60,80, 110, 140 minutes in the simulated small intestinal phase. All

This journal is ª The Royal Society of Chemistry 2013

Table 1 The macronutrient composition of the food ingredients included in the meal; salmon muscle (SM), broccoli (Br) and barley (Ba). All values are given per 100 gof raw materials (adapted from http://www.matvaretabellen.no/ (2012) written by Norwegian Directorate of Health)

Food Protein (g)

Carbohydrate (g)Lipid(g)

Energy

Calcium (mg)Sugar/starch Fibre kcal kJ

Salmon muscle 19.0 — — 15.0 211 882 19Broccoli 3.2 2.1 3.0 0.3 24 100 50Barley 11.1 59.5 12.9 1.3 294 1229 17

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lipolytic digestion experiments from 0–80 minutes wererepeated three times, with duplicate at each time point (n ¼ 6).110 and 140 minutes were repeated two times, with duplicate ateach timepoint (n ¼ 4). However, for the lipolytic studyincluding the SM + Br, 110 and 140 minutes were only repeated2 times (n ¼ 2). Samples were stored on ice and vortex-mixedwith chloroform : methanol (2 : 1) in order to stop the lipidhydrolysis, before freezing at �20 �C. Addition of the solventswas also the rst step in the lipid extraction.25

Method analysisLipid extraction and analysis

The non-aqueous fraction from the in vitro digestion wasremoved and evaporated, before lipids were redissolved inchloroform. Lipids were further separated into differentlipid classes by solid phase extraction under gravity usingamino propyl columns (300 mg, Bond Elut, Agilent). C17were used as internal standards for quantication of FAs inthe different lipid classes. Neutral lipids were eluted withchloroform : isopropanol (2 : 1 v/v) and FFA with chlor-oform : methanol : acetic acid (100 : 2 : 2 v/v). Neutral lipidswere further separated into triacylglycerols (TAG) usinghexane : dichloromethane : chloroform (80 : 10 : 2 v/v),diacylglycerols (DAG) using hexane : ethyl acetate (95 : 5 v/v)and monoacylglycerols (MAG) using chloroform : methanol(2 : 1 v/v). The solvents were removed by evaporation usingargon, and the contents of FA in the fractions were measuredas FAMEs using GC with ame ionization detection (FID).26

FAMEs were analysed using a Hewlett Packard 5890 GC andauto sampler, running GC Chemstation, equipped with a BPX-70column, 30 m � 0.22 mm i.d., 0.25 mm lm (SGE UK Ltd). Thetemperature program started at 115 �C for 3 min, increased at2 �C min�1 to 200 �C holding for 2 minutes, then increased at10 �C min�1 to 240 �C with a nal hold time of 5 minutes. Peakswere integrated with Agilent GC ChemStation soware (rev.A.05.02) (Agilent Technologies, Little Falls, DE). The hydrolysiswasmeasured as mg FFA per g lipids added, further recalculatedin % contribution of the FFA to the total lipid content (TAG +DAG + MAG + FFA).

Rheology measurement

The in vitro digestion was carried out as described above using anAR2000 controlled stress rheometer (TA instruments, Crawley,Surrey, UK), except the addition of pancreatic amylase (300 unitsper ml digestion mixture) and no titration of the pH keeping it

This journal is ª The Royal Society of Chemistry 2013

constant at 6.5. Measurements of viscosity were carried out over a230 minute period using a shear rate of 150 s�1.

Protein digestion and analysis

Samples were withdrawn at 2, 5, 10, 20, 40, 60 minutes ofgastric phase and 20, 40, 80, 140 minutes of small intestinalphase during the viscosity measurements. Proteolysis washalted by addition of 150 ml 0.5 M sodium carbonate tosamples from the gastric phase and 50 ml 0.1 M phenyl-methanesulfonyl uoride (PMSF) in the small intestinalphase. The o-phthaldialdehyde (OPA) assay was performed intriplicate by adding OPA reagent (200 ml) to sample (10 ml) in amicrotitre plate well and measuring the absorbance at 340 nmaer 10 minutes. The reagent was prepared as described byWoodward et al. (2010).27

Size distribution of lipid droplets

Nile red stock solution (0.1mgml�1 1,2-propanediol) was addedto the digesta part of the small intestinal phase, 1 : 9. 7.5 ml ofthe solution was further transferred to a cover slip and thesamples were analysed by confocal microscopy as described byMacierzanka et al. (2011),28 modied by using a SecureSealimaging spacers 9 mm diam. � 0.12 mm depth (Sigma Aldrich).Images were collected using a Leica SP-1 equipped with a 40�lens (Leica PL APO a/0.17/E). Fluorescence was excited at 488nm using an argon ion laser. Experiments were performed at 37�C in triplicate for each condition, n � X being the number oflipid droplet analysed. The results are shown as distribution ofdata points from individual measurements. The lipid surfacearea weighted mean droplet diameters D[3,2] were determinedusing Image-Pro 6.0 soware (Media Cybernetics Inc., SilverSpring, MD).

Statistical analyses

As the data are normally distributed, all values are presented asmean values with their standard error mean (SEM). Student's ttest (two-sample, assuming unequal variance) was used toestimate signicant differences (GraphPad Prism �6).

Results

Lipolysis takes place at the interface between oil and water.Therefore, the size of lipid droplets and the total surface areaare important determinants of the rate of lipid hydrolysis. Theaverage lipid droplet size and size distribution at the start of thesimulated small intestinal phase were affected by adding Br and

Food Funct., 2013, 4, 1819–1826 | 1821

Fig. 2 Effect of barley (SM + Ba) and broccoli (SM + Br), on release of total FFAfrom lipids in salmon muscle (SM). Samples were all treated with the same up tothe small intestinal phase, starting at 0 minutes. Values are given as mean oftriplicate (0–80 minutes) (n ¼ 6) and duplicate (110 and 140 minutes) (n ¼ 4) �SEM. Except for SM + Br 110 and 140 minutes (n ¼ 2).

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Ba to the SM digestion (Fig. 1A–C). Gastric in vitro digestion ofSM alone resulted in a mean diameter (D[3,2]) of the lipiddroplets of 21.2 � 0.7 mm, including Br gave a similar D[3,2] of19.5 � 0.3 mm, while including Ba led to a higher D[3,2] of30.7 � 1.6 mm, (Fig. 1D).

The effect of Br and Ba, and both of them on release of FA inthe small intestinal phase of digestion is presented in Fig. 2,showing that the extent and rate of lipid hydrolysis was inu-enced by the addition of the meal components. The lipolysis ofthe SM reached a plateau, showing no signicant release of FAfrom 40 to 140 minutes, 25.3 � 1.1% to 24.6 � 1.7%, respec-tively, with a plateau average of 23.5% contribution of FFA to thetotal lipid content. Inclusion of barley (SM + Ba) led to a similarFA release prole as for the SM (Fig. 2), reaching a plateau at40 minutes (15.8 � 2.2%) with no signicant increase in % FFAaer 140 minutes (20.1� 6.2%), giving a plateau average of 17%contribution of FFA to the total lipid content. However, therelease of FA was signicantly reduced at 20, 40, 60 and110 minutes compared to the SM; showing a 54% (P < 0.0001),24% (P ¼ 0.0054), 34% (P ¼ 0.0002) and 29% (P ¼ 0.0391)reduction, respectively. Addition of broccoli to the in vitrodigestion (SM + Br) led to a steady and signicant increase overtime from 0 to 110 min (P < 0.05). From 110 to 140 minutes theamount of released FA continued to increase from 35.4 � 8.6%to 40.4 � 6.1%, but this was not found to be signicant.Compared to the SM the release of FA was signicantly reducedat 20 minutes, 57% (P ¼ 0.0006), before releasing a similaramount of FA at 40 and 60 minutes. However, the release of FAswas increased by 10% and 64% at 80 and 140 minutes,respectively, but this was not found to be signicant. At 110minutes a 57% increase was observed, and found to be signif-icant (P ¼ 0.0064). The contribution of FFA to the total lipidcontent at endpoint 140 minutes for SM + Br was 40.4%.

Fig. 1 Confocal microscopy picture of lipid droplet distribution (red) at the startingshowing the size distribution of lipid droplets in the digesta (D), SM n¼ 67, and SM +droplet diameter (mm) is given as % of total surface area and D[3,2] is indicated by

1822 | Food Funct., 2013, 4, 1819–1826

By adding both broccoli and barley together to the SMdigestion (SM + Br + Ba) a signicant increase between all timepoints from 0 to 80 minutes was observed (P < 0.05) (Fig. 2).From 80 to 140 minutes the release of FA continued toincrease from 24.3 � 5.7% to 35.2 � 7.5%, but this was notfound to be signicant. Release of FA was signicantlyreduced, compared to the SM at 20 and 60 minutes, by 46%(P ¼ 0.0043) and 28% (P ¼ 0.0149), respectively. At 80 and 110minutes the release was higher but not signicant comparedto the SM alone, by 2% and 35%, respectively. At 140 minutesthe increase of 43% was found to be signicant (P ¼ 0.0330),giving a mean contribution of 29.5% from the FFA to the totallipid content at endpoint.

point of small intestinal phase for SM (A), SM + Ba (B) and SM + Br (C). Graph (D)Ba n¼ 66, SM + Br n¼ 263 at the start of small intestinal phase. The distribution ofthe arrow. SM, salmon muscle; Br, broccoli; Ba, barley.

This journal is ª The Royal Society of Chemistry 2013

Fig. 4 Effect of broccoli (SM + Br) and barley (SM + Ba) on viscosity in the digestaduring digestion. Values (Pa s) are shown as a mean of three different experi-ments for SM and SM + Ba (n¼ 3) and two experiments for SM + Br (n¼ 2)� SEM.The arrows indicate where the enzymes responsible for carbohydrate release andhydrolysis were added.

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With regard to specic effects on the release of the n� 3 longchain PUFAs (Fig. 3), the Ba gave an overall larger contributionof both EPA (Fig. 3A) and DHA (Fig. 3B) to the total FFA content,signicantly at 80 minutes (P ¼ 0.0352 and P ¼ 0.0421),110 minutes (P ¼ 0.0186 and P ¼ 0.0292) and 140 minutes(P ¼ 0.0065 and P ¼ 0.0078), respectively. Conversely, the Brgave an overall lower contribution of EPA and DHA to the totalFFA content. Furthermore, the Br affected the % contribution tothe total FFA content differently for the EPA and DHA, shown tobe signicantly lower at 40 (P ¼ 0.0374), 110 (P ¼ 0.0021) and140 (P¼ 0.0020) minutes for the EPA, while the DHA was shownto be signicantly lower at all time points (P < 0.01).

To check if the sampling gave a representative picture of whathappened during the in vitro digestion a comparison with the totalvessel volume was made at 140 minutes. There were no signicantdifferences in the proportions of FFA between the total vesselcontent and the samples withdrawn for any of the different mealcombinations (results not shown). To get an overview of theenvironment during the in vitro digestion samples were also ana-lysed for the effects of the meal components on the viscosity(Fig. 4). The addition of Ba led to a signicant increase in viscosity(P < 0.001) at all time points compared to the SM + Br and SM.Inclusion of Br also increased the viscosity signicantly comparedto the SM alone. Interestingly the viscosity of the SM+ Ba increasedsignicantly over the period of digestion rather than decreasing asa result of hydrolysis. Finally, protein degradation during digestionwasmeasured by the breakdown of peptide bonds (Fig. 5). Broccoli

Fig. 3 The% contribution of EPA (A) and DHA (B) to total lipid content. Values aregiven as a mean of (20–80 minutes) triplicate (n ¼ 6) and (110 and 140 minutes) asduplicate (n ¼ 4) � SEM. Except from SM-Br 110 and 140 minutes (n ¼ 2). Signif-icant differences from the SMare given as */** (P < 0.05/<0.01). SM, salmonmuscle;Br, broccoli; Ba, barley.

Fig. 5 Effect of broccoli and barley on release of peptides during the in vitrodigestion. The dotted line shows the changes in the pH during digestion and thearrows show when the enzymes responsible for protein hydrolysis are added.Values are shown as a mean of three different experiments with six measure-ments for each time point for SM and SM + Ba (n¼ 18) and two experiments withsix measurements for each time point for SM + Br (n ¼ 12) � SEM. The bars showthe amount of peptides released (mmol ml�1) � SEM, and significant differences(P < 0.05) to SM are given as *. SM, salmon muscle; Br, broccoli; Ba, barley.

This journal is ª The Royal Society of Chemistry 2013

increased the amount of hydrolysis by approximately 10% duringthe digestion, signicantly at 2, 10, 20 and 60 minutes of thegastric phase (P < 0.05) and at all time points in the small intestinalphase (P < 0.05). Barley also gave a signicant increase in therelease of peptides at 10 (P < 0.001) and 20 (P ¼ 0.0404) minutesgastric phase compared to the SM alone, but no signicantdifferences were observed at the other time points.

Discussion

The work presented in this study shows that the release of fattyacids (FAs) during in vitro gastrointestinal digestion of salmonmuscle (SM) was inuenced differently by addition of broccoli(Br) and barley (Ba). These components contain several bioac-tive compounds which might affect lipolysis, and a number ofdifferent mechanisms could explain the observed effects.

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Dietary lipid mainly consists of TAGs,29 hence there are twolipolytic enzyme systems which are of importance, the gastricand the pancreatic lipase.30,31 These enzymes are responsible forhydrolysing the FA from the specic positions on the tri-acylglycerol (TAG); sn-1, -2 and -3, and are both important forefficient lipolysis in vivo. In this study, the aim was both to studythe release of total FFA and the release of the n � 3 PUFAs, EPAand DHA. Previous studies have also described the positions ofthe FA on TAGs from marine oils,32 showing that both EPA(approx. 47%) and DHA (approx. 76%) are predominantly foundin sn-2. However, EPA is more randomly distributed in the othertwo positions compared to DHA.33 As there is no commerciallyavailable gastric lipase having the specic properties of thehuman gastric lipase; i.e. high stability and activity at low pHbut not above neutrality, resistant to bile salts for adsorption atthe lipid–water interface and a high stereo-specicity for the sn-3 position on the TAG,31,34 the decision was made to just includethe pancreatic lipase during the experiments conducted.Consequently, the pancreatic lipase was the sole enzymeresponsible for the lipolytic activity observed.

When Ba was included with the SM, both a delay and aninhibitory effect on lipolysis were observed, which could beexplained by several mechanisms including the increased sizeof the lipid droplets. Raw dehulled barley contains approxi-mately 60% starch, 13% dietary bre and 27% of a mixture ofproteins, lipids, ash, sugars and moisture.35 The hypothesizedbioactive component in barley is the solubilized dietary breb-glucan.36 Dietary bre is dened as the part of the grain that isnot digested by the endogenous enzymes of the upper gastro-intestinal tract (GIT). It is known to have several functions in theGIT in vivo, and the mechanisms seem to be dependent on thesolubility of the dietary bre.37 The soluble and solubilizeddietary bre, along with partially degraded starch, have beenshown to increase viscosity.37,38

The digestible carbohydrates are mainly broken down by theaction of two amylases, the salivary amylase from the mouth39

and the pancreatic a-amylase in the small intestine. In thelipolytic studies conducted in this study the enzyme responsiblefor the breakdown of the carbohydrates was the salivaryamylase. The salivary amylase has been shown to be protectedby oligosaccharide breakdown products, passing through thegastric phase without inactivation.40 This is supported by thedata from this experiment showing an increase in viscosityduring the gastric phase which is ascribed to the action of thesalivary amylase. However, in the viscosity measurements thepancreatic amylase was added to study the additive effect of thisenzyme on viscosity. Pre-cooking and grinding of the barleybefore the in vitro digestion would also increase viscosity bybreaking open the cells in the grain releasing the starch.41 Inanimal models, an increase in luminal viscosity has been shownto inuence physicochemical reactions that may affect diges-tion through both direct and indirect mechanisms.42 However,as this is a closed system only simulating the mechanicalactions of the in vivo digestion, only a fewmechanisms would berelevant. During in vivo digestion an increase in viscosity wouldaffect the peristaltic mixing process, delay the binding of thedigestive enzymes to their respective substrates, and would

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reduce the ability to be absorbed by the enterocytes.43 Further-more, dietary bres have also previously been shown to alter thedigestion and absorption of several nutrients affecting thepancreatic lipase directly in vitro.43,44 Both of these mechanismsmight be a relevant parameter when studying lipolysis in an invitro system, and might be plausible explanations for theobserved effect.

In this study we suggest that the observed effect of Ba onlipolysis is both due to a delay in the onset of lipolysis as well asan overall reduction in the total release of FA. The delay mightbe linked directly to the observed increase in viscosity describedabove. However, if the increase in viscosity was the only factoraffecting the change in lipolysis observed, the effect would be tolimit the rate of FA release but not affect the total release,meaning that the lipolysis would generate the same totalamount of FFA but at a later time point. The lipolysis of SM andSM + Ba both reached a plateau, which is in accordance withwhat other studies have reported.45,46 This has been hypothe-sized to be due to product inhibition because of saturation ofthe micellar solubilisation, further limiting the in vitro systemwhen studying lipolysis.45,47 This hypothesis is supported by theresults obtained from this study showing an overall lower extentof lipolysis when adding barley, suggesting that the equilibriumwas reached at a lower FFA-concentration. Cereals have previ-ously been shown to retain bile acids36 suggested to be due to aspecic binding to b-glucans, although results are inconclu-sive.48–50 Bile acids are essential for efficient lipolytic activityby emulsifying and dispersing the lipolytic products makingmixed micelles and transporting lipids to the enterocytes forabsorption.51 When less bile acids are available the formation ofmixed micelles will decrease, giving a lower thresholdfor reaching the equilibrium and thus a lower maximum extentof lipolysis is obtained.

Adding Br to the in vitro digestion of SM inuenced theenzyme kinetics differently from Ba by causing a continuousincrease in FFA between all time points so that no plateau wasreached. Broccoli also induced a signicant increase in peptiderelease during the gastric phase, suggesting a higher degree ofproteolysis. Proteins and peptides are also well known to absorbto the oil–water interface aiding formation and stability ofemulsions.52 Signicant inhibition and delay in lipolysis by Brwas also shown in the small intestinal phase. It is possible thatthe increase in surface active peptide release in the gastricphase might sterically hinder the lipase and thereby explain thedecrease in rate of lipolysis observed.53 Broccoli also has a highcontent of bioactive compounds some of which have beenshown to affect lipolysis in vitro, like polyphenol extracts54,55 andthylakoid preparations from various plants.55 The latter one hasbeen conrmed in vivo by animal studies,55 while the in vivovalidation of effects of polyphenols has been unsuccessful.56 Ifany of the constituents from Br inhibited pancreatic lipase, theinhibition might gradually be reversed by breakdown orremoval of the inhibitor, which could explain the observedincrease in the amount of FFA released with time. From 80minutes small intestinal phase of the digestion Br gave anincrease in the % contribution of the FFA to the total lipidcontent. Broccoli contains Ca2+ which has previously been

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shown to affect the lipolytic equilibrium in vitro by precipitatingFA making Ca(FA)2+-soaps. The soaps will remove the FFA fromthe interface of the lipid droplet, preventing product inhibitionof lipolysis by pushing the equilibrium towards the ionised FA(Le Chatelier's principle).57 Several studies have also reportedsignicant increases in lipolysis with increased calciumconcentration.58,59 In plant tissues Ca2+ is bound to macromol-ecules in the cell wall and is in solution in the vacuole,60 whichmight be released in the gastric phase due to the low pH andsubsequently bind to various compounds in the small intestinalphase depending on affinity. Broccoli also increased theviscosity. However, the increase was small, and we suggest thatthe observed change was mainly due to an increase of solidmaterial in the vessel. This is further supported by the obser-vation of an overall reduction in viscosity for SM and SM + Brduring the in vitro digestion, as the food-structures andmacromolecules were hydrolysed into smaller components.When both Br and Ba were included together (SM + Br + Ba), thelipid release was an average of the previously observed effect ofthe Br and Ba on release of lipids from the SM.

Several studies have investigated the transport and distri-bution of long chain FAs, including metabolism and utilisationof these FAs both aer ingestion and from the adiposetissue.61,62 However, few studies have been conducted on thespecic release of these FAs within the gastrointestinal envi-ronment. In this study we have investigated the effect of the Baand Br on the release of EPA and DHA. Results showed that thetwo food components affected the hydrolysis of these specicFAs in different ways; Ba increased the % contribution to thetotal FFA content while the Br inhibited this contribution. Anoverall variation in the release of FA at specic time pointsmight lead to masking of selective hydrolysis by pancreaticlipase. On the basis of this, the release of the specic FA wasmeasured as the % contribution of the specic FA to the totalFFA composition in the digesta. The % contribution of EPA andDHA to the FFA fraction was observed to be lower in the digestacompared to that in the total lipid of the SM. This inefficienthydrolysis of these FAs could be ascribed to several well-knownmechanisms; the position of the FA on the TAG, the pancreaticlipase position preference,61 and the position of the rst doublebond.62 Data from this study suggest that there are componentsin the Br and Ba affecting the release of EPA and DHA, and thusthe bioaccessibility of these FAs. However, the specic mecha-nism behind the observed effect and possible health implica-tion needs to be further examined.

Concluding remarks

In this study we showed that the in vitro digestion of lipids insalmon muscle was affected by including broccoli and barley tothe digestion. The results indicate that specic compoundswithin these two meal components directly inuence lipolysis.This was further supported when comparing the peptide releasepatterns, which showed a similar pattern for all meal combi-nations, although the inclusion of Br increased the amount ofpeptide release. Changes induced by the addition of barley maybe ascribed to the increase in viscosity induced by the dietary

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bre and potential binding of b-glucans to bile salts. The effectof broccoli, which differed from that of barley, could be attrib-uted to effects by chlorophyll, thylakoids and polyphenols.Finally, this could have implications for the uptake of lipids aspart of a complex meal, as well as satiety regulation through thetriggering of specic responses in the gastro intestinal tract.Furthermore, data showed that inclusion of meal componentsaffected the bioaccessibility of the long chain n � 3 PUFAs, EPAand DHA, known to be of nutritional importance. However, theobservations need to be validated in vivo, and the mechanismsand nutritional importance need to be further examined.

Conflict of interest

The authors report no conict of interest and are aloneresponsible for the content and writing of this manuscript.

Acknowledgements

The project was supported by Research Council of Norway (Pro.no. 202379/I10) and BBSRC in the UK (ISP grant BB/J004545/1)and European Framework 7 project DREAM (FP7-222654-2), andcarried out at Institute of Food Research, Norwich, UK. Finan-cial support from the Foundation for Research Levy on Agri-cultural products in Norway is also gratefully acknowledged. Wewish to thank Dagbjørn Skipnes for obtaining the salmon,Balazs Bajika for helping out doing the confocal microscopy,Olaia Martinez Gonzalez for helping out doing preliminary invitro digestion studies and Nicola Woodward for setting up theTitrando for the in vitro digestion model. The authors areparticipants in the FA1005 COST Action INFOGEST on fooddigestion.

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