Food Chemistry 172 (2015) 361–367

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Improved bioavailability of vitamin D3 using a b-lactoglobulin-basedcoagulum

http://dx.doi.org/10.1016/j.foodchem.2014.09.0540308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Faculté des Sciences de l’Agriculture et de l’Alimentation,Pavillon Paul Comtois, 2425 Rue de l’Agriculture, Université Laval, Québec, QC G1V 0A6,Canada. Fax: +1 (418) 656 3353.

E-mail address: Muriel.Subirade@fsaa.ulaval.ca (M. Subirade).

Fatoumata Diarrassouba a, Ghislain Garrait b, Gabriel Remondetto c, Pedro Alvarez a, Eric Beyssac b,Muriel Subirade a,⇑a Chaire de recherche du Canada sur les protéines, les bio-systèmes et les aliments fonctionnels, Institut de recherche sur les nutraceutiques et les aliments fonctionnels(INAF/STELA), Université Laval, Québec, QC, Canadab EA-CIDAM, Lab Biopharmacie, Faculté de Pharmacie, 28 Place Henri Dunant, 63001 Clermont-Ferrand, Francec Centre de recherche et développement, Agropur Coopérative, 4700 rue Armand Frappier, St-Hubert, QC J3Z 1G5, Canada

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 April 2014Received in revised form 4 August 2014Accepted 10 September 2014Available online 17 September 2014

Keywords:b-LactoglobulinVitamin D3

StabilitySolubilityEncapsulationBioavailability

Vitamin D3 (D3) was encapsulated within a water-soluble matrix, formed by promoting the blg/D3

complex by acidification. The capacity of the blg-based coagulum to increase the long term stability ofD3 in cold storage, upon exposure to intensive UV-light, and in the presence and absence of intestinal pro-teases, was evaluated. Additionally, the impact of the sequestration of D3 within the matrix of blg-basedcoagulum on its bioavailability was determined in vivo with force-fed rats. The water solubility,long-term storage and UV-light stability of D3 were significantly increased (p < 0.0001) due to the highencapsulation efficiency (94.5 ± 1.8%). The blg-based coagulum was not rapidly disrupted by theproteases in the intestines, leading to a slow release of D3, increased uptake of D3 and subsequentenhancement of the bioavailability of D3 in rats.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The beneficial effects of higher intake of vitamin D3 (D3) havebeen well established in countless skeletal and extra-skeletal dis-eases (Grant, Schwalfenberg, Genuis, & Whiting, 2010; Holick,2007). The annual economic burden of D3 deficiency in Canadahas been estimated to be about 14.4 $ billion, which includes directcosts, such as hospitals, drugs, physician care, as well as indirectcosts, such as mortality, long-term diseases and transient disability(Grant et al., 2010). In western Europe, adequate D3 status is asso-ciated with an estimated reduction of 187 billon € per year (Grantet al., 2009). The main indicator of D3 status is serum 25-hydrox-yvitamin D3 (25(OH)D3) (Wagner, Sidhom, Whiting, Rousseau, &Vieth, 2008), which is generally considered optimal above50–72 nM, despite some controversy about this threshold (Holick,2007). Reports indicate that benefits in disease reduction may startfrom a mean serum 25(OH)D3 level above 100 nM (Grant et al.,2009, 2010). However, for most people living in northern latitudes,irrespective of age or skin type, this threshold is not reached when

exposure to sunlight is minimum (Calvo, Whiting, & Barton, 2004;Grant, 2011; Holick, 2007; Whiting, Green, & Calvo, 2007). Despitethe fortification of staple foods, such as fluid milk, margarine orbreakfast cereals, the elevated prevalence of hypovitaminosis D3

indicates the inability of food fortification programmes to achieveadequate intake (Calvo et al., 2004). In addition to insufficient sunexposure and dietary intake of fortified foods, this failure can alsobe ascribed to the fact that the retention of D3 in food is problem-atic (Tippetts, Martini, Brothersen, & McMahon, 2012). As such,overages of 30% for D3 are usually considered consistent with goodmanufacturing practices by some trade associations (Yetley, 2007).However, this decision does not seem to be sufficient to maintainappropriate levels of D3 at the time of ingestion, indicating thatthe incorporation of adequate amounts of D3 in foods is indeedchallenging. D3 is fat-soluble, requiring the use of lipidic foodsfor fortification purposes, which is not compatible with consumers’demand for foods with low fat content. In addition, because D3 ishighly sensitive to light and oxygen, it is easily degraded. Conse-quently, deviation of label declarations from actual amounts ofD3 in foods are common (Yetley, 2007).

Attempts have been made to incorporate D3 into other dairyproducts, such as cheese, for improved retention and stability.However, prodigious amounts of D3 in powder, emulsion or oilforms are used to fortify milk with low yields in cheese, when

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compared to the initial concentration incorporated into the milkbatch (Ganesan, Brothersen, & McMahon, 2011; Tippetts et al.,2012; Wagner et al., 2008). This implies great profit losses forthe industry, given the fact that, to date, cheese fortification doesnot improve the bioavailability of D3 compared to supplements(Wagner et al., 2008). Important losses of D3 may occur due toits affinity for milk whey proteins, which are considered as cheese-making waste products (Tippetts et al., 2012; Wagner et al., 2008).Bovine milk b-lactoglobulin (blg) accounts for about 60% of theproteins in whey (Nicolai, Britten, & Schmitt, 2011). Recent reportsindicate that blg binds D3 to form a complex that is resistant to pHvariation from 1.5 to 8, including that prevailing in the gastro-intestinal tract (Diarrassouba et al., 2013). Most importantly, thehighest stability of the blg/D3 complex was observed at pH 5, closeto the isoelectric point (pI) of the protein. Therefore, in the currentwork, acid coagulation of the blg/D3 complex was triggered byslowly decreasing the pH close to the pI of blg, using a mild acidi-fying agent. GLD, a food additive, promotes the auto-aggregation ofnative beta-lactoglobulin by mild acidification, which is advanta-geous for heat-sensitive bioactive encapsulation. The blg/D3

complex was also shown to significantly improve the solubilityof D3, implying that higher amounts of the vitamin can be incorpo-rated within the coagulum matrix (Diarrassouba et al., 2013). Thisbeta-lactoglobulin-based platform exhibits a dense polypeptidenetwork, in which bioactive compounds will ultimately be bioac-cessible to digestive enzymes, offers a significant protective barrieragainst damaging environmental factors, including light andoxidative species. Hence, the D3-fortified coagulum can later beincorporated into cheese or other dairy products, such as yogurt,during processing to improve the retention, uptake and bioavail-ability of D3. This may allow cheese fortification with realisticamounts of D3 (Health, 2011; Tippetts et al., 2012). The aim ofthe present work was to investigate the capacity of the blg-basedcoagulum, produced in a mild acidic condition, to entrap and retainD3 within the protein matrix. The impact of the sequestration of D3

in the coagulum, on the long-term stability during cold storage andupon light irradiation, was evaluated. The intestinal stability of theD3-enriched coagulum and its resistance to proteolytic activitywere examined in vitro. Most importantly, an in vivo experiment,using rats, was carried out to study the impact of the sequestrationof D3, within the coagulum matrix, on the bioavailability of D3, bydosage of serum 25(OH)D3 levels in animals. To our knowledge,this work is the first report of the use of blg-based coagulum toenhance the solubility, stability and bioavailability of D3 forimproved D3 encapsulation.

2. Materials and methods

2.1. Materials

b-Lactoglobulin (blg) (B variant, purity P 90% PAGE) frombovine milk, D3 (purity P 98%, HPLC) and glucono-d-lactone(GDL, USP specified) were obtained from Sigma–Aldrich ChemicalCompany (St. Louis, MO, USA) and used without further purifica-tion. Methanol (MeOH, HPLC grade) and trifluoroacetic acid (TFA)were purchased from EMD International (Gibbstown, NJ, USA).

2.2. Sample preparation

The blg stock solution, concentrated at 20% (w/v), was madedaily by dissolving blg in MilliQ water under mild stirring condi-tions for 2 h at room temperature and then stored overnight at4 �C to allow complete rehydration. Stock solutions of D3, concen-trated at 1 mM were prepared daily by dissolving 10 mg of thevitamin in 25 ml of methanol, followed by a dilution with MilliQ

water to an initial concentration of 220 lM (Bilodeau et al.,2011). All D3-containing solutions were protected from light bypreparing them in a dark room and further protected using analuminium foil wrapping.

2.3. Encapsulation of D3 in the blg-based coagulum

The blg/D3 coagulum was first prepared by mixing the samevolumes of blg and D3 solutions in order to obtain a final concen-tration of 110 lM for the vitamin. The blg/D3 mixture was thenincubated for 2 h at room temperature, before adding GDL at aconcentration of 2% (w/v) for an additional 2 h of incubation. Theheadspace in the conical tubes containing the samples was reducedto a minimum in order to limit oxidative degradation of D3 by air.The coagulum was completely formed when phase separationoccurred around pH 4.7, with the creamy-coloured coagulum atthe bottom layer of the conical tube and clear serum at the top.The serum was separated from the coagulum after centrifugationat 15,500�g/5 min and the coagulum was disrupted by rapidlydecreasing the pH below the pI of blg, using HCl (Nicolai et al.,2011). D3 was determined in both fractions by HPLC, as previouslydescribed (Diarrassouba et al., 2013). The encapsulation efficiency(EE) was computed, using the following formula:

EE ð%Þ ¼ Total amount of D3 in the coagulumTotal amount of D3

� 100

2.4. Stability of D3 during long term storage at 4 �C

The stability of D3 entrapped in the coagulum at 4 �C was deter-mined over 5 weeks. The initial concentration of encapsulated D3

was determined after 24 h, as described in Section 3.3. Samplescorresponding to weeks 1–5 were stored at 4 �C in a fridge. Eachweek, a sample was withdrawn for determination of the amountof D3 remaining in solution by HPLC, as described elsewhere(Diarrassouba et al., 2013). Solutions of D3 diluted in MilliQ waterwere prepared and analysed under the same conditions.

2.5. Stability of D3 to UV-light irradiation

The D3-containing coagulum and D3 solutions were exposed toUV light (254 nm, 15 W) in order to assess the UV-light stability ofD3 over 24 h (Semo, Kesselman, Danino, & Livney, 2007). At eachsampling time, consisting of t 0, 1, 2, 4, 6, 8, 10 and 24 h, theamount of D3 remaining in the microspheres was determined, asindicated in the previous sections.

2.6. Kinetic release of D3 in simulated intestinal fluid

The release of D3 in simulated intestinal fluid (SIF) was investi-gated, using a USP-2 paddles apparatus (SOTAX Corporation, West-borough, MA, USA). The release media consisted of the simulatedintestinal fluid (SIF), as described in the US Pharmacopeia, withor without 1.0% pancreatin (w/v) at pH 6.8. Each of the solutionsof D3-enriched coagulum or D3, at the same concentration, wasincubated in one of two release media (100 ml) with continuousagitation (at �50 rpm) at 37 �C (Diarrassouba et al., 2014). Sampleswithdrawn at half-hour or 2-h intervals over 6 h were centrifuged(5000�g/5 min) and the concentration of D3 in the supernatantwas determined (Diarrassouba et al., 2013). A control, with andwithout enzyme, was also run.

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2.7. In vivo study of the intestinal absorption of the D3

Animal studies were conducted at the ‘Unité de StabulationAnimale, Ethic Committee-CE-42-12’ (Université d’Auvergne,Clermont-Ferrand, France). Adult male Wistar rats (n = 25; ElevageDépré, St. Doulchard, France), weighing 300 ± 20 g at the beginningof the experiment, were used. Housing characteristics, accommo-dation and the average feed intake are described elsewhere(Diarrassouba et al., 2014). After the acclimation period, two orthree rats were housed per cage for 3 weeks. The animals hadaccess to feed and water ad libitum. The rats (n = 15) were dividedinto three groups (n = 5) each receiving one of the treatments, con-sisting of MilliQ water (control), D3 or D3-enriched blg-based coag-ulum with the same final concentration of D3 (110 lM or 77 lg).Blood samples were collected at t 0 after 3 weeks in 0.5 ml conicaltubes pre-coated with 10 ll of sodium heparin (0.1 IU). The plasmawas separated by centrifugation at 1000�g for 10 min. Plasmasamples were stored at �20 �C prior to analysis of 25(OH)D3 inrat plasma, performed by using the 25-hydroxy vitamin D DirectEIA kit (IDS Diagnostics, Fountain Hills, AZ, USA) according to themanufacturer’s instructions. Additionally, the concentration of25(OH)D3 in rats fed the D3-enriched blg-based coagulum wascompared to that of rats fed the blg/D3 complex, which is the basicbuilding block for the formation of D3-entrapped coagulum. Theformation of the blg/D3 complex is described elsewhere(Diarrassouba et al., 2014).

2.8. Statistical analysis

Statistical analysis was performed using SAS version 12.0 (SASInstitute Inc., Cary, NC, USA). ANOVA and the least significantdifference test (LSD) were used to determine differences amongmeans and the significance level was fixed at p < 0.05. All measure-ments were performed in triplicate.

0

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0 1 2 3 4 5

Rec

over

y of

D3

(%)

Time (weeks)

D3 ßlg/D3 CoagulumD3 βlg/D3 Coagulum

** * **

Fig. 1. Long term stability of D3 and the D3-loaded blg-based coagulum underrefrigerated conditions (4 �C). ⁄Statically different from D3 alone (p < 0.05).

3. Results and discussion

3.1. Encapsulation efficiency of D3 in the blg-based coagulum

The bovine milk blg was used to prepare a coagulum by mildacidification. Coagulation started when the pH reached the pI ofthe protein and was terminated at pH 4.7. The capacity of theblg-based coagulum to encapsulate D3 was investigated by induc-ing the auto-association of the blg/D3 complex, using GDL at roomtemperature. The blg-based coagulum successfully entrapped D3

and the encapsulation efficiency (EE) was 94.5 ± 1.8%, the remain-ing D3 being recovered in the serum. Similar EE was also obtainedfor higher amounts of D3, ranging from 250 lM to 1 mM (data notshown). The EE level obtained in the current work is superior tothat obtained with zein-based nanoparticles without (52.2%) andwith (71.5%) a carboxymethyl chitosan coating, which increasedto 87.9% after calcium addition (Luo, Teng, & Wang, 2012). Algi-nate-based nanoparticles, with a hydrophobic core, were used toencapsulate D3 with a resulting loading efficiency ranging from45.8 ± 1.55% to 67.6 ± 2.76 (Li, Liu, Huang, & Xue, 2011). Morerecently, an EE of 96.9% was obtained, using zein-based hydrogelbeads, which is closer to that of the current work. However, thiselevated EE required the use of the toxic crosslinking agent glutar-aldehyde, in addition to a 30% alcohol concentration, which are notcompatible with food administration (Luo, Teng, Wang, & Wang,2013).

In the current work, water-soluble blg-based coagulum wasused to entrap D3 at a high EE, using food grade GDL. As a result,the solubility of the fat-soluble vitamin increased upon sequestra-tion within the protein matrix. Therefore, the D3 enriched

coagulum can be used to increase the uptake of the vitamin indairy products such as cheese or yogurt and other food types witha zero to low fat content in fortification programmes. Gelation ofwhey proteins and blg can be induced by salt, pressure,temperature or acidification (Nicolai et al., 2011). The rate of gela-tion of blg above the critical concentration increases exponentiallyand is irreversible at higher temperature (Nicolai et al., 2011). It isimportant to note that, in the current work, irreversible gelation ofnative blg occurred at refrigerated temperature (data not shown)while the milky-coloured coagulum was formed at room tempera-ture. Although the mechanisms underlining the irreversible coldgelation of non-denatured blg are still under investigation, D3

may have played a major role in the cold gelation of native blg.D3 is highly sensitive to environmental conditions, including lightirradiation and oxygen. Therefore, before proposing the D3-loadedblg-based coagulum as a functional food ingredient, it is importantto carry out stability studies under long term storage conditions.

3.2. Impact of the blg-based coagulum on the long term stability of D3

The stability of the free D3 during storage under refrigeratedconditions was compared to that of the vitamin sequestrated inthe blg-based coagulum over 5 weeks (Fig. 1). All the conical tubesused during the experiment were filled with D3-containingsolutions to reduce the headspace, in order to minimise oxidativedegradation. Nevertheless, after the first week of refrigerated stor-age, only 22.5 ± 0.2% of the free D3 remained in solution (Fig. 1).There was over 91% of vitamin loss after the third week and atthe end of the 5 week period only 8.3 ± 1.7% of the free D3

remained in solution (Fig. 1). Compared to the free vitamin, theD3 sequestered within the blg-based coagulum was significantlymore stable (p < 0.0001). At week 1, 95.2 ± 4.4% of the D3 was intact(Fig. 1). This amount decreased to 88.3 ± 2.7% at week 3 and, after5 weeks at 4 �C, 86.3 ± 3.2% of D3 still remained in the coagulum,which represents a loss of only 13.6% (Fig. 1). The coagulumdemonstrated an important capacity to protect D3 and prolongits shelf-life during cold storage.

The recovery of D3 in the coagulum was 10-fold higher thanwith the unprotected vitamin and was improved compared tocasein micelles (Haham et al., 2012). The protective effect of thecoagulum can be ascribed in part to the antioxidant activity ofthe free thiol groups of blg (Liu, Chen, & Mao, 2007). In fact, thecoagulum was prepared using a final blg concentration of 10%(w/v), which certainly encompasses a large number of free thiolgroups. In addition, mobility of D3 entrapped within the coagulummatrix might be impaired, thus provoking the immobilization of

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the vitamin, which reduces its reactivity but also reduces access ofoxidizing agents to D3 (Ron, Zimet, Bargarum, & Livney, 2010). Thisfinding implies that sequestration of the D3 in the blg-based coag-ulum can significantly improve the retention of the vitamin andprotect it from oxidative degradation over time, which may conse-quently greatly increase its uptake. Exposure to UV-light over timealso greatly damages D3-containing food products. It is thus impor-tant to evaluate the efficiency of the coagulum in protecting thevitamin during storage.

3.3. Impact of the blg-based coagulum on the UV-light stability of D3

The photochemical stability of unprotected D3 was compared tothat of the vitamin sequestrated within the blg-based coagulummatrix during 24 h of intensive UV-light irradiation. As shown inFig. 2, the protection effect of the coagulum on D3 during UV-lightirradiation was important.

Only 5.0 ± 0.7% of the unprotected D3 remained in solution afterthe first hour of exposure to light and, after the second hour,almost 99% of the free vitamin was degraded. The D3 entrappedin the coagulum remained intact, even after 10 h of irradiation(Fig. 2). After 24 h of irradiation, 94.2 ± 4.1% of D3-entrapped inthe blg-based coagulum was still intact. The good protective effectobtained in the current work is higher than that reported in the lit-erature (Luo et al., 2012). This important shielding effect againstphotochemical degradation can be ascribed to several factors.The abundant aromatic side chains and double bonds in blg-basedcoagulum might absorb much of the UV-light and, thus impair theabsorption of D3 (Luo et al., 2012; Ron et al., 2010). Furthermore,the physical immobilization of D3 within the coagulum matrixreduces its reactivity and restricts its access to oxidizing agents,as explained above.

The present work demonstrates the efficiency of the blg-basedcoagulum in protecting and improving the stability of D3 duringlong term storage in the cold and upon intensive UV-light irradia-tion, all of which occurs before ingestion. However, after ingestion,proteolytic enzymes prevailing in the stomach and intestinesmight destabilize blg and thus affect the ability of the coagulumto retain D3 within the coagulum matrix. Therefore, it is importantto study the behaviour of the D3-entrapped blg-based coagulumduring transit in the gastrointestinal (GI) tract.

3.4. Impact of the blg-based coagulum on the intestinal kinetic releaseof D3

In the present work, D3 was entrapped in the blg-basedcoagulum by inducing the self-aggregation of the blg/D3 complex

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0 1 2 4 6 8 10 24

Rec

over

y of

D3

(%)

Time (hours)

D3 ßlg/D3 CoagulumD3 βlg/D3 Coagulum* * * * *

**

Fig. 2. Photo-degradation of D3 and the D3-loaded blg-based coagulum uponexposure to UV-light (k = 254 nm). ⁄Statically different from D3 alone (p < 0.05).

upon mild acidification close to the pI of the protein (pH 4.7).The D3-entrapped blg-based coagulum might persist in thestomach due to the fact that the blg/D3 complex has improvedstability at the gastric pH (Diarrassouba et al., 2014). This is furthersupported by the fact that the structure of native blg remains intactunder the acidic conditions and pepsin concentrations in thestomach (Taulier & Chalikian, 2001). Furthermore, after foodingestion, the pH of the stomach is between 4.5 and 6.0, whichencompasses the pH of the coagulum (Keller, 2013). Therefore,the present work assumed that, when administered post-prandially,the blg-based coagulum might not be significantly affected by thegastric pH, with or without pepsin and thus, can remain stable inthe stomach. However, this may not be true for the intestinal pH inthe presence of the proteases.

The capacity of the blg-based coagulum to release D3 in thesimulated intestinal fluid (SIF), in the presence and absence of pan-creatin, was evaluated. Fig. 3 represents the kinetic release of theunprotected D3 and D3-entrapped in the blg-based coagulum dur-ing digestion. The integrity of the coagulum was well preserved,with a loss of only 3.7 ± 2.7% and 17.4 ± 0.1% in the presence andabsence of pancreatin, respectively, after 6 h in SIF (Fig. 3).

The unprotected D3 was almost completely degraded at the endof the digestion, with a loss of 90.0 ± 1.3% (Fig. 3). It is important tonote that the release of D3 was slower in the presence of pancreatinthan in its absence. A similar result was previously observed for theblg/D3 complex which was significantly more stable (p < 0.05) inthe presence of pancreatin than in its absence (Diarrassoubaet al., 2014). Pancreatin has no direct effect on the coagulum (suchas hydrolysis or degradation) so the integrity of the complex is wellpreserved. Addition of pancreatin has a direct effect on the dissolu-tion medium and especially the ionic strength. The increase ofionic strength of the dissolution medium, when the pancreatin isadded, has a direct influence on diffusion of the active substance(D3) from the complex. Indeed, this phenomenon has already beenobserved on diltiazem HCl release from hydroxypropyl methylcel-lulose matrix (Asare-Addo et al., 2013). Authors showed that anincrease in ionic strength brought about a decrease in matrixerosion rate. In our study, the resistance of the blg-based coagulumto proteolytic attack was improved. Normally, the structure of blgis modified at the intestinal pH, resulting in an increased suscepti-bility to the enzymes (Qin, Jameson, Bewley, Baker, & Creamer,1999; Taulier & Chalikian, 2001). The blg-based coagulum is awater-soluble matrix that could serve for the oral delivery of D3,

and consequently, could enhance the bioavailability of D3, as deter-mined by the serum level of its biological marker 25(OH)D3.

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D3 Coagulum ßlg/D3 Coagulum ßlg/D3 + PancreatinD3 βlg/D3 Coagulum βlg/D3 Coagulum + Pancreatin

* *

*

**

*

*

* **

Fig. 3. Stability of D3 and the D3-loaded blg-based coagulum in the simulatedintestinal fluid without and with pancreatin at 37 �C. ⁄Statically different from D3

alone (p < 0.05).

F. Diarrassouba et al. / Food Chemistry 172 (2015) 361–367 365

3.5. In vivo study of the bioavailability of D3

An in vivo experiment was carried out to evaluate the impact ofthe entrapment of D3 in the blg-based coagulum on its bioavailabil-ity. The rats received, by forced feeding, the unprotected D3,D3-entrapped blg-based coagulum and water supplemented with0.09% NaCl as the control. The effects of these treatments on thebioavailability of D3 were compared to that of the blg/D3 complexwhich, by auto-aggregation, led to the formation of theD3-entrapped blg-based coagulum (Diarrassouba et al., 2014). Aspresented in Fig. 4, there was a significant difference betweenthe mean 25(OH)D3 in the rats fed the D3-containing treatmentsand the control group (p < 0.0001), with a baseline of about0.02 nM (Fig. 4).

The serum level of 25(OH)D3 of the rats fed the D3-entrappedcoagulum (58.0 ± 11.5 � 103 nM) was significantly higher(p < 0.0001) than that in those fed the blg/D3 complex(20.9 ± 3.8 � 103 nM) and unprotected D3 (15.1 ± 11.5 � 103 nM).The enhancement of the bioavailability of D3 upon sequestrationin the blg-based coagulum can be ascribed to several factors. Firstof these is the blg-based coagulum entrapped D3 with a high EE,consequently increasing the water solubility of the vitamin(Li et al., 2011). Despite the fact that the unprotected D3, blg/D3

complex and D3-entrapped blg-based coagulum had equivalentinitial concentrations of D3, the blg-based coagulum actuallyencapsulated a higher amount of D3, corresponding to the high

25(O

H)D

3(x

103 n

mol

/L)

Fig. 4. Bioavailability of D3 in the rat experiment. Mean 25(OH)D3 (±SEM) in rats force-D3-loaded blg-based coagulum. D3 was measured by dosage of the serum level of thecoagulum. ⁄Statistically different from T0 (p < 0.05).

EE (94.5 ± 1.8%). Therefore, the intake of D3 by the animals fedthe D3 enriched coagulum was higher than that of those fed theunprotected D3 or blg/D3 complex. Another explanation might bethe increased stability of the D3-entrapped blg-based coagulumat intestinal pH and in the presence of proteases. Finally, thewater-soluble character of the blg-based coagulum is totallycompatible with the aqueous nature of the lining of the digestivetract, which can also contribute to the enhancement of thebioavailability of D3 (Li et al., 2011; Sun et al., 2012).

Reports indicate that the loss of D3 in whey proteins duringcheesemaking is problematic because it results in lower retentionof the vitamin in the curd matrix and subsequently, in cheese(Tippetts et al., 2012; Wagner et al., 2008). Herein, the high affinityof D3 for the major whey protein blg was exploited to form a D3-entrapped blg-based coagulum which in turn, significantlyincreased the uptake and bioavailability of D3 in rats. Powder, eth-anol or oil-based vehicles are usually used to evaluate the impactof the carried substances on D3 bioavailability, with a greater25(OH)D3 response for oil-based vehicles because D3 is fat-soluble(Grossmann & Tangpricha, 2010; Tippetts et al., 2012). To ourknowledge this work is the first report of the use of a water-solubleblg-based matrix to improve the bioavailability of D3. Despite thedifficulty of recovering whey protein from cheese, it has been sug-gested that coagulate whey proteins can be formed independently,followed by entrapment in a casein coagulum (Dybing & Smith,1998). The D3 entrapped in the blg-based coagulum can be used

fed the water supplemented with 0.09% NaCl (control), D3, the blg/D3 complex and25(OH)D3 and was significantly enhanced for the D3 entrapped in the blg-based

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in such a procedure to enrich casein coagulum in cheesemaking orother dairy products such as yogurts. Given that whey protein-based gels and aggregates are used in many food applications asfoaming or emulsifying agents or as, films and coatings, one couldsuggest that other types of semi-solid or solid foods and beveragesmight also be fortified using the D3-entrapped coagulum (Chen,Remondetto, & Subirade, 2006; Nicolai et al., 2011).

4. Conclusion

In the current work, D3 was successfully encapsulated in a blg-based coagulum obtained by triggering the self-aggregation of theblg/D3 complex by mild acidification. The blg-based coagulum is awater-soluble matrix that entrapped D3 with a high EE(94.5 ± 1.8%), subsequently increasing the solubility of this fat-soluble vitamin. The sequestration of D3 in the coagulum matrixsignificantly increased its stability during long term cold storage.Furthermore, D3 was stable to photochemical degradation afterovernight exposure to intensive UV-light. The compactness of thematrix of the blg-based coagulum provided a protective barrieragainst degradation by intestinal proteases, resulting in a longerresidence time for D3 in the intestines. Finally, the animal experi-ment demonstrated that the entrapment of D3 in the blg-basedcoagulum significantly enhanced its bioavailability, due toincreased water solubility of D3 and concomitant prolongedintestinal uptake. The findings presented in the current work areimportant for public health as well as for the food industry. Indeed,the high prevalence of D3 deficiency among populations of all agegroups, especially those living in northern latitudes, is well estab-lished (Hanley & Davison, 2005; Holick, 2007). This deficiency hasbeen implicated in various skeletal and non-skeletal diseases, suchas cancer, cardiovascular diseases, and diabetes and bacterialinfections (Byrdwell et al., 2008; Dawson-Hughes et al., 2005;Holick, 2007; Lips, 2006). As such, a 2.5-fold increase in the dietaryintake of D3 has been recommended to achieve adequate intake(Hanley & Davison, 2005). This work suggests that the D3-entrapped blg-based coagulum may be used for the oral deliveryand improved uptake and bioavailability of D3. This may help alle-viate the economic burden of D3 deficiency and improve a numberof disease conditions related to D3 deficiency. To a broader extent,this water-soluble matrix may be used as a food grade oral deliverysystem for fat-soluble nutraceuticals. Rheological analysis andinfra-red spectroscopic studies on the formation of the cold gela-tion of the blg/D3 complex are currently in progress in order tounderstand the impact of D3, temperature and pH on the micro-structure, in addition to the rate of gelation.

Acknowledgements

This work was supported by the Natural Sciences and Engineer-ing Research Council of Canada (NSERC) through the Vanier CanadaGraduate Scholarships (F. Diarrassouba) and the Canada researchchair in protein, biosystem and functional food physical chemistry.The authors would like to thank Mr Pascal Dubé, Ms Diane Gagnon(Institut de recherche sur les nutraceutiques et les alimentsfonctionnels, Université Laval, Québec, QC, Canada) and theEA-CIDAM’s team members (Biopharmacie lab, Faculté dePharmacie, Clermont-Ferrand, France).

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