Preparation and characterization of novelnanocarriers containing krill oil forfood application

Jiajin Zhu *, Pan Zhuang **, Lanlan Luan, Qi Sun, Feiwei CaoSchool of Biosystems Engineering & Food Science, Zhejiang University, Hangzhou 310058, China

A R T I C L E I N F O

Article history:

Received 5 March 2015

Received in revised form 4 June

2015

Accepted 5 June 2015

Available online

A B S T R A C T

The encapsulation of krill oil high in docosahexaenoic acid (DHA), eicosapentaenoic acid

(EPA) and astaxanthin as a multifunctional dietary supplement for the purpose of enhanc-

ing its physical and chemical stability and to expand its application in aqueous-based food

was attempted. Nanostructured lipid carriers (NLCs) containing high content of krill oil were

successfully prepared using palm stearin as a solid lipid and lecithin as a surfactant. The

results demonstrated that the developed NLC had spherical or ovoid structure with small

size (<150 nm), narrow polydispersity index (<0.2) and high entrapment efficiency (>96%).

DSC (differential scanning calorimetry) result showed a less-ordered crystalline structure

leading to high loading capacity. NLC was found to offer bioactives in krill oil significant

protection against photooxidation upon exposure to UV light. Good physical and chemical

stabilities were revealed by long-term storage at different temperatures. Feasibilities of pas-

teurization and lyophilization were also demonstrated, showing promise for application in

functional drinks and milk powder fortification.

© 2015 Elsevier Ltd. All rights reserved.

Keywords:

Krill oil

Nanostructured lipid carriers

DHA

EPA

Astaxanthin

1. Introduction

Krill oil has recently emerged as a new dietary supplementabundant in long chain omega-3 polyunsaturated fatty acids.Essential omega-3 fatty acids, namely eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) account for over 30%of the total fatty acids in Antarctic krill oil (Kolakowska,Kolakowski, & Szczygielski, 1994). Unlike fish oil, approxi-mately 30–65% of the fatty acids in krill oil are in thephospholipid form, providing krill oil with a proposed betterbioavailability (Schuchardt et al., 2011; Ulven et al., 2011). Krill

oil also possesses 48 times higher antioxidant potency thanfish oil on the basis of ORAC (oxygen radical absorption ca-pacity) values as a result of containing various kinds of powerfulantioxidants, such as vitamins E, A and D, astaxanthin,and canthaxanthin (Farooqui, 2009). A number of animal andhuman studies have suggested that krill oil has a variety ofbiological functions, including positive effects on cardiovas-cular disease, non-alcoholic fatty liver disease, metabolicsyndrome, premenstrual syndrome, endocannabinoids, in-flammation, colon cancer and attention deficit hyperactivitydisorder (Batetta et al., 2009; Bunea, El Farrah, & Deutsch, 2004;Deutsch, 2007; Ierna, Kerr, Scales, Berge, & Griinari, 2010;

* Corresponding author. Zhejiang Engineering Center for Food Technology and Equipment, Zhejiang Key Laboratory for Agro-Food Pro-cessing, Fuli Institute for Food Science, College of Biosystems Engineering and Food Science, Zhejiang University, No. 866, YuhangtangRoad, West Lake District, Hangzhou 310058, Zhejiang, China. Tel.: +86 571 88982732; fax: +86 571 88982732.

E-mail address: jjzhu@zju.edu.cn (J. Zhu).** Corresponding author. School of Biosystems Engineering & Food Science, Zhejiang University, Hangzhou 310058, China. Tel.: +86 571

88982732; fax: +86 571 88982732.E-mail address: 375053056@qq.com (P. Zhuang).

http://dx.doi.org/10.1016/j.jff.2015.06.0171756-4646/© 2015 Elsevier Ltd. All rights reserved.

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Sampalis et al., 2003; Vigerust et al., 2013; Zhu, Shi, Qian, Cai,& Li, 2008). It is possible that the proposed benefits of krill oilmight arise from the synergism between EPA, DHA, phospho-lipids, astaxanthin and other bioactive constituents like vitaminsand flavonoids. However, the poor solubility of krill oil haslimited its application in food, especially in aqueous-basedfoods. On the market, krill oil is sold to consumers in thecapsule form. Furthermore, due to the high unsaturation of EPA,DHA and astaxanthin, they are prone to degradation and exertan undesirable effect on sensory acceptance of enriched food-stuffs when exposed to oxygen, heat, light and metal ions.Seafood-derived products fortified with krill oil are more sus-ceptible to oxidation than those enriched with other n-3 richoils (Pietrowski, Tahergorabi, Matak, Tou, & Jaczynski, 2011).Consequently, development of a delivery system which en-hances its solubility in water and minimizes oxidation isconsidered desirable for future use of krill oil in foods.

Nanostructured lipid carriers (NLCs) derived from oil/water (O/W) nanoemulsions have great potential to serve asa carrier system for bioactive compounds of foods (Tamjidi,Shahedi, Varshosaz, & Nasirpour, 2013). Possessing small size,high entrapment efficiency and the potential of mass produc-tion, has made it very promising to the food industry (Fathi,Mozafari, & Mohebbi, 2012). NLC consists of solid lipid, liquidlipid, surfactant and water as major ingredients, thus bothsolid and liquid lipids exist in the lipid phase of NLC at roomtemperature. Müller, Radtke, and Wissing (2002a, 2002b) de-veloped partly crystallized lipid droplets which constituted aless-ordered crystalline structure for NLC, in order to getover the defects of solid lipid nanoparticles(SLN) in thelate 1990s. Since the liquid oil is incorporated into the centreof the solid lipid in NLC, the bioactives dissolved in the liquidoil are simultaneously entrapped in the solid lipid, whichresults in a higher drug loading and controlled drugrelease (Varshosaz, Eskandari, & Tabakhian, 2010). NLC wereinitially intended for pharmaceutical and cosmetic applica-tions. Nonetheless, they may improve bioavailability andnutritional value of bioactives, and increase their functional-ity (consumer acceptability, shelflife, stability and safety offoods), and offer controlled release of the entrapped nutri-ents. In recent years, NLC loaded with omega-3 polyunsaturatedfatty acids of fish oil and algal oil have been successfullyprepared (Averina, Müller, Popov, & Radnaeva, 2011; Lacatusuet al., 2013; Wang et al., 2014) while little attention has beendrawn to krill oil.

When dispersed in an aqueous medium, hydrophobicbioactive compounds require stabilization and protectionagainst adverse factors (Zimet, Rosenberg, & Livney, 2011). Ithas been shown that entrapment of n-3 unsaturated fatty acidsfor food fortification dramatically reduced its oxidation (Garg,Wood, Singh, & Moughan, 2006; Tamjidi, Nasirpour, & Shahedi,2012). The challenges to use of carotenoids as nutrition en-hancers are their poor solubility in water, low bioavailability,high melting point and chemical instability (Qian, Decker, Xiao,& McClements, 2012). For these reasons, we focussed on theastaxanthin in krill oil.

The objective of this research was to firstly study the suit-ability and the effectiveness of NLC as a delivery system toencapsulate krill oil and secondly to investigate the chemicaland physical stability of the prepared NLC.

2. Materials and methods

2.1. Materials

Antarctic krill oil (about 14.8% DHA, 22.5% EPA and 250 mg/kg astaxanthin) was purchased from Shandong KeruierBiotechnology Co., Ltd (Shandong, China). This krill oil was ob-tained by solvent extraction from fresh Antarctic krill (Euphausiasuperba) and then underwent multistage purification.The lipidclasses of this krill oil are predominantly phospholipids (50%)carrying more than 95% of total omega-3 fatty acids, polar non-phospholipids (29%) and triacylglycerols (21%) and thecomposition of astaxanthin was free astaxanthin (15%),astaxanthin monoesters (28%) and astaxanthin diesters (57%).Palm stearin (melting point 58.6 °C, iodine value <17) was pur-chased from Guangdong Jinzhi Co., Ltd (Guangdong, China). DHAmethyl ester (≥98%, capillary GC), EPA methyl ester (≥97%, cap-illary GC), astaxanthin (HPLC), lecithin (L-α-phosphatidylcholine,analytical grade) were supplied by Sigma (St. Louis, MO, USA).Isopropyl alcohol (HPLC) and hexane (GC) were purchased fromTianjin Shield Chemicals Co., Ltd (Tianjin China). All otherchemicals were purchased from Sinopharm Chemical ReagentCo., Ltd. (Shanghai, China) and were of analytical grade.

2.2. Methods

2.2.1. NLC productionNanostructured lipid carriers composed of different ratios ofkrill oil, solid lipid (palm stearin) and emulsifiers were pro-duced by the optimized hot homogenization method withultrasonication technique. Palm stearin acid and krill oil weremixed together and melted at 70 °C until the total lipid phasewas entirely melted. Meanwhile, the aqueous surfactant so-lution consisting of dispersing surfactant lecithin in doubledistilled water at the same temperature was agitated at 600 rpmby a hot plate stirrer. Then, the melted lipid phase was addedto the aqueous phase drop by drop using an agitation at 600 rpmfor 60 s and then further dispersed at 10,000 rpm for 5 min withthe aid of a high-speed stirrer (FA25, Fluko, Shanghai, China).Finally, the preemulsion was treated by an ultrasonic cell dis-ruption system (JY-92-II, Ningbo Scientz Biotechnology Co., Ltd.,Ningbo, China) for 5 min (active for 2 s at intervals of 2 s, 200 W).After recrystallization upon cooling to room temperature, NLCdispersions were prepared.

2.2.2. Formulation optimizationThe optimum formulation for the NLC was ascertained usingresponse surface methodology (RSM) and a two-factor in-scribed central composite design CCD (two factors at five levels).The krill oil content in total lipid phase (w/w) (X1) and levelof the surfactant lecithin (w/w) (X2) were the input variables.The parameter of probe-ultrasonic disruptor (200 W, 5 min) andthe total lipid concentration (10%) was applied according tomany references and our preliminary single-factor experi-ments (Averina et al., 2011; Lacatusu et al., 2013; Luo, Zhao,Zhang, & Pan, 2011; Wang et al., 2014). Altogether 13 experi-ments were conducted to evaluate the effects of the twovariables on the particle size and size distribution (polydis-persity index) of NLC as they are the most important features

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of nanocarriers dispersion that dominate the physicochemi-cal stability, solubility, turbidness, biological performance, andrelease speed (Lakshmi & Kumar, 2010). The mean diameter(Y1) and polydispersity index (Y2) of the NLCs were deter-mined by a Zetasizer Nano ZS90 (Malvern Instruments,Worcestershire, UK). The data are shown in Table 1.

2.2.3. Particle size analysisThe mean diameter, PDI (polydispersity index), and zeta po-tential of NLCs were determined by a Zetasizer Nano ZS90(Malvern Instruments). Prior to the measurements, sampleswere all diluted to different concentrations using ultrapurewater. The dynamic light scattering was operated with a scat-tering angle of 90° at 25 °C.

2.2.4. Morphology characterizationMorphology of the nanocarriers was obtained using a trans-mission electron microscopy (Hitachi H-7650, Tokyo, Japan). Allsamples were diluted 100 times in double distilled water andnegatively stained with phosphotungstic acid (2%) for 4 min,and subsequently dried on copper grids at room temperature.

2.2.5. Thermal analysisThe palm stearin and developed nanocarriers containing dif-ferent ratio of krill oil were used for thermal assessments.Differential scanning calorimetry (DSC) was performed usinga DSC 1 (Mettler-Toledo, Zurich, Switzerland) to measure meltingpoint. About 2 mg sample were placed in a standard alu-minium sample pan and analysed under nitrogen purge(100 ml/min). A heating rate of 3.5 °C/min was employed in therange of 25–100 °C. The degree of recrystallization of the lipidmatrix can be reflected by recrystallization index (RI), whichwas computed to assess the physical state of NLCs using thefollowing equation (Siekmann & Westesen, 1994):

RIH

H Concentrationbulk lipid

%( ) =×

×ΔΔ

100 (1)

where ΔH and ΔHbulk represent the melting enthalpy value ofthe nanostructured lipid carriers and the bulk palm stearin,

respectively. Concentrationlipid is the concentration of the totallipid phase in the NLC dispersions.

2.2.6. LyophilizationThe prepared NLC (1 ml) was firstly diluted 10 times.Then, pro-tective agents, 0.9 g saccharose in combination with glucosefor example, were added. The obtained solution was then putto plates and frozen at −80 °C in a refrigerator for 8 h. After-wards, the final frozen solution was lyophilized with aid of alyophilizer (Alpha1-4, Marin Christ Co., Ltd., Osterode, Germany)for 24 h at −40 °C and a pressure of 0.1 Mbar. After lyophiliza-tion, the products were mashed into powder and stored at roomtemperature.

2.2.7. Determination of entrapment efficiency and drugloadingSince DHA, EPA and astaxanthin are the most importantbioactive compounds in krill oil, their entrapment efficiency(EE) and drug loading (DL) were measured employing ultrafil-tration centrifugal filter tubes (Millipore, Billerica, USA) that cancut off a molecular weight of 30 kDa (Hsu, Cui, Mumper, & Jay,2003). Gas chromatography with a flame ionization detector(GC, Agilent 7890A, Palo Alto, CA, USA) was used to deter-mine the content of DHA and EPA in krill oil using a capillarycolumn (DB-23, 60 m, 0.25 mm i.d., 0.25 µm, AgilentTechnologies).The amounts of EPA and DHA in the lipid car-riers (total) and the ultrafiltrate (free) were measured by themethod described by Wei et al. (2008).

The content of astaxanthin was determined by first-orderderivative spectrophotometry using a spectrophotometer (T60UV/VIS Spectrophotometer, Purkinje General Co., Ltd, Beijing,China).

Briefly, a known amount of samples was mixed with amixture of a 60% (v/v) n-hexane/isopropyl alcohol and sub-jected to extractions for three times. The solvents were thenevaporated and the dry extract obtained was diluted in a knownvolume of n-hexane and its absorbance was determined at472 nm wavelength, and was quantified according to the cali-bration curve prepared with the modified method describedby Sánchez-Camargo, Martinez-Correa, Paviani, and Cabral

Table 1 – Central composite design with experimental and predicted values for mean diameter and polydispersity index.

Run order Coded variables Response 1 (Y1) Response 2 (Y2)

Mean diameter (nm) Polydispersity index

(X1) krill oilcontent (%)

(X2) Levelof lecithin (%)

Experimental Predicted Experimental Predicted

1 −1 (35.00) −1 (0.70) 288.8 288.8 0.333 0.3282 −1.41 (28.79) 0 (0.90) 293.5 294.8 0.248 0.2523 1 (65.00) 1 (1.10) 149.4 147.7 0.150 0.1544 0 (50.00) 1.41 (1.18) 167.6 170.5 0.157 0.1625 1.41 (71.21) 0 (0.90) 160.3 160.3 0.170 0.1656 0 (50.00) −1.41 (0.62) 236.3 235.0 0.326 0.3217 1 (65.00) −1 (0.70) 186.4 187.5 0.231 0.2368 −1 (35.00) 1 (1.10) 240.1 237.2 0.188 0.1849 0 (50.00) 0 (0.90) 194.8 193.5 0.183 0.181

10 0 (50.00) 0 (0.90) 197.5 193.5 0.181 0.18111 0 (50.00) 0 (0.90) 189.8 193.5 0.180 0.18112 0 (50.00) 0 (0.90) 192.8 193.5 0.185 0.18113 0 (50.00) 0 (0.90) 192.5 193.5 0.177 0.181

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(2011) and Tolasa, Cakli, and Ostermeyer (2005). Lipid phasein NLC and the ultrafiltrate (free) were extracted bypetroleum ether to remove water when determining thebioactive constituents.

The EE (%) and DL (%) of the bioactive constituents (DHA,EPA, astaxanthin) in NLC were calculated from the equationsbelow:

EEW W

Wtotal free

total

%( ) =−

× 100 (2)

DLW W

Wtotal free

lipid

%( ) =−

× 100 (3)

where Wtotal is the total amount of bioactive constituents (DHA,EPA, astaxanthin) which existed in the NLC dispersions, Wfree

represents the amount of free bioactive constituents (DHA, EPA,astaxanthin) in the NLC dispersions, and Wlipid represents theamount of the lipid phase (solid and liquid lipids) that was usedin the preparation of nanostructured lipid carriers.

2.2.8. Physical stabilityFirstly, we investigated the centrifugal stability of developedNLC. About 1 g NLC was centrifuged by a supercentrifuge at10,000 rpm (9170 g) for 30 min. A stability parameter (KE) (Tang,Xia, & Liu, 2010), as below, was used to estimate the centrifu-gal stability:

KR R

RE %( ) = − ×0

0

100 (4)

where R0 is the mean diameter of the initial NLC while R is themean diameter of the NLC in the upper fluid of the centrifu-gal tube after centrifugation. Long-term physical stability wasthen assessed by storing the NLC samples at different tem-peratures (4 °C, room temperature, 40 °C) for 70 days. Particlesize and PDI of the samples were measured every 10 days. Sta-bility of sterilization was also tested by pasteurization. Sampleswere sterilized in two ways: HTST (75–90 °C, 15–16 s) and LTLT(62–65 °C, 30 min) with the assistance of a thermostat waterbath. After sterilization, the outcome was detected from bothappearance and particle size.

2.2.9. Chemical stabilityKrill oil has been reported to have better autoxidative stabil-ity than fish oil. Nevertheless, protection is still needed to reducethe degradation that occurred during storage (Lu, Bruheim,Haugsgjerd, & Jacobsen, 2014; Thomsen et al., 2013). In thisstudy, photostability of the prepared NLC was evaluated asbioactive compounds in krill oil are sensitive to sunlight andair. Briefly, 5 g NLC (0.325 g krill oil in NLC) and 0.325 g krill oilwere placed in glass tubes, respectively, and then exposed to

UV light (dual-wavelength 253.7 nm + 185 nm) by an ultravio-let lamp tube (110W, 2G11, Shanghai Yanguang ElectronicTechnology Co., Ltd, Shanghai, China) under ventilation androom temperature for 60 h. Every 10 h, the amount of bioactiveconstituents (DHA, EPA, astaxanthin) was assayed. Long-term chemical stability was also evaluated by quantifying thebioactive constituents after 70 day storage as described insection 2.2.8.

2.2.10. Application in a beverageIn this work, the prepared NLC was added to a simulated bev-erage to study its feasibility for application in the beverageindustry. The formulation of the simulated beverage con-sisted of 15% glucose, 0.1% ethylenediaminetetraacetic aciddisodium salt (Na2 EDTA), 0.1% benzoic acid, 1% NLC and 83.9%water (w/v) which resembles common beverages consumed inour daily life (Liu, Wang, & Xia, 2012). Twenty five millilitresof this simulated beverage were placed in glass bottles and thenstored at various temperatures (4 °C, room temperature and40 °C). During the storage, the mean diameter of particles wasmeasured every 10 days, as described before. Stability of ster-ilization was investigated as described in section 2.2.8.

2.2.11. Statistical analysisSPSS 19.0 statistical program and design Expert 8.0.6.1 statis-tical software (Stat Ease, Inc., Minneapolis, MN, USA) wereemployed for analysis of the experimental data statistically.Significance of factors was calculated employing Pareto analy-sis of variance (ANOVA) at 95% confidence level (p < 0.05). Allmeasurements were repeated at least three times.

3. Results and discussion

3.1. Formulation optimization

With the purpose of preparing NLC with small particle size aswell as low polydispersity index, the formula should be opti-mized. The relationship between the response functions andthe formulation variables was identified by two-factor in-scribed central composite design. It can be seen from Table 1that the lowest value of particle size and polydispersity indexwas coincident with the same condition of X1 = 65.00%;X2 = 1.10%. The mathematical model in second order workedout from the results of experiments is shown in Table 2 ac-cording to coded factors.There was a high consistency betweenthe experimental and the predicted values, R2 values (0.9976for mean diameter, 0.9961 for PDI) and adjusted-R2 values (0.9959for mean diameter, 0.9933 for PDI). Both factors (krill oil contentand lecithin level) contributed to particle size and size distri-bution significantly (p < 0.0001). The content of krill oil had agreater influence on particle size while lecithin level played a

Table 2 – The fitted quadratic model in terms of coded variables for Y1 and Y2 responses.

Responses 2nd Order polynomialequation

Regression(p-value)

R2 R2 (adjusted) Lack of fit

Mean diameter (Y1) 193 48 47 68 22 86 2 93 17 15 4 671 2 1 2 12

22. . . . . .− − + + +X X X X X X <0.0001 0.9976 0.9959 0.4697

Polydispersity index (Y2) 0 0 0 0 0 0 0 0 0 0 0 01 2 1 2 12

22. . . . . .18 31 58 16 14 3− − + + +X X X X X X <0.0001 0.9961 0.9933 0.0829

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bigger role in narrowing polydispersity index. In terms of par-ticle size, the linear effect of krill oil content was negative whilethe quadratic influence was positive at p < 0.0001. When krilloil content was low, the particle size could be markedly di-minished by adding krill oil as the core viscosity of the lipidnanocarriers declined, promoting their fluidity (Pardeike,Hommoss, & Müller, 2009). Nevertheless, further increasing theliquid oil content would lead to aggregation and growth in size(Hu et al., 2005), which ended in wider size distribution. As forpolydispersity index, it also exhibited negative and positive in-fluence in linear and quadratic terms of lecithin level,respectively (p < 0.0001). Lacking in surfactant, lecithin was ef-ficient in reducing the surface tension to lower mean diameterand PDI. However, it has been reported that when reaching“surfactant-rich zone,” the surfactant then did not greatly affectthe mean diameter and PDI (Yang, Marshall-Breton, Leser, Sher,& McClements, 2012). The Derringer’s desirability functionmethod was applied to optimize the formulation variables. Sinceour target was to obtain smallest particle size and narrowestsize distribution for the formulation, the optimum formula-tion was determined to be krill oil content (in total lipid phase)of 65% and lecithin level of 1.07%. The responses for mean di-ameter and PDI (mean of five measurements) experimentallydetermined with this optimized formulation were 143.5 nm and0.153, respectively, which were not significantly different to thepredicted values (p > 0.05).

The optimized formulation was simple but efficient and hadno toxicity or bitter taste of emulsifiers as only lecithin wasapplied as surfactant.

3.2. Particle size, zeta potential, EE and DL analysis

Fig. 1 depicts the particle size distribution of the developed NLCby intensity. It has been reported that small particle size(143.5 nm) and low value of PDI (0.153), which showed a fairlynarrow size distribution, were important for the long-term sta-bility of nanosuspensions (Lakshmi & Kumar, 2010).

Fig. 2 illustrates the variation of zeta potential following di-lution by distilled water. The initial pH value of the NLCdispersion was 6.4 and the original zeta potential (ZP) valuewas −31.0 mV, which just meets the requirement for an elec-trostatically stabilized nanosuspension, the minimum of whichshould be ±30 mV (Lakshmi & Kumar, 2010). It could be ob-served that the ZP of NLC dispersion increased rapidly to−57.4 mV, on dilution to 5 times, whereas further dilution hadlittle impact on the zeta potential. With high negative or posi-tive ZP, particles in the suspension will repel each other and

there will be no tendency for aggregation (Wissing & Müller,2002). This suggests that the original NLC dispersion could bediluted 5 times to reinforce the stability during storage, andwhen applied in beverage (usually diluted more than 5 times)the ZP plays an important role in preventing particles from floc-culating and sedimenting.

When bioactives were naturally constituents of an oil, NLCwith high content of oil was described to have lower EE, asexcess liquid lipid would be expulsed during crystallization,which also caused aggregation and size growth (Wang et al.,2014). However, as is demonstrated in Table 3, EE of DHA, EPAand astaxanthin in our prepared NLC (65% krill oil in total oilphase) were still above 96%. This interesting phenomenon maybe due to a very large proportion of phospholipids (about 50%)in krill oil bonding with DHA, EPA and astaxanthin, which en-hanced the solubility of these constituents in the lipid phaseand consequently reduced the diffusion coefficient (Tamjidi

Fig. 1 – Size distribution diagram of developed nanocarriers.

Fig. 2 – The zeta potential of NLC dispersion and thepotential values were the mean ± SD (n = 3).

Table 3 – Entrapment efficiency and drug loading of thedeveloped NLC for bioactive compounds.

Bioactive compounds EE (%) DL (%)

EPA 96.66 ± 0.54 12.14 ± 0.07DHA 96.79 ± 0.79 5.60 ± 0.05Astaxanthin 97.29 ± 0.42 0.02 ± 0.00

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et al., 2013). Furthermore, according to previous studies,astaxanthin attached to phospholipid (PL) had increasedlipophilicity compared to monoester form (Clark, Yao, She, &Furr, 2000), therefore it was less likely to move from oil phaseto the water phase to be free, leading to high EE and drugloading. This kind of nanosuspension may be defined as mul-tiple type NLC (multiple oil-in-fat-in-water (O/F/W) carrier),usually composed of a higher content of liquid oil. It has beenreported that tiny oil nano-compartments in the solid matrixof this type of NLC made the drug solubility higher, conse-quently improving the drug loading capacity (which is shownin Table 3) and still allowing controlled drug release (Mülleret al., 2002a).

3.3. Morphology and thermal characterization

The ultrastructure of the prepared NLC determined by TEM isshown in Fig. 3.The micrograph of the lipid carriers manifested

a spherical or ovoid structure which can be attributed to thenano-droplets and with no obvious agglomeration.

The thermograms of NLC containing different amounts ofkrill oil are depicted in Fig. 4. All the endothermic peaks arebroad because palm stearin was not a free fatty acid buttriacylglycerols enriched in palmitic acid and stearic acid.However, it can still be observed that their melting points (about53, 51.7, and 50.1 °C for 40, 50, and 65% krill oil contents, re-spectively) were all below that of palm stearin (58.6 °C). Becauseof the higher content of krill oil we believe this made themelting points even lower and the melting endotherms broader.Moreover, their RIs were calculated to be 52.75, 37.86, and 11.98%for krill oil contents of 40, 50, and 65% respectively. The resultsof this work imply that the addition of liquid oil (krill oil) candegrade the crystalline order and enhance the lattice defectof the lipid matrix, which was also reported by Zhang et al.(2010). The created imperfection of the lipid crystalline struc-ture could not only prevent the solid lipid from recrystallization,but also improve drug-loading capability.

3.4. Lyophilization

To store nanoparticles, lyophilization is an excellent way asit prevents nanoparticles from Ostwald ripening and hydro-lysis as well as facilitating transport (Cui, Hsu, & Mumper, 2003;Hsu et al., 2003). It had been shown that saccharides arefavourable cryoprotectants that prevent particle aggregationduring lyophilization. Therefore, the effect of lyophilizationshould be detected for the developed NLC. Glucose, lactose andsucrose were applied to protect the NLC in our study. After ly-ophilization, formed powders were added with water toredissolve. Without a cryoprotectant, the nanoparticles of pre-pared NLC were found to aggregate together and were difficultto dissolve in water after lyophilization. Table 4 displays therecovery of particle size and size distribution using differentcombinations of saccharides. All three combinations had a

Fig. 3 – TEM morphology of NLC.

Fig. 4 – DSC curves of lipid carriers composed of 40, 50, and 65% (w/w) krill oil in lipid phase.

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protective effect in particle size, among which the group ofsucrose and glucose (1:1, w/w) at 18% (w/v) did best ascryoprotectant. However, a combination of glucose and lactosewas applied by Liu et al. (2012) for coenzyme Q10 loaded NLC,which suggests that different types of NLC may require dif-ferent excipients as cryoprotectants. With this formulation ofprotectant agent, the formed canary powder was found to beeasily dissolved and well-distributed in water with a gentleshake. In addition, when brewed by boiling water, thenanopowder behaved no differently from when dissolved inroom-temperature water. After being cooled to room tempera-ture, the NLC still manifested a small mean diameter andnarrow size distribution (Table 4), which indicates its poten-tial use for milk powder fortification.

3.5. Physical stability

The centrifugal stability of the produced NLC was firstly evalu-ated. After high speed centrifugation, no stratification wasobserved and the stability parameter KE was calculated to beonly 1.74%, indicating that gravity factors had little influenceon the stability of the lipid carriers.

Pasteurization is a widely used way of sterilization that isbelieved to result in disinfection through simple heat. Thus inthis work, the feasibility of pasteurization was investigated andthe results are shown in Table 5. No visible flocculation wasfound in any of the samples after pasteurization whether withthe HTST or LTLT method. It was obvious that smaller par-ticle size and narrower size distribution were obtained withLTLT method. This may be caused by enhanced kinetic energyof nanosuspension system with raised temperature, which ledto coagulation and flocculation. It can be concluded from theresults that the developed NLC can be pasteurized before orafter addition in beverage and LTLT method is better in main-taining the size characterization.

Long term physical stability is essential for NLC duringstorage and when added to beverages for nutrition fortifica-tion. The pH value of the simulated beverage was adjusted to3.4 to ensure the antiseptic function of benzoic acid. Fig. 5 showsthe changes of mean diameter of NLCs (original sample or inbeverage) during storage at different temperatures. Particle sizeof the NLCs showed a trend of gradual growth on the whole

which was also detected in other NLC preparations (Wang et al.,2014); furthermore, a rapid increase was observed during thefirst 20 days owing to the severe change in surrounding cir-cumstance (Liu et al., 2012). It seems that the original NLCswere fit for storage at 40 °C as little growth occurred on themean diameter but the structure of NLC may be impaired andbioactives entrapped were actually unstable (described laterin section 3.6). NLCs under low temperature such as 4 °C weremore stable than when stored at room temperature due to thekinetic energy depression of nanoparticles at lower tempera-tures, whereas NLC in simulated beverage showed slowerincrease in particle size when stored at RT than that at 4 °Cand 40 °C, which indicated that change in zeta potential atvarious temperatures was the primary factor affecting thephysical stability of NLC-beverage (Liu et al., 2012; Wang et al.,2014; Weijun et al., 2009). It can be concluded that NLC aloneand NLC in simulated beverage exhibited good physical sta-bility under storage of 70 days as size characterizations werestill acceptable on the 70th day (Zhang, Chen, Zhang, Shen,& Pan, 2011). Low temperature was preferred to maintain theparticle size of NLC. As for NLC-beverages, the maximum zetapotential of the dispersion at various temperatures should befurther studied.

3.6. Chemical stability

For various kinds of natural or processed foods, the principlecausal factor of quality deterioration is the susceptibility to oxi-dation. It was reported that nanoparticulate carriers offeredsignificantly better photoprotection than the vesicularcarriers and other tested systems (Raza et al., 2013). In ourstudy, UV light exposure and ventilation condition were appliedto evaluate the photooxidation stability of NLC sincekrill oil was supposed to be stored sealed and away fromlight. In order to determine the order of the degradation,graphical method was employed (Ourique et al., 2011). Deg-radation of DHA, EPA and astaxanthin was found to follow first-order kinetics (Fig. 6), as revealed by statistically significanthigh values of r for first order (r > 0.98, p < 0.001). The exact se-quence of degradation rate constants was observed to be:DHA in krill oil (0.02435 ± 0.00108 h−1) > EPA in krilloil (0.02332 ± 0.00095 h−1) > astaxanthin in krill oil

Table 4 – – The effect of different formulation of cryoprotectant for lyophilization.

Cryoprotectantformulation

Glucose (g) Sucrose (g) Lactose (g) Mean diameter(nm)

Polydispersityindex

1 0.9 0.9 0 162.6 ± 2.3 0.201 ± 0.0172 0 0.9 0.9 176.3 ± 2.4 0.266 ± 0.0233 0.9 0 0.9 168.1 ± 4.3 0.347 ± 0.0214 (Brewed by boiling water) 0.9 0.9 0 156.3 ± 2.4 0.235 ± 0.007

Table 5 – – The stability of pasteurization.

Sterilization method Mean diameter (nm) Polydispersity index

NLC (original sample) HTST (75–90 °C, 15–16 s) 221.3 ± 4.7 0.387 ± 0.026LTLT (62–65 °C, 30 min) 175.8 ± 5.9 0.254 ± 0.032

NLC in simulated beverage HTST (75–90 °C, 15–16 s) 185.4 ± 3.3 0.341 ± 0.029LTLT (62–65 °C, 30 min) 168.7 ± 7.5 0.268 ± 0.022

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(0.01290 ± 0.00067 h−1) > DHA in NLC (0.00581 ± 0.00028 h−1) > EPAin NLC (0.00463 ± 0.00019 h−1) > astaxanthin in NLC(0.00358 ± 0.00020 h−1). The bioactives of krill oil encapsulatedin NLC exhibited a much slower rate of degradation than whendirectly exposed to UV light. This can be ascribed to the solidphotoprotective lipid layer around the entrapped bioactives,thus protecting the same from direct light (Raza et al., 2013)and hindering the oxygen from contacting with the krill oil andoxidizing it (Lacatusu et al., 2013).

Table 6 demonstrates the long-term chemical stability ofthe NLC containing krill oil. It reveals that the bioactive com-pounds entrapped in the particle were released in a prolongedway at RT and 4 °C and the released compounds were sub-jected to faster oxidative degradation (Liu et al., 2012). After

70 day storage, the minimum decline in DL of bioactive com-pounds in NLC was observed at 4 °C while the maximumdecline was detected at 40 °C. Two possible reasons are asfollows: (1) high temperature lowered the crystallinity of thenanoparticles and turned them close to liquid–liquiddispersion system as microemulsion. Solid lipid (palm stearin)was more like liquid than solid at 40 °C, thus less capable ofdelaying drug release. (2) The oxidative degradation was ac-celerated at higher temperatures. Hence, the produced NLCwas not suitable for storage at 40 °C though the sizecharacterization was found to be well-maintained at thattemperature (Fig. 5). Overall, low temperature is favoured forenabling the good physicochemical stability of the originalNLC dispersion.

Fig. 5 – Effects of long-term storage on particle size of NLC (left) and NLC in simulated beverage (right) at differenttemperatures (4, RT and 40 °C).

Fig. 6 – The amount of DHA, EPA and astaxanthin in 0.325 g krill oil and in 5 g NLC changed with time under UV lightexposure.

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4. Conclusions

Formulating krill oil in nanostructured lipid carriers of O/Wnanodispersions is a promising approach to incorporate itsbioactive constituents into aqueous-based systems and enhanceits functionality for food fortification. The prepared NLC con-taining krill oil was shown to have a small size, narrow sizedistribution, high EE and DL of DHA, EPA, and astaxanthin. DSCand TEM results showed the morphology and the less-orderedlipid matrix of NLC. The dispersions were both physically andchemically stable during 70-day storage at RT and 4 °C. Theresults showed significantly increased protection of photooxi-dation degradation of water dispersed DHA, EPA andastaxanthin inside the particles. Moreover, the results indi-cated that the developed lipid nanocarriers were stable not onlyafter application in the simulated beverage but also after ly-ophilization into powder as well.Taking these research findingsinto account, NLC is a promising vehicle system for deliver-ing bioactive constituents of krill oil with good physical andchemical stability. This work overcame the limitation of ap-plying krill oil to aqueous-based foods such as functionalbeverages and was also conducive to prolonging shelf periodof krill oil.

Acknowledgments

Thanks are due to Hui Zhang for suggestions for writing thispaper. This research was funded by the National High-tech Re-search and Development Program of China (863 Program) (No.2011AA100804) and the Research Fund of Fuli Institute of FoodScience Zhejiang University (No. KY201403).

Appendix: Supplementary material

Supplementary data to this article can be found online atdoi:10.1016/j.jff.2015.06.017.

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0 day 70 days 0 day 70 days 0 day 70 days

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ARTICLE IN PRESS

Please cite this article in press as: Jiajin Zhu, Pan Zhuang, Lanlan Luan, Qi Sun, Feiwei Cao, Preparation and characterization of novel nanocarriers containing krill oil forfood application, Journal of Functional Foods (2015), doi: 10.1016/j.jff.2015.06.017

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