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Nanostructured lipid carriers as a favorable delivery system for β-carotene
Author’s Accepted Manuscript Nanostructured lipid carriers as a favorable delivery system for β-carotene Akram Pezeshki, Hamed Hamishehkar, Babak Ghanbarzadeh, Isa Fathollahy, Fatemeh Keivani Nahr, Maryam Khakbaz Heshmati, Maryam Mohammadi www.elsevier.com/locate/sdj
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S2212-4292(18)30087-7 https://doi.org/10.1016/j.fbio.2018.11.004 FBIO360
To appear in: Food Bioscience Cite this article as: Akram Pezeshki, Hamed Hamishehkar, Babak Ghanbarzadeh, Isa Fathollahy, Fatemeh Keivani Nahr, Maryam Khakbaz Heshmati and Maryam Mohammadi, Nanostructured lipid carriers as a favorable delivery system for βcarotene, Food Bioscience, https://doi.org/10.1016/j.fbio.2018.11.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nanostructured lipid carriers as a favorable delivery system for β-carotene Akram Pezeshki1*, Hamed Hamishehkar2, Babak Ghanbarzadeh1, 3, Isa Fathollahy4, Fatemeh Keivani Nahr1, Maryam Khakbaz Heshmati1, Maryam Mohammadi 1,5 1
Department of Food Science and Technology, Faculty of Agriculture, University of
Tabriz, Tabriz, Iran 2
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
3
Professor of Department of Food Engineering, Faculty of Engineering, Near East University, Cyprus,
Mersin 10, Nicosia, Cyprus, Turkey 4
Department of Food Science and Technology, Mamaghan branch, Islamic Azad University, Mamaghan,
Iran. 5
Biotechnology Research Center and Student Research Committee , Tabriz University of Medical Sciences,
Tabriz, Iran *Corresponding author Phone Number: +989132311958; Tel Number: +984133392033.
[email protected]
1
Abstract
Encapsulation using nano lipid carriers (NLC) is an effective way of protecting sensitive nutraceutical compounds from adverse environmental condition during production and storage. The objectives of the present study were to prepare β-carotene-loaded NLC using hot-high shear homogenization (Hot-HSH) and investigate their particle size, % encapsulation efficiency (%EE), stability and rheology. Poloxamer 407 was used as the surfactant and octyl octanoate and Precirol ATO5 were used as liquid oil and solid lipid, respectively. The optimum formulation was determined using the results of particle size obtained using different surfactant concentration (1, 2, 3 and 4% w/v) and solid lipid: liquid oil ratios (2:1, 4:1 and 10:1). Fourier transform infrared spectra (FTIR) were used to detect any possible bioactive-lipid complex formation and the results showed that there were no chemical interactions between β-carotene and NLC components and β-carotene loaded NLC was simply a physical mixture. The smallest particle size was observed in the formulation containing 2% Poloxamer 407 and solid lipid/liquid oil ratio of 10:1. The %EE of optimal sample was 97.7% (p<0.05) and remained stable for 14 days at 25 oC. Results showed that production of β-carotene-loaded NLC gave nanoscale particles which were stable over time and the established NLC could offer a system for new functional foods based on nanocarriers. Keywords: nano lipid carrier, β-carotene, Precirol ATO5, Polaxamer
2
1. Introduction The use of bioactive components in food products is often limited by the stability of these material, so the production of functional products containing these bioactive components has get a great interest in food industry. β-Carotene, a member of the carotenoids family is a widely used natural pigment in the food industry (Hejri et al., 2013; Hentschel et al., 2008). In addition to its use as a color, β-carotene provides vitamin A activity, antioxidant properties, enhancement of the immune response and protection of the body against cancer and cardiovascular diseases (Lacatusu et al., 2014). However, its use is limited due to its highly hydrophobic nature and difficult incorporation in aqueous-based foods, low bioavailability in the crystalline form, highly reactivity and instability to heat, light and oxygen (Brito-Oliveira et al., 2017). Encapsulation technologies such as polymeric nanoparticles, vesicular systems such as liposomes, niosomes, phytosomes and nanodispersions such as nanoemulsions, solid lipid nanocarriers (SLN), nano lipid carriers (NLC) may overcome these limitations and provide good solubilization and stabilization of lipophilic bioactive material (McClements et al., 2009). NLC can improve bioavailability, membrane permeability, physico-chemical stability and controlled release of core material. Also, no organic solvent is used in the production of these systems and they can be spray-dried and autoclaved (Manjunath et al., 2005). NLC are composed of solid and liquid lipids in which the physical state of the lipid matrix determines the mobility of bioactive material. The liquid oil leads to the formation of imperfect crystals, which provides higher bioactive loading capacity, minimizes expulsion of the bioactive to the outer medium and protects it from adverse environmental conditions (Tikekar and Nitin, 2011; Weiss et al., 2008). NLC are prepared using high shear or high pressure homogenization, microfluidization and sonication because this system is thermodynamically unstable and energy input is needed to generate small particle size (Gomes et al., 2017; McClements and Rao, 2011). 3
Cardamom essential oil (Keivani Nahr et al., 2018), rutin (Babazadeh et al., 2016), krill oil (Zhu et al., 2015) and lycopene (Okonogi and Riangjanapatee, 2015) are some examples of lipophilic bioactive molecules used with NLC. The nanometer diameter of NLC enhances the absorption and bioavailability of bioactive material (Muchow et al., 2008; Porter et al., 2007). Thus, NLC could be a potential carrier for lipophilic β-carotene which might increase its bioavailability, enabling incorporation into aqueous based food products and protecting against degradation in adverse environments (Liu and Wu, 2010; Muchow et al., 2008; Hentschel et al., 2008). The purpose of this study was to encapsulate a labile lipophilic compound, β-carotene, into NLC using hot high-shear homogenization (Hot-HSH) in which the aqueous surfactant solution is added to melted lipid blend (solid lipid and liquid oil + bioactive material) with homogenization. The particle size of NLC systems was studied. Also, the physical stability of the NLC systems was evaluated over a storage period of 60 days and the optimum formulation with minimum particle size and high stability was obtained. Then, encapsulation efficiency (%EE) and the rheological properties of NLC were evaluated and Fourier transform infrared spectroscopy (FTIR) also was used. This gave indications of possible strategies for improvement of the stability and bioavailability of β-carotene for further applications in food formulations. 2. Materials and methods 2.1. Material Precirol ATO5 (glycerol palmito stearate) was obtained from Gattefossè (Saint Periest Cedex, France). β-Carotene, octyl octanoate and Poloxamer 407 were purchased from Sigma Aldrich (Steinheim, Germany). Other chemicals were analytical grade and obtained from Merck Chemical Co. (Darmstadt, Germany). 2.2. Preparation of β-carotene loaded NLC
4
β-Carotene loaded NLC were prepared using hot high-shear homogenization (Hot-HSH) (Mohammadi et al., 2017). Initially, 240 µg β-carotene (C40H56, dark orange crystals, MW=536.89 g·mol−1, p = 0.94 g/cm3 and melting point of 176–184 °C) (Sigma-Aldrich Co.) was melted and mixed with liquid oil (octyl octanoate) and then the mixture was added to the melted solid lipid (Precirol ATO5). The lipid phase was then heated to 70 oC (water bath set point, which was 5 oC higher than the melting point of the solid lipid). Then, the aqueous solutions containing different concentrations of Ploxamer 407 (Table 1) were heated to 70 oC and added drop by drop into the lipid phase while being homogenized (Heidolph Instruments GmbH and Co., Schwabach, Germany) at 20000 rpm. The o/w nanoemulsions were kept at room temperature (22 °C) for recrystallization of the lipid phase to form NLC.
Table 1.
2.3. Fourier transform infrared spectroscopy (FTIR) The infrared spectra were scanned using the FTIR spectrophotometer (IRAffinity-1S, Shimadzo, Tokyo, Japan) with the sample:KBr ratio of 1:10 at 4 cm-1 resolution within the frequency ranges between 4000 and 400 cm-1 (one scan per single outcome.) (Keivani Nahr et al., 2018; Zhu et al., 2015). 2.4. Particle size and size distribution Before measuring the particle size of a system, the system was passed through an Amicon Ultracentrifugal filter (nominal cut-off of 30000 Da) (Sigma-Aldrich Co.) to remove the surfactant micelles. The average volume diameter and particle size distribution of the particles were obtained using a Wing SALD 2101 particle size analyzer (Shimadzo Corp., Tokyo, Japan) at 25 °C. The average particle size was calculated for the DeBroukere mean particle size (Equation 1) and the span value was calculated using Equation 2: 5
D [ 4 ,3 ]
n d n d i
i
Span
4 i
(1)
3 i
D90% D10%
ni: number of particles di: particle mean diameter (2)
D50%
Where D (90%) is the diameter where 90% of particles had a smaller particle size; D (10%) is the diameter where 10% had a smaller particle size and D (50%) is the diameter where 50% had a smaller particle size (Hamishehkar et al., 2009). The experiments were done in triplicate. 2.5. Physical stability Physical stability was investigated by following racing particle size and span value changes and the physical appearance of the optimal NLC formulation during storage at 25 °C for 60 days (Pezeshki at al., 2014). The measurements were done on days 1, 7, 14, 30, 45 and 60 after NLC production. 2.6. %EE and stability of β-carotene The %EE, stability of β-carotene and its release were studied for 14 days of storage using an Ultrospec 2000 spectrophotometer (Scinteck Co., Cambridge, England) at 25 oC at 453 nm. Various concentrations of β-carotene dissolved in chloroform were used to plot the standard curve. To obtain the %EE, one mL of the formulation was added to 2 ml chloroform and the mixture vigorously shaken for 10 min to extract the bioactive from the nanocarrier. Then the absorbance of the organic phase containing β-carotene was determined at 25 oC at 453 nm. The %EE was calculated using Equation 3:
(3) 6
The stability of β-carotene loaded-NLC was determined using the lost of β-carotene from the NLC after 14 days of storage at 25 oC using Equation 4: (4) 2.7. Rheological analysis The rheological characteristics of the formulation of sample one day after preparation with three replications were done at 25 oC using the physica rheometer (MCR 302, Anton Paar GmbH, Graz, Austria) with a concentric cylinder rheometer probe diameter of 2.5 cm for the inner cylinder and 2.7 cm for the external cylinder. To measure the viscosity and shear stress as a function of the shear rate and to determine the flow behavior of the samples, the shear rate increased from 2-100 S-1 within the interval of 10 min in the linear viscoelastic region. 2.8. Statistical analysis Statistical analysis was based on a complete randomized optimization after three repetitions. One-way ANOVA and Duncan’s mean comparison tests were used at 5% significance (p<0.05) with IBM SPSS Statistics for Windows, version 19.0 (IBM Corp., Armonk, NY, USA).
3. Results and discussion 3.1. Particle size and size distribution The effect of surfactant concentration on the average particle size and span value of βcarotene loaded NLC was studied and the results are shown in Fig. 1 a and b. These two parameters have important roles in determining the specifications of colloidal systems. The average size of the particles and particle size distribution (span) of β-carotene loaded NLC were within the range of 79–108 nm and 0.76–1.01, respectively, significant differences were observed in particle size among different formulations (p<0.05). As seen, at first by increasing the surfactant concentration from 1 to 2% in F1 to F2, the particle size 7
decreased and the smallest particle size was observed in formulations containing 2% Poloxamer (w/v). Additional surfactant concentration in F3 and F4 (Table 1) led to a size increase. Increasing the surfactant concentration, the depletion due to flocculation of micelles can increase the particle size. Surfactants in high concentrations form micelles and accumulation of these micelles increased the reported size, which should be discriminated from NLC size (Helgason et al., 2009). In addition, extremely high concentrations of surfactant reduce the efficiency of homogenization (thickening of the system, unplanned distribution and non-uniform distribution of force, resulting in the production of fewer particles (on a nanoscale with uniform distribution) and increasing particle size (Pezeshki et al., 2014). In dispersions, the particle size and physical stability depends on the type of surfactant, which leads to repulsion between particles and inhibits particle aggregation. As the results show, the surfactant concentration had a significant effect on the final size of NLC particles. Insufficient surfactant concentrations leads to instability of NLC (Kovačević et al., 2014; Kovacevic et al., 2011). However, high amounts of surfactant decrease the surface tension and produce smaller particles (Yang et al., 2014; Shegokar and Müller, 2010). In NLC, surfactants also control the crystallization of the lipid matrix (Bilia et al., 2014). Poloxamer 407 was used as a hydrophilic nonionic surfactant, in which the hydrophobic polypropylene oxide (PPO) chains as the “anchor chain” are adsorbed on the particle surfaces while hydrophilic copolymers of polyethylene oxide (PEO) chains are in the aqueous medium and a stabilizer layer is formed (Pezeshki at al., 2014; Bilia et al., 2014). The smallest span of β-carotene loaded NLC was observed in F2 and F4 showed the highest span value (Fig. 1a). Increasing the concentration of surfactant from 2 to 4% increased the span value of the system, significantly (p<0.05). The span values of lower than 1, a lower polydispersity of the system (Babazadeh et al., 2016).
8
[Fig. 1]
3.2. FTIR The FTIR spectra of β-carotene and NLC formulations were studied to estimate any possible chemical interactions among β-carotene and the carrier components (Fig. 2). The FTIR spectrum of pure β-carotene showed absorption bands at 1372 (CH3), 1475 (CH2), 1587–1680 (C=C) and 1100 cm−1 (C-O). Blank NLC and β-carotene-loaded NLC showed all peaks related to functional groups of β-carotene, except the C=C stretching peak that only was in the FTIR spectrum of β-carotene-loaded NLC, indicating physical entrapment of βcarotene in the lipid matrix. Also, the β-carotene-loaded NLC spectrum showed all the characteristic peaks of β-carotene and blank NLC, no significant change in the wavenumber of the existing peaks or formation of new peaks was found. Similar outcomes were also obtained for other nutraceuticals, Pezeshki et al. (2014) studied vitamin A palmitate-loaded NLC; Babazadeh et al. (2016) rutin NLC and Fathi and Varshosaz (2013) hesperetin NLC. FTIR analysis showed no chemical interactions among the nutraceuticals and excipients and only physical interactions.
[Fig. 2]
3.3. The effect of lipid/oil ratio on the production of β-carotene loaded NLC Selection of the proper ratio of lipid or oil, has an important role in the crystal structure and stability of nano particles (Kulkarni et al., 2016). The results showed that the use of a solid lipid/liquid oil ratio of 10 to 1, compared to other ratios led to a narrower particle size distribution, and the system was uniform and stable (Fig. 3). In low concentrations of liquid oil, oil molecules spread in the solid lipid matrix and the NLC would have a partial structure made up of a mixture of lipids (Tamjidi, et al., 2013). 9
In formulations containing higher concentrations of liquid oil, the incompatibility between liquid oil and solid lipid leads to phase separation during cooling and increases the size distribution of particles (Rao et al., 2013).
[Fig. 3]
3.4. Physical stability The particle size and its distribution in colloidal systems have important roles in the physiochemical properties of the systems such as physical and chemical stability, solubility, release rate and turbidity. Monitoring the changes in particle size of colloidal systems is the best way to determine the physical stability of the system (Tamjidi et al., 2013). Fig. 4 a and b show the effect of storage time on the particle size and span value of βcarotene loaded NLC (optimized formulation (F2)). Size of optimized β-carotene loaded formulation (F2) remained under 100 nm and did not change significantly at 25 oC after 60 days of storage. However, the span value increased and suggested less homogeneity. Babazadeh et al. (2017) observed significant increases in particle size and span value during storage of rutin-loaded NLC after 60 days at 25 °C. Zhu et al. (2015) observed a good physical stability of krill oil loaded NLC and the particle size was still acceptable on the 70th day of storage. Sun et al. (2014) observed the particle size distribution of quercetin loaded NLC remained under 0.3 after 28 days of storage at 4 oC which was not in accordance with the results of this research. Given that, breaking of the hydrogen bonds of surfactants at high temperature may reduce the physical stability of NLC (Sun et al., 2014), low temperatures are preferred to maintain the physical stability of NLC (Zhu et al. 2015).
[Fig. 4] 10
3.5. %EE and physical stability The %EE is associated with the specification of encapsulated material, the nano carrier constituents and the environmental factors (pH and temperature). Results showed the %EE of β-carotene in the optimal formulation of NLC with concentration of 2% Poloxamer was 97.7% (p<0.05). Its sustainability in the system and its release over a period of 14 days suggested that it remained in the NLC system and also released 8.51% of β-carotene. Due to the lipophilic structure of β-carotene, it could be used in the lipid matrix of nano particles, which is encircled with a Ploxamer layer and protect the active compound from destruction. The results suggested that an optimum concentration of 2% Poloxamer is sufficient to cover the surface of nanoparticles efficiently and %EE can be high with this concentration. In the NLC system, surfactant concentration had an important role in EE% and stability of nanoparticles. Solid particles are disc-like or needle-like and required sufficient amounts of surfactant to cover the new surfaces of the solid particles. Indeed, at higher surfactant concentrations, enough surfactant was present to cover the small lipids and prevent them from coalescence, thus the encapsulated material was preserved in the NLC (Pezeshki et al., 2014). Other researches were done with carotenoids especially βcarotene encapsulation in various nanocarrier systems and showed high %EE and stability of β-carotene. Encapsulation of β-carotene in nanoemulsions (Qian et al., 2012) and in alginate–based hydrogel beads (Zhang et al., 2016), carotenoid in liposome (Tan et al., 2016), β-carotene (Hentschel et al., 2008; Lacatusu et al., 2012), lycopene (Okonogi and Riangjanapatee, 2015) and vitamin A palmitate (Pezeshki et al., 2014) in loaded NLC are some examples.
3.6. Rheological properties of the NLC
11
The relationship between shear rate and viscosity of the optimum NLC formulation and also shear stress as a function of the shear rate was measured to determine the flow behavior of samples, at 10 min shear rate 2–100 S-1. There was a relatively linear relationship between shear stress and shear rate (Fig. 5). This behavior showed the simplest flow behavior of solutions, the Newtonian behavior. The viscosity of the fluid is measured from the slope of the shear stress–shear rate curve. By increasing the viscosity of the solution, the slope of the curve increases. The optimal NLC system had a uniform nanoscaled particle size distribution and was clot-free. The presence of Ploxamer as a thick viscoelastic layer around each of the lipid nanoparticles, as well as crystallization of the solid lipid and liquid oil phases in combination, increased the viscosity of system. [Fig. 5]
4. Conclusion Characterizes of the β-carotene-loaded NLC were studied in this research. FTIR spectroscopy confirmed the incorporation of β-carotene in NLC components and the formation of physical interactions among them. A certain surfactant concentration and an adequate lipid/oil ratio are required to produce stable nanostructured lipid carriers. The results suggest that an optimum concentration of 2% Poloxamer is sufficient to cover the surface of nanoparticles efficiently and avoid aggregation during homogenization, while statistically there was no difference among the results of 1, 2, 3, and 4 % Poloxamer. Adequate concentrations of surfactant-induced surfaces of particles well reduces aggregation among particles. Also, a 10 to 1 solid lipid/liquid oil ratio is suitable for producing stable nanoparticles. The lipophilic nature of β-carotene prevents the removal of active substances from the lipid matrix of the nanoparticles. Aknowledgment The support of the Drug Applied Research Center is greatly appreciated. 12
Conflict of Interest statement The authors have no conflict of interest. References Babazadeh A, Ghanbarzadeh B, Hamishehkar H (2016) Novel nanostructured lipid carriers as a promising food grade delivery system for rutin. J Funct Foods 26 (7):167-175 doi:http://dx.doi.org/10.1016/j.jff.2016.07.017 Babazadeh A, Ghanbarzadeh B, Hamishehkar H (2017) Formulation of food grade nanostructured lipid carrier (NLC) for potential applications in medicinal-functional foods. J Drug Deliv Sci Technol 39 (3):50-58 Bilia AR, Isacchi B, Righeschi C, Guccione C, Bergonzi MC (2014) Flavonoids loaded in nanocarriers: An opportunity to increase oral bioavailability and bioefficacy. Food Nutr Sci 5 (13):1212 Brito-oliveira TC, Molina CV, Netto FM, Pinho SC (2017) Encapsulation of β‐carotene in lipid microparticles stabilized with hydrolyzed soy protein isolate: Production parameters, alphatocopherol coencapsulation and stability under stress conditions. J Food Sci 82(3):659-669 Fathi M, Varshosaz J (2013) Novel hesperetin loaded nanocarriers for food fortification: Production and characterization. J Funct Foods 5 (3):1382-1391 Gomes GVL, Sola MR, Marostegan LFP, Jange CG, Cazado CPS, Pinheiro AC, Vicente AA, Pinho SC (2017) Physico-chemical stability and in vitro digestibility of β-carotene-loaded lipid nanoparticles of cupuacu butter (Theobroma grandiflorum) produced by the phase inversion temperature (PIT) method. J Food Eng 192 (1):93-102 doi:http://dx.doi.org/10.1016/j.jfoodeng.2016.08.001 Hamishehkar H, Emami J, Najafabadi AR, Gilani K, Minaiyan M, Mahdavi H, Nokhodchi A (2009) The effect of formulation variables on the characteristics of insulin-loaded poly (lactic-coglycolic acid) microspheres prepared by a single phase oil in oil solvent evaporation method. Colloids Surf B Biointerfaces 74 (1):340-349 13
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Table 1: Composition of β-carotene-loaded nanostructured lipid carriers Solid Lipid
Liquid oil
Surfactant
Precirol ATO5 (w/v)
octyl octanoate (w/v)
Poloxamer (w/v)
F1
2%
0.2%
1%
F2
2%
0.2%
2%
F3
2%
0.2%
3%
F4
2%
0.2%
4%
Formulation
17
t
Caption of Figures Figure 1: Effect of surfactant concentration on the (a) particle size and particle size distribution and (b) span value of β-carotene loaded NLC. Span was the range between 10 and 90% of the mean diameter. (Different letters indicate significant differences at p<0.05). Figure 2: FTIR spectra of (a) β-carotene and (b) blank NLC and β-carotene loaded NLC. Figure 3: Effect of lipid/oil ratio on the (a) particle size and (b) particle size distribution (span) of β-carotene NLC. (Different letters indicate significant differences at p<0.05). Figure 4: Stability of (a) particle size and (b) particle size distribution of optimized formulation of β-carotene NLC (formulation containing 2% w/v Poloxamer 407 solution, Precirol ATO5 2% (w/v) and β-carotene-contained oil-phase octyl octanoate (0.02%w/v)) during 60 days of storage. (Different letters indicate significant differences at p<0.05). Figure 5: (A) Shear stress–shear rate and (B) viscosity-shear rate of optimal formulation (2% w/v surfactant concentration, 10 to 1 solid lipid/liquid oil ratio) of NLC.
18
Figures:
D (4,3) nm
(a)
span
(b)
Fig 1.
19
(a)
(b)
Fig 2.
20
(a)
(b)
Fig 3.
21
(a)
(b)
Fig 4.
22
Fig 5.
23
PII: DOI: Reference:
S2212-4292(18)30087-7 https://doi.org/10.1016/j.fbio.2018.11.004 FBIO360
To appear in: Food Bioscience Cite this article as: Akram Pezeshki, Hamed Hamishehkar, Babak Ghanbarzadeh, Isa Fathollahy, Fatemeh Keivani Nahr, Maryam Khakbaz Heshmati and Maryam Mohammadi, Nanostructured lipid carriers as a favorable delivery system for βcarotene, Food Bioscience, https://doi.org/10.1016/j.fbio.2018.11.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nanostructured lipid carriers as a favorable delivery system for β-carotene Akram Pezeshki1*, Hamed Hamishehkar2, Babak Ghanbarzadeh1, 3, Isa Fathollahy4, Fatemeh Keivani Nahr1, Maryam Khakbaz Heshmati1, Maryam Mohammadi 1,5 1
Department of Food Science and Technology, Faculty of Agriculture, University of
Tabriz, Tabriz, Iran 2
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
3
Professor of Department of Food Engineering, Faculty of Engineering, Near East University, Cyprus,
Mersin 10, Nicosia, Cyprus, Turkey 4
Department of Food Science and Technology, Mamaghan branch, Islamic Azad University, Mamaghan,
Iran. 5
Biotechnology Research Center and Student Research Committee , Tabriz University of Medical Sciences,
Tabriz, Iran *Corresponding author Phone Number: +989132311958; Tel Number: +984133392033.
[email protected]
1
Abstract
Encapsulation using nano lipid carriers (NLC) is an effective way of protecting sensitive nutraceutical compounds from adverse environmental condition during production and storage. The objectives of the present study were to prepare β-carotene-loaded NLC using hot-high shear homogenization (Hot-HSH) and investigate their particle size, % encapsulation efficiency (%EE), stability and rheology. Poloxamer 407 was used as the surfactant and octyl octanoate and Precirol ATO5 were used as liquid oil and solid lipid, respectively. The optimum formulation was determined using the results of particle size obtained using different surfactant concentration (1, 2, 3 and 4% w/v) and solid lipid: liquid oil ratios (2:1, 4:1 and 10:1). Fourier transform infrared spectra (FTIR) were used to detect any possible bioactive-lipid complex formation and the results showed that there were no chemical interactions between β-carotene and NLC components and β-carotene loaded NLC was simply a physical mixture. The smallest particle size was observed in the formulation containing 2% Poloxamer 407 and solid lipid/liquid oil ratio of 10:1. The %EE of optimal sample was 97.7% (p<0.05) and remained stable for 14 days at 25 oC. Results showed that production of β-carotene-loaded NLC gave nanoscale particles which were stable over time and the established NLC could offer a system for new functional foods based on nanocarriers. Keywords: nano lipid carrier, β-carotene, Precirol ATO5, Polaxamer
2
1. Introduction The use of bioactive components in food products is often limited by the stability of these material, so the production of functional products containing these bioactive components has get a great interest in food industry. β-Carotene, a member of the carotenoids family is a widely used natural pigment in the food industry (Hejri et al., 2013; Hentschel et al., 2008). In addition to its use as a color, β-carotene provides vitamin A activity, antioxidant properties, enhancement of the immune response and protection of the body against cancer and cardiovascular diseases (Lacatusu et al., 2014). However, its use is limited due to its highly hydrophobic nature and difficult incorporation in aqueous-based foods, low bioavailability in the crystalline form, highly reactivity and instability to heat, light and oxygen (Brito-Oliveira et al., 2017). Encapsulation technologies such as polymeric nanoparticles, vesicular systems such as liposomes, niosomes, phytosomes and nanodispersions such as nanoemulsions, solid lipid nanocarriers (SLN), nano lipid carriers (NLC) may overcome these limitations and provide good solubilization and stabilization of lipophilic bioactive material (McClements et al., 2009). NLC can improve bioavailability, membrane permeability, physico-chemical stability and controlled release of core material. Also, no organic solvent is used in the production of these systems and they can be spray-dried and autoclaved (Manjunath et al., 2005). NLC are composed of solid and liquid lipids in which the physical state of the lipid matrix determines the mobility of bioactive material. The liquid oil leads to the formation of imperfect crystals, which provides higher bioactive loading capacity, minimizes expulsion of the bioactive to the outer medium and protects it from adverse environmental conditions (Tikekar and Nitin, 2011; Weiss et al., 2008). NLC are prepared using high shear or high pressure homogenization, microfluidization and sonication because this system is thermodynamically unstable and energy input is needed to generate small particle size (Gomes et al., 2017; McClements and Rao, 2011). 3
Cardamom essential oil (Keivani Nahr et al., 2018), rutin (Babazadeh et al., 2016), krill oil (Zhu et al., 2015) and lycopene (Okonogi and Riangjanapatee, 2015) are some examples of lipophilic bioactive molecules used with NLC. The nanometer diameter of NLC enhances the absorption and bioavailability of bioactive material (Muchow et al., 2008; Porter et al., 2007). Thus, NLC could be a potential carrier for lipophilic β-carotene which might increase its bioavailability, enabling incorporation into aqueous based food products and protecting against degradation in adverse environments (Liu and Wu, 2010; Muchow et al., 2008; Hentschel et al., 2008). The purpose of this study was to encapsulate a labile lipophilic compound, β-carotene, into NLC using hot high-shear homogenization (Hot-HSH) in which the aqueous surfactant solution is added to melted lipid blend (solid lipid and liquid oil + bioactive material) with homogenization. The particle size of NLC systems was studied. Also, the physical stability of the NLC systems was evaluated over a storage period of 60 days and the optimum formulation with minimum particle size and high stability was obtained. Then, encapsulation efficiency (%EE) and the rheological properties of NLC were evaluated and Fourier transform infrared spectroscopy (FTIR) also was used. This gave indications of possible strategies for improvement of the stability and bioavailability of β-carotene for further applications in food formulations. 2. Materials and methods 2.1. Material Precirol ATO5 (glycerol palmito stearate) was obtained from Gattefossè (Saint Periest Cedex, France). β-Carotene, octyl octanoate and Poloxamer 407 were purchased from Sigma Aldrich (Steinheim, Germany). Other chemicals were analytical grade and obtained from Merck Chemical Co. (Darmstadt, Germany). 2.2. Preparation of β-carotene loaded NLC
4
β-Carotene loaded NLC were prepared using hot high-shear homogenization (Hot-HSH) (Mohammadi et al., 2017). Initially, 240 µg β-carotene (C40H56, dark orange crystals, MW=536.89 g·mol−1, p = 0.94 g/cm3 and melting point of 176–184 °C) (Sigma-Aldrich Co.) was melted and mixed with liquid oil (octyl octanoate) and then the mixture was added to the melted solid lipid (Precirol ATO5). The lipid phase was then heated to 70 oC (water bath set point, which was 5 oC higher than the melting point of the solid lipid). Then, the aqueous solutions containing different concentrations of Ploxamer 407 (Table 1) were heated to 70 oC and added drop by drop into the lipid phase while being homogenized (Heidolph Instruments GmbH and Co., Schwabach, Germany) at 20000 rpm. The o/w nanoemulsions were kept at room temperature (22 °C) for recrystallization of the lipid phase to form NLC.
Table 1.
2.3. Fourier transform infrared spectroscopy (FTIR) The infrared spectra were scanned using the FTIR spectrophotometer (IRAffinity-1S, Shimadzo, Tokyo, Japan) with the sample:KBr ratio of 1:10 at 4 cm-1 resolution within the frequency ranges between 4000 and 400 cm-1 (one scan per single outcome.) (Keivani Nahr et al., 2018; Zhu et al., 2015). 2.4. Particle size and size distribution Before measuring the particle size of a system, the system was passed through an Amicon Ultracentrifugal filter (nominal cut-off of 30000 Da) (Sigma-Aldrich Co.) to remove the surfactant micelles. The average volume diameter and particle size distribution of the particles were obtained using a Wing SALD 2101 particle size analyzer (Shimadzo Corp., Tokyo, Japan) at 25 °C. The average particle size was calculated for the DeBroukere mean particle size (Equation 1) and the span value was calculated using Equation 2: 5
D [ 4 ,3 ]
n d n d i
i
Span
4 i
(1)
3 i
D90% D10%
ni: number of particles di: particle mean diameter (2)
D50%
Where D (90%) is the diameter where 90% of particles had a smaller particle size; D (10%) is the diameter where 10% had a smaller particle size and D (50%) is the diameter where 50% had a smaller particle size (Hamishehkar et al., 2009). The experiments were done in triplicate. 2.5. Physical stability Physical stability was investigated by following racing particle size and span value changes and the physical appearance of the optimal NLC formulation during storage at 25 °C for 60 days (Pezeshki at al., 2014). The measurements were done on days 1, 7, 14, 30, 45 and 60 after NLC production. 2.6. %EE and stability of β-carotene The %EE, stability of β-carotene and its release were studied for 14 days of storage using an Ultrospec 2000 spectrophotometer (Scinteck Co., Cambridge, England) at 25 oC at 453 nm. Various concentrations of β-carotene dissolved in chloroform were used to plot the standard curve. To obtain the %EE, one mL of the formulation was added to 2 ml chloroform and the mixture vigorously shaken for 10 min to extract the bioactive from the nanocarrier. Then the absorbance of the organic phase containing β-carotene was determined at 25 oC at 453 nm. The %EE was calculated using Equation 3:
(3) 6
The stability of β-carotene loaded-NLC was determined using the lost of β-carotene from the NLC after 14 days of storage at 25 oC using Equation 4: (4) 2.7. Rheological analysis The rheological characteristics of the formulation of sample one day after preparation with three replications were done at 25 oC using the physica rheometer (MCR 302, Anton Paar GmbH, Graz, Austria) with a concentric cylinder rheometer probe diameter of 2.5 cm for the inner cylinder and 2.7 cm for the external cylinder. To measure the viscosity and shear stress as a function of the shear rate and to determine the flow behavior of the samples, the shear rate increased from 2-100 S-1 within the interval of 10 min in the linear viscoelastic region. 2.8. Statistical analysis Statistical analysis was based on a complete randomized optimization after three repetitions. One-way ANOVA and Duncan’s mean comparison tests were used at 5% significance (p<0.05) with IBM SPSS Statistics for Windows, version 19.0 (IBM Corp., Armonk, NY, USA).
3. Results and discussion 3.1. Particle size and size distribution The effect of surfactant concentration on the average particle size and span value of βcarotene loaded NLC was studied and the results are shown in Fig. 1 a and b. These two parameters have important roles in determining the specifications of colloidal systems. The average size of the particles and particle size distribution (span) of β-carotene loaded NLC were within the range of 79–108 nm and 0.76–1.01, respectively, significant differences were observed in particle size among different formulations (p<0.05). As seen, at first by increasing the surfactant concentration from 1 to 2% in F1 to F2, the particle size 7
decreased and the smallest particle size was observed in formulations containing 2% Poloxamer (w/v). Additional surfactant concentration in F3 and F4 (Table 1) led to a size increase. Increasing the surfactant concentration, the depletion due to flocculation of micelles can increase the particle size. Surfactants in high concentrations form micelles and accumulation of these micelles increased the reported size, which should be discriminated from NLC size (Helgason et al., 2009). In addition, extremely high concentrations of surfactant reduce the efficiency of homogenization (thickening of the system, unplanned distribution and non-uniform distribution of force, resulting in the production of fewer particles (on a nanoscale with uniform distribution) and increasing particle size (Pezeshki et al., 2014). In dispersions, the particle size and physical stability depends on the type of surfactant, which leads to repulsion between particles and inhibits particle aggregation. As the results show, the surfactant concentration had a significant effect on the final size of NLC particles. Insufficient surfactant concentrations leads to instability of NLC (Kovačević et al., 2014; Kovacevic et al., 2011). However, high amounts of surfactant decrease the surface tension and produce smaller particles (Yang et al., 2014; Shegokar and Müller, 2010). In NLC, surfactants also control the crystallization of the lipid matrix (Bilia et al., 2014). Poloxamer 407 was used as a hydrophilic nonionic surfactant, in which the hydrophobic polypropylene oxide (PPO) chains as the “anchor chain” are adsorbed on the particle surfaces while hydrophilic copolymers of polyethylene oxide (PEO) chains are in the aqueous medium and a stabilizer layer is formed (Pezeshki at al., 2014; Bilia et al., 2014). The smallest span of β-carotene loaded NLC was observed in F2 and F4 showed the highest span value (Fig. 1a). Increasing the concentration of surfactant from 2 to 4% increased the span value of the system, significantly (p<0.05). The span values of lower than 1, a lower polydispersity of the system (Babazadeh et al., 2016).
8
[Fig. 1]
3.2. FTIR The FTIR spectra of β-carotene and NLC formulations were studied to estimate any possible chemical interactions among β-carotene and the carrier components (Fig. 2). The FTIR spectrum of pure β-carotene showed absorption bands at 1372 (CH3), 1475 (CH2), 1587–1680 (C=C) and 1100 cm−1 (C-O). Blank NLC and β-carotene-loaded NLC showed all peaks related to functional groups of β-carotene, except the C=C stretching peak that only was in the FTIR spectrum of β-carotene-loaded NLC, indicating physical entrapment of βcarotene in the lipid matrix. Also, the β-carotene-loaded NLC spectrum showed all the characteristic peaks of β-carotene and blank NLC, no significant change in the wavenumber of the existing peaks or formation of new peaks was found. Similar outcomes were also obtained for other nutraceuticals, Pezeshki et al. (2014) studied vitamin A palmitate-loaded NLC; Babazadeh et al. (2016) rutin NLC and Fathi and Varshosaz (2013) hesperetin NLC. FTIR analysis showed no chemical interactions among the nutraceuticals and excipients and only physical interactions.
[Fig. 2]
3.3. The effect of lipid/oil ratio on the production of β-carotene loaded NLC Selection of the proper ratio of lipid or oil, has an important role in the crystal structure and stability of nano particles (Kulkarni et al., 2016). The results showed that the use of a solid lipid/liquid oil ratio of 10 to 1, compared to other ratios led to a narrower particle size distribution, and the system was uniform and stable (Fig. 3). In low concentrations of liquid oil, oil molecules spread in the solid lipid matrix and the NLC would have a partial structure made up of a mixture of lipids (Tamjidi, et al., 2013). 9
In formulations containing higher concentrations of liquid oil, the incompatibility between liquid oil and solid lipid leads to phase separation during cooling and increases the size distribution of particles (Rao et al., 2013).
[Fig. 3]
3.4. Physical stability The particle size and its distribution in colloidal systems have important roles in the physiochemical properties of the systems such as physical and chemical stability, solubility, release rate and turbidity. Monitoring the changes in particle size of colloidal systems is the best way to determine the physical stability of the system (Tamjidi et al., 2013). Fig. 4 a and b show the effect of storage time on the particle size and span value of βcarotene loaded NLC (optimized formulation (F2)). Size of optimized β-carotene loaded formulation (F2) remained under 100 nm and did not change significantly at 25 oC after 60 days of storage. However, the span value increased and suggested less homogeneity. Babazadeh et al. (2017) observed significant increases in particle size and span value during storage of rutin-loaded NLC after 60 days at 25 °C. Zhu et al. (2015) observed a good physical stability of krill oil loaded NLC and the particle size was still acceptable on the 70th day of storage. Sun et al. (2014) observed the particle size distribution of quercetin loaded NLC remained under 0.3 after 28 days of storage at 4 oC which was not in accordance with the results of this research. Given that, breaking of the hydrogen bonds of surfactants at high temperature may reduce the physical stability of NLC (Sun et al., 2014), low temperatures are preferred to maintain the physical stability of NLC (Zhu et al. 2015).
[Fig. 4] 10
3.5. %EE and physical stability The %EE is associated with the specification of encapsulated material, the nano carrier constituents and the environmental factors (pH and temperature). Results showed the %EE of β-carotene in the optimal formulation of NLC with concentration of 2% Poloxamer was 97.7% (p<0.05). Its sustainability in the system and its release over a period of 14 days suggested that it remained in the NLC system and also released 8.51% of β-carotene. Due to the lipophilic structure of β-carotene, it could be used in the lipid matrix of nano particles, which is encircled with a Ploxamer layer and protect the active compound from destruction. The results suggested that an optimum concentration of 2% Poloxamer is sufficient to cover the surface of nanoparticles efficiently and %EE can be high with this concentration. In the NLC system, surfactant concentration had an important role in EE% and stability of nanoparticles. Solid particles are disc-like or needle-like and required sufficient amounts of surfactant to cover the new surfaces of the solid particles. Indeed, at higher surfactant concentrations, enough surfactant was present to cover the small lipids and prevent them from coalescence, thus the encapsulated material was preserved in the NLC (Pezeshki et al., 2014). Other researches were done with carotenoids especially βcarotene encapsulation in various nanocarrier systems and showed high %EE and stability of β-carotene. Encapsulation of β-carotene in nanoemulsions (Qian et al., 2012) and in alginate–based hydrogel beads (Zhang et al., 2016), carotenoid in liposome (Tan et al., 2016), β-carotene (Hentschel et al., 2008; Lacatusu et al., 2012), lycopene (Okonogi and Riangjanapatee, 2015) and vitamin A palmitate (Pezeshki et al., 2014) in loaded NLC are some examples.
3.6. Rheological properties of the NLC
11
The relationship between shear rate and viscosity of the optimum NLC formulation and also shear stress as a function of the shear rate was measured to determine the flow behavior of samples, at 10 min shear rate 2–100 S-1. There was a relatively linear relationship between shear stress and shear rate (Fig. 5). This behavior showed the simplest flow behavior of solutions, the Newtonian behavior. The viscosity of the fluid is measured from the slope of the shear stress–shear rate curve. By increasing the viscosity of the solution, the slope of the curve increases. The optimal NLC system had a uniform nanoscaled particle size distribution and was clot-free. The presence of Ploxamer as a thick viscoelastic layer around each of the lipid nanoparticles, as well as crystallization of the solid lipid and liquid oil phases in combination, increased the viscosity of system. [Fig. 5]
4. Conclusion Characterizes of the β-carotene-loaded NLC were studied in this research. FTIR spectroscopy confirmed the incorporation of β-carotene in NLC components and the formation of physical interactions among them. A certain surfactant concentration and an adequate lipid/oil ratio are required to produce stable nanostructured lipid carriers. The results suggest that an optimum concentration of 2% Poloxamer is sufficient to cover the surface of nanoparticles efficiently and avoid aggregation during homogenization, while statistically there was no difference among the results of 1, 2, 3, and 4 % Poloxamer. Adequate concentrations of surfactant-induced surfaces of particles well reduces aggregation among particles. Also, a 10 to 1 solid lipid/liquid oil ratio is suitable for producing stable nanoparticles. The lipophilic nature of β-carotene prevents the removal of active substances from the lipid matrix of the nanoparticles. Aknowledgment The support of the Drug Applied Research Center is greatly appreciated. 12
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Table 1: Composition of β-carotene-loaded nanostructured lipid carriers Solid Lipid
Liquid oil
Surfactant
Precirol ATO5 (w/v)
octyl octanoate (w/v)
Poloxamer (w/v)
F1
2%
0.2%
1%
F2
2%
0.2%
2%
F3
2%
0.2%
3%
F4
2%
0.2%
4%
Formulation
17
t
Caption of Figures Figure 1: Effect of surfactant concentration on the (a) particle size and particle size distribution and (b) span value of β-carotene loaded NLC. Span was the range between 10 and 90% of the mean diameter. (Different letters indicate significant differences at p<0.05). Figure 2: FTIR spectra of (a) β-carotene and (b) blank NLC and β-carotene loaded NLC. Figure 3: Effect of lipid/oil ratio on the (a) particle size and (b) particle size distribution (span) of β-carotene NLC. (Different letters indicate significant differences at p<0.05). Figure 4: Stability of (a) particle size and (b) particle size distribution of optimized formulation of β-carotene NLC (formulation containing 2% w/v Poloxamer 407 solution, Precirol ATO5 2% (w/v) and β-carotene-contained oil-phase octyl octanoate (0.02%w/v)) during 60 days of storage. (Different letters indicate significant differences at p<0.05). Figure 5: (A) Shear stress–shear rate and (B) viscosity-shear rate of optimal formulation (2% w/v surfactant concentration, 10 to 1 solid lipid/liquid oil ratio) of NLC.
18
Figures:
D (4,3) nm
(a)
span
(b)
Fig 1.
19
(a)
(b)
Fig 2.
20
(a)
(b)
Fig 3.
21
(a)
(b)
Fig 4.
22
Fig 5.
23