Conjugated linoleic acid loaded nanostructured lipid carrier as a potential antioxidant nanocarrier for food applications

Abstract The encapsulation of fatty acids in nanocarrier systems is a very effective technique in improving their biological efficiency and controlled delivery. Nanostructured lipid carrier (NLC) is a major type of lipid‐based nanoparticle. This study is focused on producing nanolipid carrier containing conjugated linoleic acid and fortifying low‐fat milk using this nanoparticle. Nanostructured lipid carriers were produced by hot high‐shear homogenization containing 1.5% Poloxamer 407, cocoa butter as solid lipid, and conjugated linoleic acid as liquid oil in ratio of 10:1. Results showed that the nanoparticles sized 81 nm with monomodular dispersity and the system was stable at 4 and 22°C for 40 days. Zeta potential and encapsulation efficiency (%EE) were −15.8 mV and 98.2%, respectively. Scanning electron microscopy (SEM) showed that the particles are in spiral form and small size and no significant aggregation was observed because of few changes in the system turbidity after storage time. The result of oxidative stability showed that using Nanostructured lipid carriers system resulted in lower malone dialdehyde production. Conjugated linoleic acid was protected at level of 3.9% of milk fatty acids in Nanostructured lipid carrier formulation during storage time. Based on these findings, Nanostructured lipid carriers system is an appropriate and stable nanocarrier system for delivery of nutraceuticals in foods and can be used in protecting them against oxidation, heating, and other processes in order to fortify foods and beverages.

influences processing of raw material and is a carrier of taste and aroma (Cieslak, Kowalczyk, Czauderna, Potkanski, & Szumacher-Strabel, 2010;Jozwik, Strzalkowska, Bagnicka, Polawska, & Horbanczuk, 2010;Strzalkowska et al., 2010). The linoleic acid (LA) present in milk is known as a potential anticarcinogen, which can be controlled through diet management (Morsy et al., 2018). Diet is the major source of milk CLA and many trials have been conducted with the aim of enhancing milk CLA content (Tripathi, 2014). CLA refers to a group of positional and geometric isomers of linoleic acid that are characterized by the presence of conjugated dienes. CLA is a natural, but minor, component of fats from ruminant animals that enters the human diet primarily through meat and dairy products (Whigham, Watras, & Schoeller, 2007). CLA is considered as an essential fatty acid which represents a group of linoleic acid isomers (18:2) each with intensive biological functions such as anticancer, anti-obesity, and anti-hypertension (MacDonald, 2000). Drug delivery systems have opened new avenues to improve the therapeutic effects of already-efficient molecules. Some drugs are poorly soluble in water and cannot be administered unless they are encapsulated as drug carriers. In other occasions, drugs cannot permeate cell membranes and as a consequence the concentration at the target site is insufficient. To overcome this, high doses of drugs are required, causing high toxicity and many undesired side effects (Limeres, Moretton, Bernabeu, Chiappetta, & Cuestas, 2019). Consequently, a targeted drug delivery system could selectively carry sufficient drug concentrations into the targeted tissue (or cell) improving its bioavailability and reducing the associated side effects due to high doses (Bayon-Cordero, Alkorta, & Arana, 2019).
Carriers are engineered vectors, which retain drugs either on the cell surface or in a subcellular compartment via physical or chemical interaction, encapsulation, and spacer moiety (Alavizadeh, Akhtari, Badiee, Golmohammadzadeh, & Jaafari, 2016).
One of the nanocarriers is nanostructured lipid carrier (NLC) with a unique structure and specific benefits such as high encapsulation efficiency (EE), stability against gravitational separation (due to highly particle density) and low release rate (Beloqui, Solinis, Rodriguez-Gascon, Almeida, & Preat, 2016;Brar & Verma, 2011). In order to compensate the lack of unsaturated fatty acids in foods, encapsulation by NLCs would be recommended. Lipid nanoparticles have many advantages in comparison to other particulate systems including ease of large scale production (Luo, Chen, Ren, Zhao, & Qin, 2006), biocompatible and biodegradable nature of the materials (Silva et al., 2011), low toxicity potential (Ghasemiyeh & Mohammadi-Samani, 2018), possibility of controlled and modified drug release, drug solubility enhancement and the possibility of both hydrophilic and lipophilic drug incorporation (Zur Muhlen, Schwarz, & Mehnert, 1998). NLCs have remarkably wide range of properties which make them useful for parenteral, dermal, pulmonary, and topical delivery of drugs. These products have been developed in order to reduce toxic side effects of the incorporated highly potent drugs and increase the efficacy of the treatment (Naseri, Valizadeh, & Zakeri-Milani, 2015). NLCs are second generation of lipid-based nanocarriers formed from mixture of solid and liquid lipids and have unstructured matrix due to the different moieties of the constituents of NLCs (Beloqui et al., 2016). Use of nutraceuticals compounds in food products is often restricted by the low stability of them against environment condition. So for human health, food fortification is very crucial (Karimi, Ghanbarzadeh, Hamishehkar, Mehramuz, & SamadiKafil, 2018;Pezeshky, Ghanbarzadeh, Hamishehkar, Moghadam, & Babazadeh, 2016) and production of functional ingredients has get an abundant attention in food industry. In recent years, using nanocarriers is a positive approach for food fortification, which can be effective in delivery and stability of hydrophobic compounds (Fathi & Varshosaz, 2013;Gonnet, Lethuaut, & Boury, 2010), also during the digestion nanocarriers offer higher bioavailability and inhibit the developement of off-flavor and off-color of the food (Mehmood, 2015;Sagalowicz & Leser, 2010;Shukla et al., 2014), The purpose of this study was to encapsulate a labile lipophilic compound, CLA, into NLC using hot high-shear homogenization (Hot-HSH) The particle size of NLC systems and physical stability of the NLC systems was evaluated over a storage period of 60 days and the encapsulation efficiency (EE %) and loading capacity (LC, %) were obtained. In final survey, the functional properties of CLA-NLC in transferring, releasing, and protecting CLA in pasteurized low-fat milk were studied.

| Preparation of NLC
Making of NLC was done by hot high-shear homogenization (Hot-HSH) (Heidolph Instruments GmbH and Co.) at 22000 g (Keivani Nahr, Ghanbarzadeh, Hamishehkar, & Samadi Kafil, 2018). At first, the lipid phase included CB and CLA in specific weight (in ratio of 10:1) in aqueous bath were heated to 80°C in a hot water bath. Then, the aqueous solution containing 1.5% Ploxamer 407 was heated to 80°C and added drop by drop into the lipid phase while being homogenized. For recrystallization of the lipid phase and form NLC, (CLA + CB)/w nanoemulsion was kept at 22°C.

| Particle size and zeta potential measurements
Wing SALD 2101 particle size analyzer (Shimadzo Corp.) was used for measurement the average volume diameter (DeBroukere mean particle size) and particle size distribution (span) of the particles at 22°C. Zeta potential of CLA loaded NLC was measured using zetasizer (Malvern Instruments) on first day after production.

| Scanning electron microscopy (SEM)
The surface morphologies of obtained NLC at 1st after production and after 40th days of storage were investigated using SEM (KYKY-EM3200 with an accelerating voltage of 26 kV). Before scanning, samples were diluted 20 times with deionized water (Klang, Matsko, Valenta, & Hofer, 2012).

| Physical stability
In order to checking physical stability of NLC system during storage, particle size changes and the physical appearance of the NLC formulation during storage at 4 and 22°C for 40 days (on days 1, 7, 14, 30 and 40th day) were studied (Mohammadi, Pezeshki, Abbasi, Ghanbarzadeh, & Hamishehkar, 2017).

| Turbidity assessment
The turbidimeter apparatus (Hach 2100p) with a series of turbidity standards in the range of 0-1,000 NTU (Nephelometric Turbidity Units) was used for turbidity measurement of diluted NLC formulations (diluted 10 times with water) in first, 30th and 60th days after production. The original sample turbidity obtained using Equation (3):

| CLA oxidative stabilization
Oxidative stability of NLC containing CLA was done using tiobarbituric acid (TBA) test during 30, 45, and 60th days of storage. 1 ml of NLC sample reagent with TBA solution (34 g of trichloroacetic acids 1.76 ml of 12 M hydrochloric acid and 8.28 ml water).

| DPPH scavenging assay
The free radical scavenging capacity of CLA-NLC was calculated using the methodology of Soleimanian, Golia, Varshosaz, & Sahafi (2) % LC = incorporated amount of CLA Total of used lipid × 100 (3) NTU = NTU sample * (volume of dilution water + sample volume taken for dilution (ml)) sample volume taken for dilution (ml) (2018). At first 2 ml of DPPH solution (0.1 mM in ethanol) was added to 0.3 ml of NLC ethanolic solution (500 ppm) and allowed to react at room temperature. After 30 min, the absorbance values were measured at 517 nm against the blank sample (0.1 mM ethanolic DPPH solution). The radical scavenging activity (inhibition percentage) was stated as percentage of DPPH radical elimination calculated according to the following equation (4) ( 4) where A control is the absorbance value of blank and A sample is the absorbance value of the sample at 517 nm.

| Preparation of enriched milk samples
Due to the 2.5 µg/ml CLA enrich to low-fat milk, 177 µl of NLC formulation, 250 µg of pure CLA without nanocarrier (control sample) was added to 100 ml raw milk samples in two tubes, separately, and then were pasteurized in 65°C for 30 min. In order to extract fatty acid profile from milk, sampling was done from pasteurized and raw milk.

| Fatty acid profiles measurement in milk
Identifying and determining the extracted fatty acids were fulfilled using Autosystem XL GC (Perkin Elmer) with UV ionization detector (GC-FID) at the following condition was used for separation, detection, and analysis of fatty acid available: capillary column, silica, 100 m length, 0.25 mm inner diameter; N2 (purity = 99.8%) used as carrier gas with a flow rate of 2 ml/min. Splitless mode injection was 50 ml/min splitting ratio in 0.75 min. The initial column temperature was 100°C and then the temperature was increased to 240°C at the 2°C/min and kept at 240°C for 15 min. The injection site temperature and detector were set on 270 and 300°C, respectively.

| Statistical analysis
One-way ANOVA (based on a complete randomized design) and

| RE SULTS AND D ISCUSS I ON
3.1 | Size particle, zeta potential and loading parameters (EE and LC%) measurements The average particle size (D 4,3 ), particle size distribution (span), zeta potential, EE, and LC % of the CLA bearing NLC formulation, on first day after production were shown in Table 1. The results showed the appropriate distributing and uniformity of particle size in nanoscale in NLC system (Figure 1). So in this research, zeta potential value of NLC formulation might be due to the composition of CLA and the presence of electrical barrier on the particles surface is responsible for the physical stability of nanodispersions by repulsion phenomenon F I G U R E 1 (a) Particle size and (b) particle size distribution 1 day after production of NLC formulation in ratio 10:1 solid liquid (cocoa butter) into liquid oil (CLA) using 1.5% (w/v) aqueous surfactant (Poloxamer407.) imperfect crystal, which had a large amount of space to load more bioactive substances .
Instability can result from interaction between poorly charged or uncharged nanoparticles, leading to the formation of aggregates. Zeta potential is a fundamental particle characteristic that can also be rapidly measured using light-scattering techniques (Brar & Verma, 2011).
Zeta potential measurements provide precise analysis of the electronic state of the nanoparticle surface, and the data obtained can be used to predict the stability of formulations containing these nanoparticles (Kaszuba, Corbett, Watson, & Jones, 2010). The small size of developed colloidal systems confirms good compatibility among compensates (Karimi et al., 2015). Choosing proper lipid matrix and the type of surfactant is an important key factor in preparing a stable and proper nanocarrier. It might be noted that use many different types of solid lipid for preparing NLC such as natural lipid (CB), semi-synthetic, and synthetic (Precirol ATO5) with various structures. Selecting a proper ratio from lipid and oil also plays a crucial role to create a crystal structure and uniform particle size distribution in a stable system (Soleimanian et al., 2018). At low concentrations of liquid oil, oil molecules would be spread solid lipid matrix and NLC will have an incomplete structure. In formulation which containing high concentration of liquid oil, the matrix of liquid oil in solid lipid structure would decrease during cooling phase separation happens, the size and distribution of particles will increase (Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2013).
Due to polymeric and bulky structure of Poloxamer 407 in NLC formulation, the stability of the nanoparticles within the storage time could be due to the steric repulsion of surfactant molecules rather than electrostatic repulsion. Trotta, Debernardi, and Caputo (2003) stated that the stability of the nanolipid carrier against aggregation is influenced by the ionic strength of the continuous phase and the charge density on the surface of water and fat. EE % of CLA in NLC system was more than 90%. The high %EE can be due to its lipophilic properties, which leads to its higher ability into lipid matrix compared to aqueous phase (Ni, Zheng, Hara, Pan, & Luo, 2015).

| Stability of particle size
Over a 40-day storage to period, the particle size of NLC stored at 22°C were similar to those stored at 4°C (Figure 3). NLC remained stable and uniform at both temperatures and the particle size of the system was not increased significantly (p > .05). The size distributions of both were monomodular. The presence of particles larger than 1 µm and a reduction in the particles number over time can show a physical instability.
Gravitational separations (Creaming or sedimentation), coalescence, and flocculation are main destabilization phenomena which affect the systems homogeneity (Araujo, Nikolic, Egea, Souto, & Garcia, 2011). A narrow particle size distribution minimizes the concentration gradient to the environment and inhibits the Ostwald ripening process (mass transfer from small particles to larger particles) (Talebi, Ghanbarzadeh, Hamishehkar, Pezeshki, & Ostadrahimi, 2019;Yang et al., 2018). According to obtained results, the CLA-NLC can be stored at 4°C and 22°C, which are common conditions for the storage of foods and beverage.

| Turbidity
There was no change of particle size after 60-day period (Figure 4), so it can be said that systems turbidity decreased during storage time. The slight decrease in turbidity after 60 days of storage, could F I G U R E 2 SEM morphology of CLA loaded NLC at 1st and 40th days of storage time be due to the loss of components by the changes of mean particle size and gravitational separation of particles in solution and the flotation of particles to the suspension surface. Also, due to presence of solid lipid (CB) in NLC formulation, the turbidity of NLC formulations was higher (Figure 4), it is known that higher refractive index (RI) will produce systems with higher turbidity.
The physical destabilization of nanocarriers may be perceived by microscopy, spectroscopy, turbidity, and particle size analysis.
Particle size reduction cause to an increase in solution clarity, colloidal stability, and also specific surface area which in turn increases the solubility and bioavailability (Keivani Nahr et al., 2018). The slight decrease in turbidity after 60 days of storage, could be due to the loss of components by the changes of mean particle size and gravitational separation of particles in solution and the flotation of particles to the suspension surface (Zhang, Bing, & Reineccius, 2016). The turbidity of NLC formulations was higher (Figure 4), it is known that higher refractive index (RI) will produce systems with higher turbidity. Since the RI of CB is 1.45 (Yunus, Fen, & Yee, 2009), these results would be expected. Considering that the usage of nanocarriers in beverages are naturally around 5% (Ni et al., 2015), the turbidity of CLA-NLC in milk would be disregarded.

| Oxidative stability of CLA
According to Figure 5, less secondary oxidation products (malonealdehydes) created during storage time and a meaningful difference was observed among control sample and NLC in terms of the rate of generated malonealdehydes.
Oxidative stability increased by injection CLA in colloidal nanocarrier systems and protecting this compound against environmental conditions, light, and oxygen. So, less secondary oxidation products (malonealdehydes) created during storage time and a meaningful difference was observed among control sample and NLC in terms of the rate of generated malonealdehydes. Surfactant layer covers the small lipids and prohibits them from coalescence, thus the encapsulated material was preserved in the NLC (Pezeshki et al., 2014). It was in accordance with Nikbakht et al. (Nikbakht Nasrabadi, Goli, & Nasirpour, 2016) about emulsion preparation containing CLA. Malonealdehydes mainly create in auto-oxidation with three or more double links. This compound is odorless and is able to contact with proteins.

| FTIR analysis
FTIR is a great technique of determining the interaction between components in the nanocarrier structure. FTIR can provide fundamental information on the molecular structure of organic and inorganic components (D'Angelo & Zodrow, 2011). FTIRIS offers unique possibilities to collect chemical information from biological samples with high spatial resolution (generally ~ 10 μm) (Lasch & Naumann, 2006). By comparing the peaks obtained from CLA, NLC and SLN, the increase in peak intensity in NLC containing CLA at 1,700 cm −1 , related to the C=C-H alkene group, weak vibrational peaks at 2,923 cm −1 of OH-free CLA groups and the expanded hydroxyl groups in the NLC were shown in 3,450.22 cm −1 , which is probably due to the binding with CLA in the NLC system ( Figure 6).
According to the present results, it can be shown the CLA in the NLC system without chemical bonding. The

| DPPH scavenging assay
The natural antioxidants have biological balance, so they are not accumulated in the body and cause the minimum side effects and tendency toward natural antioxidants has been increased (Hasrati, Govahi, & Mollaie, 2020). The antioxidant activity of NLC (at the concentration of 500 μg/ml) throughout the storage is presented in Table 2. By comparing the results of CLA and CLA encapsulated in NLC structure, there was a significant difference in the AA, which could be due to the CLA being free and exposed to environmental conditions (e.g. light and oxygen) and the degra-

| Protection of CLA in pasteurized low-fat milk during storage
CLA content in NLC-enriched low-fat milk was 3.9% of fatty acid in milk (regarding area peak (3.73) (Figure 7).
Considering NLC structure composed of solid lipid and liquid oil, a longer and more desirable release would be obtained from nanoparticles and it is possible to transmit active compound to aimed tissue. Also, the least distribution of encapsulated compound would be happened (Pezeshki et al., 2014;Shukla et al., 2014). Due to the presence of liquid oil in NLC formulation, compared to solid lipid nanoparticle structure (SLN) which only has solid lipid, outputting of active compound would lessen, highly EE %, protection of active compound, exposing it in environmental condition can be obtained (Karimi et al., 2018;Komaiko & McClements, 2014).
Also, the presence of surfactant layer (Poloxamer 407) in NLC, intensively influenced the crystal behavior of lipid, and therefore, release of CLA to medium was less. The least distribution of encapsulated compound would be happened (Pezeshki et al., 2014;Shukla et al., 2014). Due to the presence of liquid oil in NLC formulation, compared to solid lipid nanoparticle structure (SLN) which only has soild lipid, outputting of active compound would lessen, highly EE %, protection of active compound, exposing it in environmental condition can be obtained (Karimi et al., 2018;Komaiko & McClements, 2014).

| CON CLUS ION
Food grade delivery systems may be used to contain lipophilic nutraceuticals in aqueous-based foods and increase their bioavailability, functionality, and physical and chemical stability during the processing and storage time. NLC is a colloidal system that potentially can be used in fortification of food supplies. The particle size of NLC containing CLA was in nanoscale. They were stable during storage and unstable mechanism did not happen in them. By encapsulation CLA in NLC systems, the protection of CLA against oxidation was more and using NLC, reduced generating secondary oxidation products such as malonealdehydes. Also, stability of CLA in NLC formulation against thermal process, for example, pasteurization, environmental condition, and oxidation, was higher. Finally, using NLC can compensates the shortage of CLA in low-fat milk which happens by removing and reducing fat content.

ACK N OWLED G M ENTS
The support of university of Tabriz is greatly appreciated.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
The authors declare no ethical issue related with this article.

H U M A N A N D A N I M A L S TU D I E S
This article is a scientific and research type, and has no human or animal examples.