Enhancing oxidative stability of encapsulated fish oil by incorporation of ferulic acid into electrospun zein mat

LWT - Food Science and Technology 84 (2017) 82e90

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Enhancing oxidative stability of encapsulated fish oil by incorporation of ferulic acid into electrospun zein mat Huan Yang a, Kun Feng a, Peng Wen a, Min-Hua Zong a, Wen-Yong Lou a, Hong Wu a, b, * a b

School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, Guangzhou 510640, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2017 Received in revised form 22 May 2017 Accepted 22 May 2017 Available online 25 May 2017

A composite zein nanofibrous mat containing fish oil and ferulic acid was successfully fabricated by electrospinning. The process became more fluent and continuous by adding 30 g/L glycerol into the polymer solutions and using modified coaxial electrospinning. The average diameter of nanofibers was 440 nm. The loading capacity and encapsulation efficiency of fish oil were 20% and 94%, respectively. FTIR data demonstrated that fish oil and ferulic acid were successfully embedded into the nanofibers and there were interactions among the molecules of zein, fish oil and ferulic acid. Addition of ferulic acid into the nanofibers significantly improved the oxidative stability of encapsulated fish oil; moreover, it did not change the release behavior of fish oil. The release of encapsulated fish oil was controlled by a combination of diffusion and macromolecular chain relaxation. This composite nanofibrous mat with favorable oxidation stability and release property is potential in application as nutrition additive. © 2017 Published by Elsevier Ltd.

Keywords: Nanofibers Modified coaxial electrospinning Oxidation Release behavior

1. Introduction Omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA; 20:6), have shown to be beneficial to human health, but the high degree of unsaturated carbon-carbon double bonds render them sensitive to light, heat and oxygen, resulting in the poor oxidation stability which limits their application as nutrition additive (Torres-Giner, Martinez-Abad, Ocio, & Lagaron, 2010). Fish oil is one of the major sources of PUFAs, thus it is essential to protect it and improve its oxidation stability. For this purpose, encapsulation techniques have been considered for protection of fish oil, such as spray drying, liposome entrapment, coacervation, bubbles, porous inorganic capsules and layered nanocapsules, etc (Azmin, Harfield, Ahmad, Edirisinghe, & Stride, 2012; Mehta et al., 2017; Nangrejo et al., 2009). These methods typically produce micro and nano particles, which may limit the application and development of fish oil-loaded products. Electrospinning is a simple and highly versatile method that produces nanometer sized fibers, which have many structural and functional advantages (Wen et al., 2016b, 2016a). Nowadays,

* Corresponding author. School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China. E-mail address: [email protected] (H. Wu). http://dx.doi.org/10.1016/j.lwt.2017.05.045 0023-6438/© 2017 Published by Elsevier Ltd.

electrospinning is a promising method for encapsulation of bioactive compounds. The coaxial electrospinning, as a developed electrospinning technique, has emerged as an alternative to encapsulate bioactive compounds. Other volatile and sensitive materials have been encapsulated by co-axial system (Yao, Chang, Ahmad, & Li, 2016; Yao et al., 2017), however, in this study, a modified coaxial electrospinning technology was used to produce a single nanofibrous mat for encapsulation of fish oil. Zein is a protein from corn, which provides many advantages such as biocompatible and biodegradable, thus is considered as a potential candidate for encapsulation of functional ingredients. Generally, the most common approaches for embedding fish oil with zein were evaporation-induced self-assembly and liquidliquid dispersion. However, the fish oil product encapsulated by these methods is powder-like, and the oxidative stability of which needs to be improved. Ferulic acid (FA) is a polyphenolic compound that exhibits a wide range of therapeutic effects which is attributed to its antiinflammatory, antimicrobial, and anticancer properties (Panwar, Sharma, Kaloti, Dutt, & Pruthi, 2016). It was reported that ferulic acid can enhance the oxidative stability of fish oil by adding it as an antioxidant into the fish oil enriched milk (Sørensen, Lyneborg, Villeneuve, & Jacobsen, 2015). To date, there are only a few reports referring to the encapsulation of fish oil in zein fibers and beads via electrospinning (Moomand & Lim, 2014a, 2014b, 2015). It

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was found that the electrospun zein fibers provided a greater oxidative stability of encapsulated fish oil in comparison to nonencapsulated fish oil. To further improve the oxidative stability of fish oil, we previously encapsulated fish oil in the coaxial electrospun nanofibrous mat (Yang et al., 2017). The oxidative stability of encapsulated fish oil in the coaxial nanofibers was obviously enhanced compared to that in single nanofibers. However, the release behavior of encapsulated fish oil changed since less amount of fish oil released from the coaxial electrospun nanofibrous mat. Therefore, in order to enhance the oxidative stability of encapsulated fish oil while not to change its release property, ferulic acid was added into the electrospinning solution containing zein and fish oil to fabricate composite single nanofibrous mat in this study. The addition of ferulic acid into the nanofibrous mat can not only improve the oxidative stability of encapsulated fish oil, but also enhance the nutritional value of the composite nanofibrous mat due to the synergic effect of fish oil and ferulic acid. 2. Materials and methods 2.1. Materials Zein from corn (grade Z0001) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Fish oil was a kind gift from Sinomega Biotech Engineering Co., Ltd (Zhejiang, China). Pepsin, trypsin and ferulic acid were supplied by Aladdin Chemistry Co., Ltd (Shanghai, China). Anhydrous ethanol, glycerol and hexane are analytical grade (99.0%). Potassium bromide (KBr) is spectrum pure grade (99.5%), and xylenol orange sodium salt is indicator grade. All the above reagents were purchased from Sinopharm Chemical Reagent Co., Ltd (Guangzhou, China). 2.2. Electrospinning 2.2.1. Preparation of electrospinning solutions Ferulic acid solution was prepared by dissolving 3 g ferulic acid in 100 mL aqueous ethanol (ethanol:water 800:200 mL/mL) under constant magnetic stirring (RT5, IKA Group, Staufen, German) for 10 min. Then, 25 g zein was dissolved in ferulic acid solution by constant stirring for 0.5 h. After that, 7.5 g fish oil was added and mixed for 1 h. To investigate the effect of glycerol, 3 g glycerol was additionally added to the above-mentioned solution and mixed for 10 min. Before loading into syringes, all solutions were kept for half an hour to make sure no air bubble in the liquid phase was observed. 2.2.2. Electrospinning process For single electrospinning, the prepared solution was loaded in a 10 mL syringe equipped with a 22 gauge steel needle (outer diameter of 0.71 mm and inner diameter of 0.41 mm). A syringe pump (NE-300, New Era Pump Systems Inc., Farmingdale, New York, USA) was used and the feeding rate was fixed at 0.6 mL/h. The applied voltage was 16 kV, and the distance from the needle tip to the collector was 14 cm. For modified coaxial electrospinning, the polymer solution used as core solution was loaded in a 10 mL syringe. Anhydrous ethanol used as shell solution was loaded in a 10 mL syringe fitted with an 18 gauge steel needle (outer diameter of 1.27 mm and inner diameter of 0.84 mm) (Fig. 1). The electrospinning conditions were: shell flow rate 0.2 mL/h, core flow rate 0.6 mL/h, applied voltage 16 kV, and the distance 14 cm. All the experiments were carried out at 24
C ± 1
C under 45% ± 2% relative humidity.

Fig. 1. Schematic diagram of the modified coaxial electrospinning setup.

2.3. Characterization and measurement The mats obtained were left at ambient temperature (24
C ± 1
C) overnight before being investigated for their morphology using a scanning electron microscope (SEM) (EVO18, Carl Zeiss, Oberkochen, Germany). Prior to examination, the samples were coated with Pt for 40 s using a sputter coater (K550, Emitech Co., London, UK) under vacuum to render them electrically conductive. Images were recorded at an accelerating voltage of 10 kV. The fiber diameter distribution was calculated by analysis of around 100 fibers from the SEM image. Infrared spectra of samples were recorded using a Bruker Model Equinox 55 Fourier transform infrared (FTIR) spectrometer (Bruker Co., Karlsruhe, Germany) in a range of 4000e500 cm1. For electrospun nanofibrous mat, Attenuated total reflectance (ATR) was used for FTIR measurement. While for fish oil, ferulic acid and zein, KBr disks were adopted for FTIR measurement. Each measurement was an average of 16 scans at 4 cm1 resolution. 2.4. Loading capacity and encapsulation efficiency Loading capacity (LC) and encapsulation efficiency (EE) were determined by measuring the non-entrapped fish oil or ferulic acid according to Moomand and Lim (2014a) with some modifications. Mat (90 mg) was submerged in hexane or water (8 mL) for 1 min to remove the unencapsulated fish oil or ferulic acid, respectively, from the surface. The mixture was filtered, and the absorbance of the fish oil and ferulic acid was then determined using a UV-vis spectrophotometer (UV-2550, Shimadzu, Kyoto, Japan) at 260 nm and 320 nm, respectively. The EE and LC values were calculated as Eq. (1) and Eq. (2):

LC ¼ ðA  BÞ=C*100

(1)

EE ¼ ðA  BÞ=A*100

(2)

Where A was the total theoretical mass of fish oil or ferulic acid, B was the free mass of fish oil or ferulic acid in the collection solution, and C was the mass of the mat. 2.5. Oxidative stability analysis The oxidative stability of the encapsulated fish oil was tested at 25, 45 and 60
C under aerobic and anaerobic conditions. The

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unencapsulated fish oil was used as the control. For the test under aerobic condition, approximately 1 g of each nanofibrous mat was placed in an open Erlenmeyer flask, then kept in dark at 25, 45 and 60
C and taken for analysis at day 0, 1, 2, 3, 4 and 5. Under the anaerobic status, the treatment was similar to that of aerobic experiment, except all the samples were sealed packaged in Erlenmeyer flasks filled with nitrogen and taken for analysis at day 0, 5, 10, 15, 20 and 25. The peroxide value (PV) of oil of samples was measured following a ferrous oxidation in xylenol orange (FOX) method reported by Moomand and Lim (2014a). 2.6. Release behavior of encapsulated compound The simulated gastric fluid (SGF) (HCl solution, pH 1.2) and simulated intestinal fluid (SIF) (phosphate buffer, pH 6.8) were prepared according to the procedure in the Chinese Pharmacopoeia 2005. When preparing SGF and SIF containing enzyme, 20 g/L pepsin or 20 g/L trypsin was added to media, respectively. In the release behavior experiments in separate SGF or SIF, the samples were placed into serum bottle containing 15 mL SGF or SIF and incubated at 37
C in a rotation shaker with a rotation speed of 100 rpm. At appropriate intervals, 1 mL of the solution was collected from SGF or SIF, and then 1 mL of fresh medium was replaced into the test medium. For testing the release behavior of encapsulated compound in sequential SGF and SIF treatment, mats were firstly incubated in SGF for 2 h, then transferred to SIF and kept for 4 h. An aliquot (1 mL) of sample was collected at predetermined time intervals from the solution, and replaced by 1 mL of fresh medium. The concentration of fish oil and ferulic acid was determined following the same methodology as described in the EE and LC measurement. 2.7. Statistical analysis All the experiments were performed in triplicate. One-way ANOVA with Duncan significant difference test was carried out to determine the significant differences among samples. The probability of test statistic was set at 0.05. 3. Results and discussion 3.1. Fabrication of electrospun nanofibrous mats Previous study showed that single component zein nanofibers were generally fragile and difficult to handle, and the electrospinning process was easy to be clogged (Kanjanapongkul, Wongsasulak, & Yoovidhya, 2010). There are many methods which can be adopted to enhance the flexibility and improve the mechanical properties of fibres, such as using electrospinning type of printing (Wang, Zheng, Chang, Ahmad, & Li, 2017). It was reported that glycerol can serve as an excellent plasticizer which significantly facilitated the zein electrospinning process, improved zein fiber diameter uniformity and increased the zein nanofibers flexibility (Wongsasulak, Tongsin, Intasanta, & Yoovidhya, 2010). Hence, 30 g/L glycerol was added into the polymer solution to investigate its effect on the morphologies of nanofibers with or without ferulic acid. Additionally, adoption of modified coaxial electrospinning which realized by using ethanol as shell solution and polymer solution as core solution can also help to solve the clogging problem caused by the rapid evaporation of ethanol (Yu, Hu, Zhou, Chen, & Wang, 2013). Therefore, the morphologies of nanofibers containing 30 g/L glycerol prepared by single electrospinning and modified coaxial electrospinning were further compared in this study. As shown in Fig. 2, addition of glycerol

apparently promoted the formation of smoother and thicker fibers. And it was found that non-porous zein nanofibers were successfully generated smoothly and continuously without any clogging through the modified coaxial approach. Furthermore, the diameter distribution of fibers produced by coaxial electrospinning was narrower compared with that of single electrospinning. A similar phenomenon was also observed previously by Huang et al. (2013). Using unspinnable solvent as sheath fluid has a significant influence on the quality of inner nanofibers. The resulting nanofibers have smoother surface, smaller diameter and more uniform structure, compared to nanofibers produced directly from single electrospinning (Deng Guang Yu, Yu, Chen, Williams, & Wang, 2012). Therefore, modified coaxial electrospinning was chosen for the following experiments. It was worth noting that addition of ferulic acid into the nanofibers had no negative influence on production of smooth and uniform fibers albeit it slightly enlarged the fibers’ diameter. The average diameter of the resulting electrospun nanofibers with or without ferulic acid prepared by modified coaxial electrospinning was 440 nm and 390 nm, respectively. Table 1 summarized the LC and EE of encapsulated fish oil and ferulic acid in different nanofibers. The results showed that the LC of fish oil for zein/FO and zein/FO/FA nanofibers were 21.9% and 20%, respectively, and the corresponding EE were 95% and 94%, respectively. There was no statistical difference in LC and EE of fish oil for these two nanofibrous mats (p > 0.05), which indicated that addition of FA had no significant influence on LC and EE of encapsulated fish oil of the nanofibers. The LC and EE of ferulic acid for zein/FO/FA nanofibers were found to be 9.11% and 88.2%, respectively. Similar results were reported by Aceituno-Medina, Mendoza,
pez-Rubio (2015). The LC and EE of fish Rodríguez, Lagaron, and Lo oil for the zein/FO nanofibers achieved by the modified coaxial electrospinning was comparable to those prepared by single electrospinning (Moomand & Lim, 2014a), suggesting that the modified coaxial electrospinning was also an effective method to encapsulate bioactive compounds. 3.2. Characterization of the electrospun nanofibrous mats The FTIR spectra of fish oil, ferulic acid, zein, zein/FO nanofibers and zein/FO/FA nanofibers were depicted in Fig. 3. For fish oil, the peaks at 3013 and 1738 cm1 was assigned to the ¼ C-H stretching of double bonds and the C]O stretching of the carboxyl groups, respectively (Torres-Giner et al., 2010). These two bands remained to be observed in the nanofibers with or without ferulic acid, suggesting that fish oil was successfully entrapped into the zein nanofibrous mats. For ferulic acid spectrum, its characteristic bands were at 1691 and 1668 cm1, which indicated the presence of carbonyl group (C]O) in the different crystal lattice, and 3437 cm1 was related to the O-H stretching vibrations (Panwar et al., 2016). However, for the zein/FO/FA nanofibers, all the peaks mentioned above for ferulic acid disappeared, indicating that ferulic acid in the composite nanofibers did not form ferulic acid dimers that were necessary for the crystal lattice build up (Yu, Nie, Zhu, & Branford-White, 2010). On the other hand, the carbonyl group of zein and the hydroxyl group of ferulic acid might form numerous new hydrogen bonds. This result indicated that ferulic acid was also successfully embedded into the zein nanofibers. For zein spectrum, the peak at 1655 cm1 showed the C]O stretching vibration of amide I and the peak at 1534 cm1 was attributed to the N-H bending vibration. These absorptions are important in identifying proteins. The amide I band shifted to higher wavenumbers at 1657 and 1658 cm1 for zein/FO and zein/ FO/FA nanofibers. Meanwhile, there was also a small shift of N-H

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Fig. 2. SEM images and fiber diameter distributions of nanofibers with ferulic acid (A, B, C) or without ferulic acid (a, b, c) under different electrospinning conditions (a, A: without glycerol, prepared by single electrospinning; b, B: with 30 g/L glycerol, prepared by single electrospinning; c, C: with 30 g/L glycerol, prepared by modified coaxial electrospinning).

Table 1 Loading capacity and encapsulation efficiency of different fish oil based electrospun nanofibers (n ¼ 3). Bioactive

Sample

Loading capacity (%)

Encapsulation efficiency (%)

Fish oil Fish oil Ferulic acid

Zein/FO nanofibers Zein/FO/FA nanofibers Zein/FO/FA nanofibers

21.9 ± 0.1 20 ± 1 9.11 ± 0.08

95 ± 1 94 ± 1 88.2 ± 0.8

Data are reported as mean ± standard deviation. The mean has the same significant digits as the standard deviation which has one significant digit. FO: fish oil, FA: ferulic acid.

indicator peak, from 1534 cm1 to 1539 cm1 and 1543 cm1, respectively for the zein/FO and zein/FO/FA nanofibers. These displacements of indicator peaks to a higher wavenumber in the FTIR spectrum of fish oil-loaded zein fibers indicated that the interactions among fish oil, ferulic acid and zein protein altered the secondary structure of the protein.

3.3. Oxidative stability of encapsulated fish oil The oxidation of PUFAs in fish oil creates a variety of undesirable compounds. The peroxide value (PV) of lipid is a standard index to determine the deterioration degree of fat and oil. Hence, PVs of encapsulated fish oil for zein/FO and zein/FO/FA nanofibrous mat

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fish oil

ferulic acid

zein

1738

3013

3437

16911668

3325 1534 1655

zein/FO nanofibers 3312

1737

3014

1539 1657

zein/FO/FA nanofibers 1738

3014

1543

3323

4000

3500

1658

3000

2500

2000

-1

1500

1000

500

Wave number (cm ) Fig. 3. FTIR spectra of different samples.

2 1 0

0

5

10

15

20

25

Storage time (days)

9 6 3 0

0

100

5

10 15 20 Storage time (days)

10 5 0

0

5

10

15

20

25

25 0

0

15 10

60

5 0

30 0

5

10 15 20 Storage time (days)

25

0

1

0

2

3

1

4

5

2

3

4

5

2 3 4 Storage time (days)

5

Storage time (days)

b

400

60 45

300

30 15

200

0

0

1

2

3

4

5

100 0

1600

15

50

20

90

0

25

C

75

a

500

B

Lipid Peroxide (mmol/kg)

Lipid Peroxide (mmol/kg)

Lipid Peroxide (mmol/kg)

3

12

Lipid Peroxide (mmol/kg)

120

A

Lipid Peroxide (mmol/kg)

Lipid Peroxide (mmol/kg)

4

were measured in the absence or presence of oxygen at 25, 45 and 60
C. Fig. 4 showed the development of lipid peroxide values under anaerobic and aerobic conditions. As shown in Fig. 4, at all storage temperature, the PV of encapsulated fish oil was lower than that of unencapsulated fish oil, indicating that entrapment of fish oil in zein nanofibers can efficiently improve its oxidative stability. For the encapsulated fish oil, the PV of zein/FO nanofibers was greater than that of zein/FO/FA nanofibers, especially at high temperature. At 45
C, the difference was significant after storage for 15 days (p < 0.05), as to 60
C, the difference was significant after storage for 5 days (p < 0.05). This trend was more obviously under aerobic conditions and the difference between the PVs of zein/FO and zein/ FO/FA nanofibers was basically significant (p < 0.05) at all temperature except that storage at 45
C for 2 days. The results indicated that adding ferulic acid into the zein fibers remarkably improved the oxidation stability of encapsulated fish oil, which was attributed to the antioxidative property of ferulic acid. Ferulic acid can scavenge the active forms of oxygen involved in the initiation step of the oxidation, or break the oxidative chain reaction by reacting with the fatty peroxy radicals to form stable antioxidant radicals that are either too unreactive for further reactions or else form non-radical products (Supriyono, Sulistyo, Almeida, & Dias, 2015).

1

c

1200

200 150 100

800

50 0

400 0

0

0

1

2

1

3

4

5

2 3 4 Storage time (days) i / fib

5

Fig. 4. Development of lipid peroxide of unencapsulated fish oil (-), encapsulated fish oil of zein/FO nanofibers (:) and zein/FO/FA nanofibers (╳) stored at 25
C (A, a), 45
C (B, b) and 60
C (C, c) for a period of 25 days under anaerobic (A, B, C) and aerobic (a, b, c) conditions (FO: fish oil, FA: ferulic acid).

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Table 2 Numerical values of the oxidation kinetics model parameters for unencapsulated and encapsulated fish oils under anaerobic and aerobic conditions (n ¼ 3). Sample

25
C k (/d)

Anaerobic condition F 0.010 ± 0.000 A 0.005 ± 0.001 B 0.004 ± 0.001 Aerobic condition F 0.73 ± 0.02 A 0.405 ± 0.006 B 0.367 ± 0.002

45
C

60
C

Ea  103 (kJ/mol)

R2

0.995 0.979 0.999

R2

k (/d)

R2

k (/d)

R2

0.991 0.983 0.985

0.041 ± 0.001 0.032 ± 0.001 0.020 ± 0.001

0.993 0.975 0.982

0.142 ± 0.004 0.066 ± 0.001 0.060 ± 0.001

0.980 0.986 0.970

63 ± 1 65 ± 3 65 ± 5

0.984 0.986 0.997

1.047 ± 0.009 0.597 ± 0.005 0.551 ± 0.001

0.959 0.989 0.994

1.32 ± 0.02 0.80 ± 0.02 0.74 ± 0.01

0.948 0.955 0.988

14.0 ± 0.3 15.9 ± 0.8 16.5 ± 0.2

a b

0.999 0.999 0.999

Data are reported as mean ± standard deviation. The mean has the same significant digits as the standard deviation which has one significant digit. F: unencapsulated fish oil, A: encapsulated fish oil in zein/FO nanofibers, B: encapsulated fish oil in zein/FO/FA nanofibers (FO: fish oil, FA: ferulic acid). Note: The original mean data of a and b were 64.66 and 65.19, respectively.

To further understand the promoted effect of ferulic acid on the oxidative stability of encapsulated fish oil, the oxidation kinetics of encapsulated fish oil with or without ferulic acid was investigated. To determine the rate constant of peroxide formation reaction, Eq. (3) was fitted to the peroxide values data.

lnðP=P0 Þ ¼ kt

(3)

Where, P is the concentration of oxidation product, P0 is the initial value of P at t ¼ 0, k is the reaction rate constant, and t is time. Furthermore, the correlation of the reaction rate constant with temperature follows the Arrhenius equation as shown in Eq. (4) (Calligaris, Pieve, Kravina, Manzocco, & Nicoli, 2008):

k ¼ k0  expðEa =RTÞ

(4)

Where k is the reaction rate constant, k0 is the frequency factor, Ea is the activation energy (J mol1), R is the gas constant (8.314 J mol1 K1), and T is the temperature (K). By taking the natural logarithm of both sides of Eq. (4), it changed to Eq. (5):

ln k ¼ ln k0  Ea =RT

(5)

According to Eq. (5), a plot of lnk versus 1/T gives a straight line with a slope of -Ea/R, from which the value of activation energy can be determined. As shown in Table 2, the kinetics parameters for all samples whether in the presence of oxygen or not had a relatively high coefficient of determination (0.98  R2  0.94), indicating that they were satisfactorily adjusted to the model. As expected, the k values were clearly lower in anaerobic conditions. They increased with the growth of temperature and the k values of the unencapsulated fish oil were remarkably higher than those of the zein fiber-encapsulated counterparts. Furthermore, it decreased in the order of zein/FO nanofibers > zein/FO/FA Table 3 Oxidation kinetics model of unencapsulated and encapsulated fish oils under anaerobic and aerobic conditions. Sample Anaerobic condition F A B Aerobic condition F A B

Oxidation kinetics model k ¼ 1.11  109  exp (63.28  103/RT) k ¼ 1.04  109  exp (64.65  103/RT) k ¼ 9.93  108  exp (65.19  103/RT) k ¼ 2.082  102  exp (14.01  103/RT) k ¼ 2.488  102  exp (15.92  103/RT) k ¼ 2.882  102  exp (16.53  103/RT)

F: unencapsulated fish oil, A: encapsulated fish oil in zein/FO nanofibers, B: encapsulated fish oil in zein/FO/FA nanofibers (FO: fish oil, FA: ferulic acid).

nanofibers, which was consistent with the variation trend of PV. The Ea of unprotected fish oil, zein/FO nanofibers, zein/FO/FA nanofibers were 63, 64.66 and 65.19 kJ/mol under aerobic conditions. While in anaerobic storage, the corresponding values of Ea were 14.0, 15.9 and 16.5 kJ/mol, respectively, which demonstrated again that the encapsulated fish oil in the zein/FO/FA nanofibers had the best oxidative stability. Using the data previously obtained, the oxidation kinetics models were established as indicated in Table 3. From which, the shelf life of different samples at ambient temperature (20
C) were €khan Boran, Karaçam, and Boran (2006). calculated according to Go The results showed that the shelf life of unencapsulated fish oil, zein/FO nanofibers and zein/FO/FA nanofibers were 207, 374 and 501 days, respectively under anaerobic conditions. Not surprisingly, the shelf life of fish oil encapsulated in the zein/FO/FA and zein/FO nanofibers were approximately 2.4 and 1.8 times, respectively, as long as that of unencapsulated fish oil. It was noteworthy that the shelf life of zein/FO/FA nanofibers was 127 days longer than that of zein/FO nanofibers. Additionally, compared to our previous study, the shelf life of zein/FO/FA nanofibers was 62 days longer than that of coaxial zein/FO nanofibers (Yang et al., 2017). The results further demonstrated that electrospinning was an effective technique in encapsulation of fish oil. Moreover, addition of antioxidant into the electrospun nanofibers contributed to improve the oxidative stability of encapsulated fish oil. 3.4. Release behavior of encapsulated fish oil 3.4.1. Release behavior of encapsulated fish oil in SGF or SIF environment The release behavior of encapsulated fish oil in gastrointestinal tract plays an important role in its practical application. Fig. 5 depicted the release behavior of encapsulated fish oil from the nanofibers with or without ferulic acid in SGF or SIF environment. The results showed that in SGF, the total amount of fish oil released from zein/FO and zein/FO/FA nanofibers in the absence of enzyme were 60.12% and 62.93%, respectively, after 300 min. However, in the presence of pepsin, the corresponding values were 87.77% and 91.76%, respectively. In SIF, the similar release behaviors were observed for the encapsulated oil in both nanofibrous mats although their total release amount in the absence or presence of trypsin were lower than those in SGF with or without pepsin. This phenomenon was attributed to the difference of pH and enzyme in various simulated fluids. It was worth noting that there was no significant difference (p < 0.05) in the total release amount of fish oil between two nanofibrous mats in SGF or SIF environment, indicating that addition of ferulic acid had no negative effect on the release behavior of encapsulated fish oil in the composite zein nanofibers.

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100

A

80

80

60

60

% release

% release

100

40 20 0

0

50

100

150

200

250

B

40 20 0

300

0

50

100

C

80

80

60

60

% release

% release

100

40 20 0

0

50

100

150

100

150

200

250

300

250

300

Time (min)

Time (min)

200

250

D

40 20 0

300

Time (min)

0

50

100

150

200

Time (min)

Fig. 5. Release profiles of encapsulated fish oil from zein/FO nanofibers (:) and zein/FO/FA nanofibers (╳) under simulated gastrointestinal fluids. (A) SGF at pH 1.2, (B) SGF at pH 1.2 in the presence of pepsin, (C) SIF at pH 6.8 and (D) SIF at pH 6.8 in the presence of trypsin (SGF: simulated gastric fluid, SIF: simulated intestinal fluid) (FO: fish oil, FA: ferulic acid).

Table 4 Numerical values of the release kinetics model parameters for zein nanofibers in various simulated media (n ¼ 3). Simulated medium

Sample

n

SGF

A B A B A B A B

0.61 0.62 0.66 0.67 0.69 0.68 0.70 0.71

SGF with pepsin SIF SIF with trypsin

Kr (min-n) ± ± ± ± ± ± ± ±

0.05 0.04 0.01 0.03 0.02 0.01 0.04 0.02

0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01

± ± ± ± ± ± ± ±

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

R2

Mean dissolution time (min)

0.97 0.97 0.95 0.96 0.98 0.95 0.97 0.99

231 210 149 138 323 353 296 272

± ± ± ± ± ± ± ±

5 5 2 4 1 3 6 2

Data are reported as mean ± standard deviation. The mean has the same significant digits as the standard deviation which has one significant digit. A: zein/FO nanofibers, B: zein/FO/FA nanofibers (FO: fish oil, FA: ferulic acid, SGF: simulated gastric fluid, SIF: simulated intestinal fluid).

Gastric stage

100

Intestinal stage

Gastric stage

80

80

60

60

% release

% release

100

40 20 0

A 0

50

100

150

200

250

Time (min)

300

350

Intestinal stage

40 20

B 400

0

0

50

100

150

200

250

300

350

400

Time (min)

Fig. 6. Release profiles of encapsulated fish oil from zein/FO nanofibers (:) and zein/FO/FA nanofibers (╳) during an in-vitro digestion in the absence (A) and presence (B) of enzymes (FO: fish oil, FA: ferulic acid).

H. Yang et al. / LWT - Food Science and Technology 84 (2017) 82e90

In order to evaluate the release kinetics and mechanism of fish oil, the data were fitted to the Korsmeyer-Peppas model as shown in Eq. (6):

MDT ¼ ½n=ðn þ 1Þkr 1=n

(7)

The parameter MDT has been used not only to describe dissolution or residence profiles, but also to facilitate the comparison of different profiles (Podczeck, 1993). As shown in Table 4, the kinetic parameters for all samples had a relatively high coefficient of determination (0.99  R2  0.95), indicating that they were satisfactorily adjusted to the model. For electrospun nanofibers, an n value  0.45 indicates a release mechanism controlled by diffusion, and 0.45 < n < 0.89 represents a release mechanism governed by combination of diffusion and macromolecular chain relaxation (Kalu, Odeniyi, & Jaiyeoba, 2007). The n values were found to be between 0.61 and 0.70, 0.62 and 0.71 for zein/FO and zein/FO/FA nanofibers, respectively, suggesting that the release of encapsulated fish oil from both nanofibrous mats were generally controlled by a combination of diffusion and macromolecular chain relaxation. The n values of fish oil in the zein/FO and zein/FO/FA nanofibers were higher than that reported by Moomand and Lim (2014b), the possible reason was addition of glycerol during electrospinning. 3.4.2. Release behavior of encapsulated fish oil in sequential SGF and SIF treatment To better understand the release behavior of encapsulated fish oil in both nanofibrous mats, their release amount in sequential SGF and SIF were determined in the absence or presence of enzymes (Fig. 6). It was found that the amount of fish oil released in the presence of enzyme was higher than that in the absence of enzymes, which was consistent with the results achieved in separate SGF or SIF environment. As shown in Fig. 6, the majority of fish oil was released under the sequential SGF and SIF treatment in the presence of enzyme, and the cumulative amount of fish oil released from zein/FO and zein/ FO/FA nanofibers were 91.3% and 93.48%, respectively. For coaxial electrospun nanofibrous mat, the total amount of fish oil released was only 83.40% under the same conditions (Yang et al., 2017). These result demonstrated that the zein/FO/FA nanofibrous mat had excellent release property. The release profile of ferulic acid from the zein/FO/FA nanofibers under sequential SGF and SIF treatment in the presence of enzyme was also investigated. As shown in Fig. 7, on the first hour of gastric digestion, the release amount of encapsulated ferulic acid reached 40%, probably due to the existence of ferulic acid in the surface of fibers. The total amount of ferulic acid released was 98.52% in the sequential SGF and SIF environment. In a word, incorporating ferulic acid into the electrospun zein fibers had no significant influence on the release behavior of encapsulated fish oil. Additionally, nearly 100% ferulic acid was released from the composite nanofibrous mat, which was favorable for the exertion of its antioxidation and nutrition function. 4. Conclusion Ferulic acid was firstly added as an antioxidant into the electrospinning solution containing zein and fish oil to fabricate

Intestinal stage

80

(6)

Where Mt/M∞ is the fraction of fish oil released at time t, k is the kinetic constant, n is the release exponent. Using the n and k values derived from Eq. (6), the mean dissolution time (MDT) value can be calculated from Eq. (7):

Gastric stage

100

% release

Mt =M∞ ¼ kr t n

89

60 40 20 0

0

50

100

150

200

250

300

350

400

Time (min) Fig. 7. Release profile of ferulic acid from the zein/FO/FA nanofibers during an in-vitro digestion in the presence of enzymes (FO: fish oil, FA: ferulic acid).

composite nanofibrous mat to enhance the oxidative stability of fish oil. Addition of glycerol into the polymer solution and adoption of modified coaxial electrospinning made the process more fluent and continuous. The fish oil and ferulic acid were successfully embedded into the resulting nanofibers. Addition of ferulic acid into the electrospun zein fibers significantly improved the oxidative stability of encapsulated fish oil; moreover, it did not change the release behavior of fish oil. The results indicated that it is an effective strategy to improve stability of encapsulated fish oil by incorporation of antioxidants into the composite electrospun nanofibrous mat. Additionally, the nutritional quality of the composite nanofibrous mat can be also enhanced by a synergic effect of fish oil and ferulic acid, and has potential for developing innovative nutritional products. However, limitations still exist, such as the low throughput, which limits the large-scale commercial exploitation of electrospinning in the food industry. Hence, collaborations between researchers and manufactures are required to address this constraint by modifying the structural aspects of electrospinning setup in the future. Acknowledgements We acknowledge the National Natural Science Foundation of China (No. 31671852), the Natural Science Foundation of Guangdong Province (No. 2015A030313217), the National Natural Science Foundation of China (NSFC)-Guangdong Joint Foundation Key Project (No. U1501214), and the Science and Technology Project of Guangdong Province (No. 2013B010404005) for financial support. References
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