Stability enhancement of fish oil by its encapsulation using a novel hydrogel of cress seed mucilage/chitosan

ABSTRACT

Novel cress seed mucilage/chitosan (CSM/CS) hydrogels were fabricated for fish oil encapsulation and process parameters were optimized using the response surface methodology. The effect of different parameters (i.e., polysaccharide concentration, CSM/CS volume ratio and percentage of fish oil) on particle size, encapsulation load and efficiency were studied. Microcapsules were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy (FT-IR), and thermal gravimetric analysis (TGA). The oxidative test showed that produced hydrogels were efficient to postpone fish oil oxidation. The TGA results indicated that thermal stability of the prepared hydrogels increased due to CSM and CS interactions. Developed carriers could be used for encapsulation of hydrophobic compounds to protect them against oxidation and increase their solubility in aqueous media.

KEYWORDS:

• Chitosan
• Cress seed
• Delivery system
• Fish oil
• Hydrogel

Introduction

Hydrogels are three-dimensional polymeric networks cross-linked with covalent (chemical) or noncovalent (physical) interactions that absorb and hold a large amount of water and stay resistant against dissolving. In their swollen states, hydrogels offer unique physical and chemical characteristics with high mechanical resistance.^[1] Hydrogels are able to act as wall materials for the protection of active agents in their interior structure from any harsh conditions like oxidation, degradation, or destruction and have other applications in drug delivery, agriculture, water purification, and tissue engineering.^[2] Polymers with hydrophilic groups such as –OH, –COOH –CONH₂, –CONH–, and –SO₃H– interact with each other.^[3] Recently, biopolymeric hydrogels have gained great attention due to their biodegradability, biocompatibility, and nontoxicity that facilitate their applications in food industries.^[4] Carbohydrates are a group of biopolymers which are used for hydrogel preparation. Chitosan is the only polyelectrolyte biopolymer with positive charges because of NH₂ groups in its chains and therefore is a good volunteer to have a complex formation in acidic media with oppositely charged biopolymers.

Mucilage extracted from seeds is a group of water soluble biopolymers with high molecular weights. Mucilage can function as thickening, emulsifying or stabilizing agents in food formulations. Cress seeds consist of 6–15.5% mucilage with molecular mass of 540 kDa and zeta potential about −10.7870 mV which shows its anionic characteristic. Cress seed mucilage (CSM) is a new source of food hydrocolloid that semi-rigid chain conformation and intermediate flexibility between random coil and rigid rod are its characteristics.^[5]

Omega-3 fatty acids include long-chain polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These are considered as a group of substances which have health benefits and therefore are assumed as functional ingredients.^[6] Consumption of omega-3 fatty acids has many positive effects like prevention cardiovascular diseases, coronary artery disease, cancer, diabetes and inflammations. However, there are some limitations in fish oil application due to susceptibility of omega-3 fatty acids to light or oxygen and their off-odors and low water-solubility.^[7] Encapsulation serves as a delivery technology and is a good candidate for susceptible substances entrapment. It is a process that an active ingredient basically called core is entrapped within an outer layer that is called as shell material. The objectives of this paper were to produce and characterize hydrogel based on complex coacervation of CSM, as a new source of hydrocolloid with chitosan for encapsulation of fish oil.

Materials and method

Materials

Cress seeds were obtained from local market from Isfahan, Iran. Chitosan (CS; 75–85% deacetylation degree, low molecular weight) was purchased from Sigma–Aldrich Chemical Co. Ltd. (USA). Fish oil was provided from Zahravi Pharmaceutical Company (Tehran, Iran). The eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and total polyunsaturated fatty acids values of fish oil were 12, 18 and 34%, respectively. Acetic acid was obtained from Arman Sina Company (Iran). Methanol, 2-propanol, 2,2,4-trimethylepentane, n-hexane were supplied from Merck, Germany. NaOH and HCL were at least of analytical grade.

CSM extraction

CSM was obtained from cress seeds based on procedure reported by Karazhiyan, Razavi.^[8] Briefly, cress seeds were placed in preheated deionized water by 30:1 water to seed ratio, pH 10 (adjustment by NaOH solution) for 15 min at 35°C to obtain slurry contained mucilage around the seeds. Extraction from the slurry was performed by a laboratory extractor (Pars Khazar, Model P700, Iran). The mucilage was dried in a laboratory oven at 50°C. The final dried mucilage was milled to get powder and kept in a cool and dry place. The mucilage had molecular mass of 540 kDa and zeta potential of about −11 mV. It contains cellulose (18.3%), arabinose (19.4%), mannose (38.9%), fructose (6.8%), glucuronic acid (6.7%), galactose (4.7%), galacturonic acid (8%), rhamnose (1.9%), glucose (1.0%), and about 15% uronic acid (galacturonic acid and glucuronic acid).^[9]

Preparation of solutions

Desired concentrations of CSM and CS solutions (0.2%, 0.4% and 0.6% (w/v)) were prepared with CSM/CS ratios of 1:4, 1:1, and 4:1. CSM solutions were prepared by dissolving mucilage in deionized water and keeping 24 h for complete hydration in a refrigerator. Chitosan solutions were prepared by dissolving CS in 1% acetic acid solution on a magnetic stirrer for 24 h at room temperature.

Preparation of fish oil loaded hydrogel

Initial pH values of CSM solutions set to 2.5 with HCL (1%), while CS solutions had pH value of 3.6. Then CSM solution was added dropwise to fish oil and left 30 min for complete mixing (different mass ratios of fish oil to hydrogels; 10%, 20% and 30%). CSM solution was then added dropwise to prepared pre-emulsion. The pH of final turbid complex was adjusted to 4 by NaOH (1%) and dried with spray drier (DSD-02 Dorsa Tech. Iran). Drying was operated at inlet temperature of 120°C, outlet temperature of 85°C, pump feed flow rate of 5 ml min⁻¹ and air volume flow of 35 m³ h⁻¹.

Scanning electron microscopy

Morphological analysis of fish oil loaded hydrogels was analyzed using scanning electron microscopy (SEM) (Philips XL30). Produced hydrogels were placed on circular aluminum disc and coated with gold by a gold-sputter coater at an accelerating voltage of 20 kV for 250 s. Hydrogel mean diameter was determined from the SEM images by image analysis (ImageJ, National Institutes of Health).

Particle size measurement

The mean particle size of prepared hydrogel solutions was measured before spry drying by photon correlation spectroscopy (PCS) at a fixed angle of 90° (Zetasizer 3000 HS, Malvern Instrument, UK). Solution dilution was performed by distilled water before measurement.

Fourier transform infrared spectroscopy

To track possible interactions between fish oil and applied ingredients used for hydrogel shell, Fourier transform infrared (FTIR) was applied. Before analysis all powder samples were mixed separately with KBr to form pills. CS, CSM, fish oil and produced hydrogel were analyzed FTIR spectrophotometer (JASCO FT/IR-680 PLUS) at wavelength of 4000–400 cm⁻¹.

Encapsulation efficiency and encapsulation load

Encapsulation efficiency (EE) and encapsulation load (EL) were calculated based on a method mentioned by Bae and Lee^[10] with some modification. Five milliliters of n-hexane was added to 0.5 gr of dried powders, shaken by hands at room temperature for 3 min to extract surface fish oil. The mixture filtered through 0.2 μm PVDF syringe filter. The solution was left at room temperature for solvent evaporation until a constant weight was obtained. Total fish oil was assumed to be equal to initial added oil. Encapsulation efficiency and encapsulation load were calculated by Equations (1) and (2), respectively:

(1)

(2)

Oxidative stability

Oxidative stability of produced fish oil loaded hydrogel was analyzed right after spray drying as time zero and storage over two weeks by keeping in a laboratory oven (Memmert 845 schwabach) at 40°C in order to accelerate oxidation process. Extraction of fish oil was performed according to a previously reported method^[11] with some modifications. A 0.5 g of powder was weighted and placed into a test tube, suspended in 5 mL of n-hexane and shaken until complete dissolution over 15 min. A 300 µL portion of above suspension was taken and vortexed with 1.5 mL mixture of iso-octane:isopropanol (volume ratio of 2:1) three times for 10 s. The upper phase was separated after centrifugation (Hermle Labortechnik GmbH Z 36 HK) at 1500 rcf for 4 min.

Peroxide value was evaluated spectrophotometrically (PG instruments, T60UV, United Kingdom). A 200 µL portion of extraction medium was taken and added into 9.6 mL of a chloroform:methanol mixture (volume ratio of 7:3). For color formation, 50 mL of an ammonium thiocyanate/iron (II) chloride solution was added. The sample was vortexed briefly, let to react in dark place for exactly 5 min and absorption was measured at 500 nm.^[11]

Thermal gravimetric analysis (TGA)

Thermal analysis of the produced samples was evaluated by thermo gravimetric analysis in a scientific rheometric analyzer. Samples runs were carried out from 25°C up to 600°C at a constant heating rate of 10°C /min⁻¹ under argon atmosphere (10 mL/min). Differential thermogravimetric (DTG) curves were obtained by differentiating TG curves by TA Orchestrator software (Version V7.2.0.4).

Response surface methodology and statistical analysis

The Box–Behnken method was applied to investigate the effect of CSM/CS ratio, fish oil content and polysaccharide concentration on physicochemical properties of fish oil loaded CSM/CS hydrogels. Seventeen different runs were proposed by Design Expert Software version 7.0.0 (Stat-Ease Inc., Minneapolis, MN) (Table 1). The analysis of variance (ANOVA) was applied to determine significant parameters. The means were compared using Duncan’s multiple range test.

Table 1. Suggested RSM design for production of fish oil loaded CSM/CS micro-hydrogel and some physicochemical properties.

Independent variables				Dependent variables
Run	CSM/CS ratio (X1)	Fish oil content (%) (X2)	Polysaccharide concentration (%) (X3)	Encapsulation load (%)	Encapsulation efficiency (%)	Particle size (nm)
1	1:1	0.2	30	24	80	1949
2	1:4	0.4	30	26.1	87	4161
3	1:1	0.6	30	25.8	86	5579
4	4:1	0.6	20	18.4	92	2846
5	4:1	0.2	20	17.8	89	1722
6	1:1	0.4	20	18.8	94	2171
7	1:1	0.4	20	18.7	93.5	2247
8	1:1	0.6	10	8.7	87	2981
9	4:1	0.4	30	27	90	1586
10	1:1	0.2	10	8.3	83	1737
11	1:1	0.4	20	18.85	94.25	2487
12	1:4	0.6	20	18.1	90.5	5203
13	1:4	0.4	10	8.78	87.8	3473
14	1:4	0.2	20	17	85	960.3
15	1:1	0.4	20	18.8	94	1524
16	1:1	0.4	20	18.65	93.25	1940
17	4:1	0.4	10	9	90	1844

Results and discussion

Hydrogels were produced based on ionic interactions between carboxylic groups (–COO⁻) of CSM and the protonated amino groups (–NH₂) of CS in a pH range where two biopolymers have oppositely charged molecules equivalents. The effects of CSM/CS volume ratio, biopolymer concentration and fish oil content were investigated on particle size, encapsulation efficiency, and encapsulation load. Basically when the net charge of the medium is weak and electrostatic interaction is high, complex coacervation forms.^[12] The pHs were adjusted for CSM and CS solutions in 2.5 and 3.6, respectively, and final pH in 4. For higher pH values coacervation started to dissociate and large beads were formed. Bead formation can take place when CS molecules approach their pK_a (6.5), and therefore start to be insoluble and less ionized and thus precipitation occurred.^[13] On the other hand, the pK_a of carboxyl group is about 2.3 thus CSM becomes protonated and the medium became completely transparent under low pH (lower than 2.5).

Properties of produced fish oil loaded hydrogel

The effect of polysaccharide concentration (0.2 to 0.6%, X1), fish oil content (10 to 30%, X2) and CSM/CS volume ratio (1:4 to 4:1, X3) as independent variables on physicochemical properties of fish oil loaded CSM/CS hydrogels (i.e., encapsulations load, encapsulation efficiency and particle size) as response variables were investigated (Table 1). The precision of the models (evaluated by coefficient of determination) and predicting models were presented in Table 2. The best fitted models obtained by quadratic effect with polynomial equations for all response variables except the particle size that achieved by a two factor interaction (2FI) model. Table 3 illustrates the mean square error values of each factor and indicates significant parameters.

Table 2. Final equation in terms of actual factors and R².

	Model parameters
Response variables	Intercept	A	B	C	AB	AC	BC	A^2	B^2	C^2	R^2
Encapsulation load (%)	−4.91122	0	+1.09121	+15.41633	0	0	0	0	−0.014219	−19.26625	0.9985
Encapsulationefficiency (%)	+49.56389	+0.071528	0	+105.52083	0	0	0	0	−0.051125	−117.18750	0.9920
Particle size (nm)	−550.86691	0	0	+6932.72917	0	−129.94583	0	0	0	0	0.8349

A is CSM/CS ratio, B is fish oil content and C is polysaccharide concentration

Table 3. Mean square errors of dependent factors.

Process variables	Degree of freedom	Encapsulation load (%)	Encapsulation efficiency (%)	Particle size (nm)
CSM/CS volume ratio	1	0.5	14.31**	4.204E+006
Fish oil content (%)	1	282.42**	2.88	1.312E+006
Polysaccharide concentration (%)	1	1.42**	42.78**	1.311E+007**

** Significant at P < 0.01

Particle size

Mean particle sizes of hydrogels loaded fish oil determined by PCS were in the range of 960.3 nm to 5727 nm (Table 1). The size of hydrogels increased significantly by increasing polysaccharide concentration from about 1 to 3.5 μm. Similar trends were reported by Chopra, Bernela^[14] who showed that by increasing concentration of gum acacia from 0.5 mg/ml to 1.5 mg/ml the size of nanoparticles increased from 120.2 nm to 397.6 nm which could be due to solution viscosity or interaction between functional groups of cross-linker and biopolymer. In the current research by increasing CSM concentration, there would be more charged groups to interact which led to increase of hydrogel particle size.

Encapsulation load and encapsulation efficiency

Polysaccharide concentration and oil content had significant effects on encapsulation load whereas CSM/CS volume ratio and polysaccharide concentration had significant effect on encapsulation efficiency (P < 0.01). Figure 1 showed that EL increased as polysaccharide concentration increased due to more available space for fish oil to be entrapped. Data also showed a slight decrease of EL for higher concentration of hydrocolloids which could be due to the effect of viscosity of coacervates. A previous research showed that the viscosity of coacervates must be high enough to form resistant shell around oil droplets.^[15] Although for very high viscosity, oil entrapment will be suppressed because of hardship in spreading of coacervates around oil droplets, thus decreasing EL can be occurred. Increasing polysaccharide concentration affected the optimum charge density since more attractive forces can interact and therefore thick gel or precipitates were formed. Figure 2 shows the effect of CSM/CS volume ratio on EE that indicated by increasing in CSM volume ratio from 1:4 to 4:1 higher EE could be observed. The reason of this phenomenon might be attributed to inequality of charges ready to interact with each other. Burgess^[16] illustrated that electrostatic interaction as well as the extent of coacervation depend on the macromolecules mixing ratio. Fish oil concentration had significant effect on encapsulation load, which was due to sufficient capacity of shell to entrap encapsulant.

Figure 1. Effect of polysaccharide concentration and fish oil content on encapsulation load of micro-hydrogels.

Figure 2. Effect of polysaccharide concentration and CSM/CS volume ratio on encapsulation efficiency of micro-hydrogels.

Response surface optimization

Based on response surface methodology (RSM) proposed by design expert, numerical optimization was performed to achieve the optimum conditions needed for CSM/CS hydrogel production loaded with fish oil with highest encapsulation load (EL) and efficiency (EE) and lowest particle size. The optimum processing conditions predicted by RSM and their corresponding experimentally values were tabulated in Table 4. Minor differences were observed between RSM data and our laboratory data. The optimum sample was selected for future analysis (i.e., morphology (SEM), Fourier transform infrared spectroscopy (FTIR), thermal analysis (TGA), and oxidative stability).

Table 4. Optimized conditions and corresponding dependent data (model suggestion and experimentally determined).

Optimum condition				Optimum responses
No	CSM/CS ratio	Fish oil content	Polysaccharide concentration	EL (%) (mean ± SD)	EE (%) (mean ± SD)	Particle size (nm)
1	48/52	23	0.33	20.46	91.39	2327.63
2				20.47 ± 0.23	89 ± 1	2025

No1: Numerical optimized values proposed by RSM; No2: Experimental values; EL: encapsulation load; EE: encapsulation efficiency

Hydrogel morphology

Figure 3 shows morphology of CSM/CS hydrogel loaded by fish oil. Particles had spherical shape with some dents and a little roughness. The average size of microcapsule powders was about 9.4 ± 1.2 µm which was larger than emulsion particle size data obtained from PCS. Increasing in size of particles could be due to agglomeration during spray drying.^[17]

Figure 3. SEM micrographs of micro-hydrogels loaded with fish oil.

FTIR analysis

Ionic interaction between CSM and CS to form hydrogels was confirmed by FTIR analysis as shown in Figure 4. CSM main characteristic peak related to symmetric negative carbonyl (C = O) at 1620 cm⁻¹, other bands correspond to O–H stretching at 3416 cm⁻¹, symmetric and asymmetric of C–H at 2924 cm⁻¹ and stretching of C–O at 1128 cm⁻¹.^[5] CS main bands were at 1660 cm⁻¹ and 1590 cm⁻¹, associated to the characteristic band of amide and protonated amino groups, respectively.^[18] Also bands at 3431 cm⁻¹ and 1077 cm⁻¹ were related to stretching vibrations of OH groups and C–O starching, respectively.^[19] Typically the spectral region between 3090 to 2800 cm⁻¹ refers to eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and total n-3 free fatty acids. The main vibrational bands of fish oil were at 2923 cm⁻¹ and 2854 cm⁻¹ corresponding to asymmetric and symmetric stretching (CH₂) for total PUFAs, respectively. Also stretching ester carbonyl (–C = O) at 1745 cm⁻¹ and several bands at 1376 cm⁻¹, 915 cm⁻¹ and 718 cm⁻¹ were observed which were related to finger print region between 1500 and 650 cm⁻¹ can be identified in fish oil spectrum.^[20] CSM band at 1420 cm⁻¹ and CS peak at 1077 cm⁻¹ were obvious in CSM/CS hydrogel, showing existence of CSM and CS in fabricated sample. While as shown in spectrum of CSM/CS hydrogel, the peaks at 1620 cm⁻¹ and 1590 cm⁻¹ of CSM and CS were not observed and a new peak at 1553 cm⁻¹ could be detected. These peak changes could be attributed to hydrogel formation as a result of ionic interaction between the negatively charged carboxyl group of CSM and amino group of CS. The PUFAs and ester carbonyl peaks of fish oil were detectable in CSM/CS hydrogel spectrum at 2854 cm⁻¹ and 1745 cm⁻¹.

Figure 4. FTIR spectra of CSM (cress seed mucilage), CS (Chitosan), fish oil and CSM/CS micro-hydrogel loaded with fish oil.

Oxidative stability

Oxidative stability of fish oil at 40°C was evaluated during two weeks for encapsulated and unencapsulated oil. The peroxide value of pure fish oil at time zero was 5.4 ± 0.42 meq peroxide/kg oil which was less than encapsulated fish oil (11.1 ± 0.21 meq peroxide/kg oil). This phenomenon could be attributed to hot air exposure of fish oil during spray drying process (about 120°C). As can be seen in Figure 5 the CSM/CS hydrogel was effective for preventing fish oil oxidation after 14 days. Pronounced oxidative stability of optimum CSM/CS hydrogels was related to its capability to entrap high amount of fish oil (EE of 89 ± 1%).

Figure 5. Oxidative stability of produced micro-hydrogel loaded with fish oil and pure fish at 40°C.

Thermal gravimetric analyze

Thermal gravimetric analysis was conducted to evaluate thermal behavior of pure CSM, CS and CSM/CS hydrogel loaded with fish oil (Figure 6). Pure CSM represented two peaks. The first one started at around 48°C to 164°C with low weight loss of 7.02% (0.121 mg) associated to water evaporation. Water evaporation above 100°C has been assigned for formation of hydrogen bond among CSM and water molecules. While another assumption is evaporation of water entrapped within the polysaccharide chains. The second peak was around 224°C to 432°C by 51.16% weight loss (0.819 mg) that related to polysaccharide degradation process and disintegration of molecular chains.^[21] Two peaks were recorded in thermogram of pure CS. The first event occurred at around 43°C to 169°C associated to water evaporation with 8.53% weight loss (0.444 mg). Prolonging evaporation above 100°C can be due to inner hydrogen bonding in CS chains.^[22] The main CS degradation was observed at around 252°C to 428°C with 46.78% weight loss (2.212 mg). CSM/CS hydrogel curve shows three main peaks. The first peak started at around 40°C to 129°C and associated to water dehydration with 3.45% weight loss (0.077 mg). Here prolonging water evaporation above 100°C was related to formation of hydrogel network of CSM and CS which led to preventing of water molecules movement. The second and third spectrum illustrates the decomposition of wall materials (CSM and CS) around 240°C and 460°C that continues until 560°C with 40.62% weight loss (0.273 mg). It can be detected from DTG curves (Figure 7) that the thermal stability of CSM and CS were 315°C and 338°C, respectively while thermal stability of CSM/CS hydrogel enhanced to 355°C that could be due to interaction of wall materials and therefore better thermal resistance (Table 5).

Table 5. Starting point, weight loss and thermal stability of CSM, CS and CSM/CS micro-hydrogels during thermal degradation in TGA and DTG.

Samples	Events	Starting point of weight loss (°C)	Weight loss (%)	Thermal stability (°C)
CSM	1st	48	7.02	315
	2nd	224	51.16
CS	1st	43	8.53	338
	2nd	252	46.78
CSM/CS	1st	28	3.91	355
	2nd	240	70.3
	3rd	460	19.77

CSM: cress seed mucilage; CS: chitosan; CSM/CS: cress seed mucilage/chitosan micro-hydrogel loaded fish oil

Figure 6. TGA curves of cress seed mucilage, chitosan, and cress seed mucilage/chitosan micro-hydrogel loaded with fish oil.

Figure 7. DTG curves of cress seed mucilage, chitosan, and cress seed mucilage/chitosan micro-hydrogel loaded with fish oil.

Conclusion

Novel hydrogels were fabricated using cress seed mucilage and chitosan for encapsulation of fish oil and their different physicochemical features were studied. The average size of hydrogels in emulsion state was in the range of 960 to 5727 nm. Production method was optimized using RSM and optimum processing conditions were CSM/CS volume ratio of 48:52, fish oil of 23%, and polysaccharide concentration of 0.33%. FTIR analysis confirmed that CSM, CS, and fish oil exist in produced hydrogels and demonstrated that ionic interactions were formed through the association of the functional groups of CSM and CS (NH₃⁺ and ̶ COO¯). Morphological study showed that particles had spherical shape with some dents. Hydrogels were effective in protection of fish oil against oxidation. From DTG analysis, combination of CSM (with thermal stability at 315°C) and CS (with thermal stability at 338°C) led to thermal stability enhancement up to 355°C of produced hydrogel. The results of this study indicated that produced hydrogels can be used for encapsulating of susceptible food ingredients.