Polyphenolic compounds with relatively high antioxidant activity obtained from subcritical water extraction of apple pomace were assessed for encapsulation by spray drying technique, making use of polymeric substances co-extracted with the polyphenolic compounds. Comparative assessments were carried out of the directly encapsulated subcritical water extract (SWE) products with particles formed when encapsulated with the addition of hydroxyl propyl- β-Cyclodextrin (SWE + HP β-CD). The powders were characterized for their physico-chemical properties such as, moisture content, density, particle size, hygroscopicity to assess their suitability within cosmetic formulations. The SWE and SWE + HP β-CD encapsulated products resulted in different physical properties. Although the particle size was less than 4 μm for both products, the direct encapsulation (SWE) was highly hygroscopic and this property was significantly reduced with addition of HP β-Cyclodextrin (SWE + HP β-CD). Scanning electron microscopy (SEM) and Fourier Transform Infrared (FT-IR) spectroscopic were em-ployed to analyse the micronised powders to support evidence of encapsulation. Both techniques revealed the interaction between compounds in extract and the carrier HP β-Cyclodextrin suggesting successful encapsulation. The effect of storage conditions on retention of antioxidant activity of the subcritical water extract was evaluated within 35 days for extracts with and without the carrier HP β-Cyclodextrin. Hydroxyl propyl- β-Cyclodextrin offered protection against degradation of antioxidant compounds thereby potentially extending the shelf-life and making the encapsulated powder suitable for incorporation in cosmetic and pharmaceutical applications.
There is a growing concern that common synthetic preservatives such sodium benzoate, sodium sorbate, formaldehyde releasers and isothiazolinones may have hazardous effects such as hormonal and neurological disorders, human carcinogen linked to damaging long term effects [
Polyphenolic compounds including Proanthocyanidins from grape seeds and coffee extracts have been shown to be strong antioxidants in vitro when compared with Carotenoids, Vitamins C and E [
Apples are a significant source of bioavailable polyphenolic compounds and are a common fruit [
Freeze and spray drying microencapsulation technologies are extensively used in the cosmetic pharmaceutical and food industries in recent times to stabilise ingredients against oxidation, improve shelf-life, enhances solubility and bioavailability during applications [
The equipment setup generates particles from homogenized solution of active ingredients and coating materials [
Naturally occurring polymers have been successfully used as carriers when encapsulating bioactive compounds, For example, polyphenolic compounds such as Caffeic acid, Chlorogenic acid, Gallic acid, Quercetin, Kaemferol, Myricetin, and green tea flavonoids have been coated with soy protein [
The current study builds on the fact that carbohydrates and polyphenolic compounds from plant materials have been shown to interact [
Apple pomace a residue from cider production comprising Michelin, Dabinett, Yarlinton Mill, Chisel Jersey, Brown Snout, Vilberie and Harry Masters Jersey varieties, was supplied by Universal Beverages Limited (UBL) a subsidiary company of Bulmers, UK. The pomace sample was very heterogeneous comprising peels seeds, apple flesh and therefore was thoroughly mixed to ensure replicate samples were representative of the population of samples.
The dry weight of all samples was determined using AOCS (American Oil Chemist Society Standard) standard protocol using a laboratory oven (STATUS international, UK) at 103˚C ± 3˚C. The frozen wet apple pomace sample was homogenized for 30 seconds using Moulinex domestic blending machine to minimize variability in batch-to-batch analysis. Portions of the homogenize apple pomace was freeze-dried using vacuum freeze dryer (Model number EQ03 by Vacuum and Industrial Products, UK).
Subcritical water-mediated extraction of polyphenolics from wet homogenised apple pomace was conducted using a Parr instrument (model 5521), which was a 300 ml stainless steel reactor vessel with a heating jacket. The vessel was connected with temperature and pressure sensors. Magnetic stirrer (1240 rpm) with integrated cooling system was attached to help enhance mass transfer. A back pressure regulatory valve was used to control the pressure inside the vessel. A nitrogen gas cylinder was used to pressurize the reactor. The setup is illustrated in
Wet homogenised apple pomace was loaded into the reactor according to the solid-to-solvent ratio of 4.5% (w/v). The reactor vessel was first purged for 10 - 15 seconds with nitrogen, before the extraction pressure inside the vessel was set to 50 bar and the gas valve (V-3) closed. The temperature of extraction was set to 150˚C at 20 minutes residency time. Once the reaction was finished, the gas valve (V-1) was closed and the heating and stirring turned off. Cooling system to the reactor was turned on and vessel quickly removed from the heating jacket into an ice bath to allow the reactor cool below 50˚C and depressurised to allow the extract to be collected.
The extract was transferred into 500 ml Beckman centrifuge bottles and centrifuged at 4000 g for 10 minutes at a temperature 4˚C using a Beckman J2-20 centrifuge. Supernatant from the centrifugation step was further filtered under atmospheric pressure using Fisherbrand filter paper (QL 100).
Total phenolic content of the extract was determined using the Folin Ciocalteus’s micro scale method proposed by Waterhouse, (2001) [
Phenolic compounds in the subcritical water extracts were resolved according to the protocol described by [
The antioxidant activity of the extract was determined using a modification of the oxygen radical antioxidant activity assay (ORAC)protocol described by Huang et al., (2002) [
The procedure for preparing HP-β-Cyclodextrin-polyphenolic inclusion complex described by Gioxari et al., 2010 was adopted with slight modifications [
The laboratory scale spray dryer (Model-SS07, by Lab plant Ltd, UK) was employed to produce the powders. All glassware was fitted to the unit and inlet temperature set to 200˚C and allowed for 15 minutes to warm up before setting the actual spray temperature. Inlet and outlet temperatures were set at 170˚C and 84˚C respectively. Sample feed was delivered at 3.6 ml/min using a variable speed peristaltic pump into a 0.5 mm two-fluid-stainless spray nozzle with an air flow rate of 180 g/min. The subcritical water extract of the apple pomace containing 0.0283 g/ml dry solids was sprayed from 140˚C to 170˚C. The previously prepared inclusion complex mixture was also sprayed at the same condition. Feeds were continuously stirred during spraying and dry powders were separated by a cyclone and collected in an insulated sample collection bottle. A portion of the dried powder was sampled for analysis and bulk packed in seal polyethylene bags and stored in a desiccator.
A gas displacement technique was employed to determine the density of the spray dried powder using an AccuPyc II 1340 gas Pycnometer (Micrometrics Instruments Corporation). A 1 cm3 cup whose weight was previously determined using the microbalance (SART 1702 Germany) was filled with samples of the dried powder and reweighed to obtain accurate weight. The cup containing the powder was sealed in the instrument compartment and helium gas was admitted to serve as a displacement medium and expanded within the internal volume of powder. The solid phase volume of powder was computed from the changes in pressure during filling of sample chamber and that of the discharge empty chamber. Data were analysed using VI.05 software and density of powder was determined by dividing the average volume into powder weight.
Particle size and the distribution of particle size within the dried powders was determined using a HELOS/RODOS/VIBRI dispersing system (Sympatec GmbH, Clausthal-Zellerfel Germany) The setup consisted of HELOS (Helium-Neon-Laser optical system), a dry powder dispersion system RODOS, and a vibrating feeder VIBRI. Operations were controlled by software WINDOX 5 for evaluation of particle size and other analysis.
0.5 g of the dried powders (SWE and SWE + HPβ-CD) were weighed in triplicates using a microbalance instrument GR-202 (A & D Scientific Laboratory suppliers) and spread uniformly on glass petri dishes. The powder samples were kept at 23˚C in an incubator model SI-600R (Medline Scientific). 300 ml saturated solution of Sodium Chloride was placed inside the incubator to provide approximately 75.5% relative humidity and left for 7 days. Samples were reweighed after the 7 days and hygroscopicity HG of powders determined according to the equation below;
HG = Δ m / ( M + M i ) 1 + Δ m M
where Δ m was the increase in weight of powder after equilibrium. M was the initial weight of powder and M i was the free water content of powder prior to exposure to the humid environment [
Environmental Scanning Electron Microscopy (XL 30 ESEM FEG Philips, Netherlands) was used to observe the morphology of the freeze-dried and spray dried powders. The samples were spread on ESEM-stub covered with sticky carbon tape, and sputter coated under high vacuum with gold using EMSCOPE SC 500 gold sputter coater. All samples were scanned at a voltage of 15 kV using XL 30 ESEM FEG electron microscope and images captured over a range of magnifications within the sample.
FTIR analysis was performed to characterise the powders in molecular terms using Jasco FT-IR 6300 infrared spectrometer. Resolution of 4 cm−1 and 32 scans were used in a range between 4000 and 600 cm−1. A background was performed before each sample analysis to scan the environment which was subtracted from the sample spectra to avoid any interference in the results.
1.5 ml of subcritical water extract (SWE) and subcritical water extract with HPβ-Cyclodextrin (SWE + HPβ-CD) were measured into separate 2 ml Eppendorf tubes and incubated at 65˚C in a drying cabinet (Fisons Scientific instruments UK). Stability assessment in terms of antioxidant activity of all samples were determined every 7 days for 35 days by Folin Ciocalteu method. All measurements were done in triplicates and samples were not protected from external light.
Several inlet and outlet temperatures of spray drying operations were employed by trial and error to favour the generation of dried powders of the subcritical water extract. Dried powders were obtained from subcritical water extract (SWE) for inlet temperatures from 140˚C to 170˚C. There was difficulty in obtaining fine and dried powders below 140˚C and beyond 180˚C for the subcritical water extract whose solid concentration was only 2.75% (w/v). Wet products were observed below 140˚C due to insufficient drying and sticky brown products at 180˚C due to caramelisation reaction of the monomeric sugars in the extract at the high temperatures. However, spray drying of HPβ -Cyclodextrins encapsulated with the subcritical water extract (SWE + HP-β-CD) was without challenges. The incorporation of HPβ-Cyclodextrin raised the glass transition temperature of the subcritical water extracts thereby reducing the stickiness [
Colour of the directly encapsulated polyphenolic fraction (SWE) of the apple pomace with polymers co-extracted under the subcritical water-mediated hydrolysis was yellowish brown and those of the HP-β-Cyclodextrin (SWE + HPβ-CD) encapsulated reflected the colour of the directly encapsulated product in extract and were lightly brownish.
Moisture content or residual water associated with solid raw materials for nutraceutical, pharmaceutical, food and cosmetic applications can significantly affect their physico-chemical properties. Rate of dissolution, flow and compactibility of powder, as well as degradation or deterioration are all affected due to prolong exposure to moisture [
Moisture content of directly encapsulated subcritical water extract (SWE) powder was 22.6% higher than HPβ-Cyclodextrin inclusion complex (SWE + HPβ-CD). Thus suggesting that increasing the solid content through the addition of HPβ-Cyclodextrin to the subcritical water extract reduces the amount of residual water of the powder. Previous studies have shown that the moisture content of spray dried powders is dependent on the type and concentration of the carrier material used [
Hygroscopicity is defined as the estimation of the ability of a substance to absorb moisture from a relatively high humid environment and is an important property to consider during storage of powder [
The presence and concentration of low molecular weight sugars (Glucose, fructose) and organic acids (citric, malic and tartaric acid) are thought to account for the hydroscopic properties of spray dried powders. Furthermore, the high hygroscopicity, thermoplasticity, and low glass transition temperature (Tg) of these low-molecular-weight substances contribute to the stickiness of dried powders which is a phenomenon frequently encountered during spray drying [
Powder Sample | Moisture (%) | Hygroscopicity (g/100g) | Particle size (µm) |
---|---|---|---|
SWE + HPβ-CD | 5.59 ± 0.4 | 5.08 ± 0.01 | 3.46 ±0.04 |
SWE | 7.22 ± 0.01 | 9.30 ± 0.11 | 3.41 ± 0.15 |
processing and storage [
Many methods for determining particle density of solids have been applied and reported in the literature [
Analysis of the SWE spry dried powder indicated that it had a density of 1.560 ± 0.001 g/cm3 which was higher than the density of spray dried powders of the SWE + HPβ-Cyclodextrin complex (1.503 ± 0.003 g/cm3). The increase in the volume of the SWE + HPβ-Cyclodextrins powders can be attributed to the conical structure of the cyclodextrin which supports a more open matrix when compared to the compact polymers co-extracted with polyphenolics under subcritical water-mediated hydrolysis. The cumulative distribution curves for both powder samples are presented in
The mean particle size of SWE and SWE + HPβ-CD were 3.35 µm and 3.42 µm respectively and while not significant (p < 0.05) the results do reflect the differences in density which has been attributed to the architecture of the matrix within each particle. The lack of significant difference in particle size can perhaps be explained by the fact the spray drying operating conditions were similar for both samples. As it has been illustrated that different particle sizes of powders
were produced when the spray drying operating conditions are varied [
Scanning electron microscopy (SEM) was used to investigate the impact of the type of drying technique and carrier introduced. SEM images of freeze-dried and spray dried subcritical water extract (SWE) of the apple pomace revealed different morphologies. The SEM of freeze-dried subcritical water extract (
Smooth aggregates of varying sizes had formed and reflected the observations and reported for freeze-dried powders [
The particles formed under spray drying appear spherical and aggregate into a network. The morphology suggests the extract have been directly encapsulated and the “stickiness” of the particles supports the network of aggregates.
The morphology of pure HPβ-CD is shown in
SEM images of the freeze-dried encapsulated subcritical water extracts with HPβ-Cyclodextrin (
The ice formed within the encapsulated product during the freeze-drying process prevented the collapse and shrinkage of the particles [
with SWE priority and during spray drying. The SEM images clearly indicate that during processing discrete spherical particles of varying sizes are formed, creating an overall morphology distinct from that formed when the SWE is sprayed dried without cyclodextrin i.e. a free flowing powder.
However, increased magnification revealed the formation of “dents” or shrinkage in the spray dried particles. The mechanism of atomisation and combined effects of drying rates taking place at the initial drying stages could be responsible for the observed particle morphology [
FT-IR spectroscopy was employed to identify potential interactions between the carrier HP-β-CD and the subcritical water extract (SWE) complexes during both freeze drying and spray drying as a way to confirm encapsulation. The application of FT-IR was based on the fact that; vibrational spectra are unique physical properties of molecules. Therefore, it is possible to overlay spectra of pure and complexed powders to determine changes induced during encapsulation [
Infrared spectra of pure HPβ-Cyclodextrin and spray and freeze-dried HPβ-Cyclodextrin complexed with SWE, are shown in
No new peak was observed in the FTIR spectrum of binary systems, confirming that there were no new chemical interactions and no new covalent bonds had formed.
The FTIR spectrum of pure HPβ-Cyclodextrin revealed all the functional groupings recorded in
Functional group | Bond | Wavenumber (frequency) cm−1 |
---|---|---|
Alcohols and phenols | O-H stretching | 3500-3200 |
Alkanes | C-H stretching | 3000-2850 |
General carbonyls | C=O stretching | 1760-1665 |
Carboxylic acids | C=O stretching | 1760-1769 |
Esters and others | C-OH stretching | 1320-1000 |
However, the C=O was relevant in the direct encapsulated subcritical water extract (SWE) of the apple pomace and was present when complexed with HPβ-Cyclodextrin (
Carboxylic acid group (C=O) is distinctive to polyphenolic compounds and a strong peak was reported at 1740 cm−1 when quercetin was encapsulated in a surfactant Poloxamers [
The antioxidant activity determined using the ORAC assay of the liquid subcritical water extract of the apple pomace was compared to that of the spray dried powder to establish the impact of “direct” encapsulation. The spray drying had a marked impact and resulted in 46.5% loss of antioxidant activity (
A decrease in polyphenolic content of elderberry juice by 25% had been reported after spray drying and decreases further with increasing inlet temperature [
Bond | Frequency | Change Δδ | |
---|---|---|---|
HPβ-CD | SWE + HPβ-CD | ||
O-H | 3338.18 | 3341.07 | +2.89 |
C-H | 2925.48 | 2926.45 | +0.97 |
C=O (general carbonyl) | 1643.05 | 1627.63 | −15.42 |
C=O carboxyl | - | 1731.76 | +1731.76 |
C-OH | 1020.16 | 1024.98 | +4.82 |
Bond | Frequency (cm−1) | Change Δδ | |
---|---|---|---|
HPβ-CD | SWE + HPβ-CD | ||
O-H | 3338.18 | 3317.93 | −20.25 |
C-H | 2925.48 | 2928.38 | +2.90 |
C=O(general carbonyl) | 1643.05 | 1634.38 | −8.70 |
C=O carboxyl | - | 1726.94 | +1726.94 |
C-OH | 1020.16 | 1021.12 | +0.96 |
Antioxidant activity | Subcritical water extract | Percentage loss in antioxidant activity (%) | |
---|---|---|---|
Before spray drying | After spray drying | ||
TPC (mg/l) GAE | 574.1 ± 13.9 | 318.8 ± 11.2 | 44.6 |
ORAC (μmolTE/g) DW | 1517.6 ± 93 | 811.7± 20 | 46.5 |
Antioxidant activity | Subcritical water extract with HPβ-CD | Percentage loss in antioxidant activity (%) | |
---|---|---|---|
Before spray drying | After spray drying | ||
TPC (mg/l) GAE | 530.0 ± 4.4 | 513.5 ± 16 | 3.2 |
The caking nature and relatively high hygroscopicity of the directly encapsulated powder (SWE) from subcritical water extract was a potential challenge to the stability studies to be conducted in the solid form. However, liquid forms were utilised during analysis, because molecular encapsulation can form both in solid and in solution [
Changes in antioxidant activity of SWE and SWE + HPβ-CD were monitored at facilitated conditions of 60˚C to observe time effect on the retention of polyphenolic antioxidant activity. A control experiment employing Chlorogenic acid standard was set up similar to the HPβ-Cyclodextrin complex. Folin Ciocalteu method proposed among standardised assays for quality control and antioxidant activity determination was adopted and applied.
The changes in antioxidant activity overtime are presented in
Antioxidant activity decreases with time at the constant temperature 60˚C. A 44% decrease in antioxidant activity of the directly encapsulated SWE was observed compared with 25% decrease of the subcritical SWE + HPβ-CD over the 35 days period. The initial antioxidant activity of the HPβ-Cyclodextrin included complexes (both subcritical water extract and standard Chlorogenic acid) were slightly below the direct encapsulated SWE and the Chlorogenic acid standard. However, there was a sharp increase in antioxidant activity within the 7 days’ period and then decrease afterwards. One-way analysis of variance (ANOVA) of the antioxidant activity was tested against time for each treatment with post hoc Tukey comparisons at 95% confidence. The null hypothesis was, all means (antioxidant activity) were equal against the alternative hypotheses that, at least one mean (antioxidant activity) was different. There were no significant changes in antioxidant activity for subcritical water extracts within the first 7 days (p > 0.05). However, antioxidant activity varied significantly after the 7 days (p < 0.05) and no significant changes were observed between 14 - 35 days (p < 0.05). For the control experiment using the standard Chlorogenic acid, antioxidant activity significantly changed after 7 days (p > 0.05) and no significant differences in antioxidant activity measured from 14 - 28 days (p < 0.05). The 35th-day antioxidant activity of Chlorogenic acid was significantly different and may be attributed to high antioxidant activity of degradation products of Chlorogenic acid. For the HPβ-CD encapsulated complexes, antioxidant activities at day 7
were significantly higher compared to day 1 (initial) antioxidant activities (p < 0.05). No significant differences in antioxidant activities for days, 21, 28 and 35 for SWE + HPβ-CD encapsulated products were observed (p > 0.05).
Likewise, there was no significant difference in antioxidant activities for days 7, 28, 35 for HPβ-CD encapsulated Chlorogenic acid standard (p > 0.05).
Clearly, only first 7 days’ antioxidant activities were comparable for both encapsulated and non-encapsulated subcritical water extracts samples. It implies that there was no significant difference between antioxidant activities for both treatments within the first 7 days (p < 0.05).
However, antioxidant activity for day 14, 21, 28 and 35 of HPβ-CD encapsulated subcritical water extracts were significantly higher than corresponding samples without HPβ-CD (p > 0.05). Unexpectedly, only day 7 antioxidant activity of control experiment of standard Chlorogenic acid was similar to day 7 antioxidant activity of Chlorogenic acid + HPβ-Cyclodextrin (p < 0.05).
There was a significant variation of the antioxidant activity after 7 days (p < 0.05) of Chlorogenic acid with HPβ-Cyclodextrin. Antioxidant activities of days 7, 14, 21, 28 and 35 of standard Chlorogenic acid samples were significantly lower (p < 0.05), compared to antioxidant activities of days 7, 14, 21, 28 and 35 of the complex of Chlorogenic acid with HPβ-CD.
The hydroxyl Propyl Cyclodextrin offered good protection of the polyphenolic antioxidant compounds in the subcritical water extracts against degradation, and was confirmed in the control experiment using standard phenolic compound Chlorogenic acid. Cyclodextrin was considered as secondary antioxidant and had been reported to have protective effect on ascorbic acid and phenolic compound 2,2,5,7,8-pentamethylchroman-6-ol (PMC) [
Protective mechanism of the Cyclodextrin against degradation and oxidation were apparently due to the complexation of the subcritical water extracts into its hydrophobic cavity. Therefore, the HPβ-Cyclodextrin can be employed as a carrier to prolong shelf-life of the phenolic antioxidant compounds and to mask any undesired taste and colour of SWE for applications in nutraceutical, pharmaceutical industries. The polyphenolic compounds identified in the SWE is shown in
Encapsulation of subcritical water extract was successfully demonstrated by spray drying technique with and without an external carrier. Particle sizes of powders suitable for cosmetic formulations were achieved. However, spray drying methods negatively affected antioxidant activity of the extracts. Total antioxidant activity of the auto-encapsulated (SWE) was approximately 50% less than the liquid subcritical water extract. Micronised products obtained were found to be hygroscopic which would negatively affect their applications in
cosmetic and pharmaceutical formulations and cost of storage. Hydroxyl propyl-β-Cyclodextrin as a carrier has decreased the hygroscopicity of the subcritical water extract, mask the brown colour, and in addition, demonstrated protective effect against oxidation and degradation, thereby prolonging the shelf life of the antioxidants compounds. Fourier transform infra-red spectroscopy (FTIR) and Electron scanning microscopy (SEM) were selected to characterised powders to support evidence of encapsulation of the subcritical water extract into the hydrophobic cavity of the Cyclodextrin. Both techniques have revealed some level of interaction between the host HPβ-CD and subcritical water extract.
The research was supported financially by Ghana Education Trust Fund (GETFund). The authors would like to thank Universal Beverages Limited (UBL), a subsidiary company of Bulmers, UK who supplied the apple pomace sample.
The authors declare no conflicts of interest regarding the publication of this paper.
Ibrahim, S. and Bowra, S. (2019) Improving Oxidative Stability of Polyphenolic Fraction of Apple Pomace by Encapsulation Using Naturally Occurring Polymers. Journal of Encapsulation and Adsorption Sciences, 9, 83-108. https://doi.org/10.4236/jeas.2019.92005