1. Introduction
Polyphenols are natural secondary metabolites produced by plants. They are classified into different groups as phenolic acids (derivatives of benzoic acid and cinnamic acid), flavonoids (flavonols, flavones, isoflavones, flavanones, anthocyanidins and flavanols (e.g., catechins and proanthocyanidins)), stilbenes and lignans [
1]. Polyphenols are known for their strong antioxidant properties and potential health benefits, including the prevention of chronic illnesses such as cardiovascular diseases, type 2 diabetes, osteoporosis, neurodegenerative diseases and some cancers, although their protective action goes beyond the modulation of oxidative stress [
2]. They are increasingly used as nutritional supplements, nutraceuticals, as well as ingredients in foods, functional foods, pharmaceutical and cosmetic products.
Berries such as blueberries (
Vaccinium section
Cyanococcus spp.) contain abundant phenolic compounds, including anthocyanins (derived from anthocyanidins by glycosylation), flavonols and chlorogenic acids, which are mainly found in berry skin [
3,
4,
5]. Some of these compounds are pigments that impart pleasant and characteristic colours to the fruits. Berry fruits can be processed into juice, wine, jam and marmalade, among other foods. Berry processing generates large quantities of pomace, which consists of skin, seeds and some flesh [
6,
7,
8]. Berry flesh contains about 10% of the total polyphenols, while the skin and seeds contain 28–35% and 60–70%, respectively, which makes berry processing by-products an excellent source of polyphenols [
9]. According to Struck et al. [
10], processing berries into juice leaves approximately 20–30% of the fruit as pomace. Blueberry production in Canada, the second largest producer worldwide after the United States, reached 176,641 tons in 2017 [
11], with some consumed fresh and some being processed. Thus, blueberry pomace from food processing results in considerable losses in polyphenols and other valuable bioactive phytochemicals (most notably, carotenoids, vitamins and dietary fiber) if these are not recovered. Extracting these compounds from the pomace for subsequent use in foods, pharmaceuticals or fine chemicals for healthcare and lifestyle applications is considered the best approach for maximal valorisation of this by-product.
With increased awareness of food additives, functional foods and sustainable food production in recent years, consumers have become more demanding in regard to food quality. This promotes a high demand for more natural and safe sources of ingredients. Fruits, vegetables and their by-products are prime sources for the recovery of natural polyphenols with multiple functionalities. Several extraction techniques are available but the conventional ones (e.g., decoction, digestion, infusion, maceration, percolation, Soxhlet extraction, hot continuous extraction and counter-current extraction) have notorious drawbacks. They tend to be laborious, time consuming, produce diluted extracts, cause degradation of some of the desired compounds, and involve large amounts of solvents which contribute to environmental pollution and greenhouse effect. The remaining solvent residues are often flammable, volatile and toxic [
12,
13,
14,
15]. For safety, environmental and economical sustainability, green or eco-friendly processes are being developed using various methods such as microwave-assisted extraction, supercritical fluid extraction, accelerated solvent extraction, enzyme-assisted extraction and ultrasound-assisted extraction (USAE) [
16,
17]. Their main advantages include shorter extraction times, reduced energy consumption, fewer negative environmental impacts, increased safety as well as enhanced innovation and competitiveness [
18], all of which contribute to improving the sustainability of the value chain that supplies the extracts.
In this context, USAE is a particularly attractive method due to effective extraction, energy saving and the use of moderate temperatures, which is beneficial for heat-sensitive compounds [
19]. It is thus widely used to extract bioactive compounds from plant materials [
20]. The main drawback of USAE is the unavoidable use of organic solvents in some applications, yet the equipment is simpler and the overall cost is lower compared to supercritical CO
2 extraction which does not use organic solvent [
21]. Still, this limitation can be overcome by using ethanol as USAE solvent as it is safe to use in food systems, completely biodegradable, available in high purity form and at low price [
14]. Several USAE parameters affect the quality of the extracts. Among them, sonication time, temperature, solvent composition, solid/solvent ratio, particle size of the raw material, matrix parameters as well as ultrasonic irradiations (power, frequency) can affect the quantity, composition and biochemical properties of the extracts [
12,
19,
20,
22].
Several studies have examined the USAE of bioactive phytochemicals. The results tend to differ markedly among studies according to operating conditions. Moreover, each plant material has its own unique properties in terms of chemical composition, physical characteristics, processing, storage conditions, origin (e.g., genetics and growing environment) and provider, for instance [
13], which seem to affect the outcomes of USAE. Although the extraction of berry polyphenols by USAE has been studied quite extensively, extraction parameters vary widely. There is no consensus on the optimum USAE parameters and, because of the extremely diverse nature of polyphenolics and biological matrices in which they are embedded, the extraction of these compounds cannot be easily standardized or generalized [
9]. Therefore, USAE methods must be developed to be suitable for use with the plant material considered and the phenolic compounds or fractions of interest. In order to develop an extraction method well tailored for blueberry pomace, the aim of the present study was to assess the effects USAE parameters (sonication time, solid/liquid ratio, solvent composition, pH and extraction temperature) on the total phenolic, flavonoid and anthocyanin contents and total antioxidant activity of extracts prepared from blueberry wine pomace.
3. Materials and Methods
3.1. Plant Material and Chemicals
Blueberry pomace powder prepared by freeze-drying blueberry wine pomace (from Vaccinium angustifolium, lowbush blueberry) was kindly provided by Nova Agri Inc. (Centreville, NS, Canada) and stored at −30 °C prior to use. All the chemicals were of analytical reagent grade. Sodium benzoate and hydrochloric acid were obtained from Sigma Scientific (Oakville, ON, Canada). Ethanol, methanol and formic acid of HPLC grade were purchased from Caledon Laboratories (Georgetown, ON, Canada). Folin–Ciocalteu’s phenol reagent (2 N), sodium carbonate, sodium nitrite, aluminium chloride, gallic acid (GA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA). (+)-Catechin and Folin–Ciocalteu reagent were purchased from Fluka (Milwaukee, WI, USA). Anthocyanin standards in the form of anthocyanidins (cyanidin chloride, dephinidin chloride, malvidin chloride, pelargonidin chloride, peonidin chloride and petunidin chloride) and dimethyl sulfoxide were obtained from Indofine Chemical Company Inc. (Somerville, NJ, USA).
3.2. Ultrasound-Assisted Extraction (USAE)
Ultrasound-assisted extraction was performed in an ultrasonic cleaner bath (15.5 × 14 × 9 mm, Symphony 97043-932, VWR, Mississauga, ON, Canada) with a maximum operating power of 35 kHz and 64 W. Prior to USAE, a beaker half-filled with distilled water was heated to the desired extraction temperature with stirring, using an agitator hotplate equipped with a temperature probe (IKA RCT basic, Staufen, Germany, 0–1500 rpm, 0–350 °C) and the temperature was kept constant. Meanwhile, 2 g of blueberry pomace powder was poured into a 125 mL brown-coloured flask. Then, milliQ water or various ethanol-milliQ water ratios was added as the extraction solvent to reach the appropriate solid/liquid ratio and shaken for a few minutes. The flask was tightly closed to avoid solvent evaporation, then immersed by suspension into the beaker of distilled water for a few minutes so that the mixture reached the desired extraction temperature. The heated water was subsequently poured into the ultrasound bath and the flask with a weight ring was placed into the bath. USAE was carried out at maximum operating power (35 kHz) for a specified duration at the set temperature (
Table 1). The treatment was conducted in batch mode without agitation and cooling system since preliminary experiments using distilled water without any materiel immersed showed no increase in temperature (data not shown). After extraction, the resulting extracts were centrifuged at 6000 rpm for 15 min at room temperature and filtered by vacuum filtration through a 45 µm Millipore polyvinylidene difluoride (PVDF) membrane. The filtrate was transferred into a 100 mL amber glass volumetric flask, wrapped with aluminium foil to prevent degradation of bioactive compounds, and concentrated by rotary evaporation under vacuum (Büchi Rotavapor RII, Rose Scientific Ltd., Essen, Germany) at 40 °C and 100 mbars for 20 min. The filtered extract was stored in a brown-coloured bottle at 4 °C until further analyses. The USAE parameters that were varied are sonication time, solid/liquid ratio and solvent composition (% ethanol in water), pH and temperature, according to the experimental scheme summarized in
Table 1. All extractions were carried out in triplicate.
3.3. Chemicals Analyses of Extracts
All chemical analyses, except TAC which was determined by HPLC, were performed in a 96-well microplate reader Synergy 2 equipped with Gen5TM data analysis software (Biotek Instruments Inc., Winooski, VT, USA).
3.3.1. Determination of Total Phenolic Content (TPC)
Total phenolic content of the blueberry pomace extracts was determined using the method of Folin–Ciocalteu following the procedure described by Tournour et al. [
42] with slight modification. Briefly, 25 μL of either sample or standard properly diluted with milliQ water were transferred into appropriate wells. With a multichannel pipet, 125 μL of 0.2 N Folin–Ciocalteu’s reagent were added to each well, then the plate was swirled and incubated in the dark at room temperature. After 8 to 10 min, 125 μL of 7.5% sodium carbonate was added. The obtained solution was mixed thoroughly and incubated at room temperature for 30 min at least and no more than 60 min. Subsequently, the absorbance was recorded at 765 nm with a spectrophotometric microplate reader (Synergy HT Multi-Detection Microplate Reader, BioTek Instruments, Winooski, VT, USA). Absorbance was compared to a gallic acid standard curve (R
2 = 0.999) to quantify TPC in the sample. The results were expressed as milligrams of gallic acid equivalents per gram of dry matter (mg GAE/g DM). Each standard and sample solution was analysed in triplicate.
3.3.2. Determination of Total Flavonoid Content (TFC)
Total flavonoid content (TFC) of the extracts was determined according to the 96-well microplate method [
43] with some modification. A volume of 110 μL of 0.066 M sodium nitrite (NaNO
2) was added to each of the 96 wells and 25 μL of standard or properly diluted sample solution was added. The plate was gently swirled and incubated at room temperature for 5 min. Then, 15 μL of 0.75M aluminium chloride (AlCl
3) solution was added to the mixture simultaneously in each of the wells using a multichannel pipet. The plate was swirled again and incubated at room temperature. After 6 min, 100 μL of 0.5 M NaOH were added. The precipitations formed were gently dissolved using the multichannel pipet by avoiding the generation of air bubbles. Finally, absorbance was measured at 510 nm in the plate reader. All samples and standards were prepared in methanol and measured against a methanol reagent blank using the template of the microplate. Catechin (15–500 μg/mL) was used as a standard to generate a linear calibration curve (R
2 = 0.998) and results were expressed as milligrams of catechin equivalents per gram of DM (mg CE/g DM). Each standard and sample solution was analysed in triplicate.
3.3.3. Determination of Total Anthocyanin Content (TAC) and Identification of Anthocyanins
Total anthocyanin content (TAC) and individual anthocyanins were determined by HPLC-PAD using an Agilent 1100 series system equipped with a photodiode-array detector 200–800 nm (Agilent Technologies, Waldbronn, Germany). The column was a C-18 HPLC column, 5 μm, 150/4.6 mm (YMC Inc., Wilmington, NC, USA). The elution solvents were (A) 10% formic acid/milliQ water (v/v) and (B) 100% methanol. Solvent gradient was linear from 95% A/5% B to 40% A/60% B (0–20 min), isocratic at 40% A/60% B (20–23 min), linear from 40% A/60% B to 95%A/5% B (23–24 min), and isocratic at 95%A/5% B (24–28 min, run time 28 min). The detection wavelength was 520 nm. Flow rate was 0.7 mL/min, column temperature 25 °C, pressure 300 bars, sample temperature was ambient and injection volume was 40 μL.
Commercially available anthocyanidin standards of cyanidin chloride, delphinidin chloride, malvidin chloride, pelargonidin chloride, peonidin chloride and petunidin chloride were separately dissolved in 2 mL of dimethyl sulfoxide (99.9%) and used as standard stock solutions. The stock solutions were diluted in methanol (v/v) to prepare 3.125, 6.25, 12.5, 25.0 and 50 μg/mL solutions for all standards. For identification of the anthocyanins present in the extracts, these six standard solutions were separately injected into the column.
TAC of the extracts were quantified after acid hydrolysis, which enables the determination of the aglycon forms of the anthocyanins (i.e., the anthocyanidins) (
Figure 6). A 60 μL sample was transferred into a 50 mL flat-bottom centrifuge tube and 3 mL of milliQ water were added. The tube was capped and the sample was vortexed for 60 s. Then, 3.3 mL of hydrochloric acid (HCl 5N) were added. The mixture was heated in a water bath (100 °C for 60 min), then cooled to room temperature under running tap water. It was subsequently filtered through a 0.25 μm PTFE membrane filter into an HPLC vial and analysed by HPLC-PAD. Two replicates per sample were prepared. Malvidin was used as a standard to generate a linear calibration curve (R
2 = 0.997) and the results were expressed as milligrams of malvidin equivalents per gram of DM (mg ME/g DM). Standard and sample solutions were analysed in triplicate.
3.3.4. Determination of Antioxidant Activity
Antioxidant activity of the extracts was evaluated as DPPH free radical scavenging activity determined using the DPPH assay, as described by Herald et al. [
43] with some modification. The DPPH stock solution (350 mM) was prepared daily in methanol and used to prepare the working solution (350 μM). Volumes of 225 μL methanol, 25 μL of methanol plus 200 μL of DPPH working solution, and 25 μL of standards or sample plus 200 μL of DPPH were respectively added to blank wells, control wells, and standard or sample wells using a multichannel pipet. The plate was sealed with sealing tape, gently swirled then incubated for 6 h at room temperature in the dark. After incubation, absorbance was recorded at 517 nm using the above-mentioned microplate reader. The percentage of DPPH quenched was calculated using Equation. 1:
where A is the absorbance of the sample, blank or control. Trolox (62.5–1000 μM) was used as a standard to generate a calibration curve (R
2 = 0.998) and DPPH free radical scavenging activity was expressed as trolox equivalents (mg TE/g DM).
3.4. Statistical Analyses
Descriptive statistics were calculated and expressed as means ± standard deviation (SD). After checking for normality, means were compared using either one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, or the Kruskal–Wallis test followed by the Dunn’s multiple comparison test, as appropriate. Analyses were performed using Statistica version 7. Statistical significance was established at p ≤ 0.05.