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Article

Optimization of Major Extraction Variables to Improve Recovery of Anthocyanins from Elderberry by Response Surface Methodology

by
Seunghee Kim
1,†,
Hyerim Son
1,†,
So Young Pang
1,
Jin Ju Yang
1,
Jeongho Lee
1,
Kang Hyun Lee
1,
Ja Hyun Lee
2,*,
Chulhwan Park
3,* and
Hah Young Yoo
1,*
1
Department of Biotechnology, Sangmyung University, 20, Hongjimun, 2-Gil, Jongno-Gu, Seoul 03016, Republic of Korea
2
Department of Convergence Bio-Chemical Engineering, Soonchunhyang University, 22, Soonchunhyang-Ro, Asan-si 31538, Republic of Korea
3
Department of Chemical Engineering, Kwangwoon University, 20, Kwangwoon-Ro, Nowon-Gu, Seoul 01897, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Processes 2023, 11(1), 72; https://doi.org/10.3390/pr11010072
Submission received: 22 November 2022 / Revised: 15 December 2022 / Accepted: 24 December 2022 / Published: 28 December 2022
(This article belongs to the Section Environmental and Green Processes)
Browse Figures
Versions Notes

Abstract

:
Elderberry, which is well known for its richness in anthocyanin, is attracting attention in the bioindustry as a functional material with high antioxidant capacity. The aim of this study is to optimize extraction conditions to more effectively recover anthocyanins from elderberry. In a fundamental experiment to determine the suitable solvent, various GRAS reagents, such as acetone, ethanol, ethyl acetate, hexane, and isopropyl alcohol, were used, and total phenol and anthocyanin contents were detected as 9.0 mg/g-biomass and 5.1 mg/g-biomass, respectively, only in the extraction using ethanol. Therefore, ethanol was selected as the extraction solvent, and an experimental design was performed to derive a response surface model with temperature, time, and EtOH concentration as the main variables. The optimal conditions for maximal anthocyanin recovery were determined to be 20.0 °C, 15.0 min, and 40.9% ethanol, and the total anthocyanin content was 21.0 mg/g-biomass. In addition, the total phenol and flavonoid contents were detected as 67.4 mg/g-biomass and 43.8 mg/g-biomass, respectively. The very simple and economical extraction conditions suggested in this study contributed to improving the utilization potential of anthocyanin, a useful antioxidant derived from elderberry.

1. Introduction

Elderberry, belonging to the family Adoxaceae, is known to be a medicinal plant used to treat respiratory diseases [1]. Elderberry is distributed in the temperate and subtropical regions and has the characteristics of being easy to cultivate and rich in bioactive compounds [2,3]. Currently, in the food industry, elderberry is used as a colorant in the production of a variety of products, including juices, jellies, and jams [4], and the global elderberry market is forecasted to reach 214.9 million dollars by 2025 [5]. Elderberry contains polyphenols such as anthocyanins, flavonols, and phenolic acids [6]. Due to the abundant polyphenols present in elderberry, several studies have been reported on the bioactivities of elderberry, such as antioxidant, antibacterial, anti-inflammatory, and immune-stimulating activity [7,8]. In particular, elderberry contains a large number of anthocyanins, including cyanidin derivatives such as cyanidin-3-glucoside, cyanidin-3-sambubioside, and cyanidin 3-sambubioside-5-glucoside [9].
Anthocyanins are water-soluble pigments associated with the reddish to purplish color of fruits, leaves, vegetables, and flowers [10]. Anthocyanin has a molecular structure in which an alkyl group and a phenolic hydroxyl group that removes free radicals are bonded to a flavylium cation [11]. In addition, recent studies have reported that anthocyanins contribute to lowering blood sugar levels and have anti-adipogenic and anti-obesity activities in vivo [12,13]. Due to these outstanding bioactivities of anthocyanins, the global anthocyanin market, which was worth 500 million dollars in 2018, is projected to expand at a compound annual growth rate (CAGR) of 4.6% by 2026 [14]. Anthocyanins can be recovered from plant sources such as carrots, purple sweet potato, and red cabbage, and berries are known to be especially abundant anthocyanins. Among various berries, elderberry has a relatively high content of anthocyanins compared to other berries such as bilberry, cherry, and strawberry [15]. Therefore, elderberry is one of the suitable natural sources of anthocyanins.
Currently, studies on anthocyanin recovery from berries such as blueberries and blackcurrants are being actively conducted [16,17]. Acidified methanol is generally used as an anthocyanin extraction solvent [18]. In various studies, anthocyanins were recovered from blueberries, elderberries, and strawberries using 80% methanol (with 2% formic acid), 100% methanol (with 0.1% hydrochloric acid), and 100% methanol (with 1% hydrochloric acid), respectively [19,20,21]. However, the use of organic solvents raises potential health concerns due to their volatility, toxicity, and environmental pollution [22]. Since anthocyanins are mainly consumed in the food or pharmaceutical industry, it is preferable to use generally recognized as safe (GRAS) solvents, such as acetone (Ace), ethanol (EtOH), and ethyl acetate (EA) in the extraction process [23]. Accordingly, studies have recently been conducted to recover anthocyanins from berries using GRAS solvents. Coklar et al. [24] evaluated the effect of GRAS solvents, including Ace, EtOH, and EA, on anthocyanin recovery from Mahonia aquifolium berries, and confirmed that ethanol was an effective extraction solvent. In addition, Dróżdż et al. [25] recovered anthocyanins from blueberries and lingonberries using 60% ethanol. To utilize anthocyanins recovered from natural sources on an industrial scale, not only the safety of the solvent used but also the economic feasibility of the extraction process should be considered [26]. In order to design an economical extraction process, it is necessary to investigate the anthocyanin extraction efficiency for GRAS solvent types and then optimize the process variables such as temperature, time, and solvent concentration.
Response surface methodology (RSM) is a statistical analysis tool that enables the modeling and analysis of processes by evaluating the effects of various variables on the response [27]. RSM is widely applied in the chemical industry because it contributes to process optimization with minimal experimentation [28]. RSM has been used to derive optimal anthocyanin extraction conditions from various sources such as black carrot, cranberry, and raspberry by investigating process variable interactions [29,30,31].
In this study, we performed the optimization of process variables to efficiently extract anthocyanins from elderberry using RSM. The effect of GRAS solvent type on the recovery of phenols and anthocyanins from elderberry was investigated. Then, the interaction between variables such as solvent concentration, reaction temperature, and reaction time on anthocyanin recovery were analyzed and the optimal conditions for effectively extracting anthocyanins from elderberry were derived. Finally, the overall process of recovering anthocyanins from elderberry was estimated using a mass balance based on 1000 g of elderberry.

2. Materials and Methods

2.1. Materials

The freeze-dried elderberry powder was purchased from Terrasoul Superfoods (Fort Worth, TX, USA). Ethanol (EtOH) and acetone (Ace) were obtained from Samchun Chemical (Seoul, Republic of Korea). Ethyl acetate (EA), hexane (HX), and aluminum chloride (AlCl3) were purchased by Duksan Pure Chemical (Ansan, Republic of Korea). Isopropyl alcohol (IPA) and potassium chloride (KCl) were acquired from DaeJung Chmicals & Metals (Siheung, Republic of Korea). Folin–Ciocalteu reagent, sodium carbonate (Na2CO3), sodium nitrite (NaNO2), gallic acid, sodium acetate trihydrate (CH3CO2Na·3H2O), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,4,6-tripyridyl-S-triazine (TPTZ), Iron(II) sulfate (FeSO4), and Iron(III) chloride (FeCl3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents used in this study were analytical grade.

2.2. Sample Preparation and Anthocyanin Extraction Procedure

Elderberry powder was prepared in a size of 90 μm using a test sieve. As a fundamental experiment, the effect of five GRAS solvents (EtOH, Ace, EA, IPA, and HX) on anthocyanin recovery from elderberry was investigated. An amount of 0.1 g of elderberry powder was soaked in 10 mL of each solvent and reacted at 25 °C for 1 h in a water bath (BHS-2, JOANLAB, Huzhou, China). Subsequently, each extract was centrifuged at 13,000 rpm for 15 min, and the supernatants were used for total phenol and total anthocyanin content analysis. All experiments were performed in triplicate.

2.3. Experimental Design by Response Surface Methodology

To optimize anthocyanin extraction from elderberry, the central composite design (CCD), one of the RSMs, was conducted. Table 1 shows the variables (temperature, time, and EtOH concentration) and their levels. The experimental response was the total anthocyanin content of the extract.
The effects of the interactions between variables on response values were estimated through the following quadratic Equation (1):
Y = β0 + ∑ βiXi + ∑ βijXiXj + ∑ βiiXi2,
where Y is the dependent variable (total anthocyanin content), Xi and Xj are the independent variables (temperature, time, and EtOH concentration), β0 is the offset term, βi, βij, and βii are the linear, interaction, and quadratic coefficients, respectively. The analysis of variance (ANOVA) and numerical optimization were carried out using the software Design-Expert 7 (Stat-Ease, Inc., Minneapolis, MN, USA). All anthocyanin extraction experiments were performed in triplicate.

2.4. Analytical Methods

2.4.1. Total Polyphenol Content

The total phenol content (TPC) of elderberry extract was determined with a slight modification of the Folin–Ciocalteu colorimetric assay [32]. First, 10 μL of the elderberry extract was mixed with 790 μL of DW and 50 μL of Folin–Ciocalteu reagent. The mixture was reacted at 30 °C for 8 min. Subsequently, 150 μL of 20% (w/w) Na2CO3 was added to the mixture. After 1 h incubation at 25 °C, the absorbance (A) was measured at 765 nm using an ultraviolet-visible (UV) spectrophotometer (DU® 730, Beckman Coulter, Brea, CA, USA). TPC was expressed as mg gallic acid equivalent (GAE) per gram of biomass.

2.4.2. Total Flavonoid Content

The total flavonoid content (TFC) of elderberry extract was determined according to aluminum chloride colorimetric assay [33]. A total of 50 μL of the elderberry extract was mixed with 30 μL of 5% (w/w) NaNO2 and the mixture was reacted at 25 °C for 6 min. Following this, 50 μL of 10% (w/w) AlCl3 was added to the mixture and the reactant was further incubated at 25 °C for 5 min. A total of 300 μL of 1 M NaOH and 1 mL of DW were added to the reactant and reacted at 25 °C for 15 min. The absorbance (A) of the reactant was measured at 510 nm. The experiment was performed in triplicate. TFC was expressed as mg rutin equivalents (RE) per gram of biomass.

2.4.3. Total Anthocyanin Content

The total anthocyanin content (TAC) of the elderberry extract was determined using the pH differential method according to Chen et al. [34]. An appropriate dilution factor for the extract was selected through dilution with 0.025 M KCl buffer (pH 1.0) so that the absorbance at 520 nm was within the linear range of the spectrophotometer. According to the determined dilution factor, the elderberry extract was diluted with 0.025 M KCl buffer (pH 1.0) and 0.4 M sodium acetate buffer (pH 4.5), respectively. The diluted samples were reacted at 25 °C for 20 min in a water bath. Then, the absorbance (A) of each sample was measured at 520 nm and 700 nm, respectively. The TAC was calculated as cyanidin-3-glucoside equivalents (mg/g-biomass) by Equation (2):
Total anthocyanin content (mg/g-biomass) = (A × DF × MW × 103)/(ε × 1)
where A (absorbance) is (OD520nm − OD700nm) pH 1.0 − (OD520nm − OD700nm) pH 4.5, DF is dilution factor, MW is molecular weight for cyanidin-3-glucoside (449.2 g mol−1), ε is molar extinction coefficient for cyanidin-3-glucoside (26,900 L mol−1 cm–1), and 1 is path length in cm (1 cm).

2.4.4. Ferric Reducing Antioxidant Power

The ferric reducing antioxidant power (FRAP) of elderberry extract was measured according to Balciunaitiene et al. [35]. The FRAP reagent was composed of 300 mM sodium acetate buffer (pH 3.6), 10 mM TPTZ solution dissolved in 40 mM HCl, and 20 mM FeCl3·6H2O. First, 300 μL of DW was prewarmed at 37 °C for 5 min and 30 μL of elderberry extract and 900 μL of FRAP reagent were additionally blended. The blank used DW instead of elderberry extract. The mixture was reacted at 37 °C for 4 min. Finally, the absorbance of the samples was read at 593 nm. The standard curve for deriving FRAP values was linear from 0.5 to 2 mmol/L ascorbic acid.

2.4.5. ABTS Radical Scavenging Activity

The ABTS free-radical scavenging activity of elderberry extract was measured according to Lee et al. [36]. An ABTS free-radical solution was produced by blending 7 mM ABTS solution and 2.45 mM potassium persulfate. A total of 950 µL of ABTS free-radical solution and 50 µL of the elderberry extract were mixed and reacted at 25 °C for 30 min. As a control sample, 50 µL of methanol was added to 950 µL of ABTS free-radical solution. The absorbance of the samples was read at 734 nm using the UV spectrophotometer. ABTS free-radical scavenging activity was calculated by Equation (3), and the result was expressed as an IC50 (µg/mL) value, i.e., the sample concentration capable of removing free radicals to 50%.
ABTS radical scavenging activity (%) = (1 − sample OD734 nm/Control OD734 nm) × 100

2.4.6. DPPH Radical Scavenging Activity

The DPPH free-radical scavenging activity of elderberry extract was measured according to Del Pozo et al. [37]. A total of 500 μL of the elderberry extract was added to 500 μL of 0.25 mM DPPH reagent, and reacted in a water bath at 25 °C for 30 min. As a control sample, 500 μL of methanol was added to 0.25 mM DPPH reagent, instead of elderberry extract. The absorbance of the samples was measured at 517 nm using the UV spectrophotometer. DPPH free-radical scavenging activity was calculated using Equation (4) and the result was expressed as an IC50 (µg/mL) value:
DPPH radical scavenging activity (%) = (1 − sample OD517 nm/Control OD517 nm) × 100

3. Results and Discussion

3.1. Effect of Solvent Types on the Phenolic Compound Recovery from Elderberry

As a fundamental experiment to select extraction solvent, the effect of solvent type on the phenolic compound recovery was investigated. Five GRAS solvents (EtOH, Ace, EA, IPA, and HX) with different polarities were used to extract phenolic compounds from elderberry, and the TPC and TAC of the extracts were analyzed. EtOH, Ace, EA, IPA, and HX are widely used as industrial organic solvents [38,39]. As a result, phenolic compounds were detected only in the extract using EtOH, and its TPC and TAC were found to be 9.0 mg/g-biomass and 5.1 mg/g-biomass, respectively (data not shown). The solvent extraction efficiency of phenolic compounds is determined by the solubility of the compounds, which depends on the polarity of the solvent [40]. For the solvents used in the fundamental experiment, the order of polarity is EtOH > Ace > EA > IPA > HX [41]. These results are consistent with the report of Oliveira et al. [42] that the extraction efficiency increased as the solvent with relatively high polarity was used in the recovery of phenolic compounds from green coffee beans. Therefore, the TPC and TAC detected in the elderberry extract with EtOH may be due to the relatively high polarity of EtOH.
In order to recover polar phenolic compounds that are relatively soluble in polar solvents, aqueous organic solvents are usually used as extraction solvents [43]. The reduction of organic solvent consumption enables sustainable and economical process design by decreasing the overall cost [44,45]. In this regard, the effect of EtOH concentration (0, 25, 50, 75, 100%, water- EtOH mixture) was investigated on the TPC and TAC of elderberry extract and shown in Figure 1. The TPC and TAC were improved in the extract using the aqueous EtOH solvent (25, 50, 75%) compared to the extract using 0% EtOH (DW) and 100% EtOH. In particular, the TPC and TAC of the extract using 50% EtOH were the highest at 60.5 mg/g-biomass and 20.9 mg/g-biomass, respectively. The results were consistent with the other literature indicating that an aqueous EtOH solution was more effective for the recovery of phenolic compounds including anthocyanins than pure EtOH [46,47,48]. It is known that anthocyanin aglycones and their glycosides are relatively more soluble in alcohol and water, respectively [49]. The use of aqueous EtOH solution may recover both anthocyanin aglycones and their glycosides from plant sources. Numerous studies have used aqueous EtOH solution for anthocyanin extraction from plant sources such as grape pomace, purple sweet potato, and red rice bran [50,51,52]. These results demonstrated that aqueous EtOH is suitable for anthocyanin extraction from elderberry.

3.2. Optimization of Anthocyanin Extraction Conditions Using Response Surface Methodology

CCD of RSM was conducted to optimize anthocyanin extraction conditions from elderberry. The following regression equation was estimated through regression analysis on the experimental results.
Y = 21.12 − 0.50 X1 + 0.034 X2 − 1.47 X3 + 0.090 X1X2 + 0.076 X1X3 − 0.28 X2X3 − 0.32 X12 + 0.26 X22 − 2.81 X32
where Y is the predicted TAC (mg/g-biomass) and X1, X2, and X3 are the temperature, time, and EtOH concentration, respectively.
An ANOVA on Equation (5) was performed to evaluate the significance and validity of the predicted model and is shown in Table A1. The significant model terms for the predicted model were found to be X3 (p-value = 0.0056) and X32 (p-value < 0.0001). Based on the results of the ANOVA for Equation (5), the final predicted TAC model (Equation (6)) was derived by excluding ‘not significant’ model terms.
Y = 21.05 − 1.47 X3 − 2.80 X32
where Y is the predicted TAC (mg/g-biomass) and X3 is the EtOH concentration.
Table 2 shows the experimental and predicted TAC values under the suggested experimental conditions (20 runs) in the designed CCD, including only significant model terms. The TAC of the elderberry extracts ranged from 4.3 mg/g-biomass to 21.9 mg/g-biomass.
The significance and validity of the final predicted TAC model were evaluated using ANOVA, and the results are represented in Table 3. The final predicted TAC model showed an F-value of 55.25 and a p-value < 0.0001, and the high F-values (>1) and low p-values (<0.05) of the model signified the mathematical significance of the model [53]. R2 is the square of the statistical deviation between the experimental and predicted values, and a large R2 means that the response surface is closer to the actual value [54]. The R2 and adjusted R2 of the final predicted TAC model were 0.8667 and 0.8510, respectively, suggesting a high correlation between the experimental and predicted values. Low CV value (<10%) represents high reproducibility and reliability of the model [55]. AP shows signal-to-noise ratio, and a ratio of 4 or more indicates a model that can explore the design space [56]. The CV and AP of the final predicted TAC model were 7.97% and 24.377, respectively. Therefore, these results demonstrate that the final model can predict statistically significant TAC and reliably derive optimal anthocyanin extraction conditions.
A line plot showing the effect of EtOH concentration on the TAC of elderberry extracts is shown in Figure 2. TAC increased as the EtOH concentration approached about 45%, and rapidly decreased at concentrations above 45%. These results are consistent with the previous results above evaluating the effect of EtOH concentration on TAC after solvent selection. In addition, Jiang et al. [57] reported that the anthocyanin recovery of Akebia trifoliata flowers is negatively affected when the EtOH concentration is about 50% or higher.
Based on the ANOVA results, it was proved that the effect of temperature and time on TAC was insignificant. In order to design an economical process for recovering anthocyanins from elderberry, the optimal condition was derived, focusing on the EtOH concentration by setting the goals of variables and responses as follows: temperature, “minimize”; time, “minimize”; EtOH concentration, “in range”, and TAC, “maximize”. As shown in Table 4, the derived optimal conditions were a temperature of 20 °C, a time of 15 min, and an EtOH concentration of 40.9%. The TAC predicted under optimal conditions was estimated to be 21.2 mg/g-biomass, and the experimental results showed 21.0 mg/g-biomass. These results demonstrate that the model can accurately and reliably predict TAC.

3.3. Antioxidant Activity Assessment of the Elderberry Extract

Elderberry contains various polyphenolic compounds, including anthocyanins. The bioactive compound content in the elderberry extract recovered under optimal conditions was quantified and is shown in Table 5. Total polyphenol and flavonoid contents in elderberry extract were found to be 67.4 mg/g-biomass and 43.8 mg/g-biomass, respectively. In other words, the total anthocyanin content (21.0 mg/g-biomass) in elderberry extract accounts for about 31.2% of the total polyphenol in the extract. This result is similar to the report of Gagneten et al. [58] that anthocyanins account for about 31.5% of the total phenol in elderberry fruit.
The antioxidant potential can generally be identified through two mechanisms: its ability to reduce metallic species and its ability to react with free radicals [59]. The antioxidant activity of elderberry extract was evaluated using FRAP assay, ABTS assay, and DPPH assay. The FRAP assay measures the iron-reducing activity, and ABTS and DPPH assays evaluate radical-scavenging activity. Ascorbic acid, which is known to have excellent antioxidant activity, was used as a control. Table 6 shows the antioxidant activity results of ascorbic acid and elderberry extract. The FRAP values of ascorbic acid and elderberry were 63.3 mmol/L and 80.2 mmol/L, respectively. ABTS IC50 of the elderberry extract was 0.5 mg/mL, which was 125% of ascorbic acid’ (0.4 mg/mL), and DPPH IC50 was 0.8 mg/mL, which was 89% of ascorbic acid (0.8 mg/mL). Various studies have compared the antioxidant activities of ascorbic acid and berry extracts. It was reported that winter cherry, Zanthoxylum schinifolium fruit, and acai berry showed antioxidant activities of 20.81% (with FRAP assay), 22.7% (with ABTS assay), and 78.8% (with DPPH assay), respectively, compared to ascorbic acid [60,61,62]. Our study shows that elderberry extract has antioxidant activity similar to that of ascorbic acid compared to other extracts. As a result, elderberry extract has the potential to be used as a natural source of anthocyanins with antioxidant activity.

3.4. Evaluation of Overall Process for Anthocyanin Recovery from Elderberry

Figure 3 shows mass balance for the overall process of anthocyanin recovery from elderberry. Under the control condition (temperature, 25 °C; time, 60 min; solvent, 100% EtOH), the total anthocyanin content was estimated to be about 5.1 g based on 1000 g of elderberry. Under the optimal conditions (temperature, 20.0 °C; time, 15.0 min; solvent, 40.9% EtOH), the total anthocyanin content was assessed to be approximately 21.0 g based on 1000 g of biomass, which is a 4.1-fold improvement compared to the control. After anthocyanin extraction, about 266 g of solid residues were generated. Elderberry residue is presumed to consist of sugars such as glucose, xylose, and galacturonic acid that are characteristic of plant cell wall polysaccharides [63]. These sugars have the potential to be used as carbon sources for microbial fermentation. As a follow-up study, we plan to design a process to recover fermentable sugars from elderberry residues after extraction.
Table 7 summarized recent studies on anthocyanin recovery from berries. Anthocyanin was recovered from berries such as bilberry, blackberry, blue honeysuckle berry, blueberry, elderberry, and strawberry through various extraction methods. Extraction techniques such as supercritical carbon dioxide extraction, microwave-assisted extraction, ultrasound-assisted extraction, and maceration have been used to recover anthocyanins. The supercritical carbon dioxide extraction method is characterized by high efficiency, eco-friendliness, and safety [64]. However, supercritical carbon dioxide, which is non-polar, has limitations in extracting polar compounds such as anthocyanins, and an acidic solvent should be used as a co-solvent to recover anthocyanins [65]. Kerbstadt et al. [66] recovered about 13.7 mg/g-biomass of anthocyanins from bilberry through supercritical carbon dioxide extraction using acidic 50% EtOH as an extraction solvent. Microwave-assisted extraction and ultrasound-assisted extraction are methods that allow bioactive substances to dissolve in a solvent through damage to the plant cell wall [67]. These two methods may cause structural destruction of anthocyanins by locally generating excessive vibration or high temperature in the extract [68]. Furthermore, supercritical carbon dioxide extraction, microwave-assisted extraction, and ultrasound-assisted extraction have limitations that increase the operating cost of the entire process due to high temperature and high energy input [69]. Maceration, a traditional extraction method, is a relatively low-cost technique for recovering bioactive substances with poor thermal stability [70]. Despite the development of new extraction techniques as described above, studies on recovering anthocyanins by macerating berries in various solvents have been reported [20,45,71]. These studies generally used acidified methanol as the extraction solvent. In this study, EtOH, a GRAS solvent, was used as an extraction solvent without adding an acidic solution. Our elderberry extracts have the potential to be safely used in various industries such as food and cosmetics. The novel aspect of this study is that a highly efficient anthocyanin recovery process was designed by deriving mild conditions, such as low solvent consumption (even using GRAS solvent without acid addition), low temperature, and short reaction time. As a follow-up study, since elderberry extract contains unknown bioactive compounds including anthocyanin, identification of these compounds in the extract is required to approach practical industrial applications.

4. Conclusions

In this study, we proposed a statistical model for the efficient recovery of anthocyanins from elderberry. For the selection of extraction solvents, the content of phenolic compounds in elderberry extracts recovered with industrially used GRAS solvents was investigated. Among the GRAS solvents, phenolic compounds were detected only in the EtOH extract, and the total phenol content and total anthocyanin content were found to be 9.0 mg/g-biomass and 5.1 mg/g-biomass, respectively. EtOH was selected as the extraction solvent, and optimal conditions were derived by considering the correlation between the variables, such as temperature, time, and EtOH concentration. The optimal conditions were determined to be a temperature of 20.0 °C, a time of 15.0 min, and an EtOH concentration of 40.9%. Under the optimal conditions, the total anthocyanin content of the elderberry extract was 21.0 mg/g-biomass, which was a 4.1-fold improvement over the control before optimization. In addition, as a result of evaluating the antioxidant activity, the FRAP values of ascorbic acid and elderberry were 63.3 mmol/L and 80.2 mmol/L, respectively. The ABTS IC50 of ascorbic acid and elderberry extract were 0.5 mg/mL and 0.4 mg/mL, respectively, and the DPPH IC50 of ascorbic acid and elderberry were 0.8 mg/mL and 0.9 mg/mL, respectively. We confirmed that elderberry extract has antioxidant activity similar to that of ascorbic acid. The novelty of our study derives from the designing of a highly efficient anthocyanin recovery process with mild conditions, including low temperature, short reaction time, and low solvent usage. In a follow-up study, we plan to design a process to convert elderberry residues generated after anthocyanin extraction into fermentable sugars and identify bioactive compounds in the extracts for industrial applications.

Author Contributions

Conceptualization, S.K. and H.S.; methodology, S.Y.P. and J.J.Y.; software, K.H.L.; validation, J.H.L., C.P. and H.Y.Y.; formal analysis, J.L.; investigation, S.K. and H.S.; data curation, K.H.L.; writing—original draft preparation, S.K. and H.S.; writing—review and editing, J.H.L., C.P. and H.Y.Y.; visualization, S.K. and H.S.; supervision, H.Y.Y.; project administration, J.H.L., C.P. and H.Y.Y.; funding acquisition, H.Y.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT) NRF-2020R1C1C1005060.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Table
Table A1. Analysis of variance for the predicted total anthocyanin content model.
Table A1. Analysis of variance for the predicted total anthocyanin content model.
SourceSum of SquareDegree of FreedomMean SquareF-Valuep-ValueRemarks
Model258.58928.7310.280.0006significant
X14.0314.031.440.2574
X20.01910.0190.006710.9363
X334.58134.5812.370.0056significant
X1X20.06410.0640.0230.8827
X1X30.04610.0460.0160.9006
X2X30.6310.630.220.6462
X122.6412.640.940.3542
X221.6711.670.60.4568
X32199.221199.2271.27<0.0001significant
Residual27.95102.8
Lack of fit23.1854.644.860.0539not significant
Pure error4.7750.95
Total286.5419
Coefficient of determination (R2): 0.9024. Adjusted R2: 0.8146. Coefficient of variation (CV): 8.89%. Adequate precision (AP): 12.942.

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Processes 11 00072 g001 550
Figure 1. Total phenolic and total anthocyanin content recovered from elderberry for various ethanol concentrations.
Figure 1. Total phenolic and total anthocyanin content recovered from elderberry for various ethanol concentrations.
Processes 11 00072 g001
Processes 11 00072 g002 550
Figure 2. Line plot explaining the effect of EtOH concentration on total anthocyanin content.
Figure 2. Line plot explaining the effect of EtOH concentration on total anthocyanin content.
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Processes 11 00072 g003 550
Figure 3. Mass balance of recovered anthocyanin based on 1000 g of elderberry.
Figure 3. Mass balance of recovered anthocyanin based on 1000 g of elderberry.
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Table
Table 1. The selected variables and their coded levels for the central composite design.
Table 1. The selected variables and their coded levels for the central composite design.
VariablesUnitSymbolCoded Level
−2−1012
Temperature°CX12030405060
TimeminX21530456075
EtOH concentration%X30255075100
Table
Table 2. Predicted and experimental values of anthocyanin content recovered from elderberry under the designed extraction conditions.
Table 2. Predicted and experimental values of anthocyanin content recovered from elderberry under the designed extraction conditions.
RunCoded Factor LevelsResponse
X1X2X3Predicted TAC (mg/g-Biomass)Experimental TAC (mg/g-Biomass)
1−1−1−120.120.1
21−1−118.718.7
3−11−120.520.3
411−119.519.0
5−1−1117.519.6
61−1116.518.2
7−11116.918.5
811116.217.6
9−20020.819.8
1020018.818.3
110−2022.121.0
1202022.221.8
1300−212.813.9
140026.94.3
1500021.121.4
1600021.120.1
1700021.121.9
1800021.121.9
1900021.119.9
2000021.120.0
Table
Table 3. Analysis of variance for the final predicted total anthocyanin content model.
Table 3. Analysis of variance for the final predicted total anthocyanin content model.
SourceSum of SquareDegree of FreedomMean SquareF-Valuep-ValueRemarks
Model248.332124.1755.25<0.0001significant
X334.58134.5815.390.0011significant
X32213.751213.7595.11<0.0001significant
Residual38.20172.25
Lack of fit33.43122.792.920.1227not significant
Pure error4.7750.95
Total286.5419
Coefficient of determination (R2): 0.8667. Adjusted R2: 0.8510. Coefficient of variation (CV): 7.97%. Adequate precision (AP): 24.377.
Table
Table 4. Numerical optimization of anthocyanin extraction from elderberry.
Table 4. Numerical optimization of anthocyanin extraction from elderberry.
VariablesCoded ValuesActual Values
Temperature−2.020.0 °C
Time−2.015.0 min
EtOH concentration−0.36340.9%
ResponsePredictedExperimental
TAC (mg/g-biomass)21.221.0
Table
Table 5. Quantification of bioactive compounds in elderberry extract.
Table 5. Quantification of bioactive compounds in elderberry extract.
Content (mg/g-Biomass)
Total polyphenol67.4
Total flavonoid43.8
Total anthocyanin21.0
Table
Table 6. Evaluation of antioxidant activity of elderberry extract.
Table 6. Evaluation of antioxidant activity of elderberry extract.
Ascorbic AcidElderberry Extract
FRAP value (mmol/L)63.380.2
ABTS IC50 (mg/mL)0.50.4
DPPH IC50 (mg/mL)0.80.9
Table
Table 7. Summary of various literature on anthocyanin recovery from berries.
Table 7. Summary of various literature on anthocyanin recovery from berries.
BiomassExtraction MethodConditionsTAC
(mg/g-Biomass)		Ref.
SolventTemp. (°C)Time (min)S/L Ratio (g/L)
BilberrySupercritical carbon-dioxide extraction50% EtOH
(with 0.1% HCl)		505010013.7[66]
BlackberryMicrowave-assisted extraction52% EtOH–4252.2[72]
Blue honeysuckle berryMaceration0.35% HCl42302024.0[45]
BlueberryUltrasound-assisted extraction72.5% EtOH
(with 0.02% HCl)		301,4405016.2[73]
ElderberryMaceration100% MeOH
(with 1% HCl)		–20508.1[20]
ElderberryMaceration100% MeOH
(with 1% HCl)		–20509.5[71]
ElderberryUltrasound-assisted extraction100% MeOH
(with 0.1% HCl)		2490500.7[74]
StrawberryUltrasound-assisted extraction80% EtOH905–0.4[75]
ElderberryMaceration40.9% EtOH20.015.010021.0This study
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Kim, S.; Son, H.; Pang, S.Y.; Yang, J.J.; Lee, J.; Lee, K.H.; Lee, J.H.; Park, C.; Yoo, H.Y. Optimization of Major Extraction Variables to Improve Recovery of Anthocyanins from Elderberry by Response Surface Methodology. Processes 2023, 11, 72. https://doi.org/10.3390/pr11010072

AMA Style

Kim S, Son H, Pang SY, Yang JJ, Lee J, Lee KH, Lee JH, Park C, Yoo HY. Optimization of Major Extraction Variables to Improve Recovery of Anthocyanins from Elderberry by Response Surface Methodology. Processes. 2023; 11(1):72. https://doi.org/10.3390/pr11010072

Chicago/Turabian Style

Kim, Seunghee, Hyerim Son, So Young Pang, Jin Ju Yang, Jeongho Lee, Kang Hyun Lee, Ja Hyun Lee, Chulhwan Park, and Hah Young Yoo. 2023. "Optimization of Major Extraction Variables to Improve Recovery of Anthocyanins from Elderberry by Response Surface Methodology" Processes 11, no. 1: 72. https://doi.org/10.3390/pr11010072

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