Extraction Process of Polyphenols from Soybean (Glycine Max L.) Sprouts: Optimization and Evaluation of Antioxidant Activity

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Article Extraction Process of Polyphenols from Soybean (Glycine max L.) Sprouts: Optimization and Evaluation of Antioxidant Activity

Xuan Tien Le 1,*, Vo Luu Lan Vi 1, Tran Quoc Toan 2,3, Long Giang Bach 4,5, Tran Thanh Truc 6 and Pham Thi Hai Ha 7,*

1 Department of Chemical Engineering, Ho Chi Minh City University of Technology, Vietnam National University-Ho Chi Minh City, Ho Chi Minh City 700000, Vietnam 2 Vietnam Academy of Science and Technology, Graduate University of Science and Technology, Hanoi 100000, Vietnam 3 Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi 100000, Vietnam 4 NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City 700000, Vietnam 5 Center of Excellence for Biochemistry and Natural Products, Nguyen Tat Thanh University, Ho Chi Minh City 700000, Vietnam 6 Food Technology Department, College of Agriculture, Can Tho University, Can Tho City 94000, Vietnam 7 Faculty of Biotechnology, Nguyen Tat Thanh University, Ho Chi Minh City 700000, Vietnam * Correspondence: [email protected] (X.T.L.); [email protected] (P.T.H.H.)

Received: 27 June 2019; Accepted: 29 July 2019; Published: 1 August 2019

Abstract: This research aimed to optimize the total polyphenol content (TPC) extracted from soybean sprout powder under diﬀerent experimental parameters, including ethanol concentration (60–100% v/v), extraction temperature (40–80 ◦C), extraction time (15–150 min), material:solvent ratio (1:4–1:10 g/mL), the number extraction cycles (1, 2 and 3 times), the age of sprout (0–7 days), and the used part of the sprout (cotyledon, hypocotyl, or radicle). The obtained results were used in response surface methodology, in combination with a central composite design, to model the total polyphenol content (TPC) with respect to three variables, including ethanol concentration, extraction temperature, and material:solvent ratio. The experimental conditions for optimal recovery of TPC consisted of ethanol concentration of 88% (v/v), extraction temperature of 59 ◦C, material:solvent ratio of 1:6.5 g/mL, extraction time of 60 min, and 2 cycles of maceration. In addition, for maximal TPC, the sprout should undergo the germination of 5 days and the radicle fraction should be used. Based on the suggested optimum conditions, the obtained and veriﬁed TPC was 19.801 mg genistein (GE)/g dry weight (d.w.). The obtained dried extract also exhibited low antioxidant activity.

Keywords: soybean sprout; isoﬂavone; total polyphenol content; optimization; antioxidant activity

1. Introduction Due to the favorable geographic and climatic conditions, Vietnam possesses a rich source of medicinal materials and agricultural crops. One of the Far East’s oldest crops is soybean (Glycine max (L.) Merrill), a native species of China. The crop has been used in various forms as one of the most important sources of dietary protein and oil thanks to its excellent protein-producing eﬃciency per unit of land, proﬁtability, and potential use as a defense crop against famine. Soybean protein is also among plant proteins of best quality because it contains essential amino acids and holds the potential to replace proteins of animal origin [1]. Recently, the soybean has been claimed as a potential weapon against chronic diseases [2–7].

Processes 2019, 7, 489; doi:10.3390/pr7080489 www.mdpi.com/journal/processes Processes 2019, 7, 489 2 of 18

The soybean seed and sprout have been shown to possess phytochemicals, such as phenolic acids, saponins, phytosterols, isoflavones, phytates, and trypsin inhibitors, which are valuable in prevention of degenerative conditions [8]. Among them, isoflavones, phytoestrogen compounds belonging to polyphenols, have mechanism of action and function almost identical to those of female hormones. Structurally, isoflavones are oxygen heterocycles containing a 3-phenylchroman skeleton that is hydroxylated at 4 and 7 positions [9,10]. Since the structure is similar to that of estrogen, isoflavones are able to resist factors causing hormone-related cancers [11]. In vitro investigations and animal models have shown that isoflavones are a good candidate for inhibition of hormone-related cancers, such as breast, endometrial (uterine), and prostatic. Isoflavone also figures prominently due to its role as a classic dietary antioxidant. Other health benefits of isoflavone may include immunomodulation and risk reduction of chronic diseases, including cardiovascular diseases, diabetes, cancer, osteoporosis, and obesity [12–16]. Isoflavone content in the soybean sprout is generally higher than that in the soybean seed and the discrepancy can range from 20% to 50% after 24 h of germination [17]. In terms of isoflavone composition, the soybean sprout has been found to accumulate glycitein in large quantities in the hypocotyl, whereas the leaf fraction contains mostly daidzein and genistein [18]. In addition, coumestrol, an estrogen-like isoflavone, and its glucosides were also found in the soybean seed coats. These results were also confirmed in another comparative study where, among 46 different beans and bean sprout samples, coumestrol was found in soybean, black mappe, and green gram sprouts [19]. The sprouting process of the soybean is also suggested to cause total phenolic and flavonoid content to rise. To be specific, in a previous germination investigation involving six legume sprouts, the total phenolic content in the soybean was found to double after 120 h of germination, averaging 1 28.27 mg gallic acid (GAE).g− dry weight [20]. The same study also suggested that soybean sprout was ranked second only after peanut sprouts in terms of antioxidant activity and phenolic content. Another extensive review indicated that both total phenolic and flavonoid content of the soybean sprout methanolic extract far exceeded those of the cowpea and mungbean sprouts. Composition-wise, the major phenolic acid in the soybean sprout was salicylic acid, followed by ferulic acid and 3-hydroxycinnamic acid [21]. Attempts to achieve efficient recovery of biologically active compounds from plant materials could contribute to the development of food products in many ways. First, through optimization, improvements in existing manufacturing systems could be made, possibly leading to reduced material use, and in turn economic advantages. Second, successful surveys on plants that potentially produce useful compounds could propose new uses for existing species. For soybean sprout, despite its widespread culinary applications and established nutritional value, to date, industrial use of the sprout is limited and detailed investigation regarding isolation processes of valuable compounds from the material has been uncomprehensive. To our knowledge, only the isoflavone content of the soybean sprout cotyledon extract has been investigated and optimized, leaving total phenolic content unexplored [22]. Therefore, the aim of this research is to apply a centered-central composite design (CCD) subjected to response surface methodology (RSM) [23–26] to optimize the recoveries of total polyphenol (TPC). Considered optimization parameters include ethanol concentration, temperature of extraction, time of extraction, material:solvent ratio, and the number of extraction cycles. The results of this study are expected to contribute to the development of a new approach for large-scale production of phenolic compounds from soybean sprouts.

2. Materials and Methods

2.1. Plant Sample Preparation The soybean seeds (DT2010 variety) [27] were harvested at Cu Chi Commune, Ho Chi Minh City, Vietnam, in February, 2019. Soybean seeds were preserved in zip bags, placed in a dry, ventilated place, and kept away from moisture during the study. Processes 2019, 7, x 3 of 18

ProcessesThe2019 soybean, 7, 489 seeds (DT2010 variety) [27] were harvested at Cu Chi Commune, Ho Chi Minh3 of 18 City, Vietnam, in February, 2019. Soybean seeds were preserved in zip bags, placed in a dry, ventilated place, and kept away from moisture during the study. Soybean seeds were thenthen germinatedgerminated withwith thethe followingfollowing procedure.procedure. First, 2 kg of soybean seeds werewere soakedsoaked intointo 20,000 20,000 mL mL of of warm warm water water at at 35–40 35–40◦C °C for for 3–5 3–5 h. h. Afterwards, Afterwards, the the seeds seeds were were spread spread in basketsin baskets in ain dark a dark place place and and covered covered with with dark dark colored colored towels towels to avoidto avoid light. light. The The seeds seeds were were kept kept at roomat room temperature temperature (25–30 (25–30◦C) °C) and and allowed allowed to to germinate germinate for for zero zero to to 7 7 days. days. WaterWater waswas sprayedsprayed twicetwice daily toto provideprovide moisturemoisture duringduring sprouting.sprouting. At day 0, after soaking in warm water, thethe sproutssprouts hadhad aa lengthlength ofof 1.41.4 cm.cm. On the second and the thirdthird day,day, thethe lengthlength ofof thethe sproutssprouts waswas 3.33.3 cm and 7.4 cm, respectively. On the third day, the primary rootsroots appearedappeared andand thethe lengthlength reachedreached 10.310.3 cm.cm. From the fourth to the seventh day, thethe epicotylepicotyl appeared, developed, and the ﬁrstfirst leaf appeared at the sixthsixth day.day. The length of the sprouts at thethe fourth,fourth, ﬁfth,fifth, sixth,sixth, andand seventh seventh days days was was 12.5 12.5 cm, cm, 16.1 16.1 cm, cm 18.3, 18.3 cm, cm, and and 22.4 22.4 cm, cm, respectively respectively (Figure (Figure1). Soybean1). Soybean sprouts sprouts after after being being germinated germinated for for a suitablea suitable time time were were dried dried until until the the humidityhumidity waswas lessless thanthan 12%12% andand then then crushed. crushed. The The color color of of dried dried hypocotyl hypocotyl and and roots roots were were dark dark brown brown and that and of that dried of cotyledondried cotyledon was golden was golden brown. brown. Crushed Crushed soybean soybean germ powdergerm powder had dark had brown dark brown color. color.

Figure 1. Germination progress of soybean seeds over 77 days.days.

2.2. Extraction Method Maceration of plant material was employed in thisthis study.study. In brief,brief, twentytwenty gramsgrams ofof powderedpowdered soybeansoybean sprouts werewere extractedextracted under didifferentﬀerent extractionextraction parameters,parameters, suchsuch asas ethanol concentration (60–99.5%(60–99.5% vv/v/v),), extractionextraction temperaturetemperature (40–80(40–80 ◦°C),C), extraction time (30–150 min), material-solvent ratio (1:4–1:10(1:4–1:10 gg/mL),/mL), andand thethe numbernumber ofof extractionextraction cyclescycles (1,(1, 2,2, andand 33 times).times). The mixturemixture ofof powderpowder andand solventsolvent waswas agitatedagitated atat 120 120 rpm. rpm. After After extraction, extraction, the the solvent solvent was was evaporated evaporated from from the the extract extract liquid liquid at 50at 50◦C °C under under vacuum vacuum to obtainto obtain the the dried dried extract. extract.

2.3. Optimization of ExtractionExtraction Process by Response Surface Methodology (RSM)(RSM) Response Surface Methodology (RSM), in combinationcombination with central composite design (CCD), waswas adoptedadopted to to investigate investigate the the interaction interaction of experimentalof experimental variables variables and toand produce to produce a statistical a statistical model formodel the for extraction the extraction process process [28]. The [28]. factor The levelsfactor forleve thels for RSM the optimization RSM optimization process process were selected were selected based onbased single on factorsingle optimizationfactor optimizati method,on method, as shown as inshown Table in1. Table The three 1. The selected three variablesselected variables were ethanol were concentrationethanol concentration (X1), temperature (X1), temperature (X2), and the(X2), ratio and of the material ratio /ofsolvent material/ (X3 ).solvent Each variable (X3). Each consisted variable of 3consisted different of levels 3 different ranging levels from ranging low ( 1), from to medium low (−1), (0), to andmedium to high (0), ( +and1). Theto high number (+1). ofThe experiments number of k − performedexperiments was performed N = 2 + 2kwas+ 3N (N = =220k +with 2k + k 3= (N3), in= which20 with k isk the= 3), number in which of independent k is the number variables of k/4 andindependent 2k of additional variables experiments and 2k of atadditional the α. Distance experiments from center at the toα. point Distanceα = 2from(α center= 1.68 to with point k = α3). = All studies were conducted at five levels ( α, 1, 0, +1, +α). Thus, in this study, 20 experiments were − − carried out with 23 experiments of total planning, 3 replicated experiments at the center to evaluate Processes 2019, 7, 489 4 of 18 errors, and 6 additional experiments (Table2). The model that describe the contents of isoflavone as the response variable (Y) with respect to experimental parameters is established as follows:

X3 X3 Xn 1 X3 2 − Y(TPC) = β0 + βixi + βiixi + βijxiβxj i=1 i=1 i=1 j=i+1 where Y shows the response; β0, βi, βii, and βij indicate the regression coefficient for the intercept, liner, square, and interaction, respectively; xi and xj represent the independent factors; and n shows the number of factor involved. The empirical model was evaluated using analysis of variance (ANOVA) at 5% significance level. The significant terms of the model were also obtained by ANOVA. The adequacy of the model was identified based on R-squared, adj-R-squared, predicted-R-squared, F-value, and the lack-of-fitness statistic. The adequacy of the model was validated by reproducing the experiment in triplicate with obtained optimal parameters. The obtained results were then verified by comparing the predicted value with the actual values obtained from the experimental work.

Table 1. Experimental factors with their levels used in the experiment.

Level Factor Symbol 1 0 +1 − EtOH concentration (%) X1 85 90 95 Temperature (◦C) X2 55 60 65 Solvent-material ratio X3 4 6 8

Table 2. Central composite design of coded factors.

Run X1 X2 X3 1 ( 1) ( 1) ( 1) − − − 2 1 ( 1) ( 1) − − 3 ( 1) 1 ( 1) − − 4 1 1 ( 1) − 5 ( 1) ( 1) 1 − − 6 1 ( 1) 1 − 7 ( 1) 1 1 − 8 1 1 1 9 ( α) 0 0 − 10 (+α) 0 0 11 0 ( α) 0 − 12 0 (+α) 0 13 0 0 ( α) − 14 0 0 (+α) 15 0 0 0 16 0 0 0 17 0 0 0 18 0 0 0 19 0 0 0 20 0 0 0

2.4. Quantiﬁcation of Total Polyphenol Content (TPC) Based on Folin-Ciocalteu (FC) Methodology The determination of TPC in the dried extract was done using the technique described by Singleton with modiﬁcation [29,30]. First, the calibration curve was established by mixing 1 mL of aliquots with concentrations of 125, 250, 500, 750, 1000, 1250, and 1500 µg/mL genistein (GE, Sigma-Aldrich, St. Louis, MO, USA) solutions with 200 µL of Folin Ciocalteu (FC, Sigma-Aldrich, St. Louis, MO, USA) reagent, Processes 2019, 7, 489 5 of 18

3160 µL of water, and 600 µL sodium carbonate (Sigma-Aldrich, St. Louis, MO, USA) solution (20%). The absorbance was measured with a spectrophotometer (Shimadzu, Kyoto, Japan) after 30 min at 760 nm. Testing sample for quantiﬁcation of TPC was prepared as follows. First, 42 g of dried extract was dissolved in 6 mL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to produce the stock solution. Then, 40 µL of the stock solution was mixed with 200 µL of FC. The solution was sonicated for 5 min at room temperature, followed by addition of 3160 µL of water and 600 µL sodium carbonate solution (20%). The ﬁnal solution was sonicated for 30 min at room temperature. The blank sample was prepared identically, except for the stock solution, which was replaced by 40 µL of DMSO. Testing sample was measured 3 times to obtain the average value. A UV-Vis Spectrophotometer (Shimadzu, Kyoto, Japan) was used to take the absorbance of the mixture at 760 nm wavelength against DMSO blank. The standard curve (100–1000 mg/ L) was prepared from genistein to calculate the concentration of the sample. The obtained result was explicated as mg of genistein per mg of the dried extract (mg genistein/ mg). The amount of TPC was calculated using the following equation: ! mg Genistein C m TPC = 1 1 g d.w C2m where C1 is the concentration obtained from Genistein standard curve (mg GE/L DMSO), C2 is the concentration of the dried extract (mg dried extract/L DMSO), m1 is the weight of total dried extract (mg), and m is the weight of dried material (soybean sprouts powder) (g).

2.5. Antioxidation Evaluation with 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) DPPH measurements were used based on the method of Brand-Williams and co-workers and Lee and co-workers [31,32], in which the determination of antioxidant potency is based on the scavenging activity of the stable DPPH free radical. DPPH was ﬁrst mixed with methanol (Sigma-Aldrich, St. Louis, MO, USA) 80% (with OD 517 nm = 0.80 0.02) to form the solution with the concentration of 40 µg/mL. Vitamin C (Sigma-Aldrich, ± St. Louis, MO, USA), mixed in methanol 80% with concentrations of 0–100 µg/mL, was used as the positive control. To commence the experiment, the sample was ﬁrst dissolved in methanol 80%. Then, 180 µL of the prepared DPPH solution was added into 120 µL of the sample solution. The resultant solution was shaken, stored in darkness at 30 ◦C for 30 min, and then measured at a wavelength at 517 nm. Each experiment was repeated 3 times to calculate the average value. Besides, the color solution was prepared by adding 180 µL of 80% MeOH (Sigma-Aldrich, St. Louis, MO, USA) solution to 120 µL of the sample solution (120 µL). The absorbance of the color solution was measured at 517 nm. Each experience was repeated 3 times to calculate the average. The blank solution was prepared by adding 180 µL of the prepared DPPH solution into 120 µL of 80% MeOH solution. The percentage of DPPH radical scavenging is calculated according to the following formula:

A (AS AC) DPPH(%) = b − − Ab where Ab is the optical density of the blank sample; As is the optical density of sample; Ac is the optical density of pigment; and IC50 value is calculated by the graph of % inhibition.

2.6. Instruments The following instruments were used: moisture meter (MA35, Sartorius, Göttingen, Germany); UV-Vis spectrophotometer (Thermo Genesys 10S UV-Vis, Waltham, MA, USA); ultrasonic bath (Elma S 100 H, Elma, Singen, Germany); Elisa microplate reader (2100-C Optic Ivymen System, Biotech S.L., Madrid, Spain); rotary evaporator Buchi-R210 (Marshall Scientiﬁc, Hampton, NH, USA). Processes 2019, 7, 489 6 of 18 Processes 2019, 7, x 6 of 18

3. Results Results and and Discussion Discussion

3.1. Single-Factor Single-Factor Investigations Investigations with with Regard Regard to to TPC

3.1.1. Influence Influence of of Ethanol Ethanol Concentration Concentration Ethanol has been extensively used for extraction of biologically active compounds from plants due to to its its low low toxicity. toxicity. To To enhance the extraction extraction ef effificiencyciency of polyphenol polyphenol in soybean soybean sprouts, sprouts, ethanol ethanol isis usually used with water at diff different concentrations due to its high dielectric property. Figure 22 indicatesindicates thethe eeffectffectof of di differentfferent ethanol ethanol concentrations concentrations ranging ranging from from 60% 60% to 99.5% to 99.5% on recoveries on recoveries of TPC. of TPC.The TPC The recoveredTPC recovered from soybeanfrom soybean sprout sprout at 60% at ethanol 60% ethanol was 3.4 was mg 3.4 GE /mgg dry GE/g weight dry (d.w.).weight Further(d.w.). Furtherincrease increase of ethanol of ethanol concentration concentration to 80% enhanced to 80% enha thenced yield the of yield recovery of recovery to 7.1 (mg to 7.1 GE /(mgg d.w.) GE/g and d.w.) the andyield the peaked yield at peaked about 9.0 at mg about GE/g 9.0 d.w. mg at 90%GE/g concentration. d.w. at 90% However,concentration. raising However, the concentration raising pastthe concentration90% to 99.5% loweredpast 90% the to 99.5% yield to lowe 7.4red mg the GE /yieldg d.w. to 7.4 mg GE/g d.w.

10.0 9.0 9.0 8.0 7.4 7.1 7.0 5.8 6.0 5.0 4.0 3.4 mg GE/g d.w. mg GE/g 3.0 2.0 1.0 0.0 60 70 80 90 99.5 Concentration (v/v)

Figure 2. EffectEﬀect of of ethanol concentration.

Since most of the the bioactive bioactive components, components, such such as as saponins, saponins, phenolics, phenolics, and and flavonoids, flavonoids, are are highly highly polar, purepure ethanolethanol isis notnot capable capable of of fully fully extracting extracting bioactive bioactive compounds. compounds. Therefore, Therefore, water water should should be beadded added to enhanceto enhance the the recovery recovery yield. yield. However, However, excess excess addition addition of water of water to the to extractionthe extraction solvent solvent may maygenerate generate water-soluble water-soluble gums existinggums existing in soybean in soybean sprouts, sprouts, rather than rather TPC, than which TPC, might which complicate might complicatethe filtration the process filtration and reducesprocess theand isoflavone reduces yieldthe isoflavone through absorption yield through by the absorption impurities, suchby the as impurities,protein and such fat. as From protein the aboveand fat. observation, From the ab theove ethanol observation, concentration the ethanol of 90%concentration was selected of 90% for wassubsequent selected experiments. for subsequent experiments.

3.1.2. Influence Inﬂuence of of Extraction Extraction Temperature Temperature Figure3 3 illustrates illustrates the the TPC TPC in relationin relation to extraction to extraction temperature. temperature. As the temperatureAs the temperature was elevated was elevatedfrom 40 ◦ Cfrom to 60 40◦C, °C the to TPC60 °C, increased the TPC gradually increased from gradually 6.6 to the from peak 6.6 of to 11.1 the mg peak GE/ gof d.w. 11.1The mg mechanism GE/g d.w. Thethrough mechanism which temperature through which elevation temperature improves elevatio recoveryn ofimproves extractant recovery could be of explained extractant by enhancedcould be explaineddiﬀusivity by of enhanced solvent and diffusivity improved of solvent solubility and of improved phenolic compoundssolubility of inphenolic solvents. compounds In addition, in solvents.increased In mass addition, transfer increased of phenolic mass compounds, transfer of phenolic caused by compounds, reduction incaused the viscosity by reduction and surface in the viscositytension, might and surface contribute tension, to improvement might contribute of extracted to improvement phenolic compounds. of extracted However, phenolic TPC compounds. decreased However,dramatically TPC to decreased the minimum dramatically point of to 6.0 the mg mini GE/mumg when point the of temperature 6.0 mg GE/g increased when the from temperature 60 ◦C to increased80 ◦C. This from decrease 60 °C mayto 80 be °C. due This to decrease the thermal may destruction be due to the of polyphenolsthermal destruction at high temperature.of polyphenols at high temperature.

Processes 2019, 7, 489 7 of 18 ProcessesProcesses 2019 2019,, 77,, xx 77 ofof 1818

12.012.0 11.1

10.010.0 8.4 7.8 8.08.0 6.66.6 6.06.0 6.06.0

4.0

mg GE/g d.w. mg GE/g 4.0 mg GE/g d.w. mg GE/g

2.02.0

0.00.0 4040 50 60 70 80 80 Temperature (°C)

FigureFigure 3.3. EEffectffect of extraction temperature. temperature. 3.1.3. Influence of Material:Solvent Ratio 3.1.3.3.1.3. InfluenceInfluence ofof Material:SolventMaterial:Solvent Ratio Material-to-solvent ratio is an important parameter in the extraction process, since the diffusion Material-to-solventMaterial-to-solvent ratioratio is an important parameter in the extraction process,process, sincesince thethe diffusiondiffusion mechanism relies on the difference in concentrations between material and the solution. In liquid–solid mechanismmechanism reliesrelies onon thethe differencedifference in concentrations between material and thethe solution.solution. InIn liquid–liquid– extraction, the amount of substances collected depends on the amount of solvent. The ratio of material solidsolid extraction,extraction, thethe amountamount ofof substancessubstances collected depends on the amount ofof solvent.solvent. TheThe ratioratio ofof andmaterialmaterial solvent andand was solventsolvent allowed waswas to allowedallowed vary from to vary 1:04 from to 1:10. 1:04 to 1:10. FigureFigureFigure4 shows 44 showsshows that thatthat when whenwhen the the material material to to solventsolvent ratioratio increasedincreased from from 1:04 1:04 toto to 1:06,1:06, 1:06, TPCTPC TPC increasedincreased increased fromfromfrom 6.7 6.76.7 to to 12.7to 12.712.7 mg mgmg GE GE/gGE/g/g d.w. d.w.d.w. and andand reached reached the the peak peak of 13.0 of 13.0 mg GEmg/ gGE/g d.w. d.w.d.w. as the asas ratio thethe ratio reachedratio reachedreached 1:08. 1:08. This1:08. is becauseThisThis isis the becausebecause addition thethe of additionaddition solvent elevatesof solvent the elevates difference the of concentrationdifference ofof concentration gradient,concentration thus gradient, maintaininggradient, thusthus the dimaintainingffmaintainingusion until thethe equilibrium diffusiondiffusion untiluntil is reached. equilibrium At the is equilibriumreached. At the state equilibrium where the statestate amount wherewhere of the polyphenolsthe amountamount ofof in thepolyphenolspolyphenols material is in exhausted,in thethe materialmaterial the isis addition exhausted, of ethanol the addi willtion not of promoteethanol will the not TPCnot promotepromote yield any thethe longer. TPCTPC yieldyield Evidently, anyany dilonger.fflonger.erences Evidently,Evidently, of yields differencesdifferences that wereachieved ofof yieldsyields atthat ratios were of achieved 1:06, 1:08, at andratios 1:10 ofof 1:06,1:06, were 1:08,1:08, subtle andand and 1:101:10 not werewere statistically subtlesubtle significant.andand notnot statisticallystatistically As a result, significant.significant. the raw material:solvent As a result, th ratioe raw of 1:06material:solvent was more economical ratioratio ofof 1:061:06 and waswas selected moremore for subsequenteconomicaleconomical experiments. andand selectedselected forfor subsequentsubsequent experiments.

14.0 13.0 14.0 12.7 13.0 12.8 13.0 12.7 12.8 13.0 12.0 12.0 11.0 11.0 10.0 9.0 10.0 9.0 9.0 9.0 8.0 8.0 6.7 7.0 6.7 7.0 6.0 6.0

mg GE/g d.w. mg GE/g 5.0

mg GE/g d.w. mg GE/g 5.0 4.0 4.0 3.0 3.0 2.0 2.0 1.0 1.0 0.0 0.0 1:04 1:05 1:06 1:08 1:10 1:04 1:05Material:solvent 1:06 Ratio (g/mL) 1:08 1:10 Material:solvent Ratio (g/mL)

Figure 4. Effect of material:solvent ratio. FigureFigure 4.4. EEffectect of material:solvent ratio. ratio. ﬀ

Processes 2019, 7, 489 8 of 18

Processes 2019, 7, x 8 of 18 3.1.4. Influence of Extraction Time 3.1.4. Influence of Extraction Time Figure5 presents the e ffect of extraction time on the TPC. It was observed that the extraction Figure 5 presents the effect of extraction time on the TPC. It was observed that the extraction time was positively correlated with the TPC in the first 60 min, as evidenced by a steady rise of TPC time was positively correlated with the TPC in the first 60 min, as evidenced by a steady rise of TPC from 5.4 to 12.8 when prolonging the time from 15 to 60 min. From 60 to 150 min, the TPC appeared from 5.4 to 12.8 when prolonging the time from 15 to 60 min. From 60 to 150 min, the TPC appeared to be unchanged, at around 12.7 mg GE/g d.w. This could be explained by the Fick’s second law of to be unchanged, at around 12.7 mg GE/g d.w. This could be explained by the Fick’s second law of diffusion, which articulates that an eventual equilibrium between material solute and the solvent diffusion, which articulates that an eventual equilibrium between material solute and the solvent will will be achieved [33]. Therefore, prolonging the time period past a certain point had no effect on the be achieved [33]. Therefore, prolonging the time period past a certain point had no effect on the extraction of phenolic compounds. Therefore, 60 min was selected as the suitable extraction time. extraction of phenolic compounds. Therefore, 60 min was selected as the suitable extraction time.

15.0

12.8 12.9 12.4 12.7 12.0 10.4

9.0

6.0 5.4 mg GE/g d.w. mg GE/g

3.0

0.0 15 30 60 90 120 150 Extraction time (min)

FigureFigure 5. EffectEﬀect of extraction time.

3.1.5.3.1.5. InfluenceInfluence ofof NumberNumber ofof ExtractionExtraction Cycles FromFrom Figure Figure6, 6, it wasit was indicated indicated that that TPC TPC was was not no completelyt completely extracted extracted after af theter firstthe first cycle. cycle. In fact, In thefact, yield the ofyield the of second the second cycle wascycle higher was higher than that than of that the firstof the cycle first and cycle TPC and after TPC two after cycles, two totalingcycles, 12.8totaling mg GE12.8/g mg d.w. GE/g After d.w. the After third th extractione third extraction cycle, totalcycle, TPC total rose TPC from rose from 12.8 to12.8 14.0 to mg14.0 GEmg/ gGE/g d.w., suggestingd.w., suggesting that most that ofmost TPC of has TPC been has exhaustedbeen exhaus afterted theafter third the cycle.third cycle. Considering Considering the minor the minor yield enhancementyield enhancement after the after third the cycle, third extraction cycle, extraction through through two cycles two was cycles selected was asselected the suitable as the procedure suitable forprocedure subsequent for subsequent experiments. experiments.

Processes 2019, 7, 489 9 of 18 Processes 2019, 7, x 9 of 18 Processes 2019, 7, x 9 of 18 16.0 16.0 14.0 14.0 12.8 14.0 14.0 12.8 12.0 12.0 10.0 10.0 8.0 8.0 5.7 6.0

mg GE/g d.w. mg GE/g 5.7 6.0 mg GE/g d.w. mg GE/g 4.0 4.0 2.0 2.0 0.0 0.0 123 123 Number of extraction (times) Number of extraction (times)

Figure 6. Effect of the number of extraction. FigureFigure 6.6. EEffectffect of the number of extraction. 3.1.6.3.3.6. InfluenceInfluence of Germination Time 3.3.6. Influence of Germination Time Figure7 7 illustrates illustrates changes changes in in TPC TPC with with regard regard to to germination germination day. day. Within Within the the first first five five days, days, it is it Figure 7 illustrates changes in TPC with regard to germination day. Within the first five days, it clearis clear that that the the TPC TPC correlated correlated positively positively with with germination germination time time and and exhibited exhibited a turning a turning point point at the at fifth the is clear that the TPC correlated positively with germination time and exhibited a turning point at the day.fifth Withoutday. Without germination, germination, the TPC the inTPC the in soybean the soybean seeds seeds was initiallywas initially 5.5 mg 5.5 GE mg/g GE/g d.w. d.w. After After the first the fifth day. Without germination, the TPC in the soybean seeds was initially 5.5 mg GE/g d.w. After the dayfirst ofday germination, of germination, the TPC the of TPC soybean of soybean sprouts sprouts expanded expanded around 1.5around times, 1.5 reaching times, 9.4reaching mg GE 9.4/g d.w.mg first day of germination, the TPC of soybean sprouts expanded around 1.5 times, reaching 9.4 mg Afterwards,GE/g d.w. Afterwards, TPC kept continuously TPC kept continuously increasing increasing from 12.8 tofrom 16.8 12.8 mg to GE 16.8/g d.w. mg GE/g and reachedd.w. and a reached peak of GE/g d.w. Afterwards, TPC kept continuously increasing from 12.8 to 16.8 mg GE/g d.w. and reached 19.6 mg GE/g d.w. at the fifth day. However, the TPC declined from 19.6 to 14.5 mg GE/g d.w. when the aa peak peak of of 19.6 19.6 mg mg GE/gGE/g d.w.d.w. atat thethe fifthfifth day. However, the TPCTPC declineddeclined fromfrom 19.6 19.6 to to 14.5 14.5 mg mg GE/g GE/g germination period increased from 5 days to 7 days. The results followed a similar trend as reported d.w.d.w. when when thethe germinationgermination periodperiod increasedincreased from 5 days toto 77 days.days. TheThe resultsresults followedfollowed a a similar similar by Phommalth, who observed the highest isoflavone content in the whole sprout (Pungsannamulkong trendtrend as as reported reported byby Phommalth,Phommalth, whowho observedobserved the highest isoflavoneisoflavone contentcontent inin the the whole whole sprout sprout and Aga3 varieties) on the seventh day of germination [17]. After 7 days, there was a decreasing trend (Pungsannamulkong(Pungsannamulkong andand Aga3Aga3 varieties)varieties) on the seventh day ofof germinationgermination [17]. [17]. After After 7 7 days, days, there there inwaswas isoflavone a a decreasing decreasing content trend trend as in thein isoflavoneisoflavone germination content period as increased.the germination periodperiod increased.increased.

19.619.6 20.020.0

18.018.0 16.8 16.816.8 16.0 14.9 16.0 14.9 14.514.5 14.014.0 12.812.8 12.012.0 9.4 10.010.0 9.4 8.08.0 mg GE/g d.w. mg GE/g mg GE/g d.w. mg GE/g 5.55.5 6.06.0 4.04.0 2.02.0 0.00.0 0123456701234567 Germination Day (day)

FigureFigure 7.7. EffectEﬀect of germination day. day.

Processes 2019, 7, 489 10 of 18 Processes 2019, 7, x 10 of 18

3.1.7.3.3.7. Influence Influence of the Structural Parts of the Soybean Sprouts From Figure8 8,, itit waswas observedobserved thatthat thethe TPCTPC contentcontent inin thethe radicleradicle fractionfraction waswas thethe highesthighest atat 24.4 mg GE/g GE/g d.w. The The figure figure is is 1.82-times 1.82-times higher higher than than that that in in hypocotyl hypocotyl (13.4 (13.4 mg mg GE/g GE/ d.w.)g d.w.) and and 3- 3-timestimes higher higher than than that that in in cotyledon cotyledon (8.0 (8.0 mg mg GE/g GE/g d.w.). d.w.). This This is is consistent consistent with with a a previous previous study, study, where thethe isoflavoneisoflavone contentcontent in in the the hypocotyl hypocotyl was was found found at at a a higher higher level level than than that that in in the the cotyledon cotyledon of non-sproutedof non-sprouted soy-bean soy-bean seeds, seeds, while while the the radicle radicle of of germinated germinated seeds seeds showed showed thethe highesthighest isoflavoneisoflavone content [[34].34].

25.0 24.4

20.0

15.0 13.4

10.0 8.0 mg GE/g d.w. mg GE/g

5.0

0.0 Cotyledon Hypocotyl Radical

Part of sprout

Figure 8. The polyphenol content in different diﬀerent parts of soybean sprouts.

3.2. Optimization Process with RSM

3.2.1. Model Fitting Using RSM Variations ofof factors factors for optimizationfor optimization of processes of processes were selected were basedselected on singlebased factoron single experiments. factor Toexperiments. be specific, To the be factor specific, levels the for factor optimization levels for of optimization TPC included of ethanol TPC included concentration ethanol X 1concentration(85–95% v/v), extraction temperature X (55–65 C), and material:solvent ratio X (1:4–1:8 g/mL). Extraction time X1 (85–95% v/v), extraction2 temperature◦ X2 (55–65 °C), and material:solvent3 ratio X3 (1:4–1:8 g/mL). andExtraction germination time and day germination were fixed at day 60 minwere and fixed 5 days, at 60 respectively. min and 5 days, The obtained respectively. optimum The responseobtained wasoptimum 19.621 response (mg GE/ gwas d.w.), 19.621 based (mg on theGE/g average d.w.), ofba centersed on points the average (optimum of center condition). points The (optimum result of experimentalcondition). The design result based of experimental on CCD and design corresponding based on responses CCD and are corresponding shown in Table responses3. The second-order are shown polynomialin Table 3. The equation second-order of optimized polynomial condition equation for TPC of isoptimized as follows: condition for TPC is as follows: Y = 19.62 − 0.7572X − 0.3258X + 0.5415X −1.25X2 − 1.32X2 − 0.8648X2 YTPC = 19.62 0.7572X1 0.3258X2 + 0.5415X3 1.25X1 1.32X2 0.8648X3 where Y is the responses,− X1, X2, and− X3 are the factors, including− ethanol− concentration,− extraction wheretemperature, Y is the and responses, material-solvent X1,X2, and ratio, X3 respectively.are the factors, Table including 4 shows ethanol the result concentration, of analysis of extraction variance. temperature,Overall, most andof model material-solvent terms and interaction ratio, respectively. factors were Table statistically4 shows thesignificant result of(p analysis< 0.05). The of variance.coefficient Overall, of determination most of model (R2) was terms 0.9902, and interaction which indicates factors a were good statistically correlation significant between predicted (p < 0.05). Theand coeactualfficient (experimental) of determination values (R2. )The was suitability 0.9902, which of indicatesthe model a goodalso correlationrelates to a between good agreement predicted andbetween actual predicted (experimental) R2 and adjusted values. TheR2. Furthermore, suitability of for the a modelfitted model, also relates a non-significant to a good agreement lack-of-fit betweenand Adequate predicted precision R2 and value adjusted greater R2 .than Furthermore, 4 are desirable. for a fitted Thus, model, the terms a non-significant of experimental lack-of-fit design andobtained Adequate in this precision study suggested value greater that models than 4 are are reliable desirable. and Thus, have thegood terms predictability. of experimental This could design be obtainedmanually in confirmed this study by suggested examining that the models actual areand reliable predicted and response have good in Table predictability. 3. This could be manually confirmed by examining the actual and predicted response in Table3. Table 3. Central composite design of actual factors and responses based on actual and predicted values.

Processes 2019, 7, 489 11 of 18

Table 3. Central composite design of actual factors and responses based on actual and predicted values.

Factors Response Run Y (Actual) Y (Predict) X X X TPC TPC 1 2 3 (mg GE/g d.w.) (mg GE/g d.w.) 1 85 55 4 16.7 16.54 2 95 55 4 15.1 15.33 3 85 65 4 15.7 15.99 4 95 65 4 14.9 14.68 5 85 55 8 17.6 17.92 6 95 55 8 16.4 16.61 7 85 65 8 17.4 17.27 8 95 65 8 15.2 15.46 9 81.6 60 6 17.5 17.35 10 98.4 60 6 14.8 14.81 11 90 51.6 6 16.5 16.43 12 90 68.4 6 15.4 15.33 13 90 60 2.6 16.3 16.20 14 90 60 9.4 18.2 18.04 15 90 60 6 19.7 19.621 16 90 60 6 19.5 19.621 17 90 60 6 19.8 19.621 18 90 60 6 19.5 19.621 19 90 60 6 19.4 19.621 20 90 60 6 19.8 19.621

Table 4. Analysis of Variance (ANOVA) results for the quadratic model for optimization of bioactive compounds from soybean sprouts.

Factors Total Polyphenol Content (TPC) Source Sum of squares F-value p-value - Model 62.89 110.03 <0.0001 Significant X1 7.83 123.3 <0.0001 - X2 1.45 22.83 0.0007 - X3 4 63.06 <0.0001 - X1X2 0.005 0.0787 0.7847 Not significant X1X3 0.125 1.97 0.1909 - X2X3 0.005 0.0787 0.7847 - 2 X1 22.65 356.68 <0.0001 Significant 2 X2 25.28 398.71 <0.0001 - 2 X3 10.78 169.71 <0.0001 - Residual 0.6350 - - - Lack of Fit 0.4867 3.28 0.1091 Not significant Pure Error 0.1483 - - - Cor Total 63.52 - - - Coefficient of Variation 1.46 - - - PRESS 4.55 - - - R2 0.9900 - - - R2 Adjusted 0.9810 - - - R2 Predicted 0.9284 - - - Adequate Precision 277.367 - - - Processes 2019, 7, x 12 of 18

R2 Adjusted 0.9810 - - - R2 Predicted 0.9284 - - - Adequate 277.367 - - - Precision Processes 2019, 7, 489 12 of 18 3.2.2. Analysis of Response Surface

3.2.2.Figure Analysis 9 depicts of Response interaction Surface impact of ethanol concentration and temperature on the TPC at different material-solvent ratios. Apparently, both factors exerted positive impacts on the TPC Figure9 depicts interaction impact of ethanol concentration and temperature on the TPC at di ﬀerent production.material-solvent The change ratios. in the Apparently, ratio also both seemed factors to shift exerted the positive response impacts surface on upward the TPC without production. altering the surfaceThe change shape. in theThe ratio shape also of seemed the response to shift thealso response indicated surface that upwardthe yield without of TPC altering was therapidly enhancedsurface with shape. elevating The shape temperature of the response extraction also indicated and ethanol that the concentration. yield of TPC was At ratios rapidly of enhanced 1:4, 1:6, and 1:8 g/mL,with elevating further calculations temperature extractionshowed that and maximum ethanol concentration. yield was At16.7, ratios 19.7, of 1:4,and 1:6, 17.6 and mg 1:8 GE/g g/mL, d.w., respectively.further calculations showed that maximum yield was 16.7, 19.7, and 17.6 mg GE/g d.w., respectively.

(a)

(b)

Figure 9. Cont.

ProcessesProcesses 20192019, 7, x, 7, 489 13 of13 18 of 18 Processes 2019, 7, x 13 of 18

(c) (c) Figure 9. Response surface plot showing effects of extraction temperature and ethanol concentration Figureon yieldFigure 9. ofResponse 9.TPC.Response (a) surface Material-solvent surface plot plot showing showing ratio effects eofffects 1:4; of(b extractionextr) Material-solventaction temperature temperature ratio and andof ethanol 1:6; ethanol (c) concentration Material-solvent concentration onratio yieldon of yield1:8. of TPC. of TPC. (a) (Material-solventa) Material-solvent ratio ratio of of 1:4; 1:4; ( (b) Material-solvent ratio ratio of of 1:6; 1:6; (c) ( Material-solventc) Material-solvent ratioratio of 1:8. of 1:8. FigureFigure 10 presents10 presents the the response response surface surface plotplot which which gave gave the the extraction extraction yield yield of TPC of asTPC a function as a function of Figure 10 presents the response surface plot which gave the extraction yield of TPC as a function of material:solventmaterial:solvent ratioratio and and ethanol ethanol concentration. concentration. Different Different plots according plots according to fixed temperatures to fixed temperatures (55 ◦C, of(55 material:solvent°C,60 ◦60C,°C, and and 65 ◦C) 65 ratio are°C) illustrated. andare illustrated.ethanol It was concentration. indicated It was indicated that theDifferent extraction that plotsthe yield extraction according of TPC increased yield to fixed of analogouslyTPC temperatures increased (55analogously °C,with 60 the °C, material:solvent withand 65the °C) material:solvent are ratio illustrated. and ethanol ratioIt concentration.was and indicated ethanol However, that concentration. the as extraction the ethanol However, yield concentration of TPCas the beganincreased ethanol analogouslyconcentrationsurpassing with began 90% the (v /vsurpassing ),material:solvent TPC started 90% to decline.(v/v ratio), TPC Ofand thestarted ethanol three to subplots, decline.concentration. the Of plot the of threeHowever, extraction subplots, temperatureas the the ethanol plot of concentrationextractionof 60 ◦ Ctemperature had began a calculated surpassing of 60 TPC °C hadpeak90% a at (calculatedv/v 19.7), TPC mg GE startedTPC/g d.w. peak to decline. at 19.7 mg Of GE/gthe three d.w. subplots, the plot of extraction temperature of 60 °C had a calculated TPC peak at 19.7 mg GE/g d.w.

(a) (a) Figure 10. Cont.

Processes 2019, 7, 489 14 of 18 Processes 2019, 7, x 14 of 18

(b)

(c)

FigureFigure 10. Response 10. Response surface surface plot plot showing showing eeffectsﬀects of of material:solvent material:solvent ratio ratio and ethanoland ethanol concentration concentration on yield of TPC. (a) Extraction temperature of 55 C; (b) Extraction temperature of 60 C; (c) Extraction on yield of TPC. (a) Extraction temperature of 55◦ °C; (b) Extraction temperature of◦ 60 °C; (c) Extraction temperature of 65 ◦C. temperature of 65 °C. The extraction yield of TPC was investigated at diﬀerent ethanol concentrations (85%, 90%, and 95% v/v) with respect to two variables, namely material:solvent ratio and extraction temperature, as shown in Figure 11. Similar to Figure 10, the extraction yield increased with increase of ratio of The extraction yield of TPC was investigated at different ethanol concentrations (85%, 90%, and solid–liquid and extraction temperature from 55 to 60 ◦C. However, elevating the temperature past 95% v/v) with respect to two variables, namely material:solvent ratio and extraction temperature, as 60 ◦C caused extraction yield of TPC to diminish. Maximum TPC was calculated to be 17.6, 19.7, shown in Figure 11. Similar to Figure 10, the extraction yield increased with increase of ratio of solid– and 16.4 mg GE/g d.w., in which the peak was recognized at a temperature of 60 ◦C. liquid and extraction temperature from 55 to 60 °C. However, elevating the temperature past 60 °C caused extraction yield of TPC to diminish. Maximum TPC was calculated to be 17.6, 19.7, and 16.4 mg GE/g d.w., in which the peak was recognized at a temperature of 60 °C.

Processes 2019, 7, 489 15 of 18 Processes 2019, 7, x 15 of 18

(a)

(b)

(c)

Figure 11. Response surface plot showing eeffectsﬀects of material:solventmaterial:solvent ratio and extraction temperature on yield of TPC. (a)) EtOHEtOH concentrationconcentration of 85% (vv/v/v);); ((b)) EtOHEtOH concentrationconcentration of 90% (vv/v/v);); ((cc)) EtOHEtOH concentrationconcentration ofof 95%95% ((vv/v/v).).

3.2.3. Validation of the Model

Processes 2019, 7, 489 16 of 18

3.2.3. Validation of the Model Validations of the models were carried out based on optimum extraction conditions obtained fromProcesses RSM analysis. 2019, 7, x Table5 shows the parameters and the results of validation experiments.16 Triplicate of 18 attempts of this experiment gave a TPC result of 19.11 0.25 mg GE/g d.w. This indicated that there is ± a desirableValidations agreement of betweenthe models predicted were carried and out experimental based on optimum (actual) extraction values. Byconditions applying obtained the paired t-test,from no signiﬁcant RSM analysis. variance Table 5 betweenshows the actual parameters and predicted and the results values of validation (p < 0.05) experiments. was observed. Triplicate Therefore, the generatedattempts of response this experiment model gave was adequatea TPC result in of predicting 19.11 ± 0.25 the mg TPC GE/g yield. d.w. This indicated that there is a desirable agreement between predicted and experimental (actual) values. By applying the paired t-test, no significant varianceTable between 5. The actual result ofand optimum predicted point values experiment. (p < 0.05) was observed. Therefore, the generated response model was adequate in predicting the TPC yield. EtOH Extraction Material-Solvent TPC Model TPC Experiment Error with Concentration Temperature Table 5. The(g /resultmL) of optimum(mg point GE/g experiment. d.w.) (mg GE/g d.w.) Model (%) (% v/v) (◦C) EtOH Extraction Material- TPC Model TPC Experiment Error with Concentration Temperature Solvent (mg GE/g d.w.) (mg GE/g19.32 d.w.) Model (%) 2.42 88(% v/v) 59(°C) 1:6.5(g/mL) 19.801 18.88 4.65 19.3219.14 2.42 3.34 88 Average TPC59 Standard Deviation1:6.5 19.801 18.88 19.11 0.254.65 ± 19.14 ± 3.34 Average TPC ± Standard Deviation 19.11 ± 0.25 3.3. Evaluating the Antioxidant Ability of Dried Extract at Optimum Conditions Compared3.3. Evaluating with the Antioxidant the positive Ability control of Dried of ascorbic Extract at acid,Optimum the Conditions results showed that soybean sprouts extracts possessedCompared with a concentration-dependent the positive control of ascorbic relationship acid, the withresults DPPH showed scavenging that soybean assay sprouts activity (Figureextracts 12). Whenpossessed the a testconcentration-dependent sample was prepared relationship at a concentration with DPPH 1000scavengingµg/mL, assay the activity free radical (Figure 12). When the test sample was prepared at a concentration 1000 μg/mL, the free radical inhibition percentage of soybean sprouts dried extract in this study was 28.6%. The IC50 of soybean sproutsinhibition was 1350 percentageµg/mL, of lower soybean than sprouts thatof dr theied extract vitamin in Cthis by study approximately was 28.6%. The 193 IC times.50 of soybean This results sprouts was 1350 μg/mL, lower than that of the vitamin C by approximately 193 times. This results was similar with the study by Khang et al. (2016), which reported the antioxidation activity of soybean was similar with the study by Khang et al. (2016), which reported the antioxidation activity of at a concentration almost identical to our study at 26.64% [20], and indicated that the soybean sprouts soybean at a concentration almost identical to our study at 26.64% [20], and indicated that the soybean driedsprouts extract dried used extract in this used study in this possessed study possessed low antioxidant low antioxidant capacity. capacity.

100.0

90.0 y = 0.1051x - 91.812 80.0 R² = 0.9914

70.0

60.0

50.0

40.0 Inhibition (I%) 30.0

20.0

10.0

0.0 1290 1300 1310 1320 1330 1340 1350 1360 1370 1380 Concentration (μg/mL)

Figure 12. The antioxidant ability of dried extract obtained at optimum conditions. Figure 12. The antioxidant ability of dried extract obtained at optimum conditions. 4. Conclusions4. Conclusion The presentThe present study study has successfullyhas successfully performed performed the extractionthe extraction of polyphenols of polyphenols from from soybean soybean sprouts via macerationsprouts via techniquemaceration and technique investigated and investigat the relationshiped the relationship between TPCbetween and variousTPC and experimental various experimental parameters, including ethanol concentration, extraction temperature, extraction time,

Processes 2019, 7, 489 17 of 18 parameters, including ethanol concentration, extraction temperature, extraction time, material:solvent ratio, extraction cycle, number of germination days, and the used structural part of the soybean sprout. To achieve this, multiple single-factor experiments were carried out and the resultant conditions were used to further optimize the TPC via RSM. The optimization with RSM procedure proceeded with respect to three selected parameters, including material:solvent ratio, extraction temperature, and ethanol concentration. The ﬁnal optimum parameters that gave maximum TPC are as follows: extraction time of 60 min, extraction temperature of 59 ◦C, ethanol concentration of 88% (v/v), material-solvent ratio of 1:6.5 g/mL, maceration through 2 cycles, and germination period of 5 days, with the plant material being the radicle part of the sprouts. Validation experiments showed that these conditions yielded a TPC of 19.11 0.25 mg GE/g d.w. The obtained dried extract exhibited low antioxidant activity. ± Further studies should focus on scalability and feasibility assessment of the maceration production of biologically active compounds from soybean sprouts. In addition, compositional determination of the extracted products should be contemplated.

Author Contributions: Investigation, T.Q.T., L.G.B., T.T.T., and P.T.H.H.; writing—original draft, X.T.L. and V.L.L.V. Funding: This research received no external funding. Acknowledgments: This research is funded by Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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