thermal processing effects on the chemical ... - hindawi

Research ArticleThermal Processing Effects on the Chemical Constituent andAntioxidant Activity of Okara Extracts Using SubcriticalWater Extraction

Hongyi Sun Xi Yuan Zhenya Zhang Xin Su and Min Shi

Graduate School of Life and Environmental Sciences University of Tsukuba Ibaraki Japan

Correspondence should be addressed to Min Shi shimin0816gmailcom

Received 23 April 2018 Accepted 28 June 2018 Published 1 August 2018

Academic Editor Beatriz Oliveira

Copyright copy 2018 Hongyi Sun et al is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Subcritical water extraction (SWE) has been employed for the extraction of bioactive compounds from plant materials with cost-effectiveness less consuming time and environmental sustainability To explore the effects of thermal processing during SWEtotal organic content (TOC) total sugar polysaccharides total phenolic content (TPC) total flavonoid content (TFC) andantioxidant activity (ABTS andDPPH assays) of eight aqueous extracts have been quantitatively investigatede results indicatedthat elevated temperatures indeed resulted in significant changes in the constituents and antioxidant activities of okaraextracts Among them the extract obtained at 220degC exhibited the highest total phenolic flavonoid content DPPH (22-diphenyl-1-picrylhydrazyl) radical-scavenging activity and ABTS [22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonate)] radical-scavenging activity However phenolic compounds were destroyed after the treatment above 230degC suggesting that any polymerprocessing is improper to undertake at higher than this value to achieve the high antioxidant activity Moreover a significantpositive correlation between TPC or TFC and antioxidant capacity (DPPH and ABTS) values was detected

1 Introduction

Okara is rich in bioactive compounds including dietary fiberprotein polysaccharides and phenolic compounds many ofwhich have been claimed to contribute to the antioxidantactivity due to their redox properties derived from vari-ous possible mechanisms free radical-scavenging activitytransition-metal-chelating activity andor singlet-oxygen-quenching capacity [1] For instance phenolic compoundsplay an important role in stabilizing lipid peroxidation andinhibiting various types of oxidizing enzymes [2] Never-theless the differences in the structures and substitutionsmay influence the phenoxyl radical stability and impair theantioxidant properties

Subcritical water extraction (SWE) is defined as hotwater at temperatures ranging between 100degC and 374degCunder high pressure to maintain water in the liquid state [3]As one of an environmentally friendly and efficient tech-nique it has been a wide range of applications such as

extraction hydrolysis and wet oxidation of organic com-pounds [4 5] Elevated temperatures can modify the di-electric constant and ionization constant of water resultingin the possibility of tuning its polarity and acceleratinghydrolysis thereby obtaining organic compounds instead ofusing organic solvents during food industry processing [6]

It is well known that heating extraction is an essentialprocessing procedure especially during SWE processingwhich is attributed to the oxidation thermal degradationand leaching of bioactive compounds from fresh vegetables[7] Depending upon the morphology and nutritionalproperties of raw materials different heating conditionssuch as heating duration and temperatures have eitherpositive or negative effects on the antioxidant properties ofvegetables [8]

However no information is available on the effects ofthermal treatments on bioactive compounds and antioxi-dant activity in okara under subcritical water conditionserefore the objective of this study was to investigate the

HindawiJournal of ChemistryVolume 2018 Article ID 6823789 8 pageshttpsdoiorg10115520186823789

effects of different temperatures under subcritical waterextraction conditions on a range of potentially health-relatedchemical constituents and antioxidant capacities in order toevaluate the potential of SWE for the production of highbioactive extracts from okara

2 Materials and Methods

21 Chemicals and Standard Solutions Ascorbic acidFolinndashCiocalteu reagent ethanol sodium carbonate gallicacid sodium nitrite aluminium nitrate sodium hydroxidedisodium hydrogen phosphate potassium persulphatesodium dihydrogen phosphate phenol and D-glucosewere purchased fromWako Pure Chemical (Osaka Japan)11-diphenyl-2-picrylhydrazyl radical (DPPH) 22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) werepurchased from Sigma-Aldrich Inc (Saint Louis MOUSA) All the other chemical reagents were of analyticalgrade

22 Sample Preparation Okara (788 moisture content)was purchased from Inamoto Co Ltd (Tsukuba Japan)Residual water was removed by drying at 60degC for 5 hground in a high-speed disintegrator (IFM-800 IWATANIJAPAN) to obtain a fine powder (250 μm) and then refluxedwith 85 ethanol at 70degC for 4 h to defat deactivate enzymesand remove interference components e residue wasthermally dried at 50degC and vacuum packed to decrease lipidoxidation

23 Extraction Procedure A schematic diagram of thesemicontinuous SWE unit is shown in Figure 1 Subcriticalwater extraction was performed in a 200mL SUS-316stainless steel tube reactor (MMS-200 OMLABOJAPAN) with the maximum operating pressure of 20MPaand temperature of 300degC e extraction system consists ofan extraction vessel (id 385mmtimes 197mm) covered witha heating jacket a Bourdon tube pressure gauge a pressurerelease valve and a temperature sensor connected witha temperature controller by the thermocouple tomonitor thereal-time temperature In a typical run 10 g of pretreatedokara powder was loaded inside the extraction reactor Afteradding a fixed liquid-solid ratio of 30mLg the vessel waselectrically heated to each specific temperature whichgenerally took less than 8min e extraction process thenlasted 10min at the required temperature (160ndash230degC) eextracts were centrifuged at 3600 rpm for 10min and sep-arated from the soluble liquid portion through a filter paper(Whatman number 1) All of the supernatant were stored at4degC in the dark before use

24DeterminationofTotalOrganicCarbon (TOC) TOCwasmeasured by a TOC analyzer (TOC-VCSN ShimadzuJapan) after the samples being centrifuged and filtratedthrough 045 microm membrane More details on the de-termination of TOC have previously been reported [9]

25 Determination of Total Sugar e total sugar was de-termined by the phenol-sulfuric acid method with certainmodifications [10] e color reaction was initiated bymixing 1mL of solution with 05mL of a 5 phenol solutionand 25mL of concentrated sulfuric acid and the reactionmixture was incubated in a boiling water bath for 15minAfter cooling to room temperature the optical density(O D) of the mixture was determined at 490 nm and thetotal carbohydrate content was calculated with D-glucose asa standard e results were expressed as milligram ofglucose equivalent per gram of okara [11]

26 Determination of Polysaccharides e filtrate was pre-cipitated by adding dehydrated ethanol to a final concen-tration of 80 (vv) and stored at 4degC for 12 h eprecipitate was harvested as crude polysaccharides aftercentrifugation at 7500 rpm for 15min and washed tripletimes using dehydrated ethanol After being redissolved inultrapure water the aqueous solution was subjected toremove proteins by using the Sevag reagent [12] dialyzedwith deionized water for 72 h and concentrated under re-duced pressure Finally the polysaccharide product wascollected after lyophilization Polysaccharide content wasassayed using the phenol-sulfuric acid method [13] All theresults were expressed as mg of glucose equivalent per g ofthe pretreated okara

(2)

(4)

(6)

(7)

(5)

(3)

(1)

Figure 1 Experimental schematic diagram of the subcritical watersystem for crude polysaccharide production from okara (1)Pressure gauge (2) safety head (3) pressure release valve (4)temperature sensor (5) insulation jacket (6) SWT reactor and (7)temperature controller and thermocouple

2 Journal of Chemistry

27 Analysis of Total Phenolic Compounds Total phenoliccontent (TPC) was determined on the basis of the FolinndashCiocalteu colorimetric method [14] e sample (05mL)and 2mL of sodium carbonate (75 gL) were added to 25mLof 10 (wv) Folin-Ciocalteu reagent After 30min of re-action at room temperature (intermittent shaking for colordevelopment) the absorbance was measured at 765 nm eTPC was determined from the linear equation of a standardcurve prepared with gallic acid

28 Analysis of Total Flavonoid Compounds e total fla-vonoid content (TFC) in extract was measured by a colori-metric assay [15] e extract (5mL) was added to a 10mLflask and then 5 NaNO2 solution (03mL) was added Aftermixed well the solution was allowed to stand for 6min atroom temperature 5 Al(NO3)3 solution (03mL) was addedto the flask mixed well and kept for 6min at room tem-perature At last 4 NaOH solution (44mL) was addedmixed well and kept for 12min at room temperature Ab-sorbance was read on aUV spectrophotometer at 510 nm andthe total flavonoid contents () were estimated using cali-bration curves Total flavonoid content in samples was cal-culated from a calibration curve (R2 0999) using rutin andexpressed as mg of rutin equivalent (RE) per g of dry weight

29 Antioxidant Activities

291 DPPH Radical-Scavenging Activity Assay DPPHradical-scavenging activity of extractions was evaluatedaccording to a literature procedure with a slight modification[13] Aliquots (05mL) of various concentrations (03125ndash1000mgmL) of extracts were mixed with 3mL (25 microgmL)of a MeOH solution of DPPH and then were shaken vig-orously and allowed to stand in the dark for 30min eabsorbance was measured with a spectrophotometer at517 nm against a blank Decrease of the DPPH solutionabsorbance indicated an increase of the DPPH radical-scavenging activity Ascorbic acid was used as positivecontrols DPPH radical-scavenging activity was calculatedaccording to the following equation

scavenging activity () 1minusA1

A01113888 1113889 times 100 (1)

where A0 is the absorbance without samples and A1 is theabsorbance containing the samples

Results were expressed in millimole (mM) ascorbic acidequivalent (AAE) per 1 g of a sample on a dry weight basis

292 ABTS Radical-Scavenging Activity Assay ABTSradical-scavenging activity was measured using the methodswith some modifications [16] ABTS was dissolved in dis-tilled water at a final concentration of 70mM and mixedwith a potassium persulphate solution at a final concen-tration of 245mM e mixture was conserved in the darkat ambient temperature for 12ndash16 h before use For indi-vidual sample experiment prepared ABTSmiddot+ solution wasdiluted with 001M phosphate buffer saline (PBS pH 74) to

achieve its absorbance within 070plusmn 002 at 734 nm wave-length And then a constant volume (015mL) of variousconcentrations of samples (03125ndash1000mgmL) wasreacted with 285mL of ABTSmiddot+ solution by mixing vigor-ously Eventually the absorbance was measured at 734 nmafter incubation at ambient temperature for 10minAscorbic acid was used as positive controls e scavengingactivity of the ABTS free radicals was calculated using thefollowing equation

scavenging activity () 1minusA1 minusA2( 1113857

A3 minusA4( 11138571113890 1113891

times 100

(2)

where A1 is the absorbance of ABTS solution + sample A2 isthe absorbance of potassium persulphate + sample A3 is theabsorbance of ABTS solution + distilled water and A4 is theabsorbance of potassium persulphate + distilled water


210 Statistical Analysis e data obtained in this studywere expressed as the mean of three replicate determinationsplus or minus the standard deviation (SD) e content oftotal phenolic content was calculated as meanplusmn SD (n 3)and expressed as mg gallic acid equivalents (GAEs)g dryweight e Pearson correlation coefficient (R) and pvaluewere used to show correlations and their significances (SPSS190 for Windows SPSS Inc IL USA) Probability values ofplt 005 and plt 001 were considered to be statisticallysignificant and extremely significant respectively

3 Results and Discussions

31 Extraction Yields

311 General As other processing steps like millinggrinding and homogenization extraction can recover andisolate phytochemicals from plant materials and is impor-tant for obtaining extracts with acceptable yields and strongantioxidant activity [17] Generally extraction efficiency isassociated with the chemical nature of phytochemicals theextraction method used sample particle size the solventused as well as the presence of interfering substances [18]Besides the yield of extraction depends on the solvent withvarying polarity pH temperature extraction time andcomposition of the sample Among these influential factorsextraction temperature is one of the crucial parametersespecially under the subcritical condition

In the current study a reaction time was 10min anda liquid-solid ratio was 30mLg based on previous optimalextraction conditions using response surface methodology[19] e various extractions from okara at different tem-peratures were presented in Table 1 e results revealeda considerable diversity in the chemical constituents in theextracts investigated in the present study For TOC it waskept relatively stable until 180degC which produced the highestvalue of 3000plusmn 073 and then sharply decreased to

Journal of Chemistry 3

2064plusmn 073 possibly owing to a weak hydrolysis re-action pyrolysis and gasification of the organic com-pounds [3] e trend of total sugar was similar to that ofTOC With the increasing temperatures total sugar re-duced continuously from 21036plusmn 435mgmiddotGEg to 2283 plusmn044mgmiddotGEg ere was a wide range of variations in totalpolysaccharides from 030mgmiddotGAg to 14561mgmiddotGAge results were consistent with the fact that total sugarsof the aqueous phase comprise mixtures of poly- oligo-di- and monosaccharides [20]

312 Total Phenolic Content Phenolic compounds haveinhibitory effects on mutagenesis and carcinogenesis inhumans when ingested up to 1 g daily from a diet rich infruits and vegetables [21] e interests of phenolics areincreasing in the food industry because they retard oxi-dative degradation of lipids and thereby improve thequality and nutritional value of food [22] e total phe-nolic content of diverse extracts in this study was de-termined based on the aforementioned reagent methodTPC values were calculated from the gallic acid standardcalibration curve y 00056x minus 00003 with R2 09995where x is the absorbance and y is the concentration ofgallic acid equivalents expressed as mg GAEg Significantdifferences were found for TPC among the extracts atvarious subcritical temperatures e TPC of variousaqueous extracts was in the range of 2332plusmn 014mgmiddotGAEgto 7618 plusmn 045mgmiddotGAEg (increased by 327 folds) undersubcritical conditions (Table 1) e cleaving of the es-terified and glycosylated bond or the formation of theMaillard reaction during the heating process may be re-sponsible for the increase in total phenolics after heating[8 23] However when the heating temperature rose to230degC a slight decrease (7082 plusmn 065mgmiddotGAEg) occurredmainly because of accelerated interaction decomposition oftarget products under the elevated temperature And theresults are in good agreement with the published resultsrelated to the total phenolic content of canola mealachieved by subcritical water technology [24]

313 Total Flavonoid Content Flavonoids are the naturallyoccurring polyphenols representing one of the most prev-alent classes of compounds in vegetables nuts fruits andbeverages such as coffee tea and red wine [25] TFC valueswere calculated from rutin standard calibration curvey 00012x+ 00027 with R2 0992 where x is the

absorbance and y is the concentration of rutin equivalentsexpressed as mgmiddotREg e TFC of okara extracted at dif-ferent temperatures in this study is shown in Table 1 Anincreasing trend of TFC in first seven extracts was found asthe temperature elevated whereas there was a moderatedecrease in the TFC when the temperature reached to 230degCe total flavonoid contents varied from 1560plusmn 087 (160degC)to 6612plusmn 069mgmiddotREg (220degC) with the difference of 424-fold in the initial flavonoid contentse decrease in the totalflavonoid at a higher temperature could be attributed to thedegradation of flavonoids which possibly depends on thestructure of particular flavonoids [8] Our results are inagreement with the findings of Ioku et al [26] who foundthat total flavonoid content increased after heating ata certain temperature and magnitude of time whereas toomuch exposure to severe conditions reduced the content oftotal flavonoid content Moreover in most fruits and veg-etables flavonoids contain glycosidic bonds and exist asdimers and oligomers and the industrial processing such asheating or boiling results in the formation of monomers bythe hydrolysis of glycosidic bonds [27]


321 DPPH Radical-Scavenging Activity Since differenttrends have been found in numerous antioxidant activityassays it is of necessity to evaluate and compare the anti-oxidant capacity of extracts by means of multiple assaysrather than one single test Hence the total antioxidantactivity of extracts in this study needed to be measured usingthe DPPH assay and the ABTS assay respectively

DPPH radical is a stable free radical with a charac-teristic absorption maximum at 517 nm [28] e DPPHradical-scavenging activity assay is sensitive enough toaccommodate samples in a short period of time and detectactive ingredients at low concentrations [29] e DPPHscavenging radical effects of the eight different aqueousextracts from okara and standard antioxidant (ascorbicacid) were compared and shown in Figure 2 In this studyascorbic acid showed DPPH radical-scavenging activity of9730 plusmn 012 at the initial concentration of 03125mgmLexhibiting excellent scavenging ability on DPPH radicalsAnd altered concentrations of all the SWE extracts (031250625 125 25 5 and 10mgmL) showed DPPH scav-enging activities in a dose-dependent manner As shown inTable 1 all aqueous extracts on the DPPH assay followed an

Table 1 Chemical constituents and antioxidant activities of different okara extracts by using SWE

Temperature(degC) TOC () Total sugar

(mgmiddotGEg)Polysaccharides

(mgmiddotGEg)TPC

(mgmiddotGAEg)TFC

(mgmiddotREg)DPPH

(mMmiddotAAEg)ABTS

(mMmiddotAAEg)160 2983plusmn 075 21036plusmn 435 14561plusmn 309 2332plusmn 014 1560plusmn 087 085plusmn 014 3154plusmn 073170 2827plusmn 079 17402plusmn 241 9241plusmn 023 2712plusmn 037 1827plusmn 019 339plusmn 027 4500plusmn 230180 3000plusmn 073 16377plusmn 142 6186plusmn 298 3905plusmn 046 2031plusmn 040 731plusmn 035 7377plusmn 115190 2064plusmn 073 11256plusmn 614 4886plusmn 140 5050plusmn 054 2812plusmn 088 1873plusmn 115 10328plusmn 241200 2173plusmn 035 8916plusmn 181 1901plusmn 114 6725plusmn 073 4900plusmn 140 2375plusmn 036 12637plusmn 088210 2217plusmn 025 6108plusmn 209 313plusmn 0045 7114plusmn 054 5812plusmn 064 3867plusmn 114 15619plusmn 347220 2086plusmn 020 3909plusmn 093 054plusmn 00091 7618plusmn 045 6612plusmn 069 4343plusmn 058 17712plusmn 877230 2030plusmn 023 2283plusmn 044 030plusmn 0014 7082plusmn 065 6591plusmn 066 4183plusmn 153 16961plusmn 970


ascending the order of 160degClt 170degClt 180degClt 190degClt 200degClt 210degClt 230degClt 220degC And it was obvious that DPPHassays varied to a great extent ranging from 085 to 4343mMAAEg among the extract accessions when the temperaturerose from 160degC to 220degC In other words DPPH radical-scavenging activities of extracts signicantly increased(plt 005) as the temperature elevated e results indicatedthat SWE plays a positive role on promoting the quantity ofantioxidant metabolites in the obtained extracts and ad-vancing radical-scavenging activity Moreover it could beattributed to the hydrolysis of glycoside bonds of phenoliccompounds thus increasing the number of phenolic hydroxylgroups and consequently the antioxidant activity of the ex-tracts taking into account TPC and TFC content in theextracts

322 Total Antioxidant Activity Using ABTS Since thecharacteristic absorption spectrum of ABTS cation radicalcan be determined at 414 645 734 and 815 nm [30 31]because of the presence of antioxidants [32] common or-ganic radical cation (ABTS+) assay is one of the most widelyapplied methods to assess antioxidant activity [33] In ad-dition other reports indicated that ABTS radical-scavengingactivity is more of accuracy sensitivity and robustness forscreening single ingredient and other complex antioxidantmixtures such as plant extracts beverages and biologicaluids [34]

Eight extracts at various temperature levels were mea-sured and compared for their free radical-scavenging ac-tivities against ABTS radicals and the results of thescavenging ability of SWE extracts on ABTS free radicals areshown in Figure 3 Apparently all the extracts reduced theabsorbance at 734 nm and the concentration of the extractswas directly proportional to the reduction In the ABTSradical-scavenging assay noteworthy dierences amongvalues of all the extracts were found (plt 005) As thecontrol group ascorbic acid at a concentration of 03125ndash10mgmL was also available to produce excellent inhibitionof ABTS radicals To bemore specic levels of ABTS radical-scavenging activities followed a descending order of220degCgt 230degCgt 210degCgt 200degCgt 190degCgt 180degCgt 170degC and160degC with values of 7706 7294 7213 61835157 3727 1910 and 1501 at the initial concen-tration of 03125mgmL respectively When the concen-tration rose to 25mgmL almost all the samples were equalto the eect produced by ascorbic acid at the same con-centration except for the sample processed at 160degC An-other result should be pointed out that the eect ofscavenging capacity on ABTS radicals was as signicant asthat of ascorbic acid when the concentration of test solutionwas greater than 5mgmL which indicated that there was nodierence on ABTS radical-scavenging activity betweenaqueous extracts and ascorbic acid at a relatively higherconcentration

Also it should be noted that DPPH and ABTS assayswere conducted in ethanol and water media respectively[35] From a mechanistic perspective the DPPH radical-scavenging assay emphasizes the capacity of the extract

transferring electrons or hydrogen atoms while the ABTSradical-scavenging activity could reect the hydrogen do-nating and the chain-breaking capacity of the extract [36]e order of ABTS radical-scavenging activity was consis-tent with that of the DPPH radical suggesting thermaltreatment had decisive eects on scavenging radicals dis-solved in organic or aqueous solvent to some extent

33 Correlations among Constituents and AntioxidantActivities In order to evaluate the suitability and reliability

03125 0625 125 25 50 100Concentration (mgmL)

0

20

40

60

80

100

DPP

H ra

dica

l-sca

veng

ing

activ

ityof

extr

acts

()

180degC

200degC

210degCAscorbic acid160degC170degC

220degC230degC

190degC

Figure 2 DPPH radical-scavenging assays of dierent aqueousextracts from okara by SWE and standard antioxidant (ascorbicacid) Data were expressed as meanplusmn standard deviation of trip-licate samples


0

20

40

60

80

100

ABT

S ra

dica

l-sca

veng

ing

activ

ity o

f ext

ract

s (

)

Ascorbic acid

170degC160degC

210degC220degC230degC

180degC

200degC190degC

Figure 3 ABTS radical-scavenging assays of dierent aqueousextracts from okara by SWE and standard antioxidant (ascorbicacid) Data were expressed as meanplusmn standard deviation of trip-licate samples


of the antioxidant assay for measurement of total antioxi-dant activity of extracts from okara by SWE Pearson cor-relation coefficients between the antioxidant capacities(DPPH and ABTS) total organic carbon total sugarpolysaccharides total phenolic content and total flavonoidcontent for all extracts prepared under different extractiontemperatures were calculated and the results are shown inTable 2 According to the results of the present study sig-nificant correlations (plt 005) were detected in all caseswhich revealed that values of antioxidant activity de-termined by two different methods were comparable andreliable

TOC showed strongly linear relationship with total sugar(R 0899 R2 0808 plt 001) and polysaccharides(R 0815 R2 0664 plt 005) which is in accordance withthe fact that carbon element is essential in these twocompounds It should be noted that TOC total sugar andpolysaccharides were negatively correlated with two selectedin vitro assays suggesting there was no direct relation be-tween the three aforementioned carbohydrates and anti-oxidant abilities under these subcritical water conditionsToo much exposure to elevated temperatures contributed totheir degradation and inactivation e correlation co-efficient for DPPH and ABTS assay (R 0988 R2 0976plt 001) indicated that the values of antioxidant activitiesassayed by these two different methods were significantlycorrelated associating with spectrophotometry-based assaymethods and elimination ability of the radical cation [37]

Phenolic compounds in extracts like other antioxidativecompounds are believed to account for a major portion ofthe antioxidant activity in many plants due to interactionwith free radicals by acting as electron donor of hydrogenatoms Measurements of the scavenging effects of DPPH andABTS radicals showed that radical-scavenging capacity in-creased with increase in TPC Apparently TPC exhibiteda strong correlation with both DPPH (R 0962 R2 0925plt 001) and ABTS (R 0980 R2 0960 plt 001) Con-sistently as main ingredients of TPC (R 0959 R2 0920plt 001) TFC also has closed connections with both DPPH(R 0982 R2 0945 plt 001) and ABTS (R 0973R2 0947 plt 001) e high correlations obtained in thiswork could suggest that the antioxidant activities of theseextracts are resulted from phenolic compounds whichcontribute to the DPPH and ABTS scavenging activities Allthese results differ from previous studies on Amaranthusmantegazzianus [38] ree factors may bring about thedifferences between the results of this study and other

studies (1) the difference in plant matrix (2) the method andconditions of extraction (temperature time and solvents)led to differences in compositions and antioxidant activities(3) the difference of phenolic structures like possessinga higher number of hydroxyl groups [39] It is worth notingthat there must be some other soluble compounds existing inthe extracts like proteins peptides and pigments might bealso responsible for the antioxidant activity partly [40]

4 Conclusions

ermal processing had significant effects on the compo-sitions and antioxidant activities on okara extracts in thisstudy Specifically TOC total sugar and polysaccharides inextracts decreased under elevated temperature due to somereactions like pyrolysis and gasificatione highest yields ofTPC and TFC were observed in the extract obtained asextraction temperature was 220degC with the other two fixedparameters (residence time of 10min and liquid-solid ratioof 30mLg) Among the various aqueous extracts allaqueous extracts showed high antioxidant activity whichindicated SWE played important roles in the increase of theantioxidant capacities of okara extracts on DPPH scavengingactivity and ABTS assay According to correlation com-parisons the total phenolic contents of the extracts werecoherent with the antioxidant activities of the extractspending further analysis of their specific composition toexplain the underlying mechanism is finding could beuseful to the disposal processing of okara by-products in thefood industry

Data Availability

e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

References

[1] Y Yao and G Ren ldquoEffect of thermal treatment on phenoliccomposition and antioxidant activities of two celery culti-varsrdquo LWT-Food Science and Technology vol 44 no 1pp 181ndash185 2011

[2] B D Craft A L Kerrihard R Amarowicz and R B PeggldquoPhenol-based antioxidants and the in vitro methods used for

Table 2 Correlation coefficients between constituents and antioxidant activities of okara extracts by SWE

R (R2) TOC Total sugar Polysaccharides TPC TFC DPPH ABTSTOC 1 mdash mdash mdash mdash mdash mdashTotal sugar 0899 (0808)lowastlowast 1 mdash mdash mdash mdash mdashPolysaccharides 0815 (0664)lowast 0952 (0906)lowastlowast 1 mdash mdash mdash mdashTPC minus0878 (0771)lowastlowast minus0967 (0935)lowastlowast minus0959 (0920)lowastlowast 1 mdash mdash mdashTFC minus0821 (0674)lowast minus0968 (0937)lowastlowast minus0897 (0805)lowastlowast 0959 (0920)lowastlowast 1 mdash mdashDPPH minus0857 (0734)lowastlowast minus0981 (0962)lowastlowast minus0914 (0835)lowastlowast 0962 (0925)lowastlowast 0982 (0945)lowastlowast 1 mdashABTS minus0872 (0760)lowastlowast minus0993 (0986)lowastlowast minus0954 (0910)lowastlowast 0980 (0960)lowastlowast 0973 (0947)lowastlowast 0988 (0976)lowastlowast 1R correlation coefficient R2 coefficient of correlation lowastSignificant at plt 005 lowastlowastSignificant at plt 001


their assessmentrdquo Comprehensive Reviews in Food Science andFood Safety vol 11 no 2 pp 148ndash173 2012

[3] O Pourali F S Asghari and H Yoshida ldquoSub-critical watertreatment of rice bran to produce valuable materialsrdquo FoodChemistry vol 115 no 1 pp 1ndash7 2009

[4] R L Holliday B Y Jong and J W Kolis ldquoOrganic synthesisin subcritical water oxidation of alkyl aromaticsrdquo Journal ofSupercritical Fluids vol 12 no 3 pp 255ndash260 1998

[5] A Kruse and E Dinjus ldquoHot compressed water as reactionmedium and reactant properties and synthesis reactionsrdquoJournal of Supercritical Fluids vol 39 no 3 pp 362ndash3802007

[6] M Plaza and C Turner ldquoPressurized hot water extraction ofbioactivesrdquo TrAC Trends in Analytical Chemistry vol 71pp 39ndash54 2015

[7] G Joana Gil-Chavez J A Villa J Fernando Ayala-Zavalaet al ldquoTechnologies for extraction and production of bio-active compounds to be used as nutraceuticals and food in-gredients an overviewrdquo Comprehensive Reviews in FoodScience and Food Safety vol 12 no 1 pp 5ndash23 2013

[8] K Sharma E Y Ko A D Assefa et al ldquoTemperature-dependent studies on the total phenolics flavonoids anti-oxidant activities and sugar content in six onion varietiesrdquoJournal of Food and Drug Analysis vol 23 no 2 pp 243ndash2522015

[9] H Sun Extraction Identification and Antioxidant Activities ofPolysaccharides and Phenolic Compounds from Okara UsingSubcritical Water Technology University of Tsukuba TsukubaJapan 2016

[10] M Mecozzi ldquoEstimation of total carbohydrate amount inenvironmental samples by the phenolndashsulphuric acid methodassisted by multivariate calibrationrdquo Chemometrics and In-telligent Laboratory Systems vol 79 no 1-2 pp 84ndash90 2005

[11] S Min Y Yingnan G Di Y Zhang and Z Zhang ldquoBio-activity of the crude polysaccharides from fermented soybeancurd residue by Flammulina velutipesrdquo Carbohydrate Poly-mers vol 89 no 4 pp 1268ndash1276 2012

[12] C-W Ma M Feng X Zhai et al ldquoOptimization for theextraction of polysaccharides from Ganoderma lucidum andtheir antioxidant and antiproliferative activitiesrdquo Journal ofthe Taiwan Institute of Chemical Engineers vol 44 no 6pp 886ndash894 2013

[13] L Shuhong S Yaxin Z Dan Y Yang Z Lei and Z ZhangldquoOptimization of fermentation conditions for crude poly-saccharides by Morchella esculenta using soybean curd res-iduerdquo Industrial Crops and Products vol 50 pp 666ndash6722013

[14] M G Miguel S Nunes S A Dandlen A M Cavaco andM D Antunes ldquoPhenols and antioxidant activity of hydro-alcoholic extracts of propolis from Algarve South of Portu-galrdquo Food and Chemical Toxicology vol 48 no 12pp 3418ndash3423 2010

[15] J Zhishen T Mengcheng and W Jianming ldquoe de-termination of flavonoid contents in mulberry and theirscavenging effects on superoxide radicalsrdquo Food Chemistryvol 64 no 4 pp 555ndash559 1999

[16] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cation decolorization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9-10 pp 1231ndash1237 1999

[17] A Moure J M Cruz D Franco et al ldquoNatural antioxidantsfrom residual sourcesrdquo Food Chemistry vol 72 no 2pp 145ndash171 2001

[18] C D Stalikas ldquoExtraction separation and detection methodsfor phenolic acids and flavonoidsrdquo Journal of SeparationScience vol 30 no 18 pp 3268ndash3295 2007

[19] Z Zhenya and S Hongyi ldquoOptimization of okara poly-saccharides extraction using subcritical water technology andits antioxidant activityrdquo in Proceedings of the 5th InternationalConference of Bionic Engineering Ningbo China June 2016

[20] C Corradini A Cavazza and C Bignardi ldquoHigh-performanceanion-exchange chromatography coupled with pulsed elec-trochemical detection as a powerful tool to evaluate carbo-hydrates of food interest principles and applicationsrdquoInternational Journal of Carbohydrate Chemistry vol 2012Article ID 487564 13 pages 2012

[21] J Mulero G Martınez J Oliva et al ldquoPhenolic compoundsand antioxidant activity of red wine made from grapes treatedwith different fungicidesrdquo Food Chemistry vol 180 pp 25ndash312015

[22] A Wojdyło J Oszmianski and R Czemerys ldquoAntioxidantactivity and phenolic compounds in 32 selected herbsrdquo FoodChemistry vol 105 no 3 pp 940ndash949 2007

[23] L Priecina and D Karlina ldquoTotal polyphenol flavonoidcontent and antiradical activity of celery dill parsley onionand garlic dried in conventional and microwave-vacuumdryersrdquo IPCBEE vol 53 pp 107ndash112 2013

[24] M Hassas-Roudsari P R Chang R B Pegg and R TylerldquoAntioxidant capacity of bioactives extracted from canolameal by subcritical water ethanolic and hot water extractionrdquoFood Chemistry vol 114 no 2 pp 717ndash726 2009

[25] K B Pandey and S I Rizvi ldquoPlant polyphenols as dietaryantioxidants in human health and diseaserdquo Oxidative Med-icine and Cellular Longevity vol 2 no 5 pp 270ndash278 2009

[26] K Ioku Y Aoyama A Tokuno J Terao N Nakatani andY Takei ldquoVarious cookingmethods and the flavonoid contentin onionrdquo Journal of Nutritional Science and Vitaminologyvol 47 no 1 pp 78ndash83 2001

[27] C Manach A Scalbert C Morand C Remesy andL Jimenez ldquoPolyphenols food sources and bioavailabilityrdquoAmerican Journal of Clinical Nutrition vol 79 no 5pp 727ndash747 2004

[28] J R Soare T C Dinis A P Cunha and L Almeida ldquoAn-tioxidant activities of some extracts of ymus zygisrdquo FreeRadical Research vol 26 no 5 pp 469ndash478 1997

[29] Y-C Hseu W-H Chang C-S Chen et al ldquoAntioxidantactivities of Toona Sinensis leaves extracts using differentantioxidant modelsrdquo Food and Chemical Toxicology vol 46no 1 pp 105ndash114 2008

[30] M Arnao M Acosta J Del Rio and F Garcıa-CanovasldquoInactivation of peroxidase by hydrogen peroxide and itsprotection by a reductant agentrdquo Biochimica et BiophysicaActa (BBA)-Protein Structure and Molecular Enzymologyvol 1038 no 1 pp 85ndash89 1990

[31] N J Miller C Rice-Evans M J Davies V Gopinathan andA Milner ldquoA novel method for measuring antioxidant ca-pacity and its application to monitoring the antioxidant statusin premature neonatesrdquo Clinical science vol 84 no 4pp 407ndash412 1993

[32] D Villantildeo M Fernandez-Pachon A Troncoso andM C Garcıa-Parrilla ldquoe antioxidant activity of winesdetermined by the ABTS+ method influence of sample di-lution and timerdquo Talanta vol 64 no 2 pp 501ndash509 2004

[33] S Rawat I D Bhatt and R S Rawal ldquoTotal phenoliccompounds and antioxidant potential of Hedychium spica-tum Buch Ham ex D Don in west Himalaya Indiardquo Journal


of Food Composition and Analysis vol 24 no 4-5 pp 574ndash579 2011

[34] P Govindan and S Muthukrishnan ldquoEvaluation of totalphenolic content and free radical scavenging activity ofBoerhavia erectardquo Journal of Acute Medicine vol 3 no 3pp 103ndash109 2013

[35] K-X Zhu C-X Lian X-N Guo W Peng and H-M ZhouldquoAntioxidant activities and total phenolic contents of variousextracts from defatted wheat germrdquo Food Chemistry vol 126no 3 pp 1122ndash1126 2011

[36] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plantfoods oils and beverages extraction measurement and ex-pression of resultsrdquo Food Research International vol 41 no 3pp 274ndash285 2008

[37] E Skotti E Anastasaki G Kanellou M Polissiou andP A Tarantilis ldquoTotal phenolic content antioxidant activityand toxicity of aqueous extracts from selected Greek me-dicinal and aromatic plantsrdquo Industrial Crops and Productsvol 53 pp 46ndash54 2014

[38] V Castel O Andrich F M Netto L G Santiago andC R Carrara ldquoTotal phenolic content and antioxidant activityof different streams resulting from pilot-plant processes toobtain Amaranthus mantegazzianus protein concentratesrdquoJournal of Food Engineering vol 122 pp 62ndash67 2014

[39] Q D Do A E Angkawijaya P L Tran-Nguyen et al ldquoEffectof extraction solvent on total phenol content total flavonoidcontent and antioxidant activity of Limnophila aromaticardquojournal of food and drug analysis vol 22 no 3 pp 296ndash3022014

[40] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005


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effects of different temperatures under subcritical waterextraction conditions on a range of potentially health-relatedchemical constituents and antioxidant capacities in order toevaluate the potential of SWE for the production of highbioactive extracts from okara

2 Materials and Methods

21 Chemicals and Standard Solutions Ascorbic acidFolinndashCiocalteu reagent ethanol sodium carbonate gallicacid sodium nitrite aluminium nitrate sodium hydroxidedisodium hydrogen phosphate potassium persulphatesodium dihydrogen phosphate phenol and D-glucosewere purchased fromWako Pure Chemical (Osaka Japan)11-diphenyl-2-picrylhydrazyl radical (DPPH) 22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) werepurchased from Sigma-Aldrich Inc (Saint Louis MOUSA) All the other chemical reagents were of analyticalgrade

22 Sample Preparation Okara (788 moisture content)was purchased from Inamoto Co Ltd (Tsukuba Japan)Residual water was removed by drying at 60degC for 5 hground in a high-speed disintegrator (IFM-800 IWATANIJAPAN) to obtain a fine powder (250 μm) and then refluxedwith 85 ethanol at 70degC for 4 h to defat deactivate enzymesand remove interference components e residue wasthermally dried at 50degC and vacuum packed to decrease lipidoxidation

23 Extraction Procedure A schematic diagram of thesemicontinuous SWE unit is shown in Figure 1 Subcriticalwater extraction was performed in a 200mL SUS-316stainless steel tube reactor (MMS-200 OMLABOJAPAN) with the maximum operating pressure of 20MPaand temperature of 300degC e extraction system consists ofan extraction vessel (id 385mmtimes 197mm) covered witha heating jacket a Bourdon tube pressure gauge a pressurerelease valve and a temperature sensor connected witha temperature controller by the thermocouple tomonitor thereal-time temperature In a typical run 10 g of pretreatedokara powder was loaded inside the extraction reactor Afteradding a fixed liquid-solid ratio of 30mLg the vessel waselectrically heated to each specific temperature whichgenerally took less than 8min e extraction process thenlasted 10min at the required temperature (160ndash230degC) eextracts were centrifuged at 3600 rpm for 10min and sep-arated from the soluble liquid portion through a filter paper(Whatman number 1) All of the supernatant were stored at4degC in the dark before use

24DeterminationofTotalOrganicCarbon (TOC) TOCwasmeasured by a TOC analyzer (TOC-VCSN ShimadzuJapan) after the samples being centrifuged and filtratedthrough 045 microm membrane More details on the de-termination of TOC have previously been reported [9]

25 Determination of Total Sugar e total sugar was de-termined by the phenol-sulfuric acid method with certainmodifications [10] e color reaction was initiated bymixing 1mL of solution with 05mL of a 5 phenol solutionand 25mL of concentrated sulfuric acid and the reactionmixture was incubated in a boiling water bath for 15minAfter cooling to room temperature the optical density(O D) of the mixture was determined at 490 nm and thetotal carbohydrate content was calculated with D-glucose asa standard e results were expressed as milligram ofglucose equivalent per gram of okara [11]

26 Determination of Polysaccharides e filtrate was pre-cipitated by adding dehydrated ethanol to a final concen-tration of 80 (vv) and stored at 4degC for 12 h eprecipitate was harvested as crude polysaccharides aftercentrifugation at 7500 rpm for 15min and washed tripletimes using dehydrated ethanol After being redissolved inultrapure water the aqueous solution was subjected toremove proteins by using the Sevag reagent [12] dialyzedwith deionized water for 72 h and concentrated under re-duced pressure Finally the polysaccharide product wascollected after lyophilization Polysaccharide content wasassayed using the phenol-sulfuric acid method [13] All theresults were expressed as mg of glucose equivalent per g ofthe pretreated okara

(2)

(4)

(6)

(7)

(5)

(3)

(1)

Figure 1 Experimental schematic diagram of the subcritical watersystem for crude polysaccharide production from okara (1)Pressure gauge (2) safety head (3) pressure release valve (4)temperature sensor (5) insulation jacket (6) SWT reactor and (7)temperature controller and thermocouple







A01113888 1113889 times 100 (1)






A3 minusA4( 11138571113890 1113891

times 100

(2)



















(mgmiddotGEg)TPC

(mgmiddotGAEg)TFC

(mgmiddotREg)DPPH

(mMmiddotAAEg)ABTS










0

20

40

60

80

100

DPP

H ra

dica

l-sca

veng

ing

activ

ityof

extr

acts

()

180degC

200degC


220degC230degC

190degC



0

20

40

60

80

100

ABT

S ra

dica

l-sca

veng

ing

activ

ity o

f ext

ract

s (

)

Ascorbic acid

170degC160degC


180degC

200degC190degC







4 Conclusions


Data Availability




References





















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls









A01113888 1113889 times 100 (1)






A3 minusA4( 11138571113890 1113891

times 100

(2)



















(mgmiddotGEg)TPC

(mgmiddotGAEg)TFC

(mgmiddotREg)DPPH

(mMmiddotAAEg)ABTS










0

20

40

60

80

100

DPP

H ra

dica

l-sca

veng

ing

activ

ityof

extr

acts

()

180degC

200degC


220degC230degC

190degC



0

20

40

60

80

100

ABT

S ra

dica

l-sca

veng

ing

activ

ity o

f ext

ract

s (

)

Ascorbic acid

170degC160degC


180degC

200degC190degC







4 Conclusions


Data Availability




References





















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls














(mgmiddotGEg)TPC

(mgmiddotGAEg)TFC

(mgmiddotREg)DPPH

(mMmiddotAAEg)ABTS










0

20

40

60

80

100

DPP

H ra

dica

l-sca

veng

ing

activ

ityof

extr

acts

()

180degC

200degC


220degC230degC

190degC



0

20

40

60

80

100

ABT

S ra

dica

l-sca

veng

ing

activ

ity o

f ext

ract

s (

)

Ascorbic acid

170degC160degC


180degC

200degC190degC







4 Conclusions


Data Availability




References





















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls











0

20

40

60

80

100

DPP

H ra

dica

l-sca

veng

ing

activ

ityof

extr

acts

()

180degC

200degC


220degC230degC

190degC



0

20

40

60

80

100

ABT

S ra

dica

l-sca

veng

ing

activ

ity o

f ext

ract

s (

)

Ascorbic acid

170degC160degC


180degC

200degC190degC







4 Conclusions


Data Availability




References





















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








4 Conclusions


Data Availability




References





















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls



















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




thermal processing effects on the chemical ... - hindawi

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