evolution of total polyphenols content and antioxidant...

Research ArticleEvolution of Total Polyphenols Content andAntioxidant Activity in Broccoli Florets during Storage atDifferent Temperatures

Andrea Mahn and M Paz Rubio

Departamento de Ingenierıa Quımica Universidad de Santiago de Chile Avenida Libertador Bernardo OrsquoHiggins 3363Estacion Central 9170019 Santiago Chile

Correspondence should be addressed to Andrea Mahn andreamahnusachcl

Received 19 June 2017 Revised 18 August 2017 Accepted 27 August 2017 Published 3 October 2017

Academic Editor Senem Kamiloglu

Copyright copy 2017 Andrea Mahn and M Paz Rubio This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Broccoli has great potential as functional food because of its high content of bioactive compounds Polyphenols are to a great extentresponsible for the high antioxidant activity of broccoli An important challenge to keep the health promoting properties of broccoliis preservation with freezing and refrigeration being the preferred methods Despite storage at low temperature reduces the rate ofdeterioration reactions some reactions still occur thus affecting the content of bioactive compounds In this work we investigatedthe evolution of total polyphenols content and antioxidant activity in blanched and unprocessed broccoli florets during storageat different temperatures (minus45∘C to 20∘C) Both antioxidant activity and total polyphenols content increased at the beginning ofstorage at minus21 minus1 10 and 20∘C followed by a decrease Storage at minus45∘C produced no significant variations The uneven behaviorof antioxidant activity precluded modelingThe evolution of polyphenols was well described by a two-consecutive-reaction modelwith 119903 ge 086 and MSE le 01 The Ea values obtained for polyphenols formation (27ndash32 kJmol) and degradation (26ndash38 kJmol)confirm that in both unprocessed and blanched broccoli the same reactions are responsible for the evolution of polyphenolscontent Our results may contribute to design preservation strategies of broccoli

1 Introduction

Broccoli (Brassica oleracea var italica) is recognized as aldquosuper-foodrdquo because of the numerous healthy effects exertedby its high content of bioactive compounds such as glucosino-lates sulforaphanes polyphenols ascorbic acid andminerals[1] Altogether these compounds confer high antioxidantactivity to this vegetable Glucosinolates and sulforaphane arerecognized as anti-cancer compounds [2] ascorbic acid actsas cellular antioxidant and protects from cardiovascular dis-eases [3] and minerals such as selenium contribute to main-tain the homeostasis [4] Given the high antioxidant activityof polyphenols the intake of these compounds reduces therisk to developing degenerative diseases triggered by oxida-tive stress [5] They also have the capability to preventdiabetes ulcer osteoporosis and cancer [6 7]

Broccoli is a climacteric and highly perishable vegetabletherefore its preservation through processing is essentialHowever domestic or technological processing affects to adifferent extent the healthy properties of broccoli by alteringthe content and bioavailability of the bioactive compoundsFreezing and refrigeration are themost commonpreservationmethods used for vegetables Although low temperaturereduces significantly the rate of deterioration reactions somechanges still occur during storage in these conditions Inaddition preprocessing such as cutting and blanching affectsthe content of bioactive compounds in broccoli [8]

There are some studies about the evolution of polyphe-nolsrsquo content and antioxidant activity in Brassicaceae duringstorage Volden et al [9] reported that the total polyphenolsrsquocontent in cauliflower significantly decreased after blanchingand slightly decreased after storage at minus24∘C for one year

HindawiJournal of Food QualityVolume 2017 Article ID 3742183 9 pageshttpsdoiorg10115520173742183

2 Journal of Food Quality

The loss of antioxidant activity was much more markedPatras et al [10] reported that the antioxidant activity ofbroccoli subjected to blanching and freezing was significantlylower in comparison with unprocessed broccoli Blanchingand freezing did not affect significantly the polyphenolscontent Finally Amodio et al [11] proposed a kinetic modelto describe the evolution of polyphenols content in fresh-cut vegetables during storage The model considers an initialincrease of polyphenols due to the activation of phenylalanineammonia-lyase (PAL) triggered by abiotic stress followed bya decrease due to the action of polyphenol oxidase (PPO)Themodel was validatedwith experimental data obtained at 0 and5∘C

The aim of this work was to investigate the evolutionof total polyphenols content and antioxidant activity inblanched and unprocessed broccoli florets during storage attemperatures ranging from minus45∘C to 20∘C

2 Material and Methods

21 Plant Material Broccoli (Brassica oleracea var italica)heads were purchased at the local market (Santiago Chile) toa single supplier Broccoli heads were processed immediatelyafter purchasing as follows after washing they were cut intosingle florets of 5 cm length and 07ndash09 cm width (stem) andblanched at 57∘C for 13min [12] The blanching conditionswere chosen in order to keep the antioxidant activity of thefresh vegetable After that broccoli florets were put in an ice-water bath for 5min then put in sealed plastic bags andstored at different temperatures as stipulated by the experi-mental design The unprocessed broccoli heads followed thesame process without blanching

22 Experimental Design Blanched and unprocessed broc-coli florets were stored at different temperatures 20∘C (con-trolled ambient temperature) 10∘C (refrigerator)minus1∘Cminus21∘C(household freezers) and minus45∘C (industrial freezer) for upto 83 days depending on the preservation status of the veg-etable Temperaturewas checkedwith a k-type thermocoupleBroccoli florets were kept in plastic sealed bags with eachone containing approximately 50-g of the vegetable In eachcondition 39 bags with blanched broccoli and 39 bags withunprocessed broccoli were used In this way in each samplingprocedure three blanched and three unprocessed broccolibags were taken for analyses For storage at 20∘C sampleswere taken after 0 1 2 6 7 and 9 days of storage For storageat 10 and minus1∘C sampling was conducted every two days untilday 15 and every three days until day 41 when the vegetablewas decomposed For storage at minus21 and minus45∘C samples weretaken every 5 days until day 15 and once a week until day 83

23 Antioxidants Extraction The extraction of antioxidantcompounds was performed as reported by Vinson et al[13] A 200-mg aliquot of liquid nitrogen-pulverized broc-coli heads was accurately weighed in a vial and 4mL of80 20 (mL mL) methanol water solution was added Themixture was ultrasound processed through a titanium probeimmersed in the vial for three minutes at 80 duty cycle asreported by Montes-Bayon et al [14] Then the samples were

incubated at room temperature in an orbital shaker for 4 hAfter that samples were centrifuged at 12000timesg for 5min toremove the solidsThe supernatant was recovered and 80 20(mL mL) methanolwater solution was added to complete5mL extract volume

24 Total Polyphenolsrsquo Content The total polyphenolsrsquo con-tent (TPP) was determined spectrophotometrically throughthe Folin-Ciocalteu method [15] 180 120583L extract and 90120583LFolin-Ciocalteau reagent (diluted 1 1) were added to 360 120583Ldistilled water The mixture was homogenized and left in thedark for 5minThen 450120583L of a 200 g Lminus1 sodium carbonatesolution was added and left in darkness for 30min After thatsamples were centrifuged at 12000timesg for 5min to removethe precipitate and absorbance at 750 nm was measuredTheresults were expressed as mg of gallic acid equivalents per 100gram of dry matter (mg GAE (100 g)minus1 DM) The measure-ments were made in triplicate and average values werereported

25 Antioxidant Activity

251 Free Radical Scavenging Ability The free radical scav-enging ability (FRSA) of the broccoli extracts was mea-sured using the stable radical 22-diphenyl-1-picrylhydrazyl(DPPH∙) [16] 40-120583L vegetable extract (at 6 dilutions) weremixed with 1960 120583L DPPH solution (6 times 10minus5M in meth-anol) The absorbance decrease at 515 nm was continuouslyrecorded during 30 minutes The DPPH∙ concentration inthe reaction mixture at zero time and after 30 minutes wascalculated bymeans of a calibration curve and the remainingDPPH∙ concentration was obtained [17] The measurementsweremade in triplicate and the average values were reported

252 Hydrogen Peroxide Scavenging Activity The hydrogenperoxide scavenging activity (HPSA) was assessed throughthe method reported by Oktay et al [18] The methanolextracts were concentrated in a rotatory evaporator (StuartRE-300 United Kingdom Great Britain) to dryness andthen resuspended into the initial volume with HPLC-gradewater 100 120583L of aqueous antioxidants extract was added to900 120583L of a 4mMH2O2 solution in phosphate buffer (pH 74)Two control mixtures were used one contained HPLC-gradewater instead of extract and the other contained phosphatebuffer instead of H2O2 solution Absorbance was measuredat 230 nm HSPA was expressed as percentage with respect tocontrol The measurements were made in triplicate and theaverage values were reported

26 Kinetic Model The evolution of TPP content was des-cribed by the mechanism proposed by Amodio et al [11]given in (1) considering that polyphenols synthesis is medi-ated by PAL and their degradation is mediated by PPO In (1)119875pre is polyphenols precursor 119875 is polyphenols119874 is oxidizedcompounds and 1198961 and 1198962 are the rate constants

119875pre1198961997888rarr 1198751198962997888rarr 119874 (1)

Journal of Food Quality 3

The kinetic model is given by (2) where 119862pre 119862119875 and 119862119874are the concentrations of polyphenols precursors polyphe-nols and oxidized compounds respectively

minus119889119862pre119889119905= 1198961 sdot 119862pre

119889119862119875119889119905= 1198961 sdot 119862pre minus 1198962 sdot 119862119875

119889119862119874119889119905= 1198962 sdot 119862119875

(2)

The analytical solution of the system composed by (2) isgiven by (3) where 119862pre0 is the initial concentration of theprecursor and 119862119875 and 1198621198750 are the TPP content at 119905 = 119905 and119905 = 0 respectively Themodel was solved by estimating 1198961 1198962and119862pre0 from the experimental dataThe dependence of therate constants from temperature was considered to follow theArrhenius equation

1198621198751198621198750= 119890minus1198962119905 +

119862pre0119862119875011989611198962 minus 1198961(119890minus1198961119905 minus 119890minus1198962119905) (3)

27 Statistical Analyses Two-sample comparison was per-formed through Studentrsquos 119905 test at 95 confidence using thesoftware Statgraphics Centurion XVII (Statistical GraphicsCorp USA 2013) Model adjustment to the experimentaldata resulted from minimization of the sum of squaresand the fit quality was assessed by the Pearsonrsquos correlationcoefficient and mean squared error (119903 and MSE resp) usingMicrosoft Excel 2013

3 Results and Discussion

31 Evolution of the Antioxidant Activity The antioxidantactivity measured as free radical scavenging ability (FRSA)and as hydrogen peroxide scavenging activity (HPSA) wasdetermined at different intervals during storage in blanchedand unprocessed broccoli florets Figures 1 and 2 show FRSAand HPSA of broccoli florets during storage at 20∘C (a) 10∘C(b) minus1∘C (c) minus21∘C (d) and minus45∘C (e) respectively Blanch-ing significantly reduced the FRSAof broccoli florets (119901-value= 00045) agreeing with the literature [9 19] probably due tothe thermal decomposition of antioxidants such as ascorbicacid A similar behavior was obtained for HPSA however thedifferences were not statistically significant During storage at20∘C and 10∘C the FRSA of unprocessed broccoli increaseduntil 2 days of storage after that it showed a progressivedecrease This behavior agrees with the evolution of TPPcontent Then the initial increase of FRSA may obey anincrease in the activity of PAL leading to a higher TPP contentthat was reflected in the antioxidant activity The decrease ofFRSA observed after 2 days of storage may be attributed toa decrease in TPP content due to the action of PPO Besidesthe extractability of the antioxidant compounds was probablyaffected by the structural changes induced by blanchingHPSA showed no statistically significant differences alongthe storage period at 10 and 20∘C The FRSA of unprocessedflorets was significantly higher than FRSA of blanched florets

until 6 days of storage after that there were no statisticallysignificant differences in FRSA between blanched and unpro-cessed broccoli

In storage at minus1∘C minus21∘C and minus45∘C FRSA of unpro-cessed broccoli increased in time with respect to the initialcondition (119905 = 0) and in most cases the difference was sta-tistically significant On the contrary FRSA of blanchedbroccoli was similar to the initial FRSA and showed no cleartrend in time FRSA of blanched and unprocessed broccolishowed a decreasing trend until the 9th day of storage atminus1 minus21 and minus45∘C agreeing with Patras et al [10] whostudied the effect of low temperature preservation of blanchedbroccoli during 7 days From the 9th day onwards there wasno clear trend observing increase and decrease of FRSA atdifferent times This contradicts with the results of Volden etal [9] who reported that the antioxidant activity of blanchedbroccoli stored at minus24∘C decreased in time This differencemay be explained by the different span of the storage periodsused in both studies the authors measured the antioxidantproperties at 0 3 6 and 12 months of storage while inthe present study the storage period was much shorter (83days) In addition the authors quantified the antioxidantactivity through ORAC and FRAP while in the present studywe measured the FRSA using DPPH stable free radical Ingeneral FRSA of unprocessed broccoli was higher than thatof blanched broccoli during storage at freezing temperatureand it increased in time This behavior may be attributedto tissue damage produced by the formation of ice crystalsduring frozen storage Additionally enzyme activity is higherin unprocessed broccoli resulting in a higherTPP content thatwas reflected in the antioxidant activity

The HPSA of blanched broccoli was in most cases higherthan that of unprocessed broccoli during storage at minus1 minus21and minus45∘C opposing the results obtained for FRSA TheHPSA of both unprocessed and blanched broccoli showeda slight increase at the beginning of storage at minus1 minus21and minus45∘C followed by a decrease This agrees with theobservations of FRSA at the same temperatures

In summary the behavior of antioxidant activity duringstorage at different temperatures is affected by several factorssimultaneously which are difficult to isolate one from the oth-ers Besides the antioxidant activity depends on many com-pounds whose concentration and chemical properties differconsiderably This hinders the development of a kineticmodel to describe the evolution of FRSA of broccoli duringstorage

32 Evolution of the TPP Content The total polyphenols con-tent (TPP) was determined at different time intervals duringthe storage periods in blanched and unprocessed broccoliflorets Figure 3 shows the TPP content of broccoli floretsduring storage at 20∘C (Figure 3(a)) 10∘C (Figure 3(b)) minus1∘C(Figure 3(c)) minus21∘C (Figure 3(d)) and minus45∘C (Figure 3(e))In all cases blanching did not affect significantly the TPPcontent resulting in similar values at the beginning of storageThis contradicts Faller and Fialho [15] who reported signifi-cant losses of phenolic compounds in broccoli after domesticcooking This contradiction may be related to the mildblanching conditions used in the present study TPP content


05

1015202530354045

0 1 2 6 7 9Time (d)

FRSA

(m

olg

)

lowast

lowast

lowast

lowast

(a)

05

1015202530354045

0Time (d)

FRSA

(m

olg

)

2 6 9 16

lowast

lowastlowast

(b)

Time (d)0 2 6 9 16 20 27 36 41 48

05

1015202530354045

FRSA

(m

olg

)

lowast

lowast

lowast

lowast lowast

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 8305

1015202530354045

FRSA

(m

olg

)

Time (d)

lowast

lowast

lowast

lowast

lowast

lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 8305

1015202530354045

FRSA

(m

olg

)

Time (d)

lowast

lowast

lowast

lowastlowast

(e)

Figure 1 Evolution of the free radical scavenging ability (FRSA) in blanched (dark bars) and unprocessed (light bars) broccoli florets duringstorage at 20∘C (a) 10∘C (b) minus1∘C (c) minus21∘C (d) and minus45∘C (e) Significant differences (119901 lt 005) between blanched and unprocessed broccoliare highlighted with ldquolowastrdquo

suffered an initial increase followed by a decrease towardsthe end of the storage period at all temperatures exceptat minus45∘C This behavior agrees with the work of Amodioet al [11] who proposed a kinetic model to describe thephenolic content of fresh-cut broccoli during storage at 5∘CThe model considers a two-consecutive-reaction mechanismthat explains the increase in TPP content form day 0 untilday 6 and after that a decrease until day 12 The authorsattributed this behavior to an initial increase of the activityof the enzyme PAL triggered by abiotic stress followed bythe action of PPO that degrades phenolic compounds Thedifferent behavior observed at minus45∘Cmay obey the extremely

low reaction rates preventing the enzymatic degradation ofpolyphenols

In storage at 20∘C the TPP content was higher inunprocessed broccoli than in blanched broccoli along thecomplete storage period with statistically significant differ-ences after 1 and 7 days During storage at 10∘C there were nostatistically significant differences between TPP of blanchedand unprocessed broccoli during the complete period Bothblanched and unprocessed broccoli increased significantlythe TTP during storage with respect to the initial condition(at 119905 = 0) except for day 16th where TPP in blanchedbroccoli was similar to that found at the beginning of the


0

5

10

15

20

25

30

0 2 6 7 9

HPS

A (

)

Time (d)

(a)

0 2 6 9 16 200

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

(b)

0 2 6 9 16 20 27 36 41 480

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowastlowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

lowastlowast

lowast

(e)

Figure 2 Evolution of the hydrogen peroxide scavenging activity (HPSA) in blanched (dark bars) and unprocessed (light bars) broccoli floretsduring storage at 20∘C (a) 10∘C (b) minus1∘C (c) minus21∘C (d) and minus45∘C (e) Significant differences (119901 lt 005) between blanched and unprocessedbroccoli are highlighted with ldquolowastrdquo

storage period The fact that both blanched and unprocessedbroccoli florets showed no significant differences in TPPduring storage at 10∘C may be explained by the similar levelof tissue damage caused by the action of some endogenousplant or microbial enzymes which degrade vegetable wallsthus favoring extractability equally for both blanched andunprocessed florets It has to be noted that the blanchingtreatment was not intense enough to totally reduce themicrobial charge or to completely inactivate all the vegetableenzymes Hence after blanching there was residual microbialand enzymatic activity

During storage at minus1∘C the TPP content in unprocessedbroccoli was significantly higher after 9 27 and 36 days(Figure 3(c)) The data suggest that blanched broccoli hadlower TPP content during storage at this temperature Thismay be attributed to the better extractability in unprocessedbroccoli due to a higher activity of cell wall breaking enzymesIn blanched broccoli the TPP content was similar to theinitial condition (119901-value gt 005) during most of the storageperiod except at 2 and 6 days of storage when TPP wasslightly higher than the initial one This may be attributedto the action of PAL and the cell wall breaking enzymes that


0

200

400

600

800

1000

0 1 2 6 7 9Time (d)

TPP

(mg

GA

E10

0Ａ

dw)

lowast

lowast

(a)

0 2 6 9 160

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

(b)

0 2 6 9 16 20 27 36 41 480

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

lowast lowastlowast lowast

lowastlowast

lowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

Time (d)

lowast

lowast lowast

TPP

(mg

GA

E10

0Ａ

dw)

(e)

Figure 3 Evolution of the total polyphenolsrsquo content (TPP) in blanched (dark bars) andunprocessed (light bars) broccoli florets during storageat 20∘C (a) 10∘C (b) minus1∘C (c) minus21∘C (d) and minus45∘C (e) Significant differences (119901 lt 005) between blanched and unprocessed broccoli arehighlighted with ldquolowastrdquo

were only partially inactivated during blanching In unpro-cessed broccoli the TPP content was significantly higherthan the initial content except at 20 and 48 days of storageprobably due to the higher enzymatic activity A similarbehavior was observed at minus21∘C with TPP content beingsignificantly higher in unprocessed broccoli than in blanchedbroccoli during the most part of the storage period The TPPcontent in unprocessed broccoli increased during storagebeing significantly higher than the initial value (0 days ofstorage) Blanched broccoli showed no significant variationof TPP content with respect to the initial conditionThe vari-ations of TPP content in blanched and unprocessed florets

stored at minus45∘C were much less marked than in the otherstorage conditions At minus45∘C the TPP content of unprocessedbroccoli increased during storage being inmost cases signifi-cantly higher that the initial TPP contentThis agrees with theresults of Volden et al [9] who studied the effect of long-termfreezer storage (at minus24∘C) of different cauliflower varieties onthe TPP content The authors reported a slight increase ofTPP in all the cultivars after 3months of storage AdditionallyNinfali and Bacchiocca [20] reported that TPP content ofbroccoli suffered no change after commercial freezing stor-ageThis contradicts our results obtained atminus1 and minus21∘C butagrees with our observations after 62 days of storage atminus45∘C


12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00

20∘C10∘Cminus1∘C

minus21∘CModelModel

minus45∘Cminus1∘C


10∘C

20∘C

CPC

P0

ModelModelModel

(a)

12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00



minus1∘C


10∘C

20∘C

CPC

P0

ModelModel

ModelModelModel

(b)

Figure 4 Adjustment of the two-consecutive-reaction kinetic model to TPP content in (a) unprocessed and (b) blanched broccoli duringstorage at different temperatures

Kinetic Model The evolution of TPP content in unprocessedand blanched broccoli during storage at different temper-atures was described by a two-consecutive-reaction mech-anism as suggested in the literature [11] Figure 4 showsthe model adjustment for unprocessed (Figure 4(a)) andblanched (Figure 4(b)) broccoli florets The kinetic modeladjusted acceptably the experimental data showing 119903 ge086 (Table 1) The MSE values also indicate a good fit withvalues lower than 010 for blanched broccoli For unprocessedbroccoli the fit was somewhat more modest with MSE rang-ing between 00412 and 01040

The polyphenols formation and degradation were fasterat higher temperatures as shown by the rate constantsgiven in Table 1 For unprocessed broccoli the rate constantsfor polyphenols formation (1198961) fluctuated between 0025and 10 dminus1 and the degradation rate constants (1198962) variedbetween 1 sdot 10minus4 and 1 sdot 10minus2 dminus1 For blanched broccoli therate constants behaved in a similar way These results agreewithAmodio et al [11] who adjusted the evolution of phenoliccompounds in mandarin slices stored at 4∘C to the two-consecutive-reaction kineticsThe authors obtained 1198961 and 1198962equal to 024 and 004 dminus1

Table 2 shows the activation energies obtained for unpro-cessed and blanched broccoli florets For polyphenols for-mation in unprocessed broccoli Ea was 32 kJmol and fordegradation it was 38 kJmol This result suggests that poly-phenols degradation occurred faster than their formationprobably due to a higher activity of the endogenous ormicrobial polyphenol oxidase and in addition to depletionof the precursor compounds In blanched broccoli Ea forpolyphenols formation was 27 kJmol and for degradation itwas 26 kJmol both being lower than in unprocessed floretsThis factmay be related to the partial inactivation of both PALand PPO by blanching

The Ea values obtained in this work agree with the valuesreported by Cisse et al [21] who studied the degradationkinetics of anthocyanin in Roselle extracts during storage

They reported an Ea of 26 kJmol considering first orderdegradation kinetics and the Arrhenius equation

The Ea values obtained for formation and degradationof polyphenols in unprocessed and blanched broccoli weresimilar confirming that in both cases the same reactions areresponsible for the evolution of TPP content during storage

4 Conclusion

Both antioxidant activity and TPP content showed an initialincrease during storage at 20 10 minus1 and minus21∘C followed by adecrease Unprocessed broccoli florets reached higher FRSAand TPP values during storage in comparison with blanchedbroccoli Storage at minus45∘C produced no significant variationin FRSA and TPP content for both unprocessed and blanchedflorets Hence storage at minus45∘C is recommended to preserveantioxidant properties of broccoli The uneven behavior ofFRSA during storage attributed to the multifactorial natureof this property preclude modeling

The two-consecutive-reaction model represented ade-quately the evolution of TPP content in unprocessed andblanched broccoli florets during storage with 119903 ge 086and MSE le 01 The Ea values obtained for polyphenolsformation and degradation confirm that in both unprocessedand blanched broccoli the same reactions are responsible forthe evolution of TPP contentThe results obtained here couldcontribute to design preservation strategies of broccoli inorder to exploit its health-promoting properties

Nomenclature

119862119875 Concentration of total polyphenols1198621198750 Initial concentration of total polyphenols119862pre Concentration of polyphenols precursors119862pre0 Initial Concentration of polyphenols precursors119862119874 Concentration of oxidized polyphenolsDM Dry matter


Table 1 Kinetic parameters estimates (119903 and MSE) for evolution of TPP content in unprocessed and blanched broccoli stored at differenttemperatures ldquo119903rdquo is the Pearsonrsquos correlation coefficient andMSE is the mean squared error 119862pre0 was estimated from the experimental dataand it was equal to 320mg GAEg DM for unprocessed broccoli and 220mgGAEgDM for blanched broccoli

Temperature (∘C) 1198701 (dminus1) 1198702 (dminus1) 119903 MSEUnprocessed broccoli

20 100 00100 09318 0041210 098 00080 09679 00043minus1 016 00022 08815 00194minus21 012 00018 09172 00148minus45 0025 00001 09374 01040

Blanched broccoli20 0840 0010 09464 0007610 0800 0030 09827 00025minus1 0600 0018 09059 00058minus21 0300 0008 09301 00096minus45 0035 0002 08581 00991

Table 2 Activation energies and frequency factors obtained from the kinetic model through the Arrhenius equation

Ea1 (kJmol) 1198700 1 (sminus1) Ea2 (kJmol) 1198700 2 (sminus1)Unprocessed broccoli 32 5 38 080Blanched broccoli 27 1 26 002

Ea Activation energyFRSA Free radical scavenging abilityGAE Gallic acid equivalentsHPSA Hydrogen peroxide scavenging activity119896119894 Rate constantMSE Mean square error119874 Oxidized polyphenols119875 PolyphenolsPAL Phenylalanine ammonia-lyasePPO Polyphenol oxidase119903 Pearsonrsquos correlation coefficient119905 TimeTPP Total polyphenols content

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by Proyecto Basal USA 1555and Vridei 021711MO_PUBLIC Universidad de Santiago deChile

References

[1] D AMoreno M Carvajal C Lopez-Berenguer and C Garcıa-Viguera ldquoChemical and biological characterisation of nutraceu-tical compounds of broccolirdquo Journal of Pharmaceutical andBiomedical Analysis vol 41 no 5 pp 1508ndash1522 2006

[2] S A Ganai ldquoHistone deacetylase inhibitor sulforaphane Thephytochemical with vibrant activity against prostate cancerrdquoBiomedicine and Pharmacotherapy vol 81 pp 250ndash257 2016

[3] M A Moser and O K Chun ldquoVitamin C and heart health areview based on findings from epidemiologic studiesrdquo Interna-tional Journal of Molecular Sciences vol 17 no 8 pp 1328ndash13362016

[4] AMahn andA Reyes ldquoAn overview of health-promoting com-pounds of broccoli (Brassica oleracea var italica) and the effectof processingrdquo Food Science and Technology International vol18 no 6 pp 503ndash514 2012

[5] M DrsquoArchivio C Filesi R Di Benedetto R Gargiulo CGiovannini and R Masella ldquoPolyphenols dietary sources andbioavailabilityrdquo Annali dellrsquoIstituto Superiore di Sanita vol 43no 4 pp 348ndash361 2007

[6] A Scalbert C Manach C Morand and C Remesy ldquoDietarypolyphenols and the prevention of diseasesrdquo Reviews in FoodScience and Nutrition vol 45 no 4 pp 287ndash306 2005

[7] J Shi H Nawaz J Pohorly G Mittal Y Kakuda and YJiang ldquoExtraction of polyphenolics from plant material forfunctional foods - engineering and technologyrdquo Food ReviewsInternational vol 21 no 1 pp 139ndash166 2005

[8] C Cai HMiaoHQian L Yao BWang andQWang ldquoEffectsof industrial pre-freezing processing and freezing handling onglucosinolates and antioxidant attributes in broccoli floretsrdquoFood Chemistry vol 210 pp 451ndash456 2016

[9] J Volden G I A Borge M Hansen T Wicklund and G BBengtsson ldquoProcessing (blanching boiling steaming) effectson the content of glucosinolates and antioxidant-related para-meters in cauliflower (Brassica oleracea L ssp botrytis)rdquo LWT- Food Science and Technology vol 42 no 1 pp 63ndash73 2009

[10] A Patras B K Tiwari and N P Brunton ldquoInfluence of blanch-ing and low temperature preservation strategies on antioxidantactivity and phytochemical content of carrots green beans andbroccolirdquo LWT - Food Science and Technology vol 44 no 1 pp299ndash306 2011

[11] M L Amodio A Derossi and G Colelli ldquoModeling phenoliccontent during storage of cut fruit and vegetables a consecutive


reaction mechanismrdquo Journal of Food Engineering vol 140 pp1ndash8 2014

[12] C Perez H Barrientos J Roman and A Mahn ldquoOptimizationof a blanching step to maximize sulforaphane synthesis inbroccoli floretsrdquo Food Chemistry vol 145 pp 264ndash271 2014

[13] J A Vinson X Su L Zubik and P Bose ldquoPhenol antioxidantquantity and quality in foods fruitsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5315ndash5321 2001

[14] M Montes-Bayon M J D Molet E B Gonzalez and A Sanz-Medel ldquoEvaluation of different sample extraction strategies forselenium determination in selenium-enriched plants (Alliumsativum and Brassica juncea) and Se speciation by HPLC-ICP-MSrdquo Talanta vol 68 no 4 pp 1287ndash1293 2006

[15] A L K Faller and E Fialho ldquoThe antioxidant capacity and poly-phenol content of organic and conventional retail vegetablesafter domestic cookingrdquo Food Research International vol 42no 1 pp 210ndash215 2009

[16] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo LWTmdashFoodScience and Technology vol 28 no 1 pp 25ndash30 1995

[17] A Mahn M Zamorano H Barrientos and A Reyes ldquoOpti-mization of a process to obtain selenium-enriched freeze-driedbroccoli with high antioxidant propertiesrdquo LWT - Food Scienceand Technology vol 47 no 2 pp 267ndash273 2012

[18] M Oktay I Gulcin and O I Kufrevioglu ldquoDetermination ofin vitro antioxidant activity of fennel (Foeniculum vulgare) seedextractsrdquo LWT-Food Science and Technology vol 36 no 2 pp263ndash271 2003

[19] A Mahn and C Perez ldquoOptimization of an incubation stepto maximize sulforaphane content in pre-processed broccolirdquoJournal of Food Science and Technology vol 53 no 11 pp 4110ndash4115 2016

[20] P Ninfali and M Bacchiocca ldquoPolyphenols and antioxidantcapacity of vegetables under fresh and frozen conditionsrdquo Jour-nal of Agricultural and Food Chemistry vol 51 no 8 pp 2222ndash2226 2003

[21] M Cisse F Vaillant A Kane O Ndiaye and M DornierldquoImpact of the extraction procedure on the kinetics of antho-cyanin and colour degradation of roselle extracts during stor-agerdquo Journal of the Science of Food and Agriculture vol 92 no6 pp 1214ndash1221 2012

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


The loss of antioxidant activity was much more markedPatras et al [10] reported that the antioxidant activity ofbroccoli subjected to blanching and freezing was significantlylower in comparison with unprocessed broccoli Blanchingand freezing did not affect significantly the polyphenolscontent Finally Amodio et al [11] proposed a kinetic modelto describe the evolution of polyphenols content in fresh-cut vegetables during storage The model considers an initialincrease of polyphenols due to the activation of phenylalanineammonia-lyase (PAL) triggered by abiotic stress followed bya decrease due to the action of polyphenol oxidase (PPO)Themodel was validatedwith experimental data obtained at 0 and5∘C

The aim of this work was to investigate the evolutionof total polyphenols content and antioxidant activity inblanched and unprocessed broccoli florets during storage attemperatures ranging from minus45∘C to 20∘C

2 Material and Methods

21 Plant Material Broccoli (Brassica oleracea var italica)heads were purchased at the local market (Santiago Chile) toa single supplier Broccoli heads were processed immediatelyafter purchasing as follows after washing they were cut intosingle florets of 5 cm length and 07ndash09 cm width (stem) andblanched at 57∘C for 13min [12] The blanching conditionswere chosen in order to keep the antioxidant activity of thefresh vegetable After that broccoli florets were put in an ice-water bath for 5min then put in sealed plastic bags andstored at different temperatures as stipulated by the experi-mental design The unprocessed broccoli heads followed thesame process without blanching

22 Experimental Design Blanched and unprocessed broc-coli florets were stored at different temperatures 20∘C (con-trolled ambient temperature) 10∘C (refrigerator)minus1∘Cminus21∘C(household freezers) and minus45∘C (industrial freezer) for upto 83 days depending on the preservation status of the veg-etable Temperaturewas checkedwith a k-type thermocoupleBroccoli florets were kept in plastic sealed bags with eachone containing approximately 50-g of the vegetable In eachcondition 39 bags with blanched broccoli and 39 bags withunprocessed broccoli were used In this way in each samplingprocedure three blanched and three unprocessed broccolibags were taken for analyses For storage at 20∘C sampleswere taken after 0 1 2 6 7 and 9 days of storage For storageat 10 and minus1∘C sampling was conducted every two days untilday 15 and every three days until day 41 when the vegetablewas decomposed For storage at minus21 and minus45∘C samples weretaken every 5 days until day 15 and once a week until day 83

23 Antioxidants Extraction The extraction of antioxidantcompounds was performed as reported by Vinson et al[13] A 200-mg aliquot of liquid nitrogen-pulverized broc-coli heads was accurately weighed in a vial and 4mL of80 20 (mL mL) methanol water solution was added Themixture was ultrasound processed through a titanium probeimmersed in the vial for three minutes at 80 duty cycle asreported by Montes-Bayon et al [14] Then the samples were

incubated at room temperature in an orbital shaker for 4 hAfter that samples were centrifuged at 12000timesg for 5min toremove the solidsThe supernatant was recovered and 80 20(mL mL) methanolwater solution was added to complete5mL extract volume

24 Total Polyphenolsrsquo Content The total polyphenolsrsquo con-tent (TPP) was determined spectrophotometrically throughthe Folin-Ciocalteu method [15] 180 120583L extract and 90120583LFolin-Ciocalteau reagent (diluted 1 1) were added to 360 120583Ldistilled water The mixture was homogenized and left in thedark for 5minThen 450120583L of a 200 g Lminus1 sodium carbonatesolution was added and left in darkness for 30min After thatsamples were centrifuged at 12000timesg for 5min to removethe precipitate and absorbance at 750 nm was measuredTheresults were expressed as mg of gallic acid equivalents per 100gram of dry matter (mg GAE (100 g)minus1 DM) The measure-ments were made in triplicate and average values werereported

25 Antioxidant Activity

251 Free Radical Scavenging Ability The free radical scav-enging ability (FRSA) of the broccoli extracts was mea-sured using the stable radical 22-diphenyl-1-picrylhydrazyl(DPPH∙) [16] 40-120583L vegetable extract (at 6 dilutions) weremixed with 1960 120583L DPPH solution (6 times 10minus5M in meth-anol) The absorbance decrease at 515 nm was continuouslyrecorded during 30 minutes The DPPH∙ concentration inthe reaction mixture at zero time and after 30 minutes wascalculated bymeans of a calibration curve and the remainingDPPH∙ concentration was obtained [17] The measurementsweremade in triplicate and the average values were reported

252 Hydrogen Peroxide Scavenging Activity The hydrogenperoxide scavenging activity (HPSA) was assessed throughthe method reported by Oktay et al [18] The methanolextracts were concentrated in a rotatory evaporator (StuartRE-300 United Kingdom Great Britain) to dryness andthen resuspended into the initial volume with HPLC-gradewater 100 120583L of aqueous antioxidants extract was added to900 120583L of a 4mMH2O2 solution in phosphate buffer (pH 74)Two control mixtures were used one contained HPLC-gradewater instead of extract and the other contained phosphatebuffer instead of H2O2 solution Absorbance was measuredat 230 nm HSPA was expressed as percentage with respect tocontrol The measurements were made in triplicate and theaverage values were reported

26 Kinetic Model The evolution of TPP content was des-cribed by the mechanism proposed by Amodio et al [11]given in (1) considering that polyphenols synthesis is medi-ated by PAL and their degradation is mediated by PPO In (1)119875pre is polyphenols precursor 119875 is polyphenols119874 is oxidizedcompounds and 1198961 and 1198962 are the rate constants

119875pre1198961997888rarr 1198751198962997888rarr 119874 (1)





119889119862119874119889119905= 1198962 sdot 119862119875

(2)


1198621198751198621198750= 119890minus1198962119905 +











05

1015202530354045

0 1 2 6 7 9Time (d)

FRSA

(m

olg

)

lowast

lowast

lowast

lowast

(a)

05

1015202530354045

0Time (d)

FRSA

(m

olg

)

2 6 9 16

lowast

lowastlowast

(b)

Time (d)0 2 6 9 16 20 27 36 41 48

05

1015202530354045

FRSA

(m

olg

)

lowast

lowast

lowast

lowast lowast

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 8305

1015202530354045

FRSA

(m

olg

)

Time (d)

lowast

lowast

lowast

lowast

lowast

lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 8305

1015202530354045

FRSA

(m

olg

)

Time (d)

lowast

lowast

lowast

lowastlowast

(e)






0

5

10

15

20

25

30

0 2 6 7 9

HPS

A (

)

Time (d)

(a)

0 2 6 9 16 200

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

(b)

0 2 6 9 16 20 27 36 41 480

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowastlowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

lowastlowast

lowast

(e)





0

200

400

600

800

1000

0 1 2 6 7 9Time (d)

TPP

(mg

GA

E10

0Ａ

dw)

lowast

lowast

(a)

0 2 6 9 160

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

(b)

0 2 6 9 16 20 27 36 41 480

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)


lowastlowast

lowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

Time (d)

lowast

lowast lowast

TPP

(mg

GA

E10

0Ａ

dw)

(e)





12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00





10∘C

20∘C

CPC

P0

ModelModelModel

(a)

12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00



minus1∘C


10∘C

20∘C

CPC

P0

ModelModel

ModelModelModel

(b)








4 Conclusion



Nomenclature












Acknowledgments


References































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





119889119862119874119889119905= 1198962 sdot 119862119875

(2)


1198621198751198621198750= 119890minus1198962119905 +











05

1015202530354045

0 1 2 6 7 9Time (d)

FRSA

(m

olg

)

lowast

lowast

lowast

lowast

(a)

05

1015202530354045

0Time (d)

FRSA

(m

olg

)

2 6 9 16

lowast

lowastlowast

(b)

Time (d)0 2 6 9 16 20 27 36 41 48

05

1015202530354045

FRSA

(m

olg

)

lowast

lowast

lowast

lowast lowast

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 8305

1015202530354045

FRSA

(m

olg

)

Time (d)

lowast

lowast

lowast

lowast

lowast

lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 8305

1015202530354045

FRSA

(m

olg

)

Time (d)

lowast

lowast

lowast

lowastlowast

(e)






0

5

10

15

20

25

30

0 2 6 7 9

HPS

A (

)

Time (d)

(a)

0 2 6 9 16 200

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

(b)

0 2 6 9 16 20 27 36 41 480

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowastlowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

lowastlowast

lowast

(e)





0

200

400

600

800

1000

0 1 2 6 7 9Time (d)

TPP

(mg

GA

E10

0Ａ

dw)

lowast

lowast

(a)

0 2 6 9 160

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

(b)

0 2 6 9 16 20 27 36 41 480

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)


lowastlowast

lowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

Time (d)

lowast

lowast lowast

TPP

(mg

GA

E10

0Ａ

dw)

(e)





12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00





10∘C

20∘C

CPC

P0

ModelModelModel

(a)

12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00



minus1∘C


10∘C

20∘C

CPC

P0

ModelModel

ModelModelModel

(b)








4 Conclusion



Nomenclature












Acknowledgments


References































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


05

1015202530354045

0 1 2 6 7 9Time (d)

FRSA

(m

olg

)

lowast

lowast

lowast

lowast

(a)

05

1015202530354045

0Time (d)

FRSA

(m

olg

)

2 6 9 16

lowast

lowastlowast

(b)

Time (d)0 2 6 9 16 20 27 36 41 48

05

1015202530354045

FRSA

(m

olg

)

lowast

lowast

lowast

lowast lowast

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 8305

1015202530354045

FRSA

(m

olg

)

Time (d)

lowast

lowast

lowast

lowast

lowast

lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 8305

1015202530354045

FRSA

(m

olg

)

Time (d)

lowast

lowast

lowast

lowastlowast

(e)






0

5

10

15

20

25

30

0 2 6 7 9

HPS

A (

)

Time (d)

(a)

0 2 6 9 16 200

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

(b)

0 2 6 9 16 20 27 36 41 480

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowastlowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

lowastlowast

lowast

(e)





0

200

400

600

800

1000

0 1 2 6 7 9Time (d)

TPP

(mg

GA

E10

0Ａ

dw)

lowast

lowast

(a)

0 2 6 9 160

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

(b)

0 2 6 9 16 20 27 36 41 480

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)


lowastlowast

lowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

Time (d)

lowast

lowast lowast

TPP

(mg

GA

E10

0Ａ

dw)

(e)





12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00





10∘C

20∘C

CPC

P0

ModelModelModel

(a)

12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00



minus1∘C


10∘C

20∘C

CPC

P0

ModelModel

ModelModelModel

(b)








4 Conclusion



Nomenclature












Acknowledgments


References































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

5

10

15

20

25

30

0 2 6 7 9

HPS

A (

)

Time (d)

(a)

0 2 6 9 16 200

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

(b)

0 2 6 9 16 20 27 36 41 480

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowast

lowastlowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

5

10

15

20

25

30

HPS

A (

)

Time (d)

lowastlowast

lowastlowast

lowast

(e)





0

200

400

600

800

1000

0 1 2 6 7 9Time (d)

TPP

(mg

GA

E10

0Ａ

dw)

lowast

lowast

(a)

0 2 6 9 160

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

(b)

0 2 6 9 16 20 27 36 41 480

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)


lowastlowast

lowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

Time (d)

lowast

lowast lowast

TPP

(mg

GA

E10

0Ａ

dw)

(e)





12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00





10∘C

20∘C

CPC

P0

ModelModelModel

(a)

12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00



minus1∘C


10∘C

20∘C

CPC

P0

ModelModel

ModelModelModel

(b)








4 Conclusion



Nomenclature












Acknowledgments


References































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

200

400

600

800

1000

0 1 2 6 7 9Time (d)

TPP

(mg

GA

E10

0Ａ

dw)

lowast

lowast

(a)

0 2 6 9 160

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

(b)

0 2 6 9 16 20 27 36 41 480

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)

lowastlowast

lowast

(c)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

TPP

(mg

GA

E10

0Ａ

dw)

Time (d)


lowastlowast

lowast lowast

(d)

0 2 9 16 20 27 36 41 48 55 62 76 830

200

400

600

800

1000

Time (d)

lowast

lowast lowast

TPP

(mg

GA

E10

0Ａ

dw)

(e)





12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00





10∘C

20∘C

CPC

P0

ModelModelModel

(a)

12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00



minus1∘C


10∘C

20∘C

CPC

P0

ModelModel

ModelModelModel

(b)








4 Conclusion



Nomenclature












Acknowledgments


References































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00





10∘C

20∘C

CPC

P0

ModelModelModel

(a)

12

16

0 10 20 30 40 50 60Time (d)

20

08

04

00



minus1∘C


10∘C

20∘C

CPC

P0

ModelModel

ModelModelModel

(b)








4 Conclusion



Nomenclature












Acknowledgments


References































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Acknowledgments


References































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

evolution of total polyphenols content and antioxidant...

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