Three-DimensionalExcitationandEmissionFluorescence-Based ...downloads.hindawi.com/journals/jchem/2018/6758794.pdf · ResearchArticle Three-DimensionalExcitationandEmissionFluorescence-Based

Research ArticleThree-Dimensional Excitation and Emission Fluorescence-BasedMethod for Evaluation of Maillard Reaction Products in FoodWaste Treatment

Jiaze Liu12 Jun Yin 12 Xiaozheng He12 Ting Chen12 and Dongsheng Shen 12

1School of Environmental Science and Engineering Zhejiang Gongshang University Hangzhou 310012 China2Zhejiang Provincial Key Laboratory of Solid Waste Treatment and Recycling Hangzhou 310012 China

Correspondence should be addressed to Jun Yin junyin77gmailcom

Received 12 July 2018 Revised 5 September 2018 Accepted 30 September 2018 Published 19 November 2018

Guest Editor Gassan Hodaifa

Copyright copy 2018 Jiaze Liu et al-is is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hydrothermal treatment (HT) of food waste (FW) can formMaillard reaction products (MRPs) the biorefractory organic matterdue to the occurrence of Maillard reaction However the integrating qualitative and quantitative approach to assess MRPs isscarce -e goal of this study was to develop a method to characterize and quantify MRPs created by HT of FW MRPs wereidentified by molecular weight fractionation indirect spectrometric indicators and three-dimensional excitation-emissionfluorescence (3DEEM) analysis -e 3DEEM method combined with fluorescence regional integration (FRI) and parallel fac-tor (PARAFAC) analyses was able to differentiate clearly between MRPs and other dissolved organic compounds compared toother approaches-e volume of fluorescenceΦ from FRI andmaximum fluorescence intensity Fmax from PARAFACwere foundto be suitable quantitative parameters for determination of MRPs in the hydrothermal FW system -ese two parameters werevalidated with samples from hydrothermal FW under various operating temperatures and pH

1 Introduction

In China the stacking of FW has become a major issue tocause environmental problems Recently anaerobic di-gestion (AD) as an attractive waste treatment practice hasbeen used to decrease the amount of biowaste and recoverenergy [1] Due to the high biodegradability and watercontent of FW it becomes a good candidate for AD [2]During the process of the AD the hydrolysis step is generallyconsidered as the rate-limiting step for complex organicsubstrates degradation -erefore hydrothermal treatment(HT) was ordinarily used as a pretreatment to promote thesolubilization of complicated macromolecular solid organicmatters thus improving the AD process [3]

Despite the acceleration of dissolved properties of FW ithas been documented that HT is responsible for the for-mation of Maillard reaction products (MRPs) [4] On theone hand the formation of MRPs can lead to the substrateloss during the HTof FW On the other hand the influence

of MRPs themselves on AD merits further investigation [5]-erefore to optimize the HT process and enhance theefficiency of AD it is essential to provide an integratingquantitative and qualitative approach to assess the MRPsproduction

Numerous methods have been developed to characterizethe occurrence of MRPs by using the precision analysisinstrument [6 7] However the quantitative determinationcould not be achieved because there is no pure standard forthe measurement of MRPs [6] In addition these devices aretime-consuming and labor-intensive limiting their appli-cation and spreading -us some easy-to-use and conve-nient characterization techniques became most commonlyused methods including the UVA254 and color intensity[8 9]

Nowadays three-dimensional excitation and emissionfluorescence (3DEEM) is regarded as a promising tool tooffer characteristic information for signature chemicalstructures in a complex mixture of chromophores [10] And

HindawiJournal of ChemistryVolume 2018 Article ID 6758794 11 pageshttpsdoiorg10115520186758794

the qualitative characterization of MRPs has been achievedby the traditional 3DEEM method [11] However a quan-titative determination could not be realized because only oneexcitationemission intensity value can be used for analysisRecent studies have demonstrated that fluorescence regionalintegration (FRI) method and parallel factor analysis(PARAFAC) method were developed to integrate the areabeneath EEM spectra and semiquantitatively assess thespecific components in a complex system [12ndash14] Howeverthe application of 3DEEM to the semiquantitative charac-terization of MRPs in the complicated hydrothermal FWsystem is scarce -erefore the utilization of 3DEEM todistinguish between MRPs and other dissolved organicmatter under various hydrothermal conditions is supposedto be further explored

-is study aimed at developing a method to characterizeand quantify MRPs created by HTof FW Firstly MRPs werecharacterized and evaluated with different methods -enMRPs production was further assessed by the applicability of3DEEM combined with FRI and PARAFAC hence ex-ploring the suitable fluorescence parameters for semi-quantifying the MRPs in the hydrothermal FW system

2 Materials and Methods

21 Food Waste Sample Preparations -e FW containingrice (44) noodles (16) vegetables (23) meat (6) andtofu (11) was compounded based on the characteristicssimilar to FW collected from a canteen of ZhejiangGongshang University (Hangzhou China) in our previousstudy [15] -e five components came from the same vendorat Cui Yuan farmersrsquo market (Hangzhou China) -e FWwas cut into small pieces first by hand-breaking and thencrushed using a mangler -e untreated FW sample wasstored at minus18degC before the HT -e main characteristics ofthe FW are listed in Table S1

22 Hydrothermal Treatment Hydrothermal treatment ofFW was performed in an 80mL airtight pressure digestionvessel as described by our previous study [16] at separatebatch operations at each temperature During HT about 30 gcrushed FW was placed in the vessel Each batch wasprocessed for 30min In the first experiment the temper-ature manipulations were made at 110 120 130 140 150and 160degC in an oil bath to explore the effect of temperatureon MRPs production In the second experiment the dif-ferent initial pH values (30 40 50 60 70 80 90 and100) were tested at 130degC for 30min-e time wasmeasuredfrom when the oil bath reached the set temperature -evessels were cooled to ambient temperature after HT Eachtreatment was performed in triple vessels

23 Extraction of WEOM WEOM was obtained withdeionized water (solid-to-water ratio of 1 10 wv) and themixture was shaken for 1 h in a horizontal shaker at 35 plusmn 2degC-e extracts were separated from the mixture by centrifu-gation at 10000 rpm for 5min and filtered using themicrofiltration membrane (045 microm)

24 Synthetic MRPs Solution -e synthetic MRPs solutionwas made by a concentrated solution of melanoidins whichare defined as brown substances formed during the finalstage of the Maillard reaction -e formula of the concen-trate was made with a 1 1 molar ratio of glucose and glycinewith a buffer of 05M Na2CO3 according to the previousresearch [17]-e solution was heated at 120degC for 3 hours asthe record [17] -is synthetic MRPs solution has been usedas model MRPs to analyze the properties of MRPs [9] and itwas employed to examine the availability of MRPs char-acterization method in the present study

25 Analytical Methods of MRPs

251 Spectrometric Indicators (1) UVA254 UVA wasa measure of absorbance at 254 nm measured in a 1 cm pathlength quartz cell It can measure unsaturated bonds oraromaticity within dissolved organic matters [18]-ereforethis spectrometric index was useful for this study as MRPswere linked to the presence of unsaturated double bonds andaromatic compounds and expressed as cmminus1middotmLg dryweight

(2) Color Intensity A spectrophotometer at a wavelength of475 nm was used to determine color intensity in a 1 cm pathlength cell -e absorbance at this wavelength was charac-teristic of brown color Characteristic color intensity wasrecorded in a platinum-cobalt (PtCo) unit as previouslydescribed [9]

(3) Browning Index-e Browning index of the FW solid wasmeasured by an enzymatic digestion method which releasesthe brown pigments Samples were dried for 24 h andgrounded to a smaller size before use -e proposed methodwas modified based on pronase proteolysis created byPalombo et al [19]-e procedure was as follows 03 g of thedried sample was added into a test tube which contains 5mLdeionized distilled water at 45degC and mixed thoroughly-en another 04mL of pronase solution was added into themixture After that the test tubes were placed in a waterbath incubated for 120min at 45degC and then cooled in icewater and 1mL trichloroacetic acid (80 TCA) was addedto each tube Finally centrifugation (20min at 7000 rpm)and filtration were used before the spectrometric de-termination -e optical density of the filtrates was de-termined on a spectrophotometer Samples were measuredin a 1mL cuvette with 1 cm pass length -e OD of thebrown index was calculated as OD OD420nm minus OD550nmand expressed as ODg dry weight

252 Molecular Weight Fractionation Molecular weightfractionation was applied for a better separation andcharacterization of dissolved organic matters Fractionationof samples was performed using an ultrafiltration centrifugetube with different molecular weight cutoffs 3 kDa 10 kDaand 30 kDa -e samples were filtered in series from 30 kDato 3 kDa

2 Journal of Chemistry

253 3DEEM Analyses -e 3DEEM of WEOM was mea-sured in a 1 cm cuvette using a Hitachi F-4600 fluorescencespectrometer at room temperature (25 plusmn 2degC) -e scanningranges were 200ndash500 nm for excitation and 250ndash500 nm foremission Scanning was recorded at 5 nm intervals for ex-citation and 1 nm steps for emission respectively usinga scanning speed of 2400 nmmin -e Milli-Q water blankswere subtracted in order to eliminate the effect of Ramanscattering In addition exported EEMs were normalized bythe Raman area and eliminated the primary and secondRayleigh scattering

(1) Peak-Picking Method A peak-picking method is used forthe detection of the fluorescence intensity of easily-identifiable peaks and their locations within the EEMsAnd then the observed peaks were analyzed by comparingtheir fluorescence properties with the change of operatingcondition

Besides as an important humification index in EEMHIX can reflect the degree of humification of the sampleHIX was calculated by dividing the emission intensity intothe 435ndash480 nm regions by intensity in 300ndash345 nm whenexcitation intensity is at a wavelength of 255 nm and isshown as follows

HIX 1113936 I(435ndash480)

1113936 I(300ndash345)

(1)

(2) FRI Analysis Method -e FRI approach was employed inthis work to characterize the five excitation-emission regionsof EEM spectra [12] According to previous studies EEMspectra are usually divided into five areas (Table S2) aro-matic protein-like fluorophores (regions I and II) fulvicacid-like fluorophores (region III) soluble microbialproduct-like fluorophores (region IV) and humic acid-likefluorophores (region V) By normalizing the cumulativeexcitation-emission area volumes to relative regional areas(nm2) the volume of fluorescence (Φ(i)) was calculatedaccording to Chen et al [12] within each region (i) applyingthe following equation

Φ(i) MF(i) 1113944 1113944 I λexλem( 1113857ΔλexΔλem (2)

where MF(i) is a multiplication factor calculated by Equation(3) Δλex is the excitation wavelength interval (taken as5 nm) Δλem is the emission wavelength interval (taken as1 nm) and I(λexλem) is the fluorescence intensity at eachexcitation-emission pair (Raman units)

MF(i) total spectra area

specific region area(i)

(3)

And the normalized excitation-emission area volumesreferred to the value of region i -e entire region wascalculated and the percent fluorescence response was thendetermined using the following equation

Pin 100 timesΦin

ΦTn

(4)

(3) PARAFAC Component Analysis Method PARAFAC isa method that decomposes EEMs of complex WEOM intoavailable components PARAFAC modeling was performedinMATLAB followed by the procedure recommended in theDOMFluor toolbox [20]-e PARAFACmodels with two toseven components were computed and core consistency wasoften applied to select the optimal number of components[21] -e concentration scores of each component wererepresented by maximum fluorescence intensity (FmaxRU)

26 Other Analytical Methods -e TS volatile solids (VSs)dissolved organic carbon (DOC) soluble carbohydrate andsoluble protein were all determined -e diluent sampleswere operated as the same as mentioned in Section 23SCOD was analyzed using standard methods [22] -e DOCconcentrations of the WEOM solutions were determinedwith a total organic carbon analyzer (TOC-L CPH Shi-madzu Japan) Soluble protein was quantified by theLowryminusFolin method using bovine serum albumin as thestandard and carbohydrate was determined using thephenol-sulfuric acid method with glucose as the standard[23 24]

3 Results and Discussion

31 Variation of Food Waste after Hydrothermal TreatmentFW was treated under high pressure for 30min at 110120130 140 150 and 160degC -e SCOD concentrations ofthe hydrothermal-treated FW are shown in Figure S1 In-creasing the temperature from 110 to 160degC caused an in-crease in SCOD from 8555 gkg to 11991 gkg Although theincrement of SCOD was obtained color generation byMaillard reaction was also observed in the HTprocess due tothe high temperature and the presence of proteins andcarbohydrates [25] Figure S2 shows that the treated solidgradually produced a darker brown color than the untreatedFW with increasing treatment temperature -e significantcolor variation of hydrothermal FW was highly reliant onthe formation of MRPs which depends on the reactiontemperature Nevertheless owing to the complex and het-erogeneous nature of the MRPs it has not been possible toisolate or purify MRPs [26] -erefore there is a lack ofmethods for rapid effective and quantitative estimation ofMRPs in the hydrothermal FW system

32 Characterization and Evaluation of MRPs in FoodWasteafter Hydrothermal Treatment

321 Distribution of Soluble Organic Compounds in WEOMFigure 1 presents the molecular weight distribution ofWEOM before and after HT DOC of the original samplewas evenly in the MW analysis range and remained un-changed in each fraction after HT at 120degC Howevermolecular weight fractionation distribution was modified at140degC compared with 120degC -e total DOC content hada large promotion but significant increase of DOC from 12to 27 gL only occurred in the MW gt 30 kDa fraction -e

Journal of Chemistry 3

significant increment highlighted that dissolved organiccompounds residing in the MW gt 30 kDa fraction wereexpected as MRPs which have a molecular weight between40 and 70 kDa [27] Moreover it is well known that theformation of MRPs has a positive relationship with thecarbohydrates and proteins [28] In 140degC group the mostprevalent fraction of soluble carbohydrates and solubleproteins also resided in the MW gt 30 kDa fraction whichaccounts for 78 and 83 respectively In addition thecharacteristic absorbance UVA254 of WEOM in the MW gt30 kDa fraction was 8 75 and 34 cmminus1mLg for untreatedFW 120degC and 140degC groups respectively -e rise of thischaracteristic absorbance indicator showed that HT couldresult in the generation of MRPs from organic mattermixtures at 140degC Similar conclusions were obtained by

Barber [29] that MRP production occurred in the typicalreaction range of thermal hydrolysis of 140ndash165degC

322 Spectrometric Characterization of the MRPs from FoodWaste Nowadays various easy-to-use quantitative spec-trometric characterization methods have been applied todetermine the MRP production in complicated systems[19 30] -ree representative indicators (ΔUVA254Δbrowning index and Δcolor intensity) were employed todemonstrate the modification of MRPs content of hydro-thermal FW (Figure 2) and raw material was taken as thecontrol At first ΔUVA254 did not vary significantly(pgt 005) after HT at 110degC and 120degC -e ΔUVA254 in-creased significantly (plt 005) at 130degC which enhanced by

30

25

20

DO

C (g

L)

15

10

5

0lt3 3ndash10 10ndash30

Molecule weight (kDa)gt30

Untreated120degC140degC

(a)

50

40

30

20

10

0

Solu

ble c

arbo

hydr

ates

(gL

)

lt3 3ndash10 10ndash30Molecule weight (kDa)

gt30


(b)

Solu

ble p

rote

in (g

L)

5

4

3

2

1




(c)

UV

A25

4 (cm

ndash1middotm

Lg)

36

30

24

18

12

6




(d)

Figure 1 Different molecular weight distributions of the soluble organic compounds (a) DOC (b) Soluble carbohydrates (c) Solubleprotein (d) UVA254


15-fold compared with 120degC -en this surrogate indexcontinued ascending with the increase of treatment tem-perature indicating a significant number of compoundsaccumulated when the hydrothermal temperature above120degC In addition a similar tendency was found in the resultof the pronase method and the Δbrowning index wasutilized for determination of nonenzymatic browning -echanging of the Δbrowning index showed that the brownpigment production was slim to zero in 110degC and 120degCtreatments and exhibited a regular increase beyond thecritical temperature (120degC) -e tendencies of these twoparameters were consistent with the transformation of thesurface color of hydrothermal-treated FW However theaccuracy of these two indicators was still in doubt because ofthe complexity of hydrothermal FW systems Moreover theΔcolor intensity of WEOM was also recorded Neverthelessthe relative standard deviation ofΔcolor intensity was higherthan other methods and this color indicator did notdemonstrate the significant difference (pgt 005) between the130degC and 140degC groups which was different from theappearance changes of hydrothermal-treated FW Althoughspectrometric characterization could quickly verify the ac-cumulation of MRPs in an indirect way the influence ofother organic matters was still not solved

323 Characterization of MRPs by 3DEEM FluorescenceAnalysis -e objective of using 3DEEM fluorescence was tomonitor the change of fluorescence properties in WEOM andto analyze the effect of temperature on MRPs generation inthe hydrothermal FW system Typical 3DEEM spectra oftreated WEOM samples at different temperatures are shownin Figure 3 and detailed fluorescence properties are sum-marized in Table 1 by the ldquopeak-pickingrdquo method As ex-pected there were obvious differences in the 3DEEM spectra

of the WEOM with the rise of temperature and the maximaltransformation was found between 130degC and 140degC groupsMoreover Table 1 shows the change of peak intensity at thesoluble microbial region (λexλem 285357ndash361) whichdecreased from 358RU (raw material) to 010RU (160degC)and the sharp decrease trend started at the 140degC group Tothe best of our knowledge soluble microbial products (SMPs)contain various complex organic materials such as pro-teinaceous material carbohydrates humic and fulvic sub-stances and organic acids [31 32] -us the organic mattersresided in the region of soluble microbial by-products wereconsidered as soluble carbohydrates and proteins becauseother organic matters would not appear in this regionConversely the peak intensity at the humic acid-like region(λexλem 320ndash360419ndash438) increased from 043 to 334RUas the temperature increased and obvious transformationalso occurred at 140degC group-e peak ratio (VIV) ascendedwith the increase of temperature which implied that thegeneration and accumulation of humic acid-like organicfluorescentmolecules were attributed to the polymerization ofthe soluble carbohydrates and proteins during the HT Inaddition the movement of humic-like peaks location withinthe 3DEEM (λexλem) to longer wavelength (red-shifting)from λexλem 320428 to 360438 also indicated the humicacid-like fluorophores were concentrating and becomingmore refractory [33] Besides the HIX value has been appliedto evaluate the humification extent of dissolved organicmatter [34] From Table 1 the increase of HIX values after140 150 and 160degC treatments were indicative of more humicWEOM It is generally known that MRPs are highly aromaticand resemble humic substances it suggested that 3DEEMmight serve as a good descriptor to prove the MRPs pro-duction during the HT

Overall compared with the 3DEEM analysis methodspectrometric approaches cannot provide a direct

12

400 36 times 104

30 times 104

24 times 104

18 times 104

12 times 104

60 times 103

00

320

240

160

80

9

6

Δbro

wni

ng in

dex

(Abs

middotmL

g)

ΔUV

A25

4 (cm

ndash1middotm

Lg)

Δcol

or in

tens

ity (m

gPtC

omiddotm

Lg)

3

0

110 120 130Temperature (degC)

140 150 160

Δbrowning index

Δcolor intensityΔUVA254

Figure 2 -ree spectrometric indicators of MRPs production from FW at different temperatures


assessment of MRPs and potentially influence the de-termination of MRPs due to the fluctuation from other or-ganic or colored compounds -e molecular weightfractionation technique was used to eliminate the interferenceof smaller molecular weight constituents which caused theassessment of MRPs costlier and more time-consuming

Besides Figures 1(b) and 1(c) show that even though themolecular weight fractionation was used the existence oforganic macromolecular compounds (carbohydrates andproteins) might also affect the accuracy of UVA254 mea-surement Accordingly 3DEEM fluorescence analysis wassimple had good selectivity and provided a wealth of

450

400

350

300ex (n

m)

250

200200 300 350 400

em (nm)450 500

00

040812162024283236

(a)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(b)

em (nm)

450

400

350

300ex (n

m)

250

200250 300 350 400 450 500

00

040812162024283236

(c)450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(d)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(e)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(f )450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(g)

Figure 3 Typical EEM spectra of the WEOM extracted from FW treated at different temperatures (a) Untreated (b) 110degC (c) 120degC (d)130degC (e) 140degC (f ) 150degC and (g) 160degC

Table 1 Fluorescent properties of treated groups at each temperature

Sample nameRegion IV peak location Region V peak location

Peak ratio (IVV) HIXExem Fluorescence intensity (RU) Exem Fluorescence intensity (RU)

Untreated sample 280357 358 320438 043 012 049110degC treated sample 285360 324 320434 058 018 062120degC treated sample 285360 367 320436 052 014 062130degC treated sample 285359 256 325419 060 023 045140degC treated sample 285359 075 335421 096 128 088150degC treated sample 285361 042 345430 196 467 168160degC treated sample 285360 010 360438 343 3430 670


information about WEOM especially helpful in character-izing WEOM and interpreting the complex information inEEM [35] However fluorescence information was still re-stricted because only one peak location and fluorescenceintensity value was used for analysis and it should be noticedthat the direct quantification ofMRPs using 3DEEM can be ofmajor interest for investigation of WEOM from FW

33 Quantitative Characterization of MRPs Production

331 Fluorescence EEMs-Based FRI Analysis and PARAFACComponent Estimation Recently FRI analysis and PAR-AFAC component estimation were developed to providea more complete data analysis than the traditional ldquopeak-pickingrdquo technique [36] -erefore the FRI analyticalmethod was used to reveal the transformation of organiccompounds inWEOM by integration of the volume beneatheach EEM region According to the five regions of the FRIanalysis it can be found that the obvious characteristic peakswere presented in region IV and region V -e variation ofpercent fluorescence response P(in) of the WEOM is shownin Figure 4 -e P(in) values of region IV and region Vremained almost constant in 110 120 and 130degC groupsWhen temperature increased from 130 to 160degC the P(in)

value of region IV decreased from 498 to 169 and theP(in) of region V increased from 121 to 661 Moreoverthe P(in) value of region I also had a little reduction above130degC -ese results implied that humic acid-like materialregarded as MRPs in region V were efficiently accumulatedwhen HT temperature was beyond 130degC It was assumedthat the disappearance of soluble carbohydrates and pro-teinaceous products (region IV) and tryptophan-like sub-stances (region I) were the source material for thepolymerization of MRPs (region V) -erefore region IV(carbohydrates and proteinaceous products) matters andregion V (MRPs) matters can be effectively differentiatedbased on the FRI method

Furthermore PARAFAC can capture the heterogeneityof WEOM samples thereby decomposing EEM spectra intovarious individual fluorescent components Two fluorescentcomponents evaluated by PARAFAC using the COR-CONDIA procedure were the soluble microbial by-product-like component (C1) and humic acid-like component (C2)Individual components are shown in Figure 5 as contourplots -e excitation and emission loadings are also given inFigure 5 and excitation and emission characteristics of thecomponents identified in this study and probable source ofcomponents are depicted in Table S3 In Table S3 twoPARAFAC components are pointed out in detail C1 withpeaks of exem 280360 nm was associated with solublemicrobial products defined as carbohydrates and proteinswhich could be easily biodegradable [37 38] and C2 withspecific peaks of exem 360441 nm originated from humicacid-like substances [9 28] In the current study the humicacid-like component identified by PARAFAC was MRPswhich had higher exem than the other synthetic or isolatedhumic compounds [11 27 39] Furthermore the observedhumic-like compounds were forcefully identified as MRPs

by comparing its fluorescence property (exem) with syn-thetic MRPs solution created in our study (Figure S3) -emaximum fluorescence intensities (Fmax) of component C2remained constant at a low content when the temperaturewas in the range of 110degC to 130degC and then increased from2237 to 15579 RU by further increasing the temperaturefrom 130 to 160degC (Figure S4) which indicated little ac-cumulation of nonbiodegradable MRPs at temperaturesbelow 130degC and mass production of MRPs when temper-ature beyond 130degC Meanwhile the results in Figure 6 alsoshow that the Fmax of C1 had a slight reduction at 110degC120degC and 130degC groups but substantially dropped with thefurther increase of temperature Results from PARAFACwere consistent with the FRI analysis which suggestedbiodegradable organic compounds andMRPs inWEOM canbe separated by PARAFAC

As mentioned above the distribution of P(in) and the 2-component PARAFAC estimation provided complementaryinformation showing that humic acid-like matter (MRPs)and SMP materials (carbohydrates and proteins) whichdominated the WEOM underwent an increase and decreaserespectively as the operating temperature increased -eseresults proved FRI and PARAFAC can differentiate fluo-rescence characteristic disparities between MRPs and otherorganic compounds And it also can be seen that Maillardreaction (C2) was accelerated at elevated temperature byconsuming organics in C1 -erefore 3DEEM combinedwith FRIPARAFAC could be employed to analysis of MRPsgenerated from hydrothermal-treated FW

332 Semiquantitative Characterization of Fluorescent Pa-rameters for MRPs Production -e volume of fluorescence

100

80

60

40

20

0110 120 130 140

Temperature (degC)150 160

P(Vn)

P(IVn)

P(IIIn)

P(IIn)

P(In)

P (in

) (

)

Figure 4 -e distribution of FRI in WEOM from food waste afterhydrothermal treatment


Φ(V) parameter from FRI and maximum fluorescence in-tensity in the C2 component Fmax (C2) parameter fromPARAFAC were used as the characterization parameters forassessing the generation of MRPs All the data were based ondry weight and raw material was taken as control -ecorresponding results of fluorescent indictors are presentedin Figure 6 It can be seen from Figure 5 that the impact oftemperature on the ΔΦ(V) was minimal in 110 120 and130degC groups (pgt 005) staying at a low level from 51 times 104to 90 times 104 RUmiddotnm2middotmLg But then ΔΦ(V) increased sig-nificantly from 29 times 105 (140degC) to 12 times 106 RUmiddotnm2middotmLg(160degC) A similar tendency was found in ΔFmax (C2)(Figure 5) -e ΔFmax (C2) remained unchanged as tem-perature increased from 110 to 130degC and then went througha successive and relatively large increase from 335 to1413 RUmiddotmLg as temperature further increased -ese

results were consistent with the fact that MRPs are tem-perature dependent [29]

Besides the correlations between 3DEEM fluorescenceparameters and spectrometric parameters were analyzed tocheck whether fluorescence parameters could effectivelycharacterize the MRPs in FW after HT Pearson correlationsfor 3DEEM fluorescence parameters and spectrometricparameters are summarized in Table S4 Generally spec-trometric parameters were strongly correlated with 3DEEMfluorescence parameters (rgt 0880 plt 001) which in-dicated that these two fluorescent analytical techniquescould be employed to monitor the generation of MRPs andsemiquantitatively determine the content of MRPs in thehydrothermal FW system In addition these two indicatorswere expected to be tested at various situations which helptheir generalization and application

450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

Compound 2450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

ExcitationEmission

05

04

03

02Load

ings

01

00

200 250 300 350Wavelength (nm)

400 450 500

05

04

03

02Load

ings

01

00


(b)(a)

(d)(c)

400 450 500

ExcitationEmission

Compound 1

Figure 5 Two-component PARAFAC model generated from samples of WEOM (a and b) contour plots of the spectral shapes (c and d)line plots of the loadings


333 Examples of Application for MRPs Determination by3DEEM Semiquantitative Fluorescence Parameters Apartfrom temperature pH also has an impact on MRPs for-mation From previous studies pH and alkalinity were re-ported to increase the degree of polymerization therebycausing the accumulation of MRPs [40] -erefore it ismeaningful to evaluate the effect of pH onMRPs productionduring HT in FW In order to semiquantitatively determinetheir production 3DEEM-FRIPARAFAC methods wereapplied FW with initial pH from 30 to 10 was treated at130degC for 30min (Figure 7) As pH increased from 30 to 50the meanΔΦ(V) decreased from 705 times105 to 229 times105 RUmiddotnm2middotmLg and reached the minimum at pH 50 Never-theless an obvious increase of MRPs was observed froma minimum of 229 times 105 to 139 times 106 RUmiddotnm2middotmLg as thepH increased from 50 to 100 -e ΔFmax (C2) fromPARAFAC analysis which was associated with the contri-bution of MRPs had a similar tendency to ΔΦ(V) and in-creased from the minimum of 1424 RU at pH 50 to themaximum of 9004 RU at pH 10 Previous studies havestated that high initial pH (pH gt 5) could accelerate theMaillard reaction rate because Schiff-base matter formedeasily [41] Moreover low initial pH could also cause theformation of MRPs as it favors 12-enolisation reactionpathway and results in the ascent of compounds like furfuralor 5-hydroxymethyl-2-furaldehyde (HMF) [42] -ereforethe results above further verified that ΔΦ(V) and ΔFmax (C2)allow an effective evaluation for MRPs production in thehydrothermal FW system

Besides hydrothermal time and composition of FW alsoaffect the occurrence of Maillard reaction -us the effect ofthese hydrothermal conditions on MRPs production can beevaluated according to these two parameters In this way theformation of MRPs can be effectively controlled under theoptimized hydrothermal condition thus preventing thesubstrate loss and promoting limited hydrolysis efficiencysimultaneously -erefore the increase of bio-convertedenergy can be achieved by reducing substrate consumingfrom MRPs formation

4 Conclusion

Compared to traditional methods the 3DEEM analysis isproved to be a more sensitive method to estimate the oc-currence of Maillard reaction in the hydrothermal FWsystem providing valuable information when the MRPs areformed However its utility is limited to the quantifying ofthe MRPs -e FRI and PARAFAC analytic methods wereestablished to further distinguish MRPs from the otherdissolved organic compounds by integration of the volumebeneath each EEM region and capturing the heterogeneity ofsamples And ΔΦ(V) from FRI and ΔFmax (C2) fromPARAFAC can be considered liable parameters for semi-quantifying MRPs under various temperature and pH -eminimumMRPs were validated at temperature below 130degCand pH 50

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

Acknowledgments

-e authors appreciate the National Natural ScienceFoundation of China (Nos 51778580 and 51608480) Nat-ural Science Foundation of Zhejiang Province of China (NoLQ16E080001) Open Foundation of Key Laboratory forSolid Waste Management and Environment Safety (Tsing-hua University) Ministry of Education of China (SWMES2015ndash07) and China Scholarship Council (iCET 2017) forproviding the funding support for this project

Supplementary Materials

Table S1 characteristics of FW Table S2 excitation andemission (exem) wavelengths of fluorescence region and

150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

000

110 120 130

ΔΦ(v)

ΔFmax(C2)

Temperature (degC)140 150 160

180

150

120

90

60

30

0

Figure 6 Semiquantitative characterization of 3DEEM fluorescentparameters for MRPs production from FW at differenttemperatures

150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105

ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

0003 4 5 6 7 8 9 10

ΔΦ(v)

ΔFmax(C2)

pH

100

80

60

40

20

0

Figure 7 Fluorescence indicators of MRPs from FW at differenthydrothermal-treated pH value


their associated names Table S3 comparison of the fluo-rescence component peak location in the current study withthose reported in the previous literature and probable sourcedescription Table S4 Pearson correlations among spec-trometric parameters and EEM fluorescence parametersFigure S1 SCOD of hydrothermal-treated food waste atdifferent temperatures -e data were based on wet weightFigure S2 the appearance of hydrothermal-treated foodwaste (untreated 110degC 120degC 130degC 140degC 150degC and160degC treated) Figure S3 EEM spectra of the synthetic MRPsolution produced by glycine and glucose after hydrothermaltreatment -e peak location of synthetic MRP solution wasλexλem 355435 the λexλem value is higher than the otherhumic isolates tested which explains why this product wasMRPs Figure S4 Fmax values of the two components (C1 andC2) in WEOM from food waste after hydrothermal treat-ment (Supplementary Materials)

References

[1] W Wang H Hou S Hu and X Gao ldquoPerformance andstability improvements in anaerobic digestion of thermallyhydrolyzed municipal biowaste by a biofilm systemrdquo Bio-resource Technology vol 101 no 6 pp 1715ndash1721 2010

[2] Y Li Y Jin J Li H Li and Z Yu ldquoEffects of thermalpretreatment on the biomethane yield and hydrolysis rate ofkitchen wasterdquo Applied Energy vol 172 pp 47ndash58 2016

[3] B Grycova I Koutnık and A Pryszcz ldquoPyrolysis process forthe treatment of food wasterdquo Bioresource Technology vol 218pp 1203ndash1207 2016

[4] X Liu W Wang X Gao Y Zhou and R Shen ldquoEffect ofthermal pretreatment on the physical and chemical propertiesof municipal biomass wasterdquo Waste Management vol 32no 2 pp 249ndash255 2012

[5] L Ding J Cheng D Qiao et al ldquoInvestigating hydrothermalpretreatment of food waste for two-stage fermentative hy-drogen and methane co-productionrdquo Bioresource Technologyvol 241 pp 491ndash499 2017

[6] R Chandra R N Bharagava and V Rai ldquoMelanoidins asmajor colourant in sugarcane molasses based distillery ef-fluent and its degradationrdquo Bioresource Technology vol 99no 11 pp 4648ndash4660 2008

[7] GE Leiva G B Naranjo and L S Malec ldquoA study of differentindicators of Maillard reaction with whey proteins and dif-ferent carbohydrates under adverse storage conditionsrdquo FoodChemistry vol 215 p 410 2017

[8] S Pavlovic R C Santos and M B A Gloria ldquoMaillardreaction during the processing of lsquoDoce de leitersquordquo Journal ofthe Science of Food and Agriculture vol 66 no 2 pp 129ndash1322010

[9] J Dwyer D Starrenburg S Tait K Barr D J Batstone andP Lant ldquoDecreasing activated sludge thermal hydrolysistemperature reduces product colour without decreasingdegradabilityrdquoWater Research vol 42 no 18 pp 4699ndash47092008

[10] M Uchimiya J E Knoll W F Anderson and K R Harris-Shultz ldquoChemical analysis of fermentable sugars and sec-ondary products in 23 sweet sorghum cultivarsrdquo Journal ofAgricultural and Food Chemistry vol 65 no 35 pp 7629ndash7637 2017

[11] P G Coble ldquoCharacterization of marine and terrestrial DOMin seawater using excitation-emission matrix spectroscopyrdquoMarine Chemistry vol 51 no 4 pp 325ndash346 1996

[12] W Chen P Westerhoff J A Leenheer and K BookshldquoFluorescence excitation-emissionmatrix regional integrationto quantify spectra for dissolved organic matterrdquo Environ-mental Science and Technology vol 37 no 24 p 5701 2003

[13] L J Zhu Y Zhao Y N Chen et al ldquoCharacterization ofatrazine binding to dissolved organic matter of soil underdifferent types of land userdquo Ecotoxicology and EnvironmentalSafety vol 147 no 2 pp 1065ndash1072 2018

[14] L J Zhu H X Zhou X Y Xie et al ldquoEffects of floodgatesoperation on nitrogen transformation in a lake based onstructural equation modeling analysisrdquo Science of the TotalEnvironment vol 631-632 pp 1311ndash1320 2018

[15] K Wang J Yin D Shen and N Li ldquoAnaerobic digestion offood waste for volatile fatty acids (VFAs) production withdifferent types of inoculum effect of pHrdquo Bioresource Tech-nology vol 161 pp 395ndash401 2014

[16] J Yin K Wang Y Yang D Shen M Wang and H MoldquoImproving production of volatile fatty acids from food wastefermentation by hydrothermal pretreatmentrdquo BioresourceTechnology vol 171 pp 323ndash329 2014

[17] E C Bernardo R Egashira and J Kawasaki ldquoDecolorizationof molassesrsquo wastewater using activated carbon prepared fromcane bagasserdquo Carbon vol 35 no 9 pp 1217ndash1221 1996

[18] J Dwyer L Kavanagh and P Lant ldquo-e degradation ofdissolved organic nitrogen associated with melanoidin usinga UVH2O2 AOPrdquo Chemosphere vol 71 no 9 pp 1745ndash17532008

[19] R Palombo A Gertler and I Saguy ldquoA simplifiedmethod fordetermination of browning in dairy powdersrdquo Journal of FoodScience vol 49 no 6 p 1609 2010

[20] C A Stedmon and R Bro ldquoCharacterizing dissolved organicmatter fluorescence with parallel factor analysis a tutorialrdquoLimnology and Oceanography Methods vol 6 no 11pp 572ndash579 2008

[21] D Zhu B Ji H L Eum andM Zude ldquoEvaluation of the non-enzymatic browning in thermally processed apple juice byfront-face fluorescence spectroscopyrdquo Food Chemistryvol 113 no 1 pp 272ndash279 2009

[22] APHA Standard Methods for the Examination of Water andWastewater American Public Health AssociationAmericanWater Works AssociationWater Environment FederationWashington DC USA 20th edition 1998

[23] O H Lowry N J Rosebrough A L Farr et al ldquoProteinmeasurement with the Folin phenol reagentrdquo Journal of Bi-ological Chemistry vol 193 no 1 pp 265ndash275 1951

[24] D Herbert P J Philipps and R E Strange ldquoCarbohydrateanalysisrdquo in Methods in Enzymology vol 5B pp 265ndash277Elsevier Amsterdam Netherlands 1971

[25] C Bougrier J P Delgenes and H Carrere ldquoEffects of thermaltreatments on five different waste activated sludge samplessolubilisation physical properties and anaerobic digestionrdquoChemical Engineering Journal vol 139 no 2 pp 236ndash2442008

[26] A Adams K A Tehrani M Kersiene R Venskutonis andN De Kimpe ldquoCharacterization of model melanoidins by thethermal degradation profilerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4338ndash4343 2003

[27] J Dwyer P Griffiths and P Lant ldquoSimultaneous colour andDON removal from sewage treatment plant effluent alumcoagulation of melanoidinrdquo Water Research vol 43 no 2pp 553ndash561 2009

[28] S I F S Martins and M A J S van Boekel ldquoMelanoidinsextinction coefficient in the glucoseglycine Maillard re-actionrdquo Food Chemistry vol 83 no 1 pp 135ndash142 2003


[29] W P Barber ldquo-ermal hydrolysis for sewage treatmenta critical reviewrdquo Water Research vol 104 pp 53ndash71 2016

[30] J Dwyer and P Lant ldquoBiodegradability of DOC and DON forUVH2O2 pre-treated melanoidin based wastewaterrdquo Bio-chemical Engineering Journal vol 42 no 1 pp 47ndash54 2008

[31] J Li J Wei H H Ngo et al ldquoCharacterization of solublemicrobial products in a partial nitrification sequencing batchbiofilm reactor treating high ammonia nitrogen wastewaterrdquoBioresource Technology vol 249 pp 241ndash246 2017

[32] C Le and D C Stuckey ldquoImpact of feed carbohydrates andnitrogen source on the production of soluble microbialproducts (SMPs) in anaerobic digestionrdquo Water Researchvol 122 p 10 2017

[33] M Esparza-Soto S Nuntildeez-Hernandez and C Fall ldquoSpec-trometric characterization of effluent organic matter of a se-quencing batch reactor operated at three sludge retentiontimesrdquo Water Research vol 45 no 19 pp 6555ndash6563 2011

[34] J Hur and G Kim ldquoComparison of the heterogeneity withinbulk sediment humic substances from a stream and reservoirvia selected operational descriptorsrdquo Chemosphere vol 75no 4 pp 483ndash490 2009

[35] W T Li S Y Chen Z X Xu Y Li C D Shuang andA M Li ldquoCharacterization of dissolved organic matter inmunicipal wastewater using fluorescence PARAFAC analysisand chromatography multi-excitationemission scan a com-parative studyrdquo Environmental Science and Technologyvol 48 no 5 pp 2603ndash2609 2014

[36] H Wu Z Zhou Y Zhang T Chen H Wang and W LuldquoFluorescence-based rapid assessment of the biological sta-bility of landfilled municipal solid wasterdquo Bioresource Tech-nology vol 110 no 2 pp 174ndash183 2012

[37] X Jia B Xi M Li et al ldquoEvaluation of biogasification andenergy consumption from food waste using short-term hy-drothermal pretreatment coupled with different anaerobicdigestion processesrdquo Journal of Cleaner Production vol 152pp 364ndash368 2017

[38] J Sun L Guo Q Li et al ldquoStructural and functionalproperties of organic matters in extracellular polymericsubstances (EPS) and dissolved organic matters (DOM) afterheat pretreatment with waste sludgerdquo Bioresource Technologyvol 219 pp 614ndash623 2016

[39] M Zhang Z Wang P Li H Zhang and L Xie ldquoBio-refractory dissolved organic matter and colorants in cas-sava distillery wastewater characterization coagulationtreatment and mechanismsrdquo Chemosphere vol 178pp 272ndash279 2017

[40] M Coca M T Garcia G Gonzalez et al ldquoStudy of colouredcomponents formed in sugar beet processingrdquo Food Chem-istry vol 86 no 3 pp 421ndash433 2004

[41] W Jiang Y Chen X He S Hu S Li and Y Liu ldquoA study ofthe tyramineglucose Maillard reaction variables charac-terization cytotoxicity and preliminary applicationrdquo FoodChemistry vol 239 pp 377ndash384 2017

[42] C Rannou D Laroque E Renault C Prost and T SerotldquoMitigation strategies of acrylamide furans heterocyclicamines and browning during the Maillard reaction in foodsrdquoFood Research International vol 90 pp 154ndash176 2016


TribologyAdvances in

Hindawiwwwhindawicom Volume 2018


International Journal ofInternational Journal ofPhotoenergy


Journal of

Chemistry


Advances inPhysical Chemistry

Hindawiwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of


NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of

Applied ChemistryJournal of



Biochemistry Research International


Enzyme Research


Journal of

SpectroscopyAnalytical ChemistryInternational Journal of


MaterialsJournal of



BioMed Research International Electrochemistry

International Journal of


Na

nom

ate

ria

ls


Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom

the qualitative characterization of MRPs has been achievedby the traditional 3DEEM method [11] However a quan-titative determination could not be realized because only oneexcitationemission intensity value can be used for analysisRecent studies have demonstrated that fluorescence regionalintegration (FRI) method and parallel factor analysis(PARAFAC) method were developed to integrate the areabeneath EEM spectra and semiquantitatively assess thespecific components in a complex system [12ndash14] Howeverthe application of 3DEEM to the semiquantitative charac-terization of MRPs in the complicated hydrothermal FWsystem is scarce -erefore the utilization of 3DEEM todistinguish between MRPs and other dissolved organicmatter under various hydrothermal conditions is supposedto be further explored

-is study aimed at developing a method to characterizeand quantify MRPs created by HTof FW Firstly MRPs werecharacterized and evaluated with different methods -enMRPs production was further assessed by the applicability of3DEEM combined with FRI and PARAFAC hence ex-ploring the suitable fluorescence parameters for semi-quantifying the MRPs in the hydrothermal FW system

2 Materials and Methods

21 Food Waste Sample Preparations -e FW containingrice (44) noodles (16) vegetables (23) meat (6) andtofu (11) was compounded based on the characteristicssimilar to FW collected from a canteen of ZhejiangGongshang University (Hangzhou China) in our previousstudy [15] -e five components came from the same vendorat Cui Yuan farmersrsquo market (Hangzhou China) -e FWwas cut into small pieces first by hand-breaking and thencrushed using a mangler -e untreated FW sample wasstored at minus18degC before the HT -e main characteristics ofthe FW are listed in Table S1

22 Hydrothermal Treatment Hydrothermal treatment ofFW was performed in an 80mL airtight pressure digestionvessel as described by our previous study [16] at separatebatch operations at each temperature During HT about 30 gcrushed FW was placed in the vessel Each batch wasprocessed for 30min In the first experiment the temper-ature manipulations were made at 110 120 130 140 150and 160degC in an oil bath to explore the effect of temperatureon MRPs production In the second experiment the dif-ferent initial pH values (30 40 50 60 70 80 90 and100) were tested at 130degC for 30min-e time wasmeasuredfrom when the oil bath reached the set temperature -evessels were cooled to ambient temperature after HT Eachtreatment was performed in triple vessels

23 Extraction of WEOM WEOM was obtained withdeionized water (solid-to-water ratio of 1 10 wv) and themixture was shaken for 1 h in a horizontal shaker at 35 plusmn 2degC-e extracts were separated from the mixture by centrifu-gation at 10000 rpm for 5min and filtered using themicrofiltration membrane (045 microm)

24 Synthetic MRPs Solution -e synthetic MRPs solutionwas made by a concentrated solution of melanoidins whichare defined as brown substances formed during the finalstage of the Maillard reaction -e formula of the concen-trate was made with a 1 1 molar ratio of glucose and glycinewith a buffer of 05M Na2CO3 according to the previousresearch [17]-e solution was heated at 120degC for 3 hours asthe record [17] -is synthetic MRPs solution has been usedas model MRPs to analyze the properties of MRPs [9] and itwas employed to examine the availability of MRPs char-acterization method in the present study

25 Analytical Methods of MRPs

251 Spectrometric Indicators (1) UVA254 UVA wasa measure of absorbance at 254 nm measured in a 1 cm pathlength quartz cell It can measure unsaturated bonds oraromaticity within dissolved organic matters [18]-ereforethis spectrometric index was useful for this study as MRPswere linked to the presence of unsaturated double bonds andaromatic compounds and expressed as cmminus1middotmLg dryweight

(2) Color Intensity A spectrophotometer at a wavelength of475 nm was used to determine color intensity in a 1 cm pathlength cell -e absorbance at this wavelength was charac-teristic of brown color Characteristic color intensity wasrecorded in a platinum-cobalt (PtCo) unit as previouslydescribed [9]

(3) Browning Index-e Browning index of the FW solid wasmeasured by an enzymatic digestion method which releasesthe brown pigments Samples were dried for 24 h andgrounded to a smaller size before use -e proposed methodwas modified based on pronase proteolysis created byPalombo et al [19]-e procedure was as follows 03 g of thedried sample was added into a test tube which contains 5mLdeionized distilled water at 45degC and mixed thoroughly-en another 04mL of pronase solution was added into themixture After that the test tubes were placed in a waterbath incubated for 120min at 45degC and then cooled in icewater and 1mL trichloroacetic acid (80 TCA) was addedto each tube Finally centrifugation (20min at 7000 rpm)and filtration were used before the spectrometric de-termination -e optical density of the filtrates was de-termined on a spectrophotometer Samples were measuredin a 1mL cuvette with 1 cm pass length -e OD of thebrown index was calculated as OD OD420nm minus OD550nmand expressed as ODg dry weight

252 Molecular Weight Fractionation Molecular weightfractionation was applied for a better separation andcharacterization of dissolved organic matters Fractionationof samples was performed using an ultrafiltration centrifugetube with different molecular weight cutoffs 3 kDa 10 kDaand 30 kDa -e samples were filtered in series from 30 kDato 3 kDa





HIX 1113936 I(435ndash480)

1113936 I(300ndash345)

(1)






(3)


Pin 100 timesΦin

ΦTn

(4)











30

25

20

DO

C (g

L)

15

10

5




(a)

50

40

30

20

10

0

Solu

ble c

arbo

hydr

ates

(gL

)


gt30


(b)

Solu

ble p

rote

in (g

L)

5

4

3

2

1




(c)

UV

A25

4 (cm

ndash1middotm

Lg)

36

30

24

18

12

6




(d)







12

400 36 times 104

30 times 104

24 times 104

18 times 104

12 times 104

60 times 103

00

320

240

160

80

9

6

Δbro

wni

ng in

dex

(Abs

middotmL

g)

ΔUV

A25

4 (cm

ndash1middotm

Lg)

Δcol

or in

tens

ity (m

gPtC

omiddotm

Lg)

3

0


140 150 160

Δbrowning index






450

400

350

300ex (n

m)

250

200200 300 350 400

em (nm)450 500

00

040812162024283236

(a)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(b)

em (nm)

450

400

350

300ex (n

m)

250

200250 300 350 400 450 500

00

040812162024283236

(c)450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(d)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(e)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(f )450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(g)















100

80

60

40

20

0110 120 130 140


P(Vn)

P(IVn)

P(IIIn)

P(IIn)

P(In)

P (in

) (

)






450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

Compound 2450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

ExcitationEmission

05

04

03

02Load

ings

01

00


400 450 500

05

04

03

02Load

ings

01

00


(b)(a)

(d)(c)

400 450 500

ExcitationEmission

Compound 1





4 Conclusion


Data Availability




Acknowledgments




150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

000

110 120 130

ΔΦ(v)

ΔFmax(C2)


180

150

120

90

60

30

0


150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105

ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

0003 4 5 6 7 8 9 10

ΔΦ(v)

ΔFmax(C2)

pH

100

80

60

40

20

0




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls







HIX 1113936 I(435ndash480)

1113936 I(300ndash345)

(1)






(3)


Pin 100 timesΦin

ΦTn

(4)











30

25

20

DO

C (g

L)

15

10

5




(a)

50

40

30

20

10

0

Solu

ble c

arbo

hydr

ates

(gL

)


gt30


(b)

Solu

ble p

rote

in (g

L)

5

4

3

2

1




(c)

UV

A25

4 (cm

ndash1middotm

Lg)

36

30

24

18

12

6




(d)







12

400 36 times 104

30 times 104

24 times 104

18 times 104

12 times 104

60 times 103

00

320

240

160

80

9

6

Δbro

wni

ng in

dex

(Abs

middotmL

g)

ΔUV

A25

4 (cm

ndash1middotm

Lg)

Δcol

or in

tens

ity (m

gPtC

omiddotm

Lg)

3

0


140 150 160

Δbrowning index






450

400

350

300ex (n

m)

250

200200 300 350 400

em (nm)450 500

00

040812162024283236

(a)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(b)

em (nm)

450

400

350

300ex (n

m)

250

200250 300 350 400 450 500

00

040812162024283236

(c)450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(d)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(e)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(f )450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(g)















100

80

60

40

20

0110 120 130 140


P(Vn)

P(IVn)

P(IIIn)

P(IIn)

P(In)

P (in

) (

)






450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

Compound 2450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

ExcitationEmission

05

04

03

02Load

ings

01

00


400 450 500

05

04

03

02Load

ings

01

00


(b)(a)

(d)(c)

400 450 500

ExcitationEmission

Compound 1





4 Conclusion


Data Availability




Acknowledgments




150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

000

110 120 130

ΔΦ(v)

ΔFmax(C2)


180

150

120

90

60

30

0


150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105

ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

0003 4 5 6 7 8 9 10

ΔΦ(v)

ΔFmax(C2)

pH

100

80

60

40

20

0




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls







30

25

20

DO

C (g

L)

15

10

5




(a)

50

40

30

20

10

0

Solu

ble c

arbo

hydr

ates

(gL

)


gt30


(b)

Solu

ble p

rote

in (g

L)

5

4

3

2

1




(c)

UV

A25

4 (cm

ndash1middotm

Lg)

36

30

24

18

12

6




(d)







12

400 36 times 104

30 times 104

24 times 104

18 times 104

12 times 104

60 times 103

00

320

240

160

80

9

6

Δbro

wni

ng in

dex

(Abs

middotmL

g)

ΔUV

A25

4 (cm

ndash1middotm

Lg)

Δcol

or in

tens

ity (m

gPtC

omiddotm

Lg)

3

0


140 150 160

Δbrowning index






450

400

350

300ex (n

m)

250

200200 300 350 400

em (nm)450 500

00

040812162024283236

(a)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(b)

em (nm)

450

400

350

300ex (n

m)

250

200250 300 350 400 450 500

00

040812162024283236

(c)450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(d)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(e)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(f )450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(g)















100

80

60

40

20

0110 120 130 140


P(Vn)

P(IVn)

P(IIIn)

P(IIn)

P(In)

P (in

) (

)






450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

Compound 2450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

ExcitationEmission

05

04

03

02Load

ings

01

00


400 450 500

05

04

03

02Load

ings

01

00


(b)(a)

(d)(c)

400 450 500

ExcitationEmission

Compound 1





4 Conclusion


Data Availability




Acknowledgments




150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

000

110 120 130

ΔΦ(v)

ΔFmax(C2)


180

150

120

90

60

30

0


150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105

ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

0003 4 5 6 7 8 9 10

ΔΦ(v)

ΔFmax(C2)

pH

100

80

60

40

20

0




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








12

400 36 times 104

30 times 104

24 times 104

18 times 104

12 times 104

60 times 103

00

320

240

160

80

9

6

Δbro

wni

ng in

dex

(Abs

middotmL

g)

ΔUV

A25

4 (cm

ndash1middotm

Lg)

Δcol

or in

tens

ity (m

gPtC

omiddotm

Lg)

3

0


140 150 160

Δbrowning index






450

400

350

300ex (n

m)

250

200200 300 350 400

em (nm)450 500

00

040812162024283236

(a)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(b)

em (nm)

450

400

350

300ex (n

m)

250

200250 300 350 400 450 500

00

040812162024283236

(c)450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(d)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(e)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(f )450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(g)















100

80

60

40

20

0110 120 130 140


P(Vn)

P(IVn)

P(IIIn)

P(IIn)

P(In)

P (in

) (

)






450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

Compound 2450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

ExcitationEmission

05

04

03

02Load

ings

01

00


400 450 500

05

04

03

02Load

ings

01

00


(b)(a)

(d)(c)

400 450 500

ExcitationEmission

Compound 1





4 Conclusion


Data Availability




Acknowledgments




150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

000

110 120 130

ΔΦ(v)

ΔFmax(C2)


180

150

120

90

60

30

0


150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105

ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

0003 4 5 6 7 8 9 10

ΔΦ(v)

ΔFmax(C2)

pH

100

80

60

40

20

0




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






450

400

350

300ex (n

m)

250

200200 300 350 400

em (nm)450 500

00

040812162024283236

(a)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(b)

em (nm)

450

400

350

300ex (n

m)

250

200250 300 350 400 450 500

00

040812162024283236

(c)450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(d)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(e)

450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(f )450

400

350

300ex (n

m)

250

200250 300 350

em (nm)400 450 500

00

040812162024283236

(g)















100

80

60

40

20

0110 120 130 140


P(Vn)

P(IVn)

P(IIIn)

P(IIn)

P(In)

P (in

) (

)






450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

Compound 2450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

ExcitationEmission

05

04

03

02Load

ings

01

00


400 450 500

05

04

03

02Load

ings

01

00


(b)(a)

(d)(c)

400 450 500

ExcitationEmission

Compound 1





4 Conclusion


Data Availability




Acknowledgments




150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

000

110 120 130

ΔΦ(v)

ΔFmax(C2)


180

150

120

90

60

30

0


150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105

ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

0003 4 5 6 7 8 9 10

ΔΦ(v)

ΔFmax(C2)

pH

100

80

60

40

20

0




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls












100

80

60

40

20

0110 120 130 140


P(Vn)

P(IVn)

P(IIIn)

P(IIn)

P(In)

P (in

) (

)






450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

Compound 2450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

ExcitationEmission

05

04

03

02Load

ings

01

00


400 450 500

05

04

03

02Load

ings

01

00


(b)(a)

(d)(c)

400 450 500

ExcitationEmission

Compound 1





4 Conclusion


Data Availability




Acknowledgments




150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

000

110 120 130

ΔΦ(v)

ΔFmax(C2)


180

150

120

90

60

30

0


150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105

ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

0003 4 5 6 7 8 9 10

ΔΦ(v)

ΔFmax(C2)

pH

100

80

60

40

20

0




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls







450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

Compound 2450

400

350

300ex (n

m)

250

200250 300 350 400

em (nm)450 500

ExcitationEmission

05

04

03

02Load

ings

01

00


400 450 500

05

04

03

02Load

ings

01

00


(b)(a)

(d)(c)

400 450 500

ExcitationEmission

Compound 1





4 Conclusion


Data Availability




Acknowledgments




150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

000

110 120 130

ΔΦ(v)

ΔFmax(C2)


180

150

120

90

60

30

0


150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105

ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

0003 4 5 6 7 8 9 10

ΔΦ(v)

ΔFmax(C2)

pH

100

80

60

40

20

0




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






4 Conclusion


Data Availability




Acknowledgments




150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

000

110 120 130

ΔΦ(v)

ΔFmax(C2)


180

150

120

90

60

30

0


150 times 106

125 times 106

100 times 106

750 times 105

500 times 105

250 times 105

ΔΦ(v

) (R

Umiddotn

m2 middotm

Lg)

ΔFm

ax(C

2) (R

Umiddotm

Lg)

0003 4 5 6 7 8 9 10

ΔΦ(v)

ΔFmax(C2)

pH

100

80

60

40

20

0




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





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









Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




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Three-DimensionalExcitationandEmissionFluorescence-Based ...downloads.hindawi.com/journals/jchem/2018/6758794.pdf · ResearchArticle Three-DimensionalExcitationandEmissionFluorescence-Based