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Optimized Likens-Nickerson Methodology for Quantifying Honey Flavors

Amina Bouseta, and Sonia CollinJ. Agric. Food Chem., 1995, 43 (7), 1890-1897 DOI: 10.1021/jf00055a025 Publication Date (Web): 01 May 2002

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1890 J. Agric. Food Chem. 1995, 43 18904897

Optimized Likens-Nickerson Methodology for Quantifying HoneyFlavorsAmina Bouseta an d Sonia Collin*9?

Unit6 de Brasserie et des Industries Alimentaires, Universit6 Catholique de Louvain,Place Croix du Sud 2Bte 7,B-1348Louvain-la-Neuve, Belgium

Dichloromethaneextraction under an iner t atmosphere followed by simultaneous steam distillation-dichloromethane extraction appears to be a useful method for honey flavor quantification. Theorganoleptic features of extracts obtained in this way closely match those of the honey samples.Recovery factors obtained for a large number of chemicals highlight the critical impact of parameterssuch as oxygen level, extraction time, and cold finger temperature. While recovery is excellent foraround 70 tested chemicals when these optimized conditions are used, recovery factors must betaken into account for accurate quantification of hydrophilic compounds.

Keywords: Honey; flavor; terpenes; extraction; st eam distillationINTRODUCTION m

To isolate volatile components from a complex matrixsuch as honey and to obtain very representative extractsremain major challenges to flavor chemists. Over thepast 30 years, most studies in this field have beenrestricted to qualitative determinations. In the specificfield of honey, accurate quantification now appears tobe essential to evaluating flavor changes linked to newprocessing methods or long storage (microbiological orchemical degradation). Such knowledge would furtherbe helpful in ascertaining a honey's floral origin withoutthe ambiguity inherent in organoleptic tests. In thiscontext, high concentrations of hydroxy ketones havealready been reported as characteristic of Eucalyptusspp. and Banksia spp. honeys (Graddon et al., 1979).Citrus honeys (e.g. orange and lemon) are known tocontain methyl anthranilate, a compound that otherhoneys seem to contain at concentrations of less t han0.5 ppm (Serra, 1988;White, 1975). A recent study(Bouseta et al., 1992) imed at identifying the headspacecomposition of 84unifloral honeys also revealed a rangeof compounds characteristic of the floral source (alde-hydes in lavender honey; acetone in fir honey; diketones,sulfur compounds, and alkanes in eucalyptus honey).Fur ther studies on less volatile flavor compounds areneeded, however, to differentiate other k inds of honeys.Scant quantitative data have been published in thisarea, probably due to the lack of accurate extractionmethods. In 1973,Tschogowadse et al. attempted to

isolate terpenoids in honey by steam distillation. Oneyear later, Tsuneya et al. (1974)solated 8-p-menthene-172-diol rom an ether extract from 116 kg of lindenTilia spp.) honey. Simple solvent extraction followedby concentration either under nitrogen or in a rotaryevaporator was used by many workers in t he followingyears (Berahia et al., 1993;Bonaga et al., 1986;Graddonet al., 1979; teeg and Montag, 1987,1988;Wootton etal., 1978). More recently, Tan et al. (1988, 989a,b,*Author to whom correspondence should be ad-t S.C. is Chercheur Qualifi6 from the Fonds Nationaldressed.de la Recherche Scientifique.

0021 8561/95/1~3-1890 09.00/0

a bFigure 1. (a)Pre-extraction apparatus; (b)microextractor forsimultaneous steam distillation-solvent extraction.1990) roposed continuous liquid/liquid extraction withdiethyl ether for the extraction of polar phenolic andacidic substances. This method was applied by Wilkinset al. (1993)o determine linalool derivatives and otherheavy components in New Zealand honeys. Ferber andNursten (1977) valuated numerous flavor extractionprotocols before selecting vacuum distillation at 65 Cas th e method of choice. Bicchi et al. (1983)were thefirst to emphasize the importance of pre-extractingflavor compounds from sugars prior to heating. Theyproposed a two-step protocol including preliminaryacetone extraction followed by simultaneous Likens-Nickerson steam distillation and solvent extraction(Likens and Nickerson, 1964;Nickerson and Likens,1966).In the present work, we have optimized this two-stepmethod. Very good recovery factors are measured formost chemicals when very strict conditions are main-tained. his makes it possible to plan a real quantifica-tion. Possibilities and limitations of the method are

0 995 American Chemical Society


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oneyFlavors J. Agric. Food Chem. Vol. 43, No. 7, 1995 1891


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1892 J. Agric Food Chem. Vol. 43, No. 7, 1995 Bouseta and Collin

- 80z6 0 -

mz2 4 0 -8E

20 -- enzaldehyde- amphene0- fl-oirlene- rans-caryophyllene

0

- erpineolornv, acetate0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0

s leam dis t i l la l ion t ime (miniFigure 2. Mean recovery factors (percent) from triplicatesobtained by increasing steam distillation time (step 2).

100

t erbenone---- - t e r m e o l0 1 boinyl acetate

2 0 . l o 0 1 0 2 0cold finger temperature ('C)

Figure 3. Mean recovery factors (percent) from triplicatesobtained by decreasing cold finger temperature (step 2).

described. This technique has yielded organolepticallyhighly representative extracts for 220 unifloral honeys(data t o be published).MATERIALS AND METHODS

Honey Sample. A commercial Canadian honey was usedfor the extraction optimization. Pollen analyses revealed thepresence of 97 clover pollen grains.Honey Flavor Extraction. Solvent extraction (step 1 wasfirst performed t o remove the flavor compounds from the sugarmatrix, which could induce artifacts by nonenzymatic brown-ing reactions. After the vessel was purged with high-puritynitrogen, 100 g of honey and 200 mL of bidistilled dichlo-romethane were poured into th e extraction app aratus shownin Figure la . The mixture was stirred for 60 min at 140 rpmunder a 2 mu mi n nitrogen stream to avoid oxidation reactions.

= I ' I


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oneyFlavors J. Agflc. Food Chem. Vol. 43, No. 7,1995 1893

16

Figure 4. Chromatograms of honey extract with pentane (a), acetone (b), and dichloromethane (c).


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1894 J. Agric. Food Chem. Vol. 43, No. 7, 1995 Bouseta and CollinTable 4. Honey Sample Extraction with Three Different Solvents (Step 1)

pentane acetone dichloromethanecompound PN RT (min) identified by av (ppb) CV (%) av (ppb) C V ( I av (ppb) C V (90

methylfurancaproaldehydeoctanefurfuralfurfuryl alcohol1-hexanolm-xyleneacetylfuran5-methylfuraldehydebenzaldehydea p nen ephenolP-pinenebenzyl alcoholphenylacetaldehydephenethyl alcoholcamphorcoumarintrans-caryophyllene

1 5.62 8.93 9.54 9.75 10.16 11.47 12.28 13.29 16.010 16.311 16.912 17.413 19.614 21.615 22.216 29.317 33.418 66.519 68.7

G CGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSCG-MSGC-MS

362528125358983827786526211

351999182222181824182 1321913114218181019

3852416563701812247157254151196471316785325683

891922441928841218108167326

20215204675118143522230551221109709183944513

671185871051082071312910510

Peak numbering (PN) gives the order of elution through the column; RT, column retention time (min); GC, gas chromatographicretention data compared with those of authentic samples; MS, mass spectral data compared with those of library compounds and/or thoseof authentic samples. Average concentrations (ppb, calculated with a 100% recovery factor) and coefficients of variation (CV, standarddeviation x 100/mean, ) obtained for three (in acetone and pentane) and five (dichloromethane) analyses of the same sample.The dichloromethane extract was concentrated t o 1 mL in aKuderna-Danish flask maintained in a 45 C water bath.

Steam distillation-solvent extraction (step 2) was carriedout in a microextractor (Alltech 8910, Figure lb ) t o removeflavor compounds from the coextracted heavy matrix; thisyielded an extract suitable for on-column chromatographicinjection. The previously obtained 1 mL extract was trans-ferred t o flask A (see Figure l b ) with five 200 pL aliquots ofdichloromethane used for washing the vessel and 30 mL ofultrapure (Milli-Q water purification system, Millipore, Bed-ford, MA) deoxygenated water. Dichloromethane and ultra-pure, deoxygenated water ( 1 . 5 mL each) were introduced intoare a C by arm H. A few clean grains of carborundum weresuccessively introduced into flasks A and B. Prior to theprocedure, the entire system was purged with nitrogen (2-3mum in) for 5 min. Flask A was then heated in a 140 C oilbath. After 3 min, flask B was heated in a 90 C water bat h.The vapors were condensed in a rea C by means of a cold fingermaintained at -10 C by a cryostat. The entire steamdistillation-solvent extraction procedure was carried outunder a 2 mu mi n nitrogen flow. The steam distillation wasstopped after 45 min, and 2 mL of the dichloromethane extractwas removed from flask B. The dichloromethane layer in areaC was then collected in flask B by introducing 3 x 1 mL ofdichloromethane through arm H; flask B was finally washedwith 3 x 0.5 mL of dichloromethane. Fifty microliters of 1000ppm chloroheptane was added t o the combined extracts as anexternal standard. The extract was then concentrated to0.25-0.5 mL in a Snyder Kude rna and a micro-Dufton column.One microliter was analyzed by GC and GC-MS.

Gas Chromatography Analytical Conditions. For gaschromatography, we used a Hewlett-Packard Model 5890 gaschromatograph equipped with a Hewlett-Packard Model 7673automatic sample r, a cold on-column injector, a flame ioniza-tion detector, and a Shimadzu CR4A integrator. Analysis ofthe honey volatile compounds was carried out on a 50 m x0.32 mm, wall-coated, open tubular (WCOT) CP-SIL5 CBcapillary column (film thickness, 1.2 ,um). The oven temper-atur e was programmed t o rise from 30 t o 85 C a t 55 Wmin,then t o 145 C a t 1 C/min, and t o 250 C at 3 C/min. Thecarrier gas was helium at a flow rate of 1.5 mumin. Theinjector temperature was maintained at 3 C above the oventemperature. The detector temperature was 275 C. Theminimum peak are a for da ta acquisition was set a t 500 pV-s.

Gas Chromatography Mass Spectrometry AnalyticalConditions. The column (see above) was directly connectedto an HP 5988 quadrupole mass spectrometer. Electron

impact mass spectra were recorded a t 70 eV. Spectral record-ing throughout elution was automatically performed with theHP59970C software. Peaks were identified by their enhance-ment after coinjection of authentic standard compounds andwith the help of the NBS/EPA/NIH mass spectra library.RESULTS AND DISCUSSION

Simultaneous Steam Distillation-Solvent Ex-traction (Step 2) Optimization. To obtain an ac-curate method for quantifying honey flavors, we opti-mized step 2 with respect t o the distillation time, thecold finger temperature, and the oxygen level. This wasdone on a test mixture composed of the followingsuspected honey constituents: mono- and sesquiter-penes (camphene, P-pinene, and trans-caryophyllene),terpenic alcohols (terpineol), ketones (verbenone),esters(bornyl acetate), and aromatics (benzaldehyde). Recov-ery factors were checked with a 30mL standard mixtureof the above-listed compounds, diluted to concentrationsclose t o 1ppm in ultrapure, deoxygenated water. ThepH of 5.7 at the beginning of the extraction was equalt o the pH of a real honey extract [pH value obtainedafter extraction (step 1 of honey; see below].We first determined the kinetic parameters of thesteam distillation-solvent extraction step using all ofthe experimental conditions described under Materialsand Methods apart from the nitrogen flow. Results ontriplicates a re listed in Table 1. Figure 2 clearly shows

that the recovery fac to r reaches a maximum after 30min. After 45 min and probably due t o losses, theextraction efficiency slightly decreases for the mostvolatile compounds (benzaldehyde and monoterpenes;see Figure 2). With a 45 min extraction time, all of therecovery factors exceed 77 ,with 97-99 for terpinol,verbenone, bornyl acetate, and trans-caryophyllene.Next, we determined the optimal temperature of thecold finger. The data reported in Table 2 and Figure 3emphasize how critical this parameter is. A tempera-ture above -5 C significantly decreases the extractionefficiency. The three most volatile chemicals, benzal-dehyde, camphene, and P-pinene, even require a tem-perature of -10 C. In the case of monoterpenes, lessefficient condensations (boiling points under 165 C) and


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Honey Flavorsoxidation reactions are assumed to occur when th e coldfinger temperature exceeds 0 C, leading to very lowrecovery (33-42 ).As suggested above, monoterpenes must be protectedfrom oxidation. To determine the real impact of oxygen,steam distillation-solvent extraction was performedwith and without a stream of nitrogen gas. Table 3shows the favorable effect of a 2 mu mi n nitrogen flow.However, as the nitrogen flow rate is increased, therecoveries of benzaldehyde, camphene, and /3-pinenedecrease from 98, 97, and 97% (at 2 mumin) , respec-tively, to 78, 18, and 18 at 60 mumin) , respectively.As expected, higher nitrogen flow rates reduce theefficiency with which the more volatile compounds arecondensed in the cold finger. Recovery ratios exceeding97% were reproducibly obtained with a 2 mUminnitrogen flow for all standard compounds. As will bedemonstrated in the last section, however, this methodis not recommended for temperature-sensitive moleculessuch as C4-Cb lactones.

In o u r preliminary tes ts, the nature of the vessel usedt o evaporate the solvent proved also t o be of primeimportance. Rotary evaporators and nitrogen purges,both frequently used for honey extracts, lead t o the loss(up t o 90%) of many volatiles. The system used here,i.e. the Kuderna-Danish flask (for concentration to 1mL and micro-Dufton column (for concentration t o 0.25mL), avoids the loss of any compound.

Solvent Extraction (Step 1 Optimization. Thefavorable effect of a nitrogen flow was also proven atthis first step. In triplicate measurements, peak inten-sities varied significantly according t o the size of theoxidized fraction unless nitrogen was used. Therefore,all extractions described below were carried out underan inert atmosphere.Three different extracting solvents, pentane, acetone,and dichloromethane, were investigated on a Canadian

honey. All of the experimental parameters were asdescribed under Materials and Methods, including thesteam distillation-solvent extraction step (step 2). Lowyields of volatile extracts were obtained with pentane(Figure 4a), but qualitatively matching chromatograms(see Figure 4b,c) were obtained with the two othersolvents. After acetone extraction, however, the extractswere richer in furan derivatives (Table 4, P N 1 ,4 ,5 ,8 ,and 91, suggesting that nonenzymatic browning reac-tions may occur more easily. This observation can berelated t o the higher solubility of fructose and glucosein acetone than in dichloromethane. Moreover, concen-tration to 1 mL is time-consumingwhen acetone is used.The small amounts of furan derivatives detected in thedichloromethane extract were not due to a n artifact butcame from the honey sample itself, as demonstrated byanother method dedicated t o the analysis of morevolatile compounds (Bouseta et al., 1992).

We further determined on the same honey sample theoptimal dichloromethane extraction time in triplicates.The kinetic curves obtained differed considerably fromone compound t o another (see Figure 5). For allcompounds, the amount extracted increased, as ex-pected, with time up t o about 60 min, due to slowsolubilization of the flavor compounds. After this time,step 1 efficiency decreased for monoterpenes (depictedfor B-pinene in Figure 5). A similar effect was observeda bit la ter for benzyl alcohol. On the other hand, nosignificant loss was noticed until 120min for compounds

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J. Agric. Food Chem. Vol. 43, No. 7,1995 1895

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Figure 5. Honey volatile concentrations calculated with a100% recovery factor for different extracting times in step 1(experiences in triplicates).


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1896 J. Agric. food Chem. Vol. 43, No. 7, 1995 Bouseta and CollinTable 5. Coefficients of Variation (CV, Standard Deviation x 1001Mean, Percent) and Recovery Factors R, ean x100/Expected Concentration, Percent) Obtained for Five Analyses (Steps 1 and 2) of the Same Test Mixture(Concentrations around 100 ppb)

comnound RT(min) C V ( % ) R(%) comDound RT(min) C V ( % ) R(%)hydrocarbonsoctanem-xyleneo-xylenenonanea-pinene/3-citronellene

camphenesabinene/3-pinene2-carenea-phellandrene3-carenep-cymenelimoneney -terpinene1,2,3,4-tetramethylbenzenetrans-caryophyllenea-humulenehexadecane3-methyl-3-buten-1-013-methyl-2-buten-1-014-hydroxy-4-methyl-2-pentanonefurfuryl alcoholn-hexanolcyclohexanolphenol2-octanolbenzyl alcohol1,8-cineole1-phenylethyl alcoholp-cresolguaiacolphenethyl alcohollinaloolcamphor4-ethylphenolborneolmentholterpinene-4-01a-terpineol4-allylanisole3-phenylpropan-1-01/3-citronelloltrans-anetholethymolcinnamyl alcoholcarvacroleugenol

alcoholsiphenolslethers

9.612.213.414.116.616.717.519.019.521.321.322.022.623.426.035.368.771.681.27.38.210.2

10.611.512.417.519.721.723.424.325.326.829.229.233.635.036.837.638.139.439.742.443.350.250.751.051.958.7

118898359111055889710974328

298325526714637251014129101082038815510

such a s benzaldehyde, phenylethyl alcohol, or phenyl-acetaldehyde. A 60 min solvent extraction time wasselected.

Reproducibility of StandardMixture Extraction(Steps 1 and 2). The reproducibility of the optimizedmethod (see above), calculated for five consecutiveanalyses of a standard mixture, is given in Table 5. Formost low-polarity compounds (hydrocarbons, aldehydes,ketones, acyclic esters, dimethyl disulfide, terpenicalcohols, etc.), variation coefficients below 12% andrecovery factors above 70% (above 90% for 35 chemicals)are obtained. Poor recovery factors are calculated,however, for hydrophilic alcohols (low volatility) suchas 4-hydroxy-4-methyl-2-pentanone,urfuryl alcohol,phenol, benzyl alcohol, phenethyl alcohol, 4-ethylphenol,3-phenylpropan-1-01, and cinnamyl alcohol. In suchcases, recovery factors must be taken into account foraccurate quantification. As shown in Table 5 , themethod is not recommended for C4-C5 lactones.

949097838910189918792949979999210210398949597342893103431043810072749860119936210210296

105100531081011052810385

aldehydesketones3,4-hexanedionecaproaldehyde2-furaldehydetrans-2-hexenal2-heptanonehep a n a1trans-2-heptenal5-methylfurfuralbenzaldehyde2-octanoneoctanalsalicylaldehydetrans-2-octenal2-nonanoneI-fenchonethujoneo-methylacetophenonepropiophenonementhone2-decanoneverbenonetrans,trans-2,4-nonadienalp-anisaldehydecarvonepulegoneperillaldehyde2-undecanonetrans,trans-2,4-decadienal-butyrolactone-valerolactoneisoamyl butyratephenylethyl acetatelinalyl acetatebornyl acetatesulfur compounddimethyl disulfidefurans2-methylfuran2-acetylfuran

nitrogen compoundsindolemethyl anthranilate

esters

8.88.99.810.712.413.116.016.116.418.719.722.624.527.728.129.831.834.735.038.940.440.844.044.444.548.351.453.512.114.524.045.347.351.37.85.513.2

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LITERATURE CITEDBerahia, T.; Cerrati, C.; Sabatier, S.; Amiot, M. J. Gaschromatography-mass spectrometry analysis of flavonoidsin honey. Sci. Aliments 1993,13, 15-24.Bicchi, C.; Belliardo, F.; Frattini, C. Identification of thevolatile components of some piedmontese honeys. J Apic.Res. 1983,22, 130-136.Bonaga, G.; Giumanini, A. G. The volatile fraction of ches tnu thoney. J Apic. Res. 1986,25, 113-120.Bonaga, G.; Giumanini, A. G.; Gliozzi, G. Chemical compositionof ches tnu t honey: analysis of the hydrocarbon fraction. JAgric. Food Chem. 1986,34,319-326.Bouseta, A.; Collin, S.; Dufour, J. P. Characteristic aromaprofiles of unifloral honeys obtained with a dynamic head-space GC-MS system. J.Apic. Res. 1992, 31 96-109.Ferber, C. E. M.; Nursten, H. E. The aroma of beeswax. J

Sci Food Agric. 1977, 28, 511-518.Graddon, A. D.; Morrisson, J. D.; Smith, J. F. Volatileconstituents of some unifloral australian honeys. J Agric.Food Chem. 1979,27, 832-837.


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Honey FlavorsLikens, S. T.; Nickerson, G. B. Determination of certain hopoil constituents in brewing products. ASBC Proc. 1964,5-13.Nickerson, G. B.; Likens, S.T. Gas chromatographic evidencefor the occurrence of hop oil components in beer. J Chro-matogr. 1966,21, 1-5.Serra, J. Determination of methyl anthran ilat e in citrus honey(citrus sp.) of easter n sp ain and its influence on t he diastaseactivity of the honey, Alimentaria 1988, 197, 37-40.Steeg, E.; Montag, A. Proof of aromatic carboxylic acids inhoney. 2 ebensm. Unters. Forsch. 1987, 184, 17-19.Steeg, E.; Montag, A. Quantitative determination of aromaticcarboxylic acids in honey. Z. Lebensm. Unters. Forsch. 1988,Tan, S.-T., Holland, P. T.; Wilkins, A. L.; Molan, P. C.Extractives from New Zealand honeys. 1. White clover,manuka, and kanuk a unifloral honeys. J Agric. Food Chem.Tan, S. T.; Wilkins, A. L.; Molan, P. C.; Holland, P. T.; Reid,M. A chemical approach to the determination of floralsources of New Zealand honeys. J Apic. Res. 19898, 28,212-222.Tan, S.-T.; Wilkins, A. L.; Holland, P. T.; McGhie, T. K.Extractives from New Zealand honeys. 2. Degraded caro-tenoids a nd o ther substances from heather honey. J Agric.Food Chem. 1989b, 37,1217-1221.Tan, S. T.; Wilkins, A. L.; Holland, P. T.; McGhie, T. K.

Extractives from New Zealand honeys. 3. Unifloral thymeand willow honey constituent s. J Agric. Food Chem. 1990,38, 1833-1838.

187, 115-120.

1988,36,453-460.

J. Agric. foo d Chem. Vol. 43, No. 7,1995 1897Tschogowadse, S. K.; Koblianidse, G. L.; Dembizkij, A. D.Aromatic components of honeys. Lebensmitte1indu.stri.e1973,Tsuneya, T.; Shibai, T.; Yoshioka, A.; Shiga, M. The study ofshina (linden, Tilia japonica Simk.) honey flavor. KoryoWhite, J. Composition of honey. In Honey, ComprehensiveSurvey; Crane, Ed.; Heinemann: London, 1975;pp 157-206.Wilkins, A. L.; Lu, Y.; Tan, S.T. Extractives from New Zealandhoneys. 4. Linalool derivatives an d oth er components fromnodding thistle (Carduus nutans) honey. J Agric. FoodChem. l993,41,873-878.Wootton, M.; Edwards, R. A.; Faraji-Haremi, R. Effect ofaccelerated storage conditions on the chemical compositionand properties of Australian honeys. 3. Changes in volatilecomponents. J Apic. Res. 1978, 17, 167-172.

20, 225-228.

1974,109, 29-35.

Received for review December 12, 1994. Accepted April 17,1995.@JF9407026

@ Abstract published inAdvance ACS Abstracts, June1,1995.

JAgricFoodChem.43.(1995).1890-1897_Miel

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