Effects of lipase, lipoxygenase, peroxidase and free fatty acids on volatile compound found in boiled buckwheat noodles

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Research ArticleReceived: 13 December 2009 Revised: 12 February 2010 Accepted: 16 February 2010 Published online in Wiley Interscience: 23 March 2010

(www.interscience.wiley.com) DOI 10.1002/jsfa.3958

Effects of lipase, lipoxygenase, peroxidaseand free fatty acids on volatile compoundfound in boiled buckwheat noodlesTatsuro Suzuki,a∗ Sun-Ju Kim,b Yuji Mukasa,a Toshikazu Morishita,a

Takahiro Noda,a Shigenobu Takigawa,a Naoto Hashimoto,a

Hiroaki Yamauchia and Chie Matsuura-Endoa

Abstract

BACKGROUND: Relationships between buckwheat (Fagopyrum esculentum Moench) flour lipase, lipoxygenase and peroxidaseactivity, along with levels of individual free fatty acids (FFAs) and levels of headspace volatile compounds of boiled buckwheatnoodles, were investigated for 12 different buckwheat varieties. Enzyme activities and FFA levels in flour were correlatedwith their respective varietal arrays of boiled noodle headspace volatile compounds, measured by gas chromatography–massspectrometry.

RESULTS: The volatiles hexanal, tentative butanal, tentative 3-methylbutanal and tentative 2-methylbutanal showed significantpositive correlation with one another, indicating that they may be generated through similar mechanisms. These importantvolatile components of buckwheat flavor were also positively correlated with lipase and/or peroxidase activity, indicatingthat enzymatic reactions are important in flavor generation in boiled buckwheat noodles. On the other hand, pentanal, whichshowed no significant correlation with any enzyme activity, showed a significant positive correlation to the levels of C18 : 2and C18 : 3 FFAs, suggesting the existence of a ‘non-enzymatic’ and/or ‘uncertain enzymatic pathway’ for flavor generation inboiled buckwheat noodles.

CONCLUSION: Lipase and peroxidase in buckwheat flour are important for flavor generation of boiled buckwheat noodles. Thisinformation is important for increasing desirable flavor of buckwheat products as well as for selecting varieties with improvedflavor.c© 2010 Society of Chemical Industry

Keywords: buckwheat; lipase; peroxidase; flavor; fatty acid

INTRODUCTIONBuckwheat (Fagopyrum esculentum Moench) is an important cropin Japan as well as China, Korea and in some other countries.1,2 Itsunique flavor is one of the most important quality characteristics ofboiled buckwheat noodles (soba), which is important in traditionalfood items in Japan.1 Flavor components in buckwheat flour3 – 7

and dough8 include a number of volatile compounds, of whichthe most important contributors include carbonyl compoundssuch as aldehydes and ketones.3,4,6,7 Amongst these, hexanalis also known as a flavor compound in soybean (Glycine max(L.) Merr.) products.9 – 11 However, no studies have focused on themechanisms of flavor generation in buckwheat.

In soybean products, hexanal is generated through thelipoxygenase pathway, which was first proposed in rice bran12

(Fig. 1). Lipase (triacylglycerol lipase EC 3.1.1.3) (LIP) catalyzes thefirst step of lipid catabolism. Lipoxygenase (EC 1.13.11.12) (LOX)is thought to have a significant effect on flavor generation insoybean13,14 and rice (Oryza sativa L.).15 Peroxidase (EC 1.11.1.7)(POX) also plays a role in flavor-related quality in the soybean.16

A number of studies have investigated the activities of LIP,17,18

LOX19 and POX20,21 in buckwheat flour, and their effects on lipid

deterioration (hydrolysis and oxidation) during its storage havealso been investigated.22 However, no studies have focused onthe possible relation between these enzymes, their substrates, andflavor generation.

Based on previous studies, we hypothesized that LIP, LOX andPOX activities in flour influenced the volatile compounds foundin boiled buckwheat noodles. In a first step towards investigat-ing the effects of LIP, LOX and POX activities and free fattyacid (FFA) levels in buckwheat flour on volatile compounds gen-erated by boiled buckwheat noodles, we (i) quantified enzymeactivities/levels and FFA concentrations in the flour of 12 buck-

∗ Correspondence to: Tatsuro Suzuki, National Agricultural Research Center forHokkaido Region, Shinsei, Memuro, Kasai-gun, Hokkaido 082-0081, Japan.E-mail: [email protected]

a National Agricultural Research Center for Hokkaido Region, Shinsei, Memuro,Kasai-gun, Hokkaido, 082-0081 Japan

b Division of Bioresources Engineering, College of Agriculture and Life Sciences,Chungnam National University, Daejeon, 305-764 Republic of Korea

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Enzymatic effect on volatile compound in buckwheat noodle www.soci.org

(Triacylglycerol, Phospholipid)

(poly unsaturated fatty acids containing a1,4-pentadiene structure such as linoleic acid)

Conjugated hydroperoxy fatty acids

(e.g., aldehyde, ketone)

LIP

LOX

Enzymatic (e.g., POX) ornon-enzymatic reaction

Lipids

Free-fatty acids

Carbonyl compounds

Figure 1. Model of lipid degradation proposed for soybean and rice bran.LIP, lipase; LOX, lipoxygenase; POX, peroxidase.

wheat varieties/breeding lines, (ii) identified and quantified, forall 12 buckwheat varieties/breeding lines, the volatile compoundsproduced by boiled buckwheat noodles, using headspace gaschromatography–mass spectrometry (GC-MS), and (iii) analyzedcorrelations between activities and levels determined in (i) and (ii).These results are presented and discussed in the context of possi-ble mechanisms of volatile compound generation during noodlepreparation and retention in boiled buckwheat noodles.

EXPERIMENTALPlant materialsBuckwheat varieties were grown in an experimental field at theNational Agricultural Research Center for the Hokkaido region inMemuro, Hokkaido, Japan (longitude 143◦ 03′ E, latitude 42◦ 53′

N). Buckwheat seeds were sown in early June and harvested in late

August. Harvested seeds were dried, threshed and stored at 4 ◦Cuntil used for experiments. Fourteen of 46 buckwheat varietieswere selected after screening to obtain a wide range of variationin LIP activity, POX activity, LOX protein concentration and rutinconcentration.

Measurement of volatile compounds in the headspaceof boiled buckwheat noodlesThe boiled buckwheat noodles from which headspace volatileswere to be sampled were made by milling seeds, making dough,making noodles, and boiling them. To achieve a sufficientlyhigh accuracy and reproducibility, time- and equipment-specificexperimental procedures were devised. The use of a specialmilling machine, capable of operating at 4 ◦C, minimized enzymeinactivation and fatty acid oxidation. Dehulled buckwheat seedswere ground for 4 min at 4 ◦C using a low-temperature millingmachine (model 5080RE, Takachiho-seiki Co., Ltd, Saitama, Japan).Two minutes after milling ended, 4.7 mL deionized water wasadded to 10 g buckwheat flour, and the mixture was homogenizedfor 4 min in a mixer operating at 140 rpm. Raw noodles weremade using a noodle-making machine with a 1.2 mm aperture(Takachiho-seiki Co., Ltd). Newly made noodles were immediatelyboiled in 5 L boiling water for 25 s, and then washed with ice-colddeionized water for 20 s.

Immediately after boiling and washing, 1.5 g boiled noodleswas weighed and placed in a headspace vial, to which 2 ngd8-toluene was added as an internal standard. The vial wasthen sealed. It took only 17 min from the start of millingto the sealing of the samples in the headspace vial. Volatilecompounds in boiled buckwheat noodles were analyzed usinga gas chromatograph (TurboMatrix 40 Trap Headspace GCSeries) with a mass spectrometric detector (MSD) PerkinElmerClarus500 Mass Spectrometer (PerkinElmer Co., Ltd, Waltham,MA, USA). Column: VOCOL, 60 m × 0.25 mm i.d., film thickness1.5 µm (Supelco, Bellefonte, PA, USA). Temperature program:starting temperature 40 ◦C (5 min), heating rate 5 ◦C min−1, finaltemperature 240 ◦C (12 min). Tinj 250 ◦C; Tdet 280 ◦C; injectionvolume 1 µL. Carrier gas: He, flow rate 1 mL min−1. MS conditions:

Table 1. Lipase (LIP) and peroxidase (POX) activities, ratio (across varieties/lines) of lipoxygenase 1 and 2 (LOX1 and LOX2), individual and total FFAcontent and rutin content in flours of 12 buckwheat cultivars/lines

LIP POX LOX1 LOX2 C16 : 0 C18 : 0 C18 : 1 C18 : 2 C18 : 3 Total FAb Rutin

� A405 min−1 g−1 d.w. � A492 min−1 g−1 d.w. Ratioa g kg−1 d.w.

Kitawasesoba 1.33 2.99 46.9 38.5 6.64 0.124 6.02 4.02 0.69 17.5 0.0981

Kitayuki 1.31 2.96 73.2 49.8 8.42 0.177 4.31 5.04 0.98 18.9 0.0845

Hokkai 6 1.23 2.00 62.0 53.1 9.10 0.426 4.12 5.61 1.33 20.6 0.0805

Kantoh 1 1.51 6.08 63.8 52.6 10.2 0.074 5.07 5.30 1.06 21.7 0.0643

Shirataki-nishi 1.42 3.54 57.3 56.8 8.05 0.080 4.45 5.09 1.02 18.7 0.0788

Sumchanka 1.48 3.32 53.1 56.3 6.83 0.056 3.42 4.45 1.06 15.8 0.0640

cv. Monteneuf 1.72 3.63 58.7 48.4 7.67 0.503 5.37 5.54 1.21 20.3 0.0856

HC10 1.67 6.86 80.8 63.4 8.42 0.345 3.63 5.14 1.26 18.8 0.0661

Tanno-hiushinai 1.44 3.03 72.8 54.9 7.41 0.092 3.26 4.60 1.04 16.4 0.0558

mh-1 1.49 1.03 84.0 51.6 6.97 0.062 2.96 3.91 0.99 14.9 0.0951

Gan-chao 1.89 4.01 85.0 40.4 10.4 0.125 4.29 4.62 1.02 20.4 0.0143

w/sk86GF 1.67 1.63 100 46.0 11.2 0.085 3.40 4.44 1.01 20.1 0.0445

a w/sk86GF is defined as 100.b C16 : 0 + C18 : 0 + C18 : 1 + C18 : 2 + C18 : 3.Data are means of two independent experiments.

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electron impact mode, total ion current (TIC) recorded. The peaksinvestigated in this study were tentatively identified by comparingthe mass spectra of the compounds to spectra from the NIST02spectral library. In addition, hexanal and pentanal were identifiedby retention time and mass spectra as well as by comparing themto commercially available standards. Relative content of eachvolatile compound was quantified using a standard curve derivedfrom the internal standard (d8-toluene).

Free fatty acid and rutin determinationFFAs were extracted according to the method of Folch et al.23

One gram of buckwheat flour was mixed with 10 mL chloro-form–methanol (2 : 1, v/v) (CM solution) and maintained at 37 ◦Cfor 60 min. A crude extract was obtained by centrifugation, andthe extract was washed three times with deionized distilledwater. The chloroform layer was concentrated with a vacuumevaporator. FFAs were dissolved in 1 mL methanol, and then an-alyzed using a YMC FFA analysis kit (YMC Co., Ltd, Kyoto, Japan).Briefly, FFAs were separated and detected using high-performanceliquid chromatography (HPLC) following their labeling with2-nitrophenylhydrazide. To extract rutin from buckwheat flour,100 mg flour was homogenized in 1 mL methanol–0.1% (v/v)phosphoric acid (9 : 1) and extracted at 80 ◦C for 3 h. The cen-trifuged supernatant was passed through a filter and analyzed byHPLC according to the method of Suzuki et al.24

Assay of in vitro LIP and POX activityBuckwheat flour (100 mg) was mixed with 1 mL of 50 mmol L−1

acetate–LiOH buffer (pH 5.0) for 20 min on ice. A crudeprotein extract was obtained by centrifugation at 4 ◦C and usedimmediately. LIP activity was determined spectrophotometricallyby the modified procedure of Suzuki18 using p-nitrophenyllaurate (pNPC12) as a substrate. The assay mixture contained0.5 mmol L−1 pNPC12 in a 200 mmol L−1 acetate–LiOH buffer (pH6.0) containing 0.3% Triton X-100, 4% (v/v) acetone and 50 µLcrude protein extract in a total volume of 0.3 mL. Assay wasinitiated by addition of the enzyme. Activities were determinedat 22 ◦C by measuring the initial rate of increase in absorbance at405 nm. POX activity was determined spectrophotometrically bythe modified procedure of Suzuki21 using guaiacol as a substrate.POX activities were determined at 22 ◦C by measuring the initialrate of increase in absorbance at 492 nm. The assay mixturecontained 0.05 mmol L−1 substrate, 200 mmol L−1 acetate–LiOHbuffer (pH 5.0) and 50 µL crude protein extract in a total volumeof 0.3 mL. Assay was initiated by addition of the enzyme.

LOX protein concentrationIt is difficult to assay in vitro LOX activity in buckwheat seeds.19

Thus we performed immunoblotting analysis with a LOX-specificantibody to determine the LOX protein concentration.

Preparation of anti-LOX antibodyA polyclonal antibody raised against soybean LOX was preparedusing the purified soybean LOX (a mixture of three LOXisozymes; LOX1, LOX2 and LOX3). Soybean LOX was purifiedto homogeneity using (NH4)2SO4 precipitation, ion-exchangechromatography, gel-filtration chromatography and preparativenative polyacrylamide gel electrophoresis (PAGE). Two rat pupswere used as the immune animals. After preparation of immunesera, anti-LOX-IgG was affinity purified with the antigen.

Electrophoresis and electroblotting

Sodium dodecyl sulfate–PAGE was carried out using 6% polyacry-lamide gels.25 Electroblotting was carried out using a tank blottingapparatus (Bio-Rad, Hercules, CA, USA) with a minor modification(electroblot for 14 h at 30 mV at 4 ◦C.

Immunoblotting analysis of LOX proteinImmunoblotting was performed with the affinity-purified LOX-specific IgG. The immunoreacted protein was incubated with agoat anti-rat IgG horseradish peroxidase conjugate (GE HealthcareUK Ltd, Little Chalfont, UK) and visualized with an ECL+Plusdetection kit (Amersham Pharmacia Biotech) on X-ray film(X-OMAT, Kodak). The intensity of LOX1 (higher molecularweight) and LOX2 (lower molecular weight) were quantitateddensitometrically. We have confirmed that this antibody ismonospecific to LOX protein.19

StatisticsFor individual cultivars/lines the two measurements of LIP, POX,LOX1 and LOX2, total FFAs and rutin did not differ from the meanby more than 6.65%, 9.50%, 14.1%, 10.9% and 14.7%, respectively.Similarly, for the volatiles detected in the headspace of boiledbuckwheat noodles, the two measurements of unidentified 1,unidentified 2, unidentified 3, tentative butanal, unidentified 4,tentative 3-methylbutanal, tentative 2-methylbutanal, pentanal,hexanal, and tentative heptanal did not differ from the mean bymore than 13.0%, 38.3%, 15.1%, 19.7%, 19.1%, 13.6%, 31.6%, 20.3%,23.1%, and 22.6%, respectively. We deemed such differences notto pose any problem in terms of correlation analysis betweenthese parameters. The correlation matrix (Pearson’s correlationcoefficient) was calculated using Microsoft Excel (Microsoft OfficeExcel 2003 SP3).

RESULTS AND DISCUSSIONLIP, POX activity, LOX1, and LOX2 protein concentrations, FFAand rutin concentration in buckwheat flourTable 1 shows mean (n = 2) LIP and POX activities, LOX1 andLOX2 concentrations, as well as FFA and rutin concentrationsin the buckwheat flours analyzed. The LIP activity ranged from1.89 to 1.23 �A405 min−1 g−1 (d.w.), and POX activity from 6.86 to1.03 �A492 min−1 g−1 (d.w.). Relative LOX protein concentrationsranged from 100% to 38.5% across cultivars/lines. In terms ofFFAs, C16 : 0 FFAs ranged from 11.2 to 6.64 g kg−1 (d.w.), C18 : 0from 0.503 to 0.056 g kg−1 (d.w.), C18 : 1 from 2.96 to 6.02 g kg−1

(d.w.), C18 : 2 from 5.61 to 3.91 g kg−1 (d.w.), C18 : 3 from 1.33to 0.69 g kg−1 (d.w.), total FFAs from 21.7 to 14.9 g kg−1 (d.w.),and rutin concentrations from 0.0981 to 0.0143 g kg−1 (d.w.).The major FFAs of buckwheat seeds have been shown to beC16 : 0, C18 : 1 and C18 : 2,26,27 consistent with those FFAs foundto predominate in this study (Table 1). The ranges of relative LIPactivity, POX activity, and of LOX and rutin concentrations, acrosscultivars/lines tested, ran from 100% to 61%, 15%, 38.5% and14.6%, respectively, across the different cultivars/lines, comparedto 100% to 43%,23 38%,23 14%,28 and 35%28 in previous studiesof buckwheat flour. Differences between ranges in these reportsand those reported in this paper may be attributable to varietaldifferences and environmental effects.

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0 10 20 30 40 50

inte

nsity

retention time (min)

blank (water + d8 toluene)

boiled noodle

A

B

C

D

E F

G

H

K

I JI(internalstandard)

Figure 2. Gas chromatogram of boiled buckwheat noodle headspace volatiles. A, unidentified 1; B, unidentified 2; C, unidentified 3; D, tentative butanal;E, unidentified 4; F, tentative 3-methylbutanal; G, tentative 2-methylbutanal; H, pentanal; I, d8-toluene; J, hexanal; K, tentative heptanal.

Table 2. Amounts of major volatile compounds in the headspace of boiled buckwheat noodles made from the flour of 14 buckwheat cultivars/lines,expressed as a ratio to the cultivar/line in which it is most abundant

Unidentified1

Unidentified2

Unidentified3

Tentativebutanal

Unidentified4

Tentative3-methylbutanal

Tentative2-methylbutanal Pentanal Hexanal

Tentativeheptanal

Kitawasesoba 90.9 58.0 31.4 40.8 64.7 49.6 41.6 22.6 20.6 100

Kitayuki 51.5 46.6 44.8 31.1 16.5 40.3 27.4 52.3 30.2 32.2

Hokkai 6 50.3 37.8 49.8 39.9 28.4 39.1 28.1 69.4 53.8 45.6

Kantoh 1 100 100 100 53.0 85.7 69.9 58.4 59.0 60.2 29.6

Shirataki-nishi 68.8 74.4 61.5 47.1 34.9 50.6 32.4 49.3 35.1 36.5

Sumchanka 69.6 73.9 73.6 70.1 30.7 56.2 49.2 54.5 31.8 22.0

cv. Monteneuf 89.9 47.2 78.3 100 100 100 100 100 100 46.1

HC10 68.9 81.2 91.7 77.4 77.3 72.2 72.1 67.5 85.4 41.9

Tannno-hiushinai 43.1 26.7 48.7 42.8 21.3 40.9 37.6 38.8 38.2 25.9

mh-1 82.6 83.2 80.0 42.3 40.9 47.1 25.3 52.2 53.4 26.9

Gan-chao 60.6 81.5 70.0 70.5 40.3 77.5 66.3 51.6 56.3 32.8

w/sk86GF 61.9 65.4 69.9 41.1 42.9 48.6 36.0 50.6 42.0 32.1

Data are means of two independent experiments.

Volatile compounds found in buckwheat noodlesIn a typical GC-MS chromatogram of boiled buckwheat noodlehead space (Fig. 2), similar peaks were present to those re-ported in studies of buckwheat flour and dough.3 – 8 Since thepurpose of this study was to investigate effects of enzymeson the volatile profile of buckwheat noodles, it was neithernecessary to identify every volatile compound, nor to employevery volatile compound in the correlation analysis. Therefore,we selected and tentatively identified only those volatile com-pounds important as flavor components in buckwheat aroma.These included hexanal, pentanal, butanal, 2-methylbutanal and3-methylbutanal.3 – 8 Along with these, the peak intensity of fourfurther unidentified volatile compounds (labeled 1–4) were alsoused in the correlation analysis. Table 2 shows the mean (n = 2)relative quantities of volatile compounds in boiled buckwheatnoodle headspace. The ratio of the volatile compounds acrosscultivars/lines ranged from 100% to 43.1% (unidentified 1), 26.7%(unidentified 2), 31.4% (unidentified 3), 31.1% (tentative butanal),16.5% (unidentified 4), 39.1% (tentative 3-methylbutanal), 25.3%(tentative 2-methylbutanal), 22.6% (pentanal), 20.6% (hexanal)and 22.0% (tentative heptanal).

Among the volatile compounds tested, hexanal, pentanal, ten-tative 2-methylbutanal, tentative 3-methylbutanal, unidentified 4,

tentative butanal, and unidentified 3 tended to have a signifi-cant positive correlation with each other (Table 3). Among them,butanal-related compounds (e.g., tentative butanal, tentative3-methylbutanal and tentative 2-methylbutanal) showed a strongand significant positive correlation with each other (R ≥ 0.920).Therefore, some volatile compounds such as butanal-related com-pounds can be grouped together and should be regulated bysimilar mechanisms. On the other hand, although unidentified 1and unidentified 2 were significantly correlated with each other,they were weakly correlated to other volatile compounds (Ta-ble 3). Tentative heptanal did not show a significant correlationwith any other volatile compound (Table 3). These data indi-cate that the volatile compounds present in the headspace ofboiled buckwheat noodles are likely regulated by several differentmechanisms.

Relationships between volatile compounds in the headspaceof boiled buckwheat noodles, and LIP and POX activity,LOX1 and LOX2 proteins, FFAs, and rutin concentrationsin buckwheat flourThe activity of LIP in flour showed significant positive correlationswith unidentified 3, tentative butanal, tentative 3-methylbutanal,tentative 2-methylbutanal and hexanal (Table 4). The activity of


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Table 3. Pearson’s correlations coefficients and the significance of correlations between volatile compounds in the headspace of noodles madefrom flours of 14 cultivars/lines of buckwheat

Unidentified1

Unidentified2

Unidentified3

Tentativebutanal

Unidentified4



Tentativeheptanal

Unidentified 1 –

Unidentified 2 0.585∗ –

Unidentified 3 0.455 0.722∗∗ –

Tentative butanal 0.348 0.203 0.553∗ –

Unidentified 4 0.799∗∗ 0.366 0.556∗ 0.655∗ –


0.529 0.336 0.599∗ 0.920∗∗ 0.802∗∗ –


0.429 0.175 0.512 0.939∗∗ 0.791∗∗ 0.967∗∗ –

Pentanal 0.156 −0.027 0.530 0.694∗∗ 0.491 0.658∗ 0.645∗ –

Hexanal 0.292 0.149 0.663∗∗ 0.771∗∗ 0.709∗∗ 0.793∗∗ 0.789∗∗ 0.860∗∗ –

Tentative heptanal 0.360 −0.180 −0.512 −0.069 0.338 0.015 0.071 −0.292 −0.150 –

Significant at: ∗ 5% level; ∗∗ 1% level.

Table 4. Pearson’s correlations coefficients and the significance of correlations between levels of buckwheat noodle headspace volatiles levels andenzyme activities/content, individual and total FFA levels and rutin content of the flour from which

Unidentified1

Unidentified2

Unidentified3

Tentativebutanal

Unidentified4



Tentativeheptanal

LIP 0.167 0.419 0.585∗ 0.683∗∗ 0.423 0.747∗∗ 0.690∗∗ 0.339 0.568∗ −0.256

POX 0.294 0.441 0.529 0.497 0.563∗ 0.551∗ 0.602∗ 0.222 0.444 −0.005

LOX1 −0.349 0.167 0.302 −0.125 −0.178 −0.055 −0.105 0.012 0.177 −0.471

LOX2 −0.192 0.086 0.423 0.137 −0.047 −0.080 −0.019 0.305 0.260 −0.505

C16 : 0 −0.147 0.272 0.321 −0.063 0.056 0.138 0.072 0.148 0.176 −0.291

C18 : 0 0.564∗ −0.010 −0.221 0.176 0.556∗ 0.389 0.381 0.050 0.065 0.728∗∗

C18 : 1 −0.260 −0.114 0.452 0.435 0.146 0.290 0.337 0.800∗∗ 0.696∗∗ −0.452

C18 : 2 −0.080 −0.173 0.228 0.331 0.290 0.355 0.391 0.720∗∗ 0.550∗ −0.157

C18 : 3 −0.018 −0.399 0.042 0.504 0.426 0.445 0.527 0.774∗∗ 0.715∗∗ 0.226

Total FAa 0.101 0.104 0.227 0.190 0.402 0.416 0.385 0.433 0.402 0.054

Rutin 0.363 −0.213 −0.259 −0.207 0.150 −0.227 −0.224 0.053 −0.066 0.436

Significant at: ∗ 5% level; ∗∗ 1% level.a C16 : 0 + C18 : 0 + C18 : 1 + C18 : 2 + C18 : 3.

POX showed a significant positive correlation to unidentified4, tentative 3-methylbutanal and tentative 2-methylbutanal,indicating that LIP and POX were likely important componentsin the enzymatic generation of volatile compounds. On the otherhand, neither LOX1 nor LOX2 showed a significant correlationto any volatile compound. In soybean, among the enzymes ofthe lipoxygenase pathway (Fig. 1), the enzymic action of LOX iskey in generating hexanal, which is the major source of ‘beany’flavor.13,14 In addition, LOX is a key enzyme in the generationof unfavorable volatile compounds during storage in rice.15

Therefore, in buckwheat, the key enzyme which generates volatilecompounds such as hexanal may be different from those insoybean and rice.

Levels of C18 : 1, C18 : 2 and C18 : 3 FFAs in flour showedsignificant correlations with the volatile compounds pentanal andhexanal (Table 4). These FFA are the product of LIP activity, andthe substrate of POX or other enzymatic and/or non-enzymaticreactions, which result in the generation of volatile compounds.12

When water was added to the flour during the dough-makingprocess, the lipoxygenase pathway, which generates volatilecompounds from triacylglycerol, and is catalyzed by enzymes

such as LIP and POX in each step, was likely activated. Incontrast to enzyme-catalyzed generation of volatile compounds(such as tentative 3-methylbutanal, tentative 2-methylbutanaland hexanal), correlations presented in Table 4 suggest that somevolatile compound generation occurred without the action ofLIP, LOX or POX. This was particularly the case for unidentified1, unidentified 2 and tentative heptanal, whose levels showeda weak correlation to LIP and POX activity as well as the majorFFA (C16 : 0, C18 : 1 and C18 : 2) levels, suggesting the existenceof other enzymatic or non-enzymatic pathways. Further studiesare required to address this issue. From our results, it is clearthat enzymatic reactions catalyzed by LIP and POX are importantin generating volatile compounds detected in the headspaceof boiled buckwheat noodles. Some volatile compounds suchas hexanal, 3-methylbutanal and 2-methylbutanal are importantcontributors to the unique flavor of buckwheat.3 – 8 Therefore,LIP and POX should also be important factors in generatingthe organoleptic qualities of boiled buckwheat noodles. Toclarify the role of these enzymes on flavor, further analysis offlavor compounds using sensory analysis (organoleptic evaluation)would be required.

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These results are also useful in the breeding of buckwheatvarieties with particularly desirable flavor traits. To enhance flavorin buckwheat varieties it may be possible to breed for increasedLIP and POX activity in the seed. However, LIP and POX are alsoimportant in quality deterioration of buckwheat flour; flours ofhigh LIP and POX varieties tend to deteriorate quickly, particularlyin terms of triacylyglycerol hydrolysis and fatty acid oxidation.Therefore, in developing a flavorful variety, one may have toalso consider flour deterioration. On the other hand, the factthat rutin levels showed no significant correlation with levelsof any of the volatiles measured (Table 4), but that high rutinlevels have been linked to reduced flour deterioration,22 indicatesthat there exists the possibility of developing a variety whoseflavor is enhanced, but whose flour does not deteriorate easily.Elsewhere, finding that rutin can inhibit LIP activity in vitro,22 wehypothesized that rutin could prevent LIP activity in flour duringstorage. The lack of correlation between rutin concentration andlevels of volatile compounds suggests that LIP activity involvedin flour deterioration during storage and that involved in flavorgeneration may be regulated by different mechanisms. When LIPand rutin are in contact in flour during storage, the rutin should beable to prevent lipid hydrolysis of lipids; however, some volatilecompounds or their immediate precursors may be generatedthrough the action of lipase and stored during seed maturation,when compartmentalization of LIP and rutin differs. Therefore, indeveloping varieties that are high in both rutin and LIP activity,one may be able to develop varieties whose flavor is enhanced,but whose flour does not deteriorate easily. Further studies arerequired to clarify the mechanisms as to when and where volatilecompounds are generated in buckwheat seeds and flour.

ACKNOWLEDGEMENTSWe thank Mr K. Abe, Mr T. Takakura, Mr T. Hirao, Ms K. Fujii, Ms M.Hayashida and Ms T. Ando for their technical assistance. We alsothank Dr A. Horigane and Ms S. Yamada for their useful suggestion.This study was partly supported by the NIAS Genebank Project ofthe National Institute of Agrobiological Sciences and the AkiyamaFoundation.

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Effects of lipase, lipoxygenase, peroxidase and free fatty acids on volatile compound found in boiled buckwheat noodles