Zeitschrift fLIr

LebensmittelUntersuchung und-Forschung

Z. Lebensm. Unters.-Forsch. 164, 171--176 (1977)

@ J. F. Bergmann-Verlag 1977

Enzymatic Oxydation of Linoleic Acid: Formation of Bittertasting Fatty Acids Christiane Baur, Werner Grosch, Herbert Wieser, and Harald Jugel* Deutsche Forschungsanstalt ftir Lebensmittelchemie, D-8046 Garching, Federal Republic of Germany

Enzymatische Oxydation yon LinoMiure: Bildung yon Fettsiiuren mit Bittergeschmack Zusammenfassung. Linols~iure wurde mit einer Proteinfraktion aus Sojabohnen oxydiert (25°C; 2 Std), in der Lipoxygenase- und Peroxydaseaktivit~iten vorkamen. Die gebildeten Fetts~iuren wurden isoliert und nach Emulgierung mit einem Zuckerester auf Bittergeschmack verkostet. Hauptkomponente der bitter schmeckenden Fraktion war ein Gemisch aus der 9,12,13-Trihydroxyoctadec-10-und der 9,10,13-Trihydroxyoctadec-ll-ensiiure. Die Geschmacksschwelle liegt im Bereich 0,6~3,9 gmol/ml. Zwei weitere Trihydroxys~iuren und zwei Ketodihydroxys~iuren wurden aul3erdem in der bitter schmeckenden Fraktion identifiziert. Summary. Linoleic acid was oxidized with a protein fraction from soya beans (25°C; 2 h), in which lipoxygenase and peroxydase activities occurred. The fatty acids formed were isolated and, after emulsification with a sugar ester, were evaluated for bitter taste. The main components of the bitter-tasting fraction was a mixture of 9.12.13-trihydroxyoctadec-10- and 9.10.13trihydroxyoctadec-ll-enoic acids.The taste threshold lies in the range 0.6-0.9 ~tmol/ml. Two further trihydroxy-acids and two oxodihydroxy-acids were also identified in the bitter-tasting fraction.

Introduction When ground, oat grains or soya beans can only be kept for a limited time since they become bitter. Enzymatic-oxidative changes of the lipid is one reason for the decrease in quality F1, 2]. For oats, the involvement of the enzymes lipase [3], lipoxygenase [1, 4] and peroxidase [5] were discussed. Unsaturated fatty

acids set free iipolytically are thought to be peroxidized either enzymatically or, with the participation of haem catalysts, converted into bitter substances. Two compounds having bitter tastes were isolated from autoxidized oat oil, of which one could be characterised as a dihydroxy-acidwith a tetrahydrofuran ring [4]. Also, it was shown that such compounds arise in the performic acid oxidation of Iinoleic acid [4]. In stored soya flour the bitter taste was localized to one iysolecithin and two lecithin fractions, which together represented at least 0.08% of the defatted flour [6, 7]. Further experiments suggested that it was not the phosphocholine residue that gave the bitter taste but the oxidized fatty acids [6]. There are also indications that for soya beans a relationship exists between the lipoxygenase activity and the occurence of the bitter taste [8]. Until now only the taste of products (mainly hydroperoxides) have been tested, which can form the alkaline iipoxygenase L 1. A grassy, bean-like flavor was found [9]. However the soya beans also contain neutral lipoxygenases that can oxidize not only free linoleic and linolenic acids, but also those bound in triglycerides and probably also in phospholipids [10, 11]. These neutral lipoxygenases, as shown by experiments with a corresponding enzyme from peas, form a wide range of oxidized fatty acids through co-oxidation of the substrate [12]. This work deals with the enzymatic oxidation of linoleic acid by use of a protein fraction of soya beans that contains lipoxygenase and peroxidase activity, and also with the first attempts at identification of the bitter-tasting fatty acids thus arising.

Experimental Section Material Soya beans were donated by Harburger ()lwerke, Brinckman & Mergell. Linoleic acid was obtained from Nu Chek Prep. Sucrose palmitate stearate [i5] was purchased from Serva. Tween 80 was from Schuchardt, and Sephadex G-50 from Pharmacia. Silica gel HF2s 4 and silica gel ,,Fertigplatten" were from Merck. 9.10-dihydroxystearic acid and 9.10.12.13-tetrahydroxystearic acid were prepared from oleic acid and linoleic acid respectively I-15].

Methods Enzyme Assays. The lipoxygenase acticity was measured spectro-

* We are grateful to the Deutsche Forschungsgemeinschaft for supporting this work

photometrically [13]. Peroxydase was detected according to Bergmeyer [27].

172

Protein Determination. Protein concentrations were calculated from absorbance values obtained at 280 nm by use of the relationship that 0.7 mg/ml protein gives an absorbance of A21~ = 1.0 [16]. Thin-Layer Chromatography (TLC). The fatty acids and the methyl esters were chromatographed on silica gel plates (0.5 ram). The chromatograms of the acids were twice developed for a distance of 12 cm with CHC1jmethanol/acetic acid (65 + 2.5 + 0.5). The solvent system for the methyl esters was iso-octane/ethylacetate/H20 (1 + 1 +2). The compounds were visualized in UV radiation or by spraying a small strip on each side of the plate with 5% phosphomolybdic acid in 96% ethanol and by heating the strips with a hairdryer. Derivatives of the Fatty Acids. Diazomethane was used to esterify the carboxyl group [19]. Hydroxy groups were silylated [20]. Double-bonds were hydrogenated with palladium on Norite as catalyst. O-methyloxime derivatives of carbonyl groups were prepared according to [25]. The methyl esters of fatty acids were reduced with LiA1H4 or LiA1D 4 [26]. Oxidative ozonolysis of double-bonds as described [22]. Periodate Oxidation. The fatty acid methyl ester (ca. 1 rag) in a little methanol was dissolved in 2.5 ml of a citrate-phosphate buffer pH 2.2, which was made up from 2.1% citric acid and 0.07% NazHPO 4 [17]. After the addition of 0.25 ml of 20 mM-KJO4, the mixture was stored under N 2 at room temperature for 20 min. The pH was then raised to 8 with N-NaOH, the solution was extracted with 2 x 10 ml of pentane, and the pentane phase was dried over Na2SO,. The analysis on the cleavage products followed by gaschromatography (GLC) and mass spectrometry (MS). Varian Aerograph Series 2700 gas chromatograph; conditions: 1.5m x 1/8 inch column of 3% of Silicone OV-275 on Chromosorb G-AW-DMCS (100-120 mesh), the temperature was programmed from 60 to 200°C at 4°C/min. Varian CH 7 mass spectrometer temperature of the ion source 200°C, energy 70 eV, Biemann-Watson separator. The periodate oxidation was also carried out as described by Graveland [18], and the resulting carbonyl compounds were determined as their 2.4-dinitrophenylhydrazones. 7~tration of the Fatty Acids. The fatty acid dissolved in methanol was titrated with methanolic 5 mM-KOH against bromthymol blue as indicator. The titrimetric factor of the methanolic KOH was determined with heptadecanoic acid. Spectrophotometric and Densitometric Measurements.The TLC plate was sprayed with a 5% solution of phosphomolybdic acid in 96% ethanol and heated for 15 min at. 120°C. The intensity of the coloured zones was measured with the Chromatogram Spektrophotometer (C.Zeiss) at 650 nm. IR spectra were obtained with a Beckman IR Acculab-3 from a film of the fatty acid methyl esters on a NaC1 crystal. MS was used in tandem with GLC. Conditions: 1 m x 1.2 mm column of 3% of silicone JXR on Gas-chrom Q (100120 mesh), the temperature was programmed from 170 to 2300C at 2°C/rain. Mass spectrometer operated as above. Taste Analysis. The fatty acid and the emulsifier (10% by weight relative to the fatty acid) were dissolved in a few ml of methanol. After removal of the solvent in vacuo, the residue was suspended in tap-water adjusted to pH 8 with NaOH) and emulsified for 30 s in a homogeniser (Ultra-Turrax). The taste of 1 ml of the solution was evaluated as described in 1-21]. The control samples contained tap water at pH 8 and the emulsifier. All samples were coded.

Incubation Experiment Crude Lipoxygenase PreparaHon: Ground soya beans (100 g) (defated with light petroleum) were extracted with 11 of 10 raM-sodium phosphate buffer of pH 6.8. The supernatant liquid was treated with

Chr. Baur et al. : Enzymatic Oxydation of Linoleic Acid (NH4)2SO,, and the fraction precipitating between 30 and 60% saturation was dissolved in 150 ml of 10 mM-sodium phosphate buffer of pH 6.8. The crude enzyme preparation was freed from (NH~)2SO 4 by gel filtration on a Sephadex G-50 column (70 cm x 7 cm) equilibrated with the same buffer.

Substrate: 480 mg of linoleic acid and 320 pg of Tween 80 were dissolved in 200 ml of 20 mM-sodium phosphate buffer of pH 6.8 as described in [14]. Incubation: 200 ml of substrate were mixed with the lipoxygenase preparation (7000 units, 3.5 g of protein) dissolved in 200 ml of 10 raM-sodium phosphate buffer ofpH 6.8. For two hours the mixture was continuously stirred under air at 22°C in an open vessel. Isolation of the Products : After incubation, the mixture was acidified with dilute HC1 to pH 4.0 and extracted with 3 x 800 ml of ethyl ether. The combined ether phase was washed with water and dried over NazSO4. The solution of the fatty acids was concentrated and the fatty acids were separated by TLC. The compounds were located on the plates, scraped off and extracted with methanol

Results Taste Testing For taste testing, the oxidation products separated by TLC were emuslified with sucrose palmitate stearate. This emulsifier ist tasteless, and in the concentration range 0.001~3.1% does not influence the threshold value of the standard bitter substance caffeine (1-1.5 ~tmol/ml). Preliminary investigations showed that 2 mg/ml of 9.10.12.13-tetrahydroxystearic acid but not 4mg/ml of 9.10-dihydroxystearic acid tasted bitter. The taste analysis of the oxidized fatty acids separ a t e d b y T L C (Fig. 1) s h o w e d a b i t t e r t a s t e f o r t h e fractions 1 and 3--5, with the threshold concentrations lying in the region of 0.02~3.05%.

Chemical Structure and Taste o f Fraction i T h e m a i n c o m p o n e n t of t h e b i t t e r - t a s t i n g s u b s t a n c e s ( F r a c t i o n 1) w a s c o n c e n t r a t e d . O n e h a l f o f t h e m a t e r i a l w a s set a s i d e f o r d e t e r m i n a t i o n of t h e t a s t e t h r e s h old, t h e o t h e r f o r s t r u c t u r a l d e t e r m i n a t i o n . Fraction 1 consisted of a mixture of 9.12.13-trihydroxy-octadec-trans-10-enoic acid and 9.10.13-trihydroxy-octadec-trans-ll-enoic acid. T h e I R s p e c t r u m of t h e m e t h y l a t e d F r a c t i o n 1 s h o w e d t h e p r e s e n c e o f a m e t h y l e s t e r (1740 a n d 1175 c m - 1 ) , a t r a n s d o u b l e b o n d (980 c m - 1) a n d a h y d r o x y - g r o u p (3400 c m - 1). The mass spectrum of the compounds (methylated and s i l y l a t e d ) s h o w e d s i g n i f i c a n t i o n s a t m / e 545 ( M - C H 3 ) 4 6 0 ( M - h e x a n a l ) , 387, 3 0 1 , 2 9 7 , 259, 211, 173, a n d 155. The data are in accordance with those obtained by G r a v e l a n d [-18]. A f t e r h y d r o g e n a t i o n t h e m / e o f t h e i o n s s h i f t e d t o 547 ( M - C H 3 ) , 4 6 2 ( M - h e x a n a l ) , 399, a n d 303, b u t t h e m / e 2 5 9 a n d 173 i o n s r e m a i n e d u n c h a n g e d . T h e d i f f e r e n c e s in t h e s p e c t r a b e f o r e a n d after hydrogenation showed a double-bond between C-9 a n d C-13. T h e m e t h y l a t e d a n d h y d r o g e n a t e d F r a c t i o n 1 w a s o x i d i s e d w i t h p e r i o d i c acid. A f t e r G L C s e p a r a t i o n , t h e P r o d u c t s B, C, a n d D o f F i g u r e 2 w e r e i d e n t i -

Chr. Baur et al. : EnzymaticOxydation of LinoleicAcid Fraction w i t h i

173

bitter t ~ s t e

199~ - T - ~ 3 6 1 2~--0 ~271 I

i

CH3-(CH2)z-CH= C H - C H -' CH Ti C H - (CH2) 7- COOCH 3 f , I OTMS OIMS E OTMS i

L _ ~259

5

271

i

i

1o

8

2

0

200

DistQnce (cm)

r

300

470

4()0

m/e

Fig. 1. Densitogram of the isolated acids after TLC on "Fertigplatten" (Merck). Solvent system: CHC13/methanol/acetic acid (65 + 2.5 + 0.5 v/v/v). Spray. 5% phosphomolybdic acid in 96% ethanol

560

I

I

si~0

Fig. 3. Mass spectrum of Fraction 3 after methylation and silylation 173 ~ - T - ~ 387 i

CH3-[CH214-CH 4- CH + ICH - CH = CH - (CH2)?- COOCH 3

OrMS OTMSOTMS

CH 3- (CH2)4- CH - CH - C H 2 - C H 2 - C H -(CH2)7-COOCH ? I

I

I

OH

OH

OH

I

185 ~ -_90 _ 275 ~ - J- - ~ 285

I KIO~ CH 3- (CH2lz,- CHO

OC H - CH2-CH2-CH-iCH2)?- CO0C H 3 OH

@

®

CH3- (CH2)4- C H - CH2-CH2- CH - C H - (CH2)7- COOCH 3 I

I

I

OH

OH

OH

275

100 I (%)

185

1i 3

KIO CH3-(CH2)CICH- CH2-CH2-CHO OH

358

2,,5

x 10 FI I 445 3871399 429 464

2i9 OCH- (CH2)7-COOCH3

©

i

i

200

300

ill , I l147o,s29, s~sP6° 400 500

m/e

Fig. 2. Periodate oxidation of methylated and hydrogenated Fraction l. -- B= tl-formyl-9-hydroxy-undecanoate (MS: m/e 226 195--194--166--150--101--81--74). -- C=4-hydroxy-nonanal (MS: m/e 141--140--101--84 83 81). -- D = 8-formyl-octanoate(MS:m/e 158--155 143 111--87--83 74)

Fig. 4. Mass spectrum of Fraction 4 after methylation and silylation

fled by MS analysis. Product A in Figure 2, hexanal, was characterised as its 2.4-dinitrophenylhydrazone. After a titrimetric concentration determination, mixtures of the 9.12.13- and 9.10.13-trihydroxy acids were emulsified with sucrose palmitate stearate. A bitter taste was found with a threshold concentration in the range of 0.6-4).9 gmol/ml. Changing the concentration of the emulsifier did not influence the taste.

CH3(CH2)4CH(OTMS)CHO. The m/e 387i 285, 275, 185, and 173 peaks can be derived from the formulae in Figure 4. After hydrogenation, the m/e of the ions shifted to 562 (M), 547 (M-CH3), 531 (M-OCH3), 389, and 287 (Fig. 5). The differences in the spectra before and after hydrogenation showed a double-bond in a position lower than C-11. The oxidative ozonolysis of the acetylated Fraction 4 and G L C analysis of the methyl and dimethyl esters of the mono- and dicarboxylic acids gave azelaic acid and an unidentified peak. Therefore, the double-bond is located, between C-9 and C-10 (Fig. 4). The methylated and hydrogenated Fraction 4 was oxidized with periodic acid. Cleavage products were identified as hexanal (as, the 2.4-dinitrophenylhydrazone) and ll-oxoundecanoic acid (Fig. 6). The result of the periodic oxidation experiment agrees with the structure of fraction 4 given in Figure 4.

Mass-spectral analysis of the Fractions 3--5 The mass spectrum of Fraction 3, methylated and silylated (Fig. 3) shows a small parent peak (m/e 560). Other important peaks are m/e 362, 361,271,259, and 199. These peaks can be derived from the formulae given in Figure 3. The mass spectrum of Fraction 4, methylated and silylated (Fig. 4) shows a small parent peak (m/e 560). Peaks are also present at m/e 545 (M-CH3) , 529 (MOCH3), 470 (M-TMSOH), and 358 (M-

174

Chr. Baur et al. : Enzymatic Oxydation of Linoleic Acid 173--

T ~389 i

CH3-(CH2)L-CH ~- CH - CH - (CH2)9-COOCH3

r--~ 387

r - ~ 2 5 9 Z31~- 228

[ [

I i

CH~-ICH2)~-CTCH- CH= CH+i CH - (C,2)7-COOCH~ I

OTMS OTMS OTMS

O I OTMS

]

I i -H,

275~- ± - ~287

L__ 173

OTMS ~386

287

;(°L -

17a-- ~-Hz314 I i

CH3-(CH2)4 - C H t CH= CH - CH ÷ C - CH2)7-COOCH 3 OTMS

207

288 259275299

389

II ,I II 1,3, 200

462

I

I

53115471 ,5B2 100

i

',00

OTMS 0 I -H.l 300~ - J

500

~00

386

-

I(%)

300

m/e

173

Fig. 5. Mass spectrum of Fraction 4 after methylation, hydrogenation and silylation

1y

2193013i,,4

228

387

x10

iF I

Ox

/0 NC - (CH2)9 - C"/

H/ 74.,

55

200

\OCH3

i

400

3OO

I

5{)0

m/e

87

[9

57

Fig. 7. Mass spectrum of Fraction 5 after methylation and silylation

139

I

CH3- {CH2)/'-Cll - CH - CH = CH ~ ICH - (CH2)7-COOCH 3

~i~ ,~11,~,

i

100

1,50

r-~259 I

OTMS 171

50

471

4394i5486

NOCH 3

OTMS

200

m/e

~2/,,3 2-3!--211

• I

Fig. 6. Periodate oxidation of methylated and hydrogenated Fraction 4. Mass spectrum of a cleavageproduct

TMS-O I

I

CH 3- (CH2) 4 -CH -- CH = CH - CH + C - (CH2)?-COOCH 3 I i ~ i II OTMS ]

' NOCH 3 I

I

I

173~ J

Fraction 5 was located on T L C plates by spraying the edges with a 2.4-dinitrophenylhydrazine solution. The methyl ester of Fraction 5 showed UV absorption (in methanol) in the region of 210 nm. The mass spectrum of Fraction 5, methylated and silylated (Fig. 7) shows the small ions m/e 486 (M), 471 (M-CH3), 455 (M-OCH3) which establish the molecular weight of 486. In Figure 7 rearrangement ions dominate (m/e 386, 314, 300). These ions and the ions m/e 387, 301, 259, 228, and 173 can be derived from the chemical structures given in Figure 7. The methylated Fraction 5 was converted to O-methyloxime trimethylsilyl ether derivatives. The mass spectrum (Fig. 8) shows the molecular ions (m/e 515) and ions due to elimination of 15 (CH3), 31(OCH3), 45(-NOCH3) and 135(-NOCH3; -TMSOH). Ion m/e 214 establishes the position of the C O - g r o u p at C-9 in one isomer of Fraction 5. Ions m/e 243 and 211 are characteristic for the occurence of an 9.10-Ketol group in this isomer. Before silylation, the methyl esters of Fractions 5 and 1 were reduced with LiA1H4 or LiA1D 4. Reduction with LiA1H 4 gave for Fractions 1 and 5 the same mass spectra (Fig. 9). Therefore the double-bonds in the two isomers of

!

100 (%

L_ ~214

211

-

14243

109143 149 129 173 , 1~71

H3

259

xl0 F - 358 I

380 470,8,500

I

100

200

300

400

500

m/e

Fig.8. Mass spectrum of the O-methyloxime trimethylsilyl ether derivatives of Fraction 5

Fraction 5 have the same positions as the isomers of Fraction 1. After reduction with LiA1D4, the mass spectra differed (Fig. 10). The shifts of the ions m/e 433--*434, 305--*306, and 1734174 agree with the occurence of a carbonyl group at C-9 and C-13 in the isomers of Fraction 5. Fraction 5 is therefore a mixture of 9-oxo-10.13-dihydroxy-octadec-ll-enoic and 13-oxo-9.12-dihydroxy-octadec- 10-enoic acids.

Chr. Baur et al. : Enzymatic Oxydation of Linoleic Acid 173~--T i

.--/.31

• i i

175

~303

CH3-(CH2) 4- CHj +, CHI - CH = CH 4- CH - (CH2)7-CH2OTMS OTMS OTMS

OTMS

173~-] 301~ T ~303 i C H3- (CH2)4- CHI ~ CH : CH - CHI ~ CH - (CH2) 7- CH2OTMS 173

OTMS

OTMS OTMS

303

[

275 3011i

i

200

300

I1431

4£)0

m/e

I ,514

500

,

600

Fig. 9. Mass spectrum of the Fractions 1 and 5 after reduction with LiA1H 4 and silylation 174~

T-~433 D~F I CH -oH: CH3- (CHz)z'- C + SMTO

• i I

~305

OH t c.

OTMS

D I - (CH2)7-CI - D

OTMS

OTMS

(a) F-- ~306

,

:?

Y

CH3-(CH2)~-CH 4- CH = CH - CH -- CI - (CH2)7- IC - D I , OTMS OTMS OTMS OTMS

l

173 ~

±-~434

173

(a)

xlO I-174 212 204,111231 100-

305306 !i 3/*5

2891

II 3'ffil134s

433 ,42811143/*

5oii

173 (b)

](°/I°)

191 204

, T ITM 200

305

xl0 r-

3/.4

427

, I 300

[ 1/.33

i

i m/e

400

501 )

500

Fig. 10. Mass spectra of fractions 5 (a) and 1 (b) after reduction with LiA1D 4 and silylation

Discussion

Sessa et al [-6] have shown that the bitter taste of stored soya flour ist localised in the lecithin and lysolecithin fractions: This leads to the assumption that the emulsifying action of the lecithin promotes contact between the slightly water-soluble bitter substances and the taste receptors. Below pH 8, fatty acids are still difficult to dissolve even when they have 4 hydroxy-groups, and they thus appear to be tasteless substances. If the hydroxy-fatty acids are emulsified

with soya lecithin then a bitter taste can be established. However soya lecithin is unsuitable for systematic taste analysis, as it also has a perceptible taste, even after chromatographic purification. In the search for an emulsifier for hydrophobic compounds that does not affect the taste analysis we decided upon a fatty acid sucrose ester. After emulsification with the ester, the taste of the compounds is examined. These compounds arise from the oxidation of linoleic acid with a protein fraction of soya bean possessing lipoxygenase and peroxydase activities. The main bitter substance was identified as a mixture of 9.12.13-trihydroxy-octadectrans-10-enoic acid and 9.10.13-trihydroxy-octadectrans-11-enoic acid. The taste threshold (on molar basis) is of the same order as that of caffeine and benzamide, but lower than that of L-trypthophan and Ltyrosine, which are particularly bitter tasting aminoacids [21]. The mixture of 9.12.13- and 9.10.13-trihydroxy fatty acids identified here as main bitter substances, are formed when linoleic acid ist incubated with a suspension of flow from wheat [18], barley, rye and oats [20] or peas [-12]. However, they also arise when linoleic acid hydroperoxides react with a protein fraction from oats [23] or potatoes [24]. In addition to this, they appear when linoleic acid is incubated with a relatively high concentration of a purified pea lipoxygenase [12]. On the basis of these observations and of the results reported here it is possible that the 9.12.13and 9.10.132trihydroxy acids, in combination with naturally occuring emulsifiers, contribute to the formation of a bitter taste in leguminosae and cereals. In addition to the two trihydroxy acids already described in the literature, two further trihydroxy acid isomers (with vicinal OH-groups) and a mixture of oxodihydroxy-acids were identified in the bitter-tasting fraction. The occurence of at least three oxygen functions in the alkyl chain is possibly necessary for the appearance of a bitter taste in fatty acids. The fact that 9.10-dihydroxystearic acid (4mg/ml) does not taste bitter agrees with this assumption. Our results also agree with the identification [28] of an alkene with three hydroxy-groups (1.2.4-trihydroxy-heptadeca-16-ene) as a compound that contributes to the heat-induced bitter off-flavor in avocados. Acknowledgements. The authors express their thanks to Dr. W. Mohr, Institut fiJr Lebensmitteltechnologie und Verpackung, Miinchen, for helpful advice. The mass spectrometric analysis by K.-H. Fischer and G. Sz6ni is grateful acknowledged.

References 1. Rothe, M., Woelm, G.: Nahrung 11, 149 (1967) 2. Sessa, D.J., Honig, D.H., Rackis,J.J.: Cereal Chem. 46, 675 (1969)

176 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Chr. Baur et al. : Enzymatic Oxydation of Linoleic Acid Rohrlich, M., Benschke, H.: Chemiker-Z. 75, 52 and 65 (1951) Mohr, W.: Dissertation, Univ. Miinchen 1952 Diemair, W., Janecke, H., Idstein,J. : St~irke 2, 243 (1950) Sessa, D.J., Warner, K., Honig, D.H.: J. Food Sci. 39, 69 (1974) Sessa, D.J., Warner, K., Rackis,J.J. :J. Agric. Food Chem. 24, 16 (1976) Rackis~J.J., Honig, D.H., Sessa, D.J., Moser, H.A.: Cereal Chem. 49, 586 (1972) Kalbrener, J.E., Warner, K, Eldridge, A.C.: Cereal Chem. 51, 406 (1974) Christopher,J, Pistorius, E., Axelrod, B.: Biochim. Biophys. Acta 198, 12 (1970) Morrison, W.R., Panpaprai, R.: J. Sci. Food Agric. 26, 1225 (1975) Arens, D., Grosch, W.: Z. Lebensm. Unters.-Forsch. 156, 292 (1974) Grosch, W., Laskawy, G.: J. Agrie. Food Chem. 23, 791 (1975) Surrey, K. : Plant Physiol. 39, 65 (1964) Lapworth, A., Mottram, E.N.: J. Chem. Soc. 127, 1628 (1925) Christopher,J.P., Pistorius, E.K., Axelrod, B.: Biochim. Biophys. Acta 284, 54 (1972)

17. Veldink, G.A., Vliegenthardt, J. F.G., Boldingh, J. : Biochem. J. 120, 55 (1970) 18. Graveland, A.: J. Amer. Oil Chem. Soc. 47, 352 (1970) 19. Schlenk, H., Gellerman, J. L. : Anal. Chem. 32, 1412 (1960) 20. Graveland, A.: Lipids 8, 599 (1973) 21. Wieser, H., Belitz, H.-D.: Z. Lebensm. Unters.-Forsch. 159, 65 (1975) 22. BartheI, G., Groseh, W. : J. Amer. Oil Chemists Soc. 51,540 (1974) 23, Heimann, W., Dresen, P., Klaiber, V.: Z. Lebensm. Unters.Forsch. 153, 1 (1973) 24. Galliard,T., Phillips, D.R., Matthew, J.A. : Biochim. Biophys. Acta 409, 157 (1975) 25. Green, K. : Chem. Phys. Lipids 3, 254 (1969) 26. Esselman, W.J., Clagett, C. O.: J. Lipid Res. 15, 173 (1974) 27. Bergmeyer, H.U.: Methoden der enzymatischen Analyse, 2. Aufl. Weinheim/Bergstr.: Verlag Chemie 1970 28. Ben-et, G., Dolev, A., Tata, D.: J. Food Sci. 38, 546 (1973)

Received January 26, 1977