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Carotenoid Dependent Inhibition of Durum Wheat Lipoxygenase
Journal of Cereal Science 29 (1999) 99–102 Article No. jcrs.0215, available online at http://www.idealibrary.com on
RESEARCH NOTE
Carotenoid Dependent Inhibition of Durum Wheat Lipoxygenase D. Trono∗, D. Pastore† and N. Di Fonzo∗ ∗Istituto Sperimentale per la Cerealicoltura, Sezione Operativa di Foggia S.S. 16 Km 675, 71100 Foggia, Italy; †Dipartimento di Scienze Animali, Vegetali e dell’ Ambiente, Universita` del Molise, Ed. Facolta` di Agraria, Loc. Vazzieri – 86100 Campobasso, Italy Received 15 January 1998 Keywords: lipoxygenase, b-carotene, durum wheat, semolina bleaching.
The yellow colour of pasta products, made from durum wheat semolina, is due to the presence of carotenoids mainly lutein (xantophyll)1 and is considered by consumers an important feature. In the light that carotenoids can reduce oxidative damage linked to ageing processes, not only in plants but also in animal systems2, an increase in their amount in pasta product is a valuable goal to be pursued. Unfortunately, during pasta-making a loss of colour is often observed, probably due to the lipoxygenase (LOX)-linoleate system which is responsible for carotenoid oxidation3. Although other enzymes, such as peroxidases and polyphenol oxidases, can contribute to semolina bleaching4, a major role appears to be played by LOX (EC 1.13.11.12), which catalyses the hydroperoxidation of the polyunsaturated fatty acids containing 1,4-cis, cis pentadiene structures5,6; in particular, free linoleate in durum wheat semolina is oxidised7, thus causing semolina bleaching. A reduction of LOX activity, during pastamaking, is of interest for commercial and nutritional purposes, thus requiring a detailed investigation aimed to find out whether and how
: LOX=lipoxygenase; ..=standard deviation.
Corresponding author: D. Trono. Tel: +39 0881 742972; Fax: +39 0881 713150; E-mail: [email protected] 0733–5210/99/010099+04 $30.00/0
semolina bleaching can be prevented. Since inhibition of LOX activity by b-carotene has already been reported in both animal and plant systems8, the purpose of this study was to investigate the LOX inhibition by carotenoid compounds to improve semolina and pasta quality. All reagents used were purchased from SIGMA Chemical Co. (St. Louis, MO). Linoleate solution was prepared daily by dissolving 45 mg of sodium linoleate and 150 lL of Tween 20 in 5 mL of deoxygenated 50 m sodium borate buffer (pH 9·00). Linoleate concentration was determined polarographically using a reaction mixture containing 10 lL of linoleate solution dissolved in 1·5 mL of 50 m sodium borate buffer (pH 9·00). The reaction was started by the addition of 2 units of purified SIGMA soybean LOX, type IV. b-carotene solution was prepared daily by dissolving 1 mg of b-carotene (SIGMA type I) in 10 mL of absolute ethanol with the concentration spectrophotometrically determined at 453 nm, using an e value of 140·6/m·cm9. The crude LOX extract was prepared from semolina of different cultivars of Triticum durum Desf., according to McDonald3. Protein content was determined according to Lowry10. LOX activity was measured spectrophotometrically at 25°C by a Perkin Elmer k 18 UV/VIS Spectrometer at 234 nm, according to Grossman and Zakut11. The control sample contained linoleate at different concentrations in 2 mL of 50 m sodium 1999 Academic Press
D. Trono et al.
phosphate buffer (pH 6·60); e value was 28/ m·cm12. Control was made that the e value remained unchanged following b-carotene addition. Since b-carotene was dissolved in ethanol, the test sample contained the same amount of ethanol (20 lL) whatever b-carotene concentration was used. The reaction was started by adding 25 lL of the semolina extract (0·2 mg of protein). Because of the very low LOX activity of the semolina samples from cultivars Cosmodur, Simeto and Plinio different instrumental conditions were employed. The assay was started by the addition of 0·8 mg of protein and the reaction was monitored by a high sensitive dual-wavelength spectrophotometer JASCO v560/v520 using the wavelength pair 234-280 nm. In these conditions, the e value for the conjugated diene remained 28/ m·cm. One unit of LOX activity corresponded to the production of one lmol of conjugated diene per min. Interpolation of data and kinetic parameter calculations were obtained by means of GRAFIT 3·0 (ERITHACUS) software. Total lipid extraction of semolina was obtained by suspending 5 g of semolina in 15 mL of a methanol:chloroform (2:1) solution. The suspension was shaken for 1 h, the chloroformic phase was dried by vacuum for 15 min at room temperature and the dry residue was dissolved in 2 mL of absolute ethanol. The total fatty acid content was obtained by saponification of the ethanolic solution according to Desai13, using butylated hydroxytoluene (BHT) as an antioxidant instead of ascorbate. The concentration of both free and total 1,4-cis, cis pentadiene structures was assayed spectrophotometrically at 234 nm. The assay mixture contained 5–10 lL of the ethanolic solution dissolved in 2 mL of 50 m sodium borate buffer (pH 9·00). The reaction was started by the addition of 4 units of SIGMA soybean LOX, type IV. The dough used to obtain pasta discs was made by mixing 250 g of semolina with the quantity of distilled water required to bring semolina moisture to 60% in an alveograph mixer. After 10 min mixing at 25°C, the dough was rolled and cut by a circular cutter in order to obtain 2 mm thick pasta discs. The discs were dried at 50°C for 12 h and milled by means of a Cyclotec mill, which was also used to mill durum wheat seeds in order to obtain the same granulation (60 mesh) for both semolina and ground pasta discs. Endogenous carotenoids were extracted from semolina and ground discs following the AACC water-saturated butanol (WSB) method14, with the exception of
1.0
Units/g of semolina
100
0.8
0.6
0.4
0.2
0
50
100 [Linoleate] (µM)
150
200
Figure 1 The dependence of the rate of linoleate oxidation by semolina LOX as a function of linoleate concentration either in the presence or in the absence of added b-carotene. Vertical bars represent±.. (n=3). Β, control; Μ, [b-carotene]=0·7 l; Κ, [b-carotene]=1·4 l.
extraction time, which was 3 h in this experiment. A set of experiments was carried out to ascertain whether LOX reaction in semolina could be inhibited by b-carotene. Consequently, enzyme extract from the cultivar Tresor, containing a high LOX activity and intermediate carotenoid content (see below), was used. The dependence of the rate of linoleate hydroperoxidation on the linoleate concentration shows a sigmoidal curve (Fig. 1). bcarotene inhibited the rate of the reaction, and this inhibition decreased with increased substrate concentration. In Table I the Tukey’s test analysis of data is showed in detail. Statistically significant differences among the reaction rates in the absence and in the presence of either 0·7 or 1·4 l bcarotene were found when the linoleate concentration was 50, 75 or 100 l. In particular, at 75 l linoleate and 1·4 l b-carotene (substrate/ inhibitor ratio: 54/1) 50% of the control LOX reaction rate was measured. The linoleate K0,5 was found to increase in the presence of b-carotene (Control: K0,5=81 l; 0·7 l b-carotene: K0,5=96 l; 1·4 l b-carotene: K0,5=106 l). Conversely, the Vmax was not affected (Vmax=1·1 units/g of semolina). In order to determine the amount of both the substrate of LOX and of compounds which are similar to the b-carotene in semolina, the endogenous contents of 1,4-cis, cis pentadiene structures in fatty acids and of carotenoids in Tresor semolina were assayed. They were 600±80 (..,
Carotenoid dependent inhibition of durum wheat lipoxygenase
Table I
101
Statistical analysis of data reported in Figure 1
b-carotene (l)
0·0 0·7 1·4
Linoleate (l) 25
50
75
100
150
200
a
h l m
f g i
c d e
b b b
a a a
m m m
a
Different letters indicate significant differences at the 0·001% probability level using Tukey’s test. Table II
LOX activity in 12 semolina samples and pigment loss due to pasta processing
Semolina sample
Primadur Tresor Trinakria Brindur Ambral Capeiti Dauno Altar Duilio Cosmodur Simeto Plinio
LOX activity (unitsa/g)
High
Medium
Low
Very low
Semolina carotenoid content (nmol/g)
Pasta discs carotenoid content (nmol/g)
(%)
(D nmol/g)
16·7±0·09 9·8±0·16c 8·1±0·00 16·3±0·11 11·9±0·16 9·6±0·13 12·7±0·14 10·4±0·11 8·0±0·07 13·0±0·14 11·0±0·07 8·0±0·02
12·0±0·05 6·8±0·02 5·3±0·02 13·0±0·03 9·1±0·00 7·0±0·13 10·8±0·18 8·6±0·13 6·3±0·11 13·0±0·14 10·4±0·07 7·5±0·07
28 30 34 20 23 27 15 17 21 0 5 6
4·7 3·0 2·8 3·3 2·8 2·6 1·9 1·8 1·7 0·0 0·6 0·5
1·3±0·05b 1·2±0·06 1·4±0·04 0·6±0·01 0·6±0·01 0·6±0·03 0·3±0·08 0·4±0·04 0·3±0·00 0·05±0·005 0·04±0·006 0·04±0·008
Carotenoid loss
a One unit is defined as 1 lmol of conjugated diene formed/min at 25°C. The semolina sample contained 500 l linoleate in 2 mL of 50 m sodium phosphate buffer (pH 6·60). The reaction was started by adding 0·2 mg of protein (about 25 lL of the semolina extract). For further details see text. b Values are means±.. (n=3). c n=4.
n=4) nmol/g of semolina and 3600±900 (.., n=4) nmol/g of semolina for pentadiene structures in free and total fatty acids, respectively. The carotenoid amount was 9·8±0·16 (.., n=4) nmol/g of semolina. Since many LOXs have low reactivity towards esterified fatty acids15 and no reports of lipase activity during pasta-making exist, it is reasonable to assume that the LOX substrate concentration in semolina corresponds to the amount of pentadiene present in free fatty acids. Consequently, the substrate/inhibitor ratio in semolina is about 60/1. Because of both the inhibition and the substrate/ inhibitor ratio observed in Tresor semolina, it’s possible that during pasta-making a high carotenoid content could inhibit the bleaching process. To examine this in more detail, 12 semolina samples were divided into four groups having similar LOX activities, but different carotenoid content (Table II). Different varieties were sampled
in order to obtain a range in LOX activity, carotenoid content, in both semolina and pasta discs, and carotenoid loss during pasta processing. In agreement with McDonald3, a highly significant correlation between LOX activity and carotenoid loss was found (linear regression analysis; r=0·90 and p<0·001, r=0·85 and p<0·001, respectively). The result was the same when carotenoid loss was expressed as a percentage or when carotenoid loss was expressed as nmol lost per g of semolina. Conversely, no significant correlation was found between LOX activity and the carotenoid content of either semolina (r=0·15) or pasta discs (r= −0·26). The results reported here indicate that, in each group, the percent carotenoid loss was inversely related to the initial carotenoid content. Since the samples do not differ from each other with respect to their LOX activity, the variations of carotenoid loss may be affected by its carotenoid content.
D. Trono et al.
102
Thus, this result indicates that in our experimental conditions the extent of carotenoid bleaching could depend only on the carotenoid content of the semolina itself. This study shows that semolina LOX reaction may be inhibited by the carotenoid compound b-carotene and, consistently, a lower semolina bleaching is observed in the samples having a higher carotenoid content. On the basis of these results, we suggest that semolina LOX may be inhibited by endogenous carotenoids. Therefore, a high carotenoid content of semolina is desirable because it gives a good yellow-amber colour and also, perhaps, it could partially prevent carotenoid bleaching during pasta processing.
4.
5. 6. 7. 8.
9.
Acknowledgements This research was supported by funds for the project ‘Miglioramento Cerealicolo per Innovazioni Agroindustriali’ financed by the Ministero delle Risorse Agricole, Alimentari e Forestali (MIRAAF). The authors wish to thank Prof P.R. Shewry for the critical reading of the manuscript and Prof S. Passarella for the scientific suggestions.
REFERENCES 1. Lepage, M. and Sims, R.P.A. Carotenoids of wheat flour: their identification and composition. Cereal Chemistry 45 (1968) 600–604. 2. Donnelly, J.K. and Robinson, D.S. Free radicals in foods. Free Radical Research 22(2) (1995) 147–176. 3. McDonald, C.E. Lipoxygenase and lutein bleaching ac-
10. 11.
12.
13. 14. 15.
tivity of durum wheat semolina. Cereal Chemistry 56(2) (1979) 84–89. Taha, S.A. and Sagi, F. Relationships between chemical composition of durum wheat semolina and macaroni quality. II. Ash, carotenoid pigments and oxidative enzymes. Cereal Research Communication 15(2–3) (1987) 123– 129. Siedow, J.N. Plant Lipoxygenase: structure and function. Annual Review of Plant Physiology and Plant Molecular Biology 42 (1991) 145–188. Gardner, H.W. Lipoxygenase pathway in cereals. Advances in Cereal Sciences 9 (1988) 161–215. Matsuo, R.R., Bradley, J.W. and Irvine, G.N. Studies on pigment destruction during spaghetti processing. Cereal Chemistry 47 (1970) 1–5. Lomnitski, L., Bar-Natan, R., Sklan, D. and Grossman, S. The interaction between b-carotene and lipoxygenase in plant and animal systems. Biochimica et Biophysica Acta 1167 (1993) 331–338. Davies, B.H. Carotenoids. In ‘Chemistry and biochemistry of plant pigments’, (T.W. Goodwin, ed.), Academic Press, London, New York (1976) pp 38–165. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193 (1951) 265–275. Grossman, S. and Zakut, R. Determination of the activity of LOX (Lipoxidase) In ‘Methods of Biochemical Analysis’, (D. Glick, ed.), J. Wiley and Sons, New York (1979) pp 303–329. Privett, O.S., Christense, N. and Lundberg, W.O. Products of the lipoxidase-catalyzed oxidation of sodium linoleate. Journal of American Oil Chemistry Society 32 (1955) 505–511. Desai, I.D. Vitamin E analysis methods for animal tissues. In ‘Methods in Enzymology’, (L. Packer, ed.), Academic Press, London, New York (1984) pp 138–147. American Association of Cereal Chemists. Approved Methods of the AACC. Method 14-50, approved April 13, 1961. The Association. St. Paul, MN. Hildebrand, D.F. Lipoxygenases. Physiologia Plantarum 76 (1988) 249–253.
RESEARCH NOTE
Carotenoid Dependent Inhibition of Durum Wheat Lipoxygenase D. Trono∗, D. Pastore† and N. Di Fonzo∗ ∗Istituto Sperimentale per la Cerealicoltura, Sezione Operativa di Foggia S.S. 16 Km 675, 71100 Foggia, Italy; †Dipartimento di Scienze Animali, Vegetali e dell’ Ambiente, Universita` del Molise, Ed. Facolta` di Agraria, Loc. Vazzieri – 86100 Campobasso, Italy Received 15 January 1998 Keywords: lipoxygenase, b-carotene, durum wheat, semolina bleaching.
The yellow colour of pasta products, made from durum wheat semolina, is due to the presence of carotenoids mainly lutein (xantophyll)1 and is considered by consumers an important feature. In the light that carotenoids can reduce oxidative damage linked to ageing processes, not only in plants but also in animal systems2, an increase in their amount in pasta product is a valuable goal to be pursued. Unfortunately, during pasta-making a loss of colour is often observed, probably due to the lipoxygenase (LOX)-linoleate system which is responsible for carotenoid oxidation3. Although other enzymes, such as peroxidases and polyphenol oxidases, can contribute to semolina bleaching4, a major role appears to be played by LOX (EC 1.13.11.12), which catalyses the hydroperoxidation of the polyunsaturated fatty acids containing 1,4-cis, cis pentadiene structures5,6; in particular, free linoleate in durum wheat semolina is oxidised7, thus causing semolina bleaching. A reduction of LOX activity, during pastamaking, is of interest for commercial and nutritional purposes, thus requiring a detailed investigation aimed to find out whether and how
: LOX=lipoxygenase; ..=standard deviation.
Corresponding author: D. Trono. Tel: +39 0881 742972; Fax: +39 0881 713150; E-mail: [email protected] 0733–5210/99/010099+04 $30.00/0
semolina bleaching can be prevented. Since inhibition of LOX activity by b-carotene has already been reported in both animal and plant systems8, the purpose of this study was to investigate the LOX inhibition by carotenoid compounds to improve semolina and pasta quality. All reagents used were purchased from SIGMA Chemical Co. (St. Louis, MO). Linoleate solution was prepared daily by dissolving 45 mg of sodium linoleate and 150 lL of Tween 20 in 5 mL of deoxygenated 50 m sodium borate buffer (pH 9·00). Linoleate concentration was determined polarographically using a reaction mixture containing 10 lL of linoleate solution dissolved in 1·5 mL of 50 m sodium borate buffer (pH 9·00). The reaction was started by the addition of 2 units of purified SIGMA soybean LOX, type IV. b-carotene solution was prepared daily by dissolving 1 mg of b-carotene (SIGMA type I) in 10 mL of absolute ethanol with the concentration spectrophotometrically determined at 453 nm, using an e value of 140·6/m·cm9. The crude LOX extract was prepared from semolina of different cultivars of Triticum durum Desf., according to McDonald3. Protein content was determined according to Lowry10. LOX activity was measured spectrophotometrically at 25°C by a Perkin Elmer k 18 UV/VIS Spectrometer at 234 nm, according to Grossman and Zakut11. The control sample contained linoleate at different concentrations in 2 mL of 50 m sodium 1999 Academic Press
D. Trono et al.
phosphate buffer (pH 6·60); e value was 28/ m·cm12. Control was made that the e value remained unchanged following b-carotene addition. Since b-carotene was dissolved in ethanol, the test sample contained the same amount of ethanol (20 lL) whatever b-carotene concentration was used. The reaction was started by adding 25 lL of the semolina extract (0·2 mg of protein). Because of the very low LOX activity of the semolina samples from cultivars Cosmodur, Simeto and Plinio different instrumental conditions were employed. The assay was started by the addition of 0·8 mg of protein and the reaction was monitored by a high sensitive dual-wavelength spectrophotometer JASCO v560/v520 using the wavelength pair 234-280 nm. In these conditions, the e value for the conjugated diene remained 28/ m·cm. One unit of LOX activity corresponded to the production of one lmol of conjugated diene per min. Interpolation of data and kinetic parameter calculations were obtained by means of GRAFIT 3·0 (ERITHACUS) software. Total lipid extraction of semolina was obtained by suspending 5 g of semolina in 15 mL of a methanol:chloroform (2:1) solution. The suspension was shaken for 1 h, the chloroformic phase was dried by vacuum for 15 min at room temperature and the dry residue was dissolved in 2 mL of absolute ethanol. The total fatty acid content was obtained by saponification of the ethanolic solution according to Desai13, using butylated hydroxytoluene (BHT) as an antioxidant instead of ascorbate. The concentration of both free and total 1,4-cis, cis pentadiene structures was assayed spectrophotometrically at 234 nm. The assay mixture contained 5–10 lL of the ethanolic solution dissolved in 2 mL of 50 m sodium borate buffer (pH 9·00). The reaction was started by the addition of 4 units of SIGMA soybean LOX, type IV. The dough used to obtain pasta discs was made by mixing 250 g of semolina with the quantity of distilled water required to bring semolina moisture to 60% in an alveograph mixer. After 10 min mixing at 25°C, the dough was rolled and cut by a circular cutter in order to obtain 2 mm thick pasta discs. The discs were dried at 50°C for 12 h and milled by means of a Cyclotec mill, which was also used to mill durum wheat seeds in order to obtain the same granulation (60 mesh) for both semolina and ground pasta discs. Endogenous carotenoids were extracted from semolina and ground discs following the AACC water-saturated butanol (WSB) method14, with the exception of
1.0
Units/g of semolina
100
0.8
0.6
0.4
0.2
0
50
100 [Linoleate] (µM)
150
200
Figure 1 The dependence of the rate of linoleate oxidation by semolina LOX as a function of linoleate concentration either in the presence or in the absence of added b-carotene. Vertical bars represent±.. (n=3). Β, control; Μ, [b-carotene]=0·7 l; Κ, [b-carotene]=1·4 l.
extraction time, which was 3 h in this experiment. A set of experiments was carried out to ascertain whether LOX reaction in semolina could be inhibited by b-carotene. Consequently, enzyme extract from the cultivar Tresor, containing a high LOX activity and intermediate carotenoid content (see below), was used. The dependence of the rate of linoleate hydroperoxidation on the linoleate concentration shows a sigmoidal curve (Fig. 1). bcarotene inhibited the rate of the reaction, and this inhibition decreased with increased substrate concentration. In Table I the Tukey’s test analysis of data is showed in detail. Statistically significant differences among the reaction rates in the absence and in the presence of either 0·7 or 1·4 l bcarotene were found when the linoleate concentration was 50, 75 or 100 l. In particular, at 75 l linoleate and 1·4 l b-carotene (substrate/ inhibitor ratio: 54/1) 50% of the control LOX reaction rate was measured. The linoleate K0,5 was found to increase in the presence of b-carotene (Control: K0,5=81 l; 0·7 l b-carotene: K0,5=96 l; 1·4 l b-carotene: K0,5=106 l). Conversely, the Vmax was not affected (Vmax=1·1 units/g of semolina). In order to determine the amount of both the substrate of LOX and of compounds which are similar to the b-carotene in semolina, the endogenous contents of 1,4-cis, cis pentadiene structures in fatty acids and of carotenoids in Tresor semolina were assayed. They were 600±80 (..,
Carotenoid dependent inhibition of durum wheat lipoxygenase
Table I
101
Statistical analysis of data reported in Figure 1
b-carotene (l)
0·0 0·7 1·4
Linoleate (l) 25
50
75
100
150
200
a
h l m
f g i
c d e
b b b
a a a
m m m
a
Different letters indicate significant differences at the 0·001% probability level using Tukey’s test. Table II
LOX activity in 12 semolina samples and pigment loss due to pasta processing
Semolina sample
Primadur Tresor Trinakria Brindur Ambral Capeiti Dauno Altar Duilio Cosmodur Simeto Plinio
LOX activity (unitsa/g)
High
Medium
Low
Very low
Semolina carotenoid content (nmol/g)
Pasta discs carotenoid content (nmol/g)
(%)
(D nmol/g)
16·7±0·09 9·8±0·16c 8·1±0·00 16·3±0·11 11·9±0·16 9·6±0·13 12·7±0·14 10·4±0·11 8·0±0·07 13·0±0·14 11·0±0·07 8·0±0·02
12·0±0·05 6·8±0·02 5·3±0·02 13·0±0·03 9·1±0·00 7·0±0·13 10·8±0·18 8·6±0·13 6·3±0·11 13·0±0·14 10·4±0·07 7·5±0·07
28 30 34 20 23 27 15 17 21 0 5 6
4·7 3·0 2·8 3·3 2·8 2·6 1·9 1·8 1·7 0·0 0·6 0·5
1·3±0·05b 1·2±0·06 1·4±0·04 0·6±0·01 0·6±0·01 0·6±0·03 0·3±0·08 0·4±0·04 0·3±0·00 0·05±0·005 0·04±0·006 0·04±0·008
Carotenoid loss
a One unit is defined as 1 lmol of conjugated diene formed/min at 25°C. The semolina sample contained 500 l linoleate in 2 mL of 50 m sodium phosphate buffer (pH 6·60). The reaction was started by adding 0·2 mg of protein (about 25 lL of the semolina extract). For further details see text. b Values are means±.. (n=3). c n=4.
n=4) nmol/g of semolina and 3600±900 (.., n=4) nmol/g of semolina for pentadiene structures in free and total fatty acids, respectively. The carotenoid amount was 9·8±0·16 (.., n=4) nmol/g of semolina. Since many LOXs have low reactivity towards esterified fatty acids15 and no reports of lipase activity during pasta-making exist, it is reasonable to assume that the LOX substrate concentration in semolina corresponds to the amount of pentadiene present in free fatty acids. Consequently, the substrate/inhibitor ratio in semolina is about 60/1. Because of both the inhibition and the substrate/ inhibitor ratio observed in Tresor semolina, it’s possible that during pasta-making a high carotenoid content could inhibit the bleaching process. To examine this in more detail, 12 semolina samples were divided into four groups having similar LOX activities, but different carotenoid content (Table II). Different varieties were sampled
in order to obtain a range in LOX activity, carotenoid content, in both semolina and pasta discs, and carotenoid loss during pasta processing. In agreement with McDonald3, a highly significant correlation between LOX activity and carotenoid loss was found (linear regression analysis; r=0·90 and p<0·001, r=0·85 and p<0·001, respectively). The result was the same when carotenoid loss was expressed as a percentage or when carotenoid loss was expressed as nmol lost per g of semolina. Conversely, no significant correlation was found between LOX activity and the carotenoid content of either semolina (r=0·15) or pasta discs (r= −0·26). The results reported here indicate that, in each group, the percent carotenoid loss was inversely related to the initial carotenoid content. Since the samples do not differ from each other with respect to their LOX activity, the variations of carotenoid loss may be affected by its carotenoid content.
D. Trono et al.
102
Thus, this result indicates that in our experimental conditions the extent of carotenoid bleaching could depend only on the carotenoid content of the semolina itself. This study shows that semolina LOX reaction may be inhibited by the carotenoid compound b-carotene and, consistently, a lower semolina bleaching is observed in the samples having a higher carotenoid content. On the basis of these results, we suggest that semolina LOX may be inhibited by endogenous carotenoids. Therefore, a high carotenoid content of semolina is desirable because it gives a good yellow-amber colour and also, perhaps, it could partially prevent carotenoid bleaching during pasta processing.
4.
5. 6. 7. 8.
9.
Acknowledgements This research was supported by funds for the project ‘Miglioramento Cerealicolo per Innovazioni Agroindustriali’ financed by the Ministero delle Risorse Agricole, Alimentari e Forestali (MIRAAF). The authors wish to thank Prof P.R. Shewry for the critical reading of the manuscript and Prof S. Passarella for the scientific suggestions.
REFERENCES 1. Lepage, M. and Sims, R.P.A. Carotenoids of wheat flour: their identification and composition. Cereal Chemistry 45 (1968) 600–604. 2. Donnelly, J.K. and Robinson, D.S. Free radicals in foods. Free Radical Research 22(2) (1995) 147–176. 3. McDonald, C.E. Lipoxygenase and lutein bleaching ac-
10. 11.
12.
13. 14. 15.
tivity of durum wheat semolina. Cereal Chemistry 56(2) (1979) 84–89. Taha, S.A. and Sagi, F. Relationships between chemical composition of durum wheat semolina and macaroni quality. II. Ash, carotenoid pigments and oxidative enzymes. Cereal Research Communication 15(2–3) (1987) 123– 129. Siedow, J.N. Plant Lipoxygenase: structure and function. Annual Review of Plant Physiology and Plant Molecular Biology 42 (1991) 145–188. Gardner, H.W. Lipoxygenase pathway in cereals. Advances in Cereal Sciences 9 (1988) 161–215. Matsuo, R.R., Bradley, J.W. and Irvine, G.N. Studies on pigment destruction during spaghetti processing. Cereal Chemistry 47 (1970) 1–5. Lomnitski, L., Bar-Natan, R., Sklan, D. and Grossman, S. The interaction between b-carotene and lipoxygenase in plant and animal systems. Biochimica et Biophysica Acta 1167 (1993) 331–338. Davies, B.H. Carotenoids. In ‘Chemistry and biochemistry of plant pigments’, (T.W. Goodwin, ed.), Academic Press, London, New York (1976) pp 38–165. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193 (1951) 265–275. Grossman, S. and Zakut, R. Determination of the activity of LOX (Lipoxidase) In ‘Methods of Biochemical Analysis’, (D. Glick, ed.), J. Wiley and Sons, New York (1979) pp 303–329. Privett, O.S., Christense, N. and Lundberg, W.O. Products of the lipoxidase-catalyzed oxidation of sodium linoleate. Journal of American Oil Chemistry Society 32 (1955) 505–511. Desai, I.D. Vitamin E analysis methods for animal tissues. In ‘Methods in Enzymology’, (L. Packer, ed.), Academic Press, London, New York (1984) pp 138–147. American Association of Cereal Chemists. Approved Methods of the AACC. Method 14-50, approved April 13, 1961. The Association. St. Paul, MN. Hildebrand, D.F. Lipoxygenases. Physiologia Plantarum 76 (1988) 249–253.