Article pp.209-221 du Vol.23 n°2 (2003)

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SCIENCES DES ALIMENTS, 23(2003) 209-221

ARTICLE ORIGINAL ORIGINAL PAPER

Simplified method for the determination of lipolytic activity in low moisture media

F. El Amrani, O. Fayol, R. Drapron, J. Potus et J. Nicolas*¹

RÉSUMÉ

Méthode simplifiée de mesure de l’activité lipolytique en milieux peu hydratés Une nouvelle méthode de détermination de l’activité lipolytique dans les produits de mouture des céréales est décrite. Celle-ci est basée sur la titration à l’aide d’un pHstat de la quantité d’acides gras libérés par l’activité lipolytique lors de l’incubation d’un mélange d’un produit de mouture délipidé (grain entier ou fraction de mouture) et d’huile d’olive pendant 72 h dans des conditions déterminées de tem- pérature et d’activité de l’eau (A_w). Les résultats donnés par cette méthode ont été comparés systématiquement à ceux donnés par la méthode classique de mesure de l’activité lipolytique dans les milieux peu hydratés basée sur la déter- mination des acides gras libres par chromatographie en phase gazeuse (GC) après séparation par chromatographie sur couche mince. Les deux méthodes ont été utilisées dans différentes conditions expérimentales au cours desquelles les effets de la température, de l’A_w et de l’addition de lipase exogène ont été étu- diés. Dans les fractions de mouture du blé testées (germe, son, farine et blé entier), l’activité lipolytique déterminée par la méthode pHstat est corrélée à celle obtenue par la méthode GC (r = 0,99). Par le pHstat, l’activité lipolytique mesurée est égale à 0,054, 0,75, 0,29 et 0,51 µmol.h^-1.g^-1 (ms) vs 0,053, 0,79, 0,27 et 0,48 µmol.h^-1.g^-1 (ms) par GC pour les fractions farine, son, germe et blé entier, respectivement. Après avoir fait varier les conditions d’incubation, les activités lipolytiques mesurées à l’aide du pHstat sont corrélées à celles déterminées par GC, à la suite d’une modification de l’A_w (de 0,17 à 0,87) ou de la température (30 à 40 °C) au cours de l’incubation du son (r = 0,95) ou après ajout de lipase exo- gène à la farine (r = 0,97). Cette méthode de détermination de l’activité lipolytique plus rapide que la méthode classique peut présenter un grand intérêt tant pour améliorer les conditions de stockage des produits de mouture des céréales que pour juger de l’efficacité de traitements thermiques visant à la dénaturation de l’activité lipolytique endogène de ces produits.

Mots clés

lipase ; farine de blé ; germe ; son ; pHstat.

1. Chaire de biochimie industrielle et agro-alimentaire, Conservatoire national des arts et métiers, 292, rue Saint-Martin, 75141 Paris cedex 03, France

* Correspondance : nicolasj@cnam.fr

3-El Amrani (209-222) Page 209 Mercredi, 11. juin 2003 12:50 12

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210 Sci. Aliments 23(2), 2003 F. El Amrani et al.

SUMMARY

A new method for the determination of lipolytic activity (LA) in milled products from cereals is described. This method is based on the use of the pHstat titration of free fatty acids (FFA) released by LA after incubating a mixture of defatted wholemeal or milling wheat fraction and olive oil during 72 hours at selected conditions of temperature and activity of water (A_w).

The results given by this method have been compared systematically to those given by the time-consuming and classic one of the determination of the LA in low moisture media based upon the FFA determination by gas chromatography (GC) after separation by thin layer chromatography. The two methods have been used under various experimental conditions in order to study the effects of temperature, A_w and addition of exogenous lipase in different wheat milled products. For the germ, bran, flour and whole meal fractions at 30°C and A_w = 0.8, LA determined by the titration pHstat method was correlated to the values obtained by the GC method (r = 0.99).

By pHstat, LA was found equal to 0.054, 0.75, 0.29 and 0.51 µmol.h^-1.g^-1 (dm) vs 0.053, 0.79, 0.27 and 0.48 µmol.h^-1.g^-1 (dm) by GC for the flour, bran, germ and wholemeal fraction respectively. When experimental conditions were modified, LA determined by pHstat was correlated with LA obtained by GC either when A_w was varied for the bran fraction between 0.17 and 0.87 or temperature between 30 and 40°C (r = 0.95) or after addition of exogenous lipase to wheat flour (r = 0.97). This less time-consuming method for the determination of LA may be of a great interest to improve the storage conditions of cereal milled products as well as to estimate the efficiency of heat treatments on the denaturation of endogenous lipase in these products.

Key words:

lipase ; wheat flour ; bran ; germ ; pHstat.

1 – INTRODUCTION

Due to both economical and nutritional interests, lipase activity (LA) is an important parameter for numerous cereal products since lipases are able to facilitate the initiation of the oxidative rancidity catalysed by lipoxygenase. After milling, in the beginning of storage of wheat flour, a slight hydrolysis of the lipid fraction followed by the free polyunsaturated fatty acid oxidation by wheat lipoxygenase improved the baking performance of flour (Bellenger and Godon, 1972; Berger, 1991; Castello et al., 1998). Conversely, a long term storage of flour resulted in higher levels of hydrolysis and oxidation of the lipid fraction which lead to rancid products with decreased baking performance (Cuendet, 1954; Gracza, 1965; Warwick et al., 1979).

Actual nutritional recommendations for an increasing consumption of dietary fiber, polyphenols, minerals and vitamins, promote an increasing demand for cereal wholemeal, bran and germ enriched products. After milling and due to their high endogenous lipase and lipoxygenase activities (Galliard, 1986; Tait

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Simplified method for the determination of lipolytic activity in low moisture media 211

and Galliard, 1988), these products are unstable and their sensory and nutritive values may be rapidly lost following unsuitable storage conditions. During mixing of wheat flour, the lipoxygenase acts on the the polyunsaturated fatty acids released by lipase and catalyses their peroxidation by oxygen to fatty acid hydroperoxides responsible of a large cascade of co-oxidation reactions which affect the quality of the final product (Brockman and Acker, 1977; Nicolas and Drapron, 1983; Nicolas and Potus, 1994).

Nowadays, industry used the fat acidity measurement to evaluate the hydrolysis level in the lipid fraction of cereal products. This method is rapid but gives only an idea of the general quality of the product without any information concerning its LA (Guilbot, 1960; Drapron and Berger, 1976). Fat acidity increases during storage due to the LA present in the milled products or in the contaminating micro-organisms (Gracza, 1965; Galliard, 1983). This variation of the fat acidity is dependent on the LA content of the milled products, the year, the varieties and the storage conditions (Gracza, 1965; Molteberg et al., 1995).

Therefore, an accurate measurement of the LA is needed in order to control the evolution of these products.

The LA in cereal products is not measurable by direct methods due to their low values in low or high moisture conditions (Drapron, 1985 and 1986). In 1969, Drapron and Sclafani have developped a method to quantify the LA in low moisture media in selected conditions close to those used for the storage of cereal products. In this method, the defatted milled cereal products were incubated with a substrate (olive oil) in definite conditions of temperature and activity of water (A_w). The free fatty acids (FFA) released by hydrolysis of the triglycerides of olive oil were extracted, separated by thin layer chromatography (TLC) and quantified by gas chromatography (GC) in the presence of an internal standard. This technic is very specific but is time-consuming.

The purpose of this work was to develop a technic based on similar conditions of incubation but less-time-consuming for the estimation of the FFA. In this new method, FFA are quantified by a pHstat titration avoiding the two chromatographic steps (TLC and GC). The results obtained by this new method were compared to those obtained by the original method.

2 – MATERIAL AND METHODS

2.1 Products

All chemical reagents were provided by Sigma (St-Quentin, France) or by Prolabo (Paris, France). Olive oil was a commercial pure virgin olive oil and was used without any further treatment. Lipase Novozym 677 BG was obtained from Novonordisk (Bagsgvaerd, Denmark) with an activity of 40000 lipase units (LU) per gram. According to the Novo datasheet, 1 LU corresponds to the amount of enzyme that releases 1 µmol of titratable butyric acid per minute from tributyrin under the conditions described by Greenhough et al. (1996).

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2.2 Preparation of wheat fractions

The wheat grains and three milling fractions – germ, bran and flour – were provided by Les Moulins Soufflet (Nogent-sur-Seine, France). Wheat grains were milled (< 0.5 mm) using a laboratory mill (Chopin, France) in frozen conditions. All these milled fractions were stored at – 20°C before use. In these conditions, their LA remained constant for at least six months.

2.3 Moisture content

The moisture content was determined according to the ISO 712 norm, using a Chopin apparatus thermostated at 130°C. Moisture contents were 10.7, 13.1, 15.3 and 13.7% (wet basis) for the flour, germ, wheat and bran fractions respectively.

2.4 Delipidation

The milled fractions were defatted by extraction with n-hexane at room temperature. Three successive extractions were carried out during 2 × 60 min and 1 × 90 min by stirring (3 × 200 mL of n-hexane for 10 g of sample). The samples were dried under ventilated host in order to remove the traces of solvent. The defatted samples were stored a – 20°C until use.

2.5 Preparation of the incubation medium

The method of Drapron and Sclafani (1969) was used with minor modifica- tions. Two grams of the defatted samples were thoroughly mixed with 100 mg of olive oil previously diluted with 10 mL of n-hexane containing butylated hydroxytoluene (0.01%) to avoid oxidation of the polyunsaturated fatty acids.

0.1 mg of heptadecanoic acid (C₁₇) in 1 mL of n-hexane was added to the mixture as an internal standard. After mixing, the solvent was eliminated by evaporation under host at room temperature. In routine conditions, the mixture was incubated during 72 h at 30°C into sorption tubes under vacuum maintained at an A_w of 0.8 by a sulfuric acid solution suitably diluted. In some experiments with the bran fraction, the incubation conditions were varied either by changing the A_w between 0.17 and 0.87 (by variation of the sulfuric acid concentration) or by increasing the temperature at 40°C. In other experiments with wheat flour, an exogenous lipase (Lipase Novozym 677 BG) was added to the wheat flour before the mixture preparation. Different doses of exogenous lipase were tested between 20 and 100 LU.g^-1 of flour.

2.6 Lipolytic activity measurement by GC analysis

Lipid extraction: After incubation, the mixture was poored in a centrifuge tube with 25 mL of n-hexane to extract the lipids. After 10 min of stirring, the samples were centrifuged for 10 min at 500 × g, the organic liquid phase containing lipids was collected and evaporated under vacuum at 30°C using a rotavapor.

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Separation of FFA by TLC: The lipid extracts dissolved in 100 µL of chloro- form were applied in line on silica gel plates (Durasil-60 25, Macherey-Nagel, Düren, Germany) which have been activated for 1 hour at 110°C prior use.

Plates were developped in a mixture of petroleum ether / diethyl ether / formic acid (70: 30: 1) to separate the different classes of lipid. After 1 h, the plates were observed under UV light after spraying with primuline (0.01 %).

Preparation and analysis of fatty acid methyl ester. The bands corresponding to the FFA were scrapped into screw-capped tubes. FFA were resuspended in 3 ml of borontrifluoride methanol. The sealed tubes were then heated for 15 min at 70°C to convert FFA into fatty acid methyl esters (FAME). After cooling, 2 mL of water and 3 mL of pentane were added to extract the FAME. After collecting the pentane fraction, the solvent was evaporated under a nitrogen flow and FAME were solubilized with 100 µL of heptane. The samples were stored at – 20°C prior to GC analysis. FAME were separated and quantified using a GC STAR 3400CX gas chromatograph (Varian, Les Ulis, France) equipped with a flame ionization detector and a CP Wax 52 CB Chrompack (25 m × 0.25 mm, 0.25 µm of thick- ness) column. The injector, column and detector temperatures were 250, 190 and 270°C, respectively. The flow rates were 30 and 300 mL.min^-1 for H₂ and air in the detector. The column flow rate was 1.5 mL.min^-1 and the septum purge flow rate 40 mL.min^-1. After calibration of the detector by injection of a mixture of pure pal- mitic, heptadecanoic, stearic, oleic, linoleic and linolenic acid methyl esters, the extracted FAME were quantified following the procedure described by Castello et al. (1998).

2.7 Lipolytic activity measurement by pHstat titration

The method is adapted from the ISO/DIN 73025 norm for the determination of fat acidity in cereals. Automatic titration was carried out using pHstat (Titrino SM 702, Metrohm) linked to a computer for data analysis. The modification included the use of an automatic end point titration at pH 10.0 instead of phe- nolphtalein as indicator.

Lipid extraction. After incubation of the defatted milling fractions in the presence of the olive oil, lipids were extracted either by n-hexane, method pHstat (a), or by ethanol, method pHstat (b). The samples were poored in centrifuge tubes with 20 mL of n-hexane or ethanol (95% vol) and stirred for 10 min. After centrifugation (15 min at 500 × g), the hexane extract was evaporated using rotavapor and the residue was redissolved in 20 mL of ethanol before titration, method pHstat (a). The ethanol extract was directly titrated, method pHstat (b).

pHstat titration. 7.5 ml of the extracts were added to 7.5 ml of water in the pHstat reactor. After 3 min of stabilisation, a solution of sodium hydroxide (0.01 N) was used for the automatic end point titration. Results of FFA content were given in µmole.g^-1 dry matter. In both cases (GC and pHstat titration), lipase activity (LA) was expressed in µmole.h^-1.g^-1 dry matter:

where X₀ is the FFA content (µmole.g^-1 dry matter) before incubation and X_t is the FFA content (µmole.g^-1 dry matter) after an incubation time of t hours.

A chart representing the three methods of LA determination, GC and pHstat (a) methods on the lipid extract by n-hexane and pHstat (b) method on the lipid

LA ⁼

∑

(X_t–X₀)⁄t

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extract by ethanol, is given in figure 1. In each method, assays for the LA determination of the milled fractions in standard conditions (30°C and A_w = 0.8) have been repeated 5 times whereas assays of LA in various conditions of incubation have been duplicated.

2.8 Statistical analysis

Data were analyzed using the Statgraphics Plus software (version 3.0).

Anova test was used, differences between samples are reported to be not sig- nificant when p > 0.05.

Milled fractions

Lipid extraction

(n-hexane)

Defatted fractions

+ Olive oil

+ C_17:0 (internal standard)

Mixture

Incubation (72 h)

Ethanol Extract n-Hexane Extract

Evaporation of n-hexane Thin-layer

Addition of ethanol chromatography

Ethanol Extract Heptane extract

Titration Titration Gas

chromatography

pH Stat (b) pH Stat (a) Method of Drapron

and Sclafani (1969)

Figure 1

Flowchart of the three methods of lipase activity measurement.

Diagramme des 3 méthodes de mesure de l’activité lipolytique.

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3 – RESULTS AND DISCUSSION

3.1 Comparison of the methods

Because lipase plays a key role in the quality of cereals and especially milled cereals, it is important to be able to evaluate rapidly its activity. In commercial products, only the test of acidity (ISO 7305) is used to estimate the fat acidity of the milled fractions. This test presents the advantage to be rapid but cannot allow to estimate the lipase activity.

To our knowledge, the method of Drapron and Sclafani (1969) is the only one which is reliable for the measurement of LA in low moisture media. How- ever, due to the two chromatographic steps needed to quantify the FFA released during incubation, this method is time-consuming. To simplify this part of the method, we propose to quantify the FFA by a pHstat titration.

In a first step, the extraction conditions of FFA by n-hexane were not changed. The amounts of FFA were quantified by TLC and GC according to the method of Drapron and Sclafani (1969) from the one hand, and by pHstat titration, after evaporation of n-hexane and redissolution of the lipid extract in ethanol (method pHstat a), from the other hand (Figure 1). For the comparison of the two methods, four different milled wheat products, wheat flour, germ, bran and wholemeal, were incubated during 72 h at 30°C and an A_w of 0.8, conditions proposed by Drapron and Sclafani (1969). When the results given by the method pHstat a were plotted versus those given by the method of Drapron and Sclafani (1969), a straight line was obtained for the four samples (Figure 2).

The slope close to unity as well as the correlation coefficient higher than 0.98 indicate that similar values of LA are obtained by the two methods whatever the sample tested.

In a second step and from the same samples, FFA directly extracted by ethanol were quantified by pHstat titration (method pHstat b). The results obtained by this method are given in table 1 together with those given by the two other methods. For a same sample, the three methods gave the same value of LA.

Our results confirm that among the milled fractions tested, wheat flour has the weakest LA (close to 0.05 µmol.h^-1.g^-1 dm whereas the LA found in germ, wholemeal and bran were approximately 6, 10 and 15 times higher, respectively. Therefore for the routine conditions of incubation selected by Drapron and Sclafani (1969), it is possible to determine accurately the LA of these milled fractions using a simplified method (pHstat method b). Our results are also in agreement with those obtained by Galliard (1986) who showed that among the wheat milled fractions, LA was highest in the bran fraction.

3.2 Influence of A_w and temperature

Since LA is very dependent on A_w and temperature, the validity of the pHstat method has been checked when these two parameters of incubation were varied.

For this purpose, the bran fraction was incubated under varied A_w between 0.17 and 0.87 at 30 and 40°C. Whatever the temperature tested, LA increased when A_w increased from 0.17 to 0.74 and then decreased at an A_w of 0.87 (Figure 3). In addition, for a same value of A_w, LA was always higher at 40 than at 30°C. These results

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Table 1

Lipase activities found for the milled fractions from wheat (bran, germ, wholemeal and flour) by the 3 methods tested

(All values are given in µmol of FFA.h^-1.g^-1 dry matter).

Tableau 1

Activités lipolytiques des différentes fractions meunières (son, germe, blé entier et farine) obtenues par les 3 méthodes testées (Toutes les valeurs sont exprimées en µmol d’acides gras libérés.h^-1.g^-1 matière sèche)

Bran Germ Wholemeal Flour

Method of Drapron and

Sclafani (1969) 0.79 ± 0.08â 0.28 ± 0.03^b 0.49 ± 0.01^c 0.052 ± 0.003^d pHstat method a 0.75 ± 0.05â 0.30 ± 0.09^b 0.50 ± 0.09^c 0.052 ± 0.002^d pHstat method b 0.76 ± 0.05â 0.29 ± 0.05^b 0.51 ± 0.05^c 0.050± 0.003^d a, b,c and d: a same letter indicates that values are not significantly different (p > 0.05 Anova test). The values are given as the mean of 5 determinations ± standard deviation.

a, b, c et d : une même lettre indique que les valeurs ne sont pas significativement différentes (p > 0,05 test Anova). Les valeurs sont données comme la moyenne de 5 déterminations ± l’écart type.

y = 0.99 x R² = 0.98

0 0.2 0.4 0.6 0.8

0 0.2 0.4 0.6 0.8 1

bran

germ

wholemeal

flour LA by pHstat (b) (µmol FFA.h-1.g-1dm)

LA by Drapron and Sclafani method (b) (µmol FFA.h^-1.g^-1dm) Figure 2

Correlation between the lipase activities determined by the pHstat method (b) and the method of Drapron and Sclafani (1969) in bran, germ, wholemeal and flour.

(The values are the mean of 5 determinations ± standard deviation).

Corrélation entre les activités lipolytiques mesurées selon la méthode pHstat (b) et celle de Drapron et Sclafani (1969) dans le son, le germe, le blé entier et la farine.

(Les valeurs sont la moyenne de 5 déterminations ± l’écart type).

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are similar to those given for stored cereals by Joffe and Small (1964) who have established that the rate of increase of fat acidity increased when the moisture and temperature conditions increased during storage. Similarly, Drapron and Sclafani (1969) have shown that for low A_w, LA in flour was directly related to its the sorption isotherm. LA in wheat flour was measurable at an A_w as low as 0.025 and increased proportionnaly to the A_w between 0.025 and 0.25 (Caillat and Drapron, 1974; Drap- ron, 1986). The same authors found a maximum of wheat LA when A_w was close to 0.8 (Drapron and Sclafani, 1969; Caillat and Drapron, 1974; Drapron, 1986).

On the same bran samples, LA was also measured by the method of Drap- ron and Sclafani (1969). When the values obtained by this method are compared to those given by the pH method, an excellent correlation was found whatever the A_w and the temperature tested between 0.17 and 0.87 from the one hand and 30 and 40°C from the other hand (Figure 4).

0 0.2 0.4 0.6 0.8 1.2

1

0 0.2 0.4 0.6 0.8 1

Lipase activity (µmol FFA.h-1.g-1dm)

A_w

30°C 40°C

Figure 3

Effect of A_w on the lipase activity of bran incubated at 30 and 40°C. LA was measured by the pHstat method (b). (The values are the mean of 2 determinations).

Effet de l’A_w sur l’activité lipolytique du son incubé à 30 et 40 °C. L’activité lipolytique a été mesurée par la méthode pHstat (b). (Les valeurs sont la moyenne de 2 déterminations).

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3.3 Effect of exogenous lipase

Exogenous lipase was added to the wheat flour in order to increase its LA and check the validity of the method for different LA present in wheat flour. The amount of added lipase was varied between 0 and 100 LU.g^-1. The amounts of FFA released after 72 h of incubation increased when the exogenous lipase was increased untill 40 LU.g^-1 and then reached a plateau for higher values of exogenous lipase (Figure 5a). Similar results were obtained when the same samples were analysed by the method of Drapron and Sclafani (1969) as shown by the good correlation coefficient (Figure 5b). In addition, the percentages of satu- rated or unsaturated FFA were not modified whatever the levels of exogenous lipase added (data not shown) which means that there is no difference of spe- cificity for the wheat and exogenous lipases. This observation is in agreement with the results obtained by Castello et al. (1998). Moreover, compared to the fatty acid composition of olive oil, there was no difference in the FFA released by lipase hydrolysis. This indicates that no oxidation of lipid occured during the incubation and therefore that BHT was efficient during the 72 hours incubation even when high amounts of FFA were released.

0.0 0.2 0.4 0.6 0.8 1.0 1.2

X 40°C O 30°C y = 1.01 x

R² = 0.96

LA by pHstat (b) (µmol FFA.h-1.g-1dm)

LA by Drapron and Sclafani method (µmol FFA.h^-1.g^-1dm) Figure 4

Correlation between the lipase activities determined by the pHstat method (b) and the method of Drapron and Sclafani (1969) in bran incubated at 30 and 40°C

and different A_w. (The values are the mean of 2 determinations).

Corrélation entre les activités lipolytiques déterminées par la méthode pHstat (b) et celle de Drapron et Sclafani (1969) dans le son incubé à 30 et 40 °C pour différentes A_w. (Les valeurs sont la moyenne de 2 déterminations).

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0.05

0.03 0.07 0.09 0.11

0 50 100 150

A

0.04 0.06 0.08 0.10 0.12

0.00 0.05 0.10 0.15

(0 LU)

(20 LU)

(80 LU)

(40 LU) (60 LU)

(100 LU) B

y = 1.01 x R² = 0.96

LA by pHstat (b) (µmol FFA.h-1.g-1dm)LA by pHstat (b) (µmol FFA.h-1.g-1dm)

LA by Drapron and Sclafani method (b) (µmol FFA.h^-1.g^-1dm) Exogenous lipase added (LU.g^-1dm)

Figure 5a

Lipase activity determined by the pHstat method (b) in wheat flour supplemented by different amounts of exogenous lipase and incubated 72 hours at A_w 0.8 and 30°C.

Activité lipolytique déterminée par la méthode pHstat (b) dans la farine de blé supplémentée par différentes quantités de lipase exogène et incubée

pendant 72 h à 30 °C et une A_w de 0,8.

Figure 5b

Correlation between the lipase activities determined by the pHstat method (b) and the method of Drapron and Sclafani (1969). (The values are the mean of 2 determinations).

Corrélation entre les activités lipolytiques déterminées par la méthode pHstat (b) et celle de Drapron et Sclafani (1969). (Les valeurs sont la moyenne de 2 déterminations).

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4 – CONCLUSIONS

During storage and transformation of cereal and foodstuffs in general, lipase plays an important role. Moreover, wheat flours or other cereal milled fractions may contain additives which can affect their stability during storage such as exogenous lipase. Therefore, it would be of interest to study the influence of the storage conditions such as A_w and temperature on the LA in these mixes. It is the reason why the possibility to determine LA in the milled cereal fractions by a rapid and reliable technic presents a great interest for the industry. For this purpose, the pHstat method that we propose can be used to simplify the method of Drapron and Sclafani (1969).

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