Simplified method for the determination of lipolytic ...

SCIENCES DES ALIMENTS, 23(2003) 209-221

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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 produitsde mouture des céréales est décrite. Celle-ci est basée sur la titration à l’aide d’unpHstat de la quantité d’acides gras libérés par l’activité lipolytique lors de l’incuba-tion d’un mélange d’un produit de mouture délipidé (grain entier ou fraction demouture) 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 ontété comparés systématiquement à ceux donnés par la méthode classique demesure 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 ontété utilisées dans différentes conditions expérimentales au cours desquelles leseffets 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 bléentier), l’activité lipolytique déterminée par la méthode pHstat est corrélée à celleobtenue par la méthode GC (r = 0,99). Par le pHstat, l’activité lipolytique mesuréeest égale à 0,054, 0,75, 0,29 et 0,51 µmol.h

-1

.g

-1

(ms) vs 0,053, 0,79, 0,27 et0,48 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 activitéslipolytiques mesurées à l’aide du pHstat sont corrélées à celles déterminées parGC, à la suite d’une modification de l’A

w

(de 0,17 à 0,87) ou de la température (30à 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 lipolytiqueplus rapide que la méthode classique peut présenter un grand intérêt tant pouraméliorer les conditions de stockage des produits de mouture des céréales quepour juger de l’efficacité de traitements thermiques visant à la dénaturation del’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 : [email protected]

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

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SUMMARY

A new method for the determination of lipolytic activity (LA) in milled prod-ucts from cereals is described. This method is based on the use of thepHstat titration of free fatty acids (FFA) released by LA after incubating amixture of defatted wholemeal or milling wheat fraction and olive oil during72 hours at selected conditions of temperature and activity of water (A

w

).The results given by this method have been compared systematically tothose given by the time-consuming and classic one of the determination ofthe LA in low moisture media based upon the FFA determination by gaschromatography (GC) after separation by thin layer chromatography. Thetwo methods have been used under various experimental conditions in orderto study the effects of temperature, A

w

and addition of exogenous lipase indifferent wheat milled products. For the germ, bran, flour and whole mealfractions at 30°C and A

w

= 0.8, LA determined by the titration pHstatmethod 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 conditionswere modified, LA determined by pHstat was correlated with LA obtained byGC either when A

w

was varied for the bran fraction between 0.17 and 0.87or temperature between 30 and 40°C (r = 0.95) or after addition of exoge-nous lipase to wheat flour (r = 0.97). This less time-consuming method forthe determination of LA may be of a great interest to improve the storageconditions of cereal milled products as well as to estimate the efficiency ofheat 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 animportant parameter for numerous cereal products since lipases are able tofacilitate the initiation of the oxidative rancidity catalysed by lipoxygenase. Aftermilling, in the beginning of storage of wheat flour, a slight hydrolysis of the lipidfraction followed by the free polyunsaturated fatty acid oxidation by wheatlipoxygenase improved the baking performance of flour (Bellenger and Godon,1972; Berger, 1991; Castello

et al.

, 1998). Conversely, a long term storage offlour resulted in higher levels of hydrolysis and oxidation of the lipid fractionwhich lead to rancid products with decreased baking performance (Cuendet,1954; Gracza, 1965; Warwick

et al.

, 1979).

Actual nutritional recommendations for an increasing consumption of dietaryfiber, polyphenols, minerals and vitamins, promote an increasing demand forcereal wholemeal, bran and germ enriched products. After milling and due totheir 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

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and Galliard, 1988), these products are unstable and their sensory and nutritivevalues may be rapidly lost following unsuitable storage conditions. During mix-ing of wheat flour, the lipoxygenase acts on the the polyunsaturated fatty acidsreleased by lipase and catalyses their peroxidation by oxygen to fatty acidhydroperoxides responsible of a large cascade of co-oxidation reactions whichaffect the quality of the final product (Brockman and Acker, 1977; Nicolas andDrapron, 1983; Nicolas and Potus, 1994).

Nowadays, industry used the fat acidity measurement to evaluate thehydrolysis level in the lipid fraction of cereal products. This method is rapid butgives only an idea of the general quality of the product without any informationconcerning its LA (Guilbot, 1960; Drapron and Berger, 1976). Fat acidityincreases during storage due to the LA present in the milled products or in thecontaminating micro-organisms (Gracza, 1965; Galliard, 1983). This variation ofthe 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 theevolution of these products.

The LA in cereal products is not measurable by direct methods due to theirlow values in low or high moisture conditions (Drapron, 1985 and 1986). In1969, Drapron and Sclafani have developped a method to quantify the LA inlow moisture media in selected conditions close to those used for the storageof cereal products. In this method, the defatted milled cereal products wereincubated with a substrate (olive oil) in definite conditions of temperature andactivity of water (A

w

). The free fatty acids (FFA) released by hydrolysis of thetriglycerides of olive oil were extracted, separated by thin layer chromatography(TLC) and quantified by gas chromatography (GC) in the presence of an internalstandard. This technic is very specific but is time-consuming.

The purpose of this work was to develop a technic based on similar condi-tions of incubation but less-time-consuming for the estimation of the FFA. Inthis new method, FFA are quantified by a pHstat titration avoiding the two chro-matographic steps (TLC and GC). The results obtained by this new methodwere 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 byProlabo (Paris, France). Olive oil was a commercial pure virgin olive oil and wasused without any further treatment. Lipase Novozym 677 BG was obtained fromNovonordisk (Bagsgvaerd, Denmark) with an activity of 40000 lipase units (LU)per gram. According to the Novo datasheet, 1 LU corresponds to the amount ofenzyme that releases 1 µmol of titratable butyric acid per minute from tributyrinunder 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 – wereprovided by Les Moulins Soufflet (Nogent-sur-Seine, France). Wheat grainswere milled (< 0.5 mm) using a laboratory mill (Chopin, France) in frozen condi-tions. All these milled fractions were stored at – 20°C before use. In these con-ditions, their LA remained constant for at least six months.

2.3 Moisture content

The moisture content was determined according to the ISO 712 norm, usinga 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 fractionsrespectively.

2.4 Delipidation

The milled fractions were defatted by extraction with

n

-hexane at room tem-perature. Three successive extractions were carried out during 2

×

60 min and1

×

90 min by stirring (3

×

200 mL of

n

-hexane for 10 g of sample). The sampleswere dried under ventilated host in order to remove the traces of solvent. Thedefatted 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 mgof olive oil previously diluted with 10 mL of

n

-hexane containing butylatedhydroxytoluene (0.01%) to avoid oxidation of the polyunsaturated fatty acids.0.1 mg of heptadecanoic acid (C

17

) in 1 mL of

n-

hexane was added to the mix-ture as an internal standard. After mixing, the solvent was eliminated by evapo-ration under host at room temperature. In routine conditions, the mixture wasincubated during 72 h at 30°C into sorption tubes under vacuum maintained atan A

w

of 0.8 by a sulfuric acid solution suitably diluted. In some experimentswith the bran fraction, the incubation conditions were varied either by changingthe A

w

between 0.17 and 0.87 (by variation of the sulfuric acid concentration) orby increasing the temperature at 40°C. In other experiments with wheat flour,an exogenous lipase (Lipase Novozym 677 BG) was added to the wheat flourbefore the mixture preparation. Different doses of exogenous lipase were testedbetween 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 centrifugetube with 25 mL of

n

-hexane to extract the lipids. After 10 min of stirring, thesamples were centrifuged for 10 min at 500

×

g, the organic liquid phase con-taining lipids was collected and evaporated under vacuum at 30°C using arotavapor.


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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 / formicacid (70: 30: 1) to separate the different classes of lipid. After 1 h, the plateswere observed under UV light after spraying with primuline (0.01 %).

Preparation and analysis of fatty acid methyl ester

. The bands correspondingto the FFA were scrapped into screw-capped tubes. FFA were resuspended in3 ml of borontrifluoride methanol. The sealed tubes were then heated for 15 minat 70°C to convert FFA into fatty acid methyl esters (FAME). After cooling, 2 mL ofwater and 3 mL of pentane were added to extract the FAME. After collecting thepentane fraction, the solvent was evaporated under a nitrogen flow and FAMEwere solubilized with 100 µL of heptane. The samples were stored at – 20°C priorto GC analysis. FAME were separated and quantified using a GC STAR 3400CXgas chromatograph (Varian, Les Ulis, France) equipped with a flame ionizationdetector 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 and270°C, respectively. The flow rates were 30 and 300 mL.min

-1

for H

2

and air in thedetector. The column flow rate was 1.5 mL.min

-1

and the septum purge flow rate40 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, theextracted FAME were quantified following the procedure described by Castello

etal.

(1998).

2.7 Lipolytic activity measurement by pHstat titration

The method is adapted from the ISO/DIN 73025 norm for the determinationof fat acidity in cereals. Automatic titration was carried out using pHstat (TitrinoSM 702, Metrohm) linked to a computer for data analysis. The modificationincluded 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 pres-ence 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 centrifugetubes with 20 mL of

n

-hexane or ethanol (95% vol) and stirred for 10 min. Aftercentrifugation (15 min at 500

×

g), the hexane extract was evaporated usingrotavapor 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 thepHstat 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 contentwere 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 X

0

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 Xt X0–( ) t⁄∑=


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extract by ethanol, is given in figure 1. In each method, assays for the LA deter-mination of the milled fractions in standard conditions (30°C and A

w

= 0.8) havebeen repeated 5 times whereas assays of LA in various conditions of incubationhave 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 + C17:0 (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 milledcereals, it is important to be able to evaluate rapidly its activity. In commercialproducts, only the test of acidity (ISO 7305) is used to estimate the fat acidity ofthe milled fractions. This test presents the advantage to be rapid but cannotallow to estimate the lipase activity.

To our knowledge, the method of Drapron and Sclafani (1969) is the onlyone which is reliable for the measurement of LA in low moisture media. How-ever, due to the two chromatographic steps needed to quantify the FFAreleased during incubation, this method is time-consuming. To simplify this partof 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 notchanged. The amounts of FFA were quantified by TLC and GC according to themethod of Drapron and Sclafani (1969) from the one hand, and by pHstat titra-tion, after evaporation of

n

-hexane and redissolution of the lipid extract in etha-nol (method pHstat a), from the other hand (Figure 1). For the comparison of thetwo methods, four different milled wheat products, wheat flour, germ, bran andwholemeal, were incubated during 72 h at 30°C and an A

w

of 0.8, conditionsproposed by Drapron and Sclafani (1969). When the results given by themethod pHstat a were plotted versus those given by the method of Drapronand 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 0.98indicate that similar values of LA are obtained by the two methods whatever thesample tested.

In a second step and from the same samples, FFA directly extracted by eth-anol were quantified by pHstat titration (method pHstat b). The results obtainedby this method are given in table 1 together with those given by the two othermethods. 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 theweakest 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, respec-tively. Therefore for the routine conditions of incubation selected by Drapronand Sclafani (1969), it is possible to determine accurately the LA of these milledfractions using a simplified method (pHstat method b). Our results are also inagreement with those obtained by Galliard (1986) who showed that among thewheat milled fractions, LA was highest in the bran fraction.

3.2 Influence of A

w

and temperature

Since LA is very dependent on Aw and temperature, the validity of the pHstatmethod has been checked when these two parameters of incubation were varied.For this purpose, the bran fraction was incubated under varied Aw between 0.17 and0.87 at 30 and 40°C. Whatever the temperature tested, LA increased when Awincreased from 0.17 to 0.74 and then decreased at an Aw of 0.87 (Figure 3). In addi-tion, for a same value of Aw, 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 ± 0.08a 0.28 ± 0.03b 0.49 ± 0.01c 0.052 ± 0.003d

pHstat method a 0.75 ± 0.05a 0.30 ± 0.09b 0.50 ± 0.09c 0.052 ± 0.002d

pHstat method b 0.76 ± 0.05a 0.29 ± 0.05b 0.51 ± 0.05c 0.050± 0.003d

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

pH

stat

(b

) (µ

mo

l FF

A.h

-1.g

-1d

m)

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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Simplified method for the determination of lipolytic activity in low moisture media 217©

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are similar to those given for stored cereals by Joffe and Small (1964) who haveestablished that the rate of increase of fat acidity increased when the moisture andtemperature conditions increased during storage. Similarly, Drapron and Sclafani(1969) have shown that for low Aw, LA in flour was directly related to its the sorptionisotherm. LA in wheat flour was measurable at an Aw as low as 0.025 and increasedproportionnaly to the Aw between 0.025 and 0.25 (Caillat and Drapron, 1974; Drap-ron, 1986). The same authors found a maximum of wheat LA when Aw was close to0.8 (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 com-pared to those given by the pH method, an excellent correlation was foundwhatever the Aw and the temperature tested between 0.17 and 0.87 from theone 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

Lip

ase

acti

vity

(µm

ol F

FA

.h-1

.g-1

dm

)

Aw

30°C

40°C

Figure 3

Effect of Aw 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’Aw 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 LAand check the validity of the method for different LA present in wheat flour. Theamount of added lipase was varied between 0 and 100 LU.g-1. The amounts ofFFA released after 72 h of incubation increased when the exogenous lipase wasincreased untill 40 LU.g-1 and then reached a plateau for higher values of exog-enous lipase (Figure 5a). Similar results were obtained when the same sampleswere analysed by the method of Drapron and Sclafani (1969) as shown by thegood correlation coefficient (Figure 5b). In addition, the percentages of satu-rated or unsaturated FFA were not modified whatever the levels of exogenouslipase added (data not shown) which means that there is no difference of spe-cificity for the wheat and exogenous lipases. This observation is in agreementwith the results obtained by Castello et al. (1998). Moreover, compared to thefatty acid composition of olive oil, there was no difference in the FFA releasedby lipase hydrolysis. This indicates that no oxidation of lipid occured during theincubation and therefore that BHT was efficient during the 72 hours incubationeven when high amounts of FFA were released.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0 0.2 0.4 0.6 0.8 1.0 1.2

X 40°CO 30°C

y = 1.01 x

R2 = 0.96

LA

by

pH

stat

(b

) (µ

mo

l FF

A.h

-1.g

-1d

m)

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 Aw. (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 Aw. (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) By = 1.01 x

R2 = 0.96

LA

by

pH

stat

(b

) (µ

mo

l FF

A.h

-1.g

-1d

m)

LA

by

pH

stat

(b

) (µ

mo

l FF

A.h

-1.g

-1d

m)

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 Aw 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 Aw 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, lipaseplays an important role. Moreover, wheat flours or other cereal milled fractionsmay contain additives which can affect their stability during storage such asexogenous lipase. Therefore, it would be of interest to study the influence of thestorage conditions such as Aw and temperature on the LA in these mixes. It isthe reason why the possibility to determine LA in the milled cereal fractions by arapid and reliable technic presents a great interest for the industry. For this pur-pose, the pHstat method that we propose can be used to simplify the methodof Drapron and Sclafani (1969).

REFERENCES

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Simplified method for the determination of lipolytic ...

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