TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of preparing an oil-in-water emulsion, said method comprising combining oil, water, pulse flour and optionally further ingredients. Examples of oil-in-water emulsions that can suitably be produced by the present method include mayonnaise and dressings. The invention also relates to an oil-in-water emulsion that is obtained by the

aforementioned method.

BACKGROUND OF THE INVENTION

The emulsion stability of oil-in-water emulsions is affected adversely by a number of different changes that may occur in the structure of these emulsions as time progresses.

There are several ways in which the structure of an emulsion of liquid droplets in a continuous medium can change: 1 . Creaming/Sedimentation: No change in droplet size (or droplet size distribution), but build-up of an equilibrium droplet concentration gradient within the emulsion. This phenomenon results from external force fields, usually gravitational, acting on the system. "Creaming" is the special case in which the droplets collect in a concentrated layer at the top of an emulsion. "Sedimentation" occurs when the droplets collect in a concentrated layer at the bottom of the emulsion.

2. Flocculation: Again, no change in basic droplet size or distribution but the build-up of aggregates of droplets within the emulsion. The individual droplets retain their identity. This process of flocculation results from the existence of attractive forces between the droplets.

3. Coalescence: Flocculated droplets in the bulk of the emulsion, or alternatively,

droplets within a close-packed array resulting from sedimentation or creaming, coalesce to form larger droplets. This results in a change of the initial droplet size distribution. The limiting state here is the complete separation of the emulsion into the two immiscible bulk liquids. Coalescence thus involves the elimination of the thin liquid film (of continuous phase) which separates two droplets in contact in an aggregate or a close-packed array. The forces to be considered here are therefore the forces acting within thin-liquid films in general. 4. Ostwald ripening: An alternative way in which the average droplet size in an emulsion can increase, without the droplets coalescing, occurs if the two liquids forming the disperse phase and the continuous phase, respectively, are not totally immiscible. This is the case in reality because all liquid pairs are mutually miscible to some finite extent. If one starts with a truly monodisperse emulsion system, then no effects arising from this mutual solubility will arise. However, if the emulsion is polydisperse, larger droplets will form at the expense of the smaller droplets owing to the process known as Ostwald Ripening. In principle, the system will tend to an equilibrium state in which all the droplets attain the same size (this may be, of course, that state when we have just one single large drop). The process of Ostwald ripening results from the difference in solubility between small and large droplets.

5. Phase inversion: A further way in which the structure of an emulsion may change is for the emulsion to "invert", e.g. for an o/w emulsion to change to a w/o emulsion. This may be brought about by a change in temperature or concentration of one of the components or by the addition of a new component to the system.

6. Syneresis: Yet another way in which emulsions may change is the separating off of one of the main liquid components of the emulsion. In oil-in-water emulsions both oil syneresis and water syneresis may occur.

When oil-in-water emulsions are stored for prolonged periods of time under varying temperature conditions, as is the case for retail products such as dressings and mayonnaise, the aforementioned destabilizing processes have to be slowed down. In order to achieve this, emulsifiers and water structuring agents are commonly employed in these emulsions.

Phospholipids are an example of an emulsifier that is widely used to stabilize oil-in-water emulsions. Egg yolk contains appreciable levels of phospholipids and is widely used as an oil-in-water emulsifier, e.g. in mayonnaise and dressings. Examples of water structurants include modified celluloses, starches (modified or non- modified), gums such as xanthan, agar, gelatin, carrageenan (iota, kappa, lambda), gellan, galactomannans (guar, tara, cassia, locust bean gum), konjac glucomannan, gum arabic, pectins, milk proteins, alginate, chitosan and cellulosic fibres.

WO 01/52670 describes a process of preparing a food product, the process comprising : (a) forming a mixture of a starch and protein containing pea or lentil flour and liquid,

wherein the flour starch is at least partially gelatinised and the protein flour is at least partially denatured and coagulated; and

(b) optionally allowing the mixture to set.

Protein coagulation is achieved by inclusion of a protein coagulating agent, especially a calcium or magnesium salt.

WO 2012/089448 describes a process of preparing an oil-in-water emulsion comprising 15-80 wt.% of a continuous aqueous phase and 20-85 wt.% of a dispersed oil phase, said process comprising:

• preparing an aqueous dispersion containing 0.1 -8 wt.% of finely ground pulse seed by mixing pulse flour and water and/or by shearing or milling a mixture of water and pulse seeds;

· adding oil to the aqueous dispersion to produce an oil-and-water mixture; and

• mixing the oil-and-water mixture to produce an oil-in-water emulsion comprising 80- 100 vol.% of oil droplets having a diameter of less than 10 μηι;

wherein 50-100 wt.% of the starch comprised in the aqueous dispersion is gelatinized prior to the addition of the oil. The examples of the international patent application describe the preparation of a mayonnaise using flours of lentil, black gram, mung bean and chick pea.

SUMMARY OF THE INVENTION

The inventors have found that the stability of oil-in-water emulsions containing ground pulse seed (pulse flour) can be improved significantly if at least a part of the pulse flour used in the preparation of the emulsion has previously been subjected to a heat treatment. More particularly, it was found that native (non-heat-treated) pulse flour can be replaced by heat-treated pulse flour to reduce syneresis without adverse effect on product texture. Whereas native pulse flour typically has a lipoxygenase activity well in excess of 20 U per gram of flour, the lipoxygenase activity in the heat-treated pulse flour has been reduced to less than 10 U per gram of flour as a result of the heat treatment.

Thus, the present invention provides a method of preparing an oil-in-water emulsion having an oil content of 5-69 wt.% and a water content of 30-92 wt.%, said method comprising combining the following ingredients in the indicated amounts:

• 5-69 parts by weight of oil;

• 30-92 parts by weight of water;

• 0.5-15 parts by weight of heat-treated pulse flour having a mass weighted

diameter of 10-500 μηι and a lipoxygenase activity of less than 10 U/g;

• 0-14.5 parts by weight of native pulse flour having mass weighted diameter of 10- 500 μηι and a lipoxygenase activity of more than 20 U/g and

• 0-30 parts by weight of one or more further ingredients;

wherein the combined amount of the heat-treated pulse flour and the native pulse flour is in the range of 1 to 15 parts by weight.

Although the inventors do not wish to be bound by theory, it is believed that the heat- treated pulse flour employed in accordance with the present invention differs from native (non heat-treated) pulse flour in that the pulse proteins contained therein have lost at least some of their capacity to build protein bridges between adjacent oil droplets. These protein bridges contribute substantially to the firmness of oil-in-water emulsions. Thus, heat-treated pulse flour can be applied to reduce syneresis in oil-in-water emulsions, be it that the heat-treated pulse flour contributes substantially less to the firmness of the emulsion than in the case native pulse flour is used. Consequently, the heat-treated pulse flour can be used to prepare oil-in-water emulsion that combine high stability against syneresis with excellent texture.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, one aspect of the present invention relates to a method of preparing an oil-in- water emulsion having an oil content of 5-69 wt.% and a water content of 30-92 wt.%, said method comprising combining the following ingredients in the indicated amounts:

• 5-69 parts by weight of oil;

• 30-92 parts by weight of water;

· 0.5-15 parts by weight of heat-treated pulse flour having a mass weighted

diameter of 10-500 μηι and a lipoxygenase activity of less than 10 U/g; • 0-14.5 parts by weight of native pulse flour having mass weighted diameter of 10-500 μηι and a lipoxygenase activity of more than 20 U/g and

• 0-30 parts by weight of one or more further ingredients;

wherein the combined amount of the heat-treated pulse flour and the native pulse flour is in the range of 1 to 15 parts by weight.

The term "pulse" as used herein refers to an annual leguminous crop yielding from one to twelve seeds of variable size, shape, and colour within a pod and is reserved for crops harvested solely for the dry seed. This excludes fresh green beans and fresh green peas, which are considered vegetable crops. Also excluded are crops that are mainly grown for oil extraction (oilseeds like soybeans and peanuts), and crops which are used exclusively for sowing (clovers, alfalfa). Just like words such as "bean" and "lentil", the word "pulse" may also refer to just the seed, rather than the entire plant The term "pulse flour" as used herein refers to a finely ground seed. The pulse flour is suitably produced by milling or grinding dehulled or non-dehulled pulse seeds. The pulse seeds may be milled or ground as such, or they may be milled or ground in the presence of water, e.g. to produce an aqueous slurry or paste. Unless indicated otherwise, the term "pulse flour" as used herein refers to heat-treated pulse flour as well as native pulse flour. The term "pulse flour", unless indicated otherwise, may also refer to combinations of two or more pulse flours.

The term "heat-treated pulse flour" as used herein refers to a pulse flour that has been subjected to a heating regime that has reduced lipoxygenase activity to less than 10 U per g.

Whenever reference is made herein to an enzyme activity that is expressed in Units per gram (U/g), unless indicated otherwise, this equates to the Units of enzyme activity per gram of flour.

The term "native pulse flour" as used herein refers to a pulse flour that has not been heat- treated as evidenced by a lipoxygenase activity of more than 20 U per g. The term "starch" as used herein, unless indicated otherwise, refers to starch that has not been chemically or enzimatically modified (e.g. by chemical reactions such as esterification or enzymatic hydrolysis, respectively). Starch consists of two types of molecules: the linear and helical amylose and the branched amylopectin.

The term "gelatinized starch" as used herein refers to starch that has undergone gelatinization. Starch gelatinization is a process that breaks down the intermolecular bonds of starch molecules in the presence of water and heat, allowing the hydrogen bonding sites to engage more water. This irreversibly dissolves the starch granule.

Penetration of water increases randomness in the general starch granule structure and decreases the number and size of crystalline regions. Under the microscope in polarized light starch loses its birefringence and its extinction cross during gelatinization. Some types of unmodified native starches start swelling at 55 °C, other types at 85 °C. The gelatinisation temperature of the starch is in general influenced by the fine structure of the amylopectin. The term "protein" as used herein refers to a linear polypeptide comprising at least 10 amino acid residues. Preferably, said protein contains more than 20 amino acid residues. Typically, the protein contains not more than 35,000 amino acid residues.

The term "albumin" as used herein refers to a protein that is soluble in water and in moderately concentrated salt solutions and that experiences heat coagulation. Reference is made to the Osborne protein classification system (T.B. Osborne, The Vegetable Proteins, Monographs in Biochemistry, London; Longmans, Green and Co., 1924).

The term "globulin" as used herein refers to a protein that is insoluble in water, but soluble in saline solutions.

The term "oil" as used herein refers to lipids selected from the group of triglycerides, diglycerides, monoglycerides, phospholipids and free fatty acids. The term "oil" encompasses lipids that are liquid at ambient temperature as well as lipids that are partially or wholly solid at ambient temperature.

The term "dietary fiber" as used herein refers to indigestible non-starch polysaccharides such as arabinoxylans, cellulose, lignin, pectins and beta-glucans. The term "phospholipid" as used herein refers to a lipid comprising a glycerol bound to one or two fatty acids and a phosphate group. The term "sugars" as used herein refers to mono- and disaccharides.

The term "diameter" as used herein in relation to the droplet size of the dispersed oil phase, unless otherwise specified, refers to the diameter as determined with the help of confocal laser scanning microscopy.

Lipoxygenase (LOX) activity as referred to herein is determined using the methods of Ben-Aziz (A. Ben Aziz et al., Linoleate oxidation induced by lipoxygenase and heme proteins: A direct spectroscopic assay. Anal. Biochem. 34, p. 88-100 (1 970)) with

200 nmol of linoleic acid, 0.2 mg Tween-20 and 10 μηιοΙ Tris-HCI pH 7 in 1 ml, 25 °C. Reaction is started with the addition of 10 μΙ linoleic acid from stock solution (20 mM in ethanol). The changes in absorbance at 234 nm are recorded with Shimadzu UV-1 60 spectrophotometer. The molar extinction coefficient for the conjugated diene is

25 mM ^" -cm ^"1 . The specific activity of LOX is expressed in units (U) defined as: 1 unit of LOX activity is the amount of enzyme that catalyses the conversion of 1 μηιοΙ linoleic acid per minute (λ 234 nm, 25 °C, 1 cm cuvet).

Peroxidase (PO) activity as referred to herein is measured by monitoring the increase in the absorbance at 414 nm with 2,2'-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid

(ABTS) as substrate according to Shindler and Bardsley (J.S. Shindler et al., Steady state kinetics of lactoperoxidase with ABTS as chromogen., Biochem. Biophys. Res. Commun. 67, p. 1307 (1975)). The reaction mixture consists of 2 mM - ABTS and 1 mM H ₂ 0 ₂ in 50 mM Na-acetate buffer pH 5, 25 °C. Reaction is started with the addition of 10 μΙ 0.1 M H ₂ 0 ₂ . The optical density (OD/absorbance) at 414 nm is recorded in time for 3 min. The specific activity of PO is expressed in units (U) defined as: 1 unit of peroxidase activity is the amount of enzyme that catalyses the conversion of 1 μηιοΙ ABTS per minute under the indicated conditions. The molar extinction coefficient for ABTS is 31 .1 mM ^"1 -cm ^"1 (λ 414 nm, 25 °C, 1 cm cuvet).

Esterase activity as referred to herein is determined by monitoring the increase in the absorbance at 410 nm with p-nitrophenyl acetate (pNA) as substrate based on the method as described by Khalameyzer et al. (Khalameyzer et al., Appl. Environ. Microbiol. 65 (2), p. 477-482 (1999)). The measurement is performed using a 96 well plate and in each well 150 or 1 80 μΐ phosphate buffer (50 mM, pH 7.4) and 20 or 50 μΐ extract (enzyme) preparation is pipetted. The enzymatic reaction starts after adding 40 μΐ 10 mM pNA (dissolved in DMSO) using a multipipette. The final volume per well is 240 μΙ_. The absorbance is measured at 410 nm for 120 seconds at 25 °C. The blank represents the autohydrolysis of pNA without enzyme. Esterase activity (U): one unit will hydrolyze 1 .0 μηιοΙ of p-nitrophenol (pNP) per minute at pH 7.4 at 25 °C. For the conversion of OD values into μηιοΙ product an extinction coefficient of 41 OD per μηιοΙ pNP was used.

The pulse flour (heat-treated pulse flour as well as native pulse flour) that is employed in accordance with the present invention may be obtained from dehulled and/or non- dehulled pulse seed. The water-structuring and emulsifying properties of the pulse flour are believed to be largely attributable to the starch and protein components. Since the hulls of pulse seed predominantly consist of dietary fibre, dehulling does not significantly affect the functionality of the pulse flour in the present emulsion. Preferably, the pulse flour employed is obtained from dehulled pulse seed. The water content of the heat-treated or native pulse flour typically does not exceed 20 wt.%. More preferably the water content of the pulse flour does not exceed 1 5 wt.%. Most preferably, the water content of the pulse flour does not exceed 1 0 wt.%

The starch content of the pulse flour typically is within the range of 20-75 wt.%, more preferably in the range of 25-70 wt.% and most preferably in the range of 30-60 wt.%.

Typically, the protein content of the pulse flour is in the range of 1 0-40 wt.%, more preferably of 12-38 wt.% and most preferably of 1 5-35 wt.%. The pulse flour typically contains starch and protein in a weight ratio of 1 :2 to 5:1 , more preferably of 2:3 to 3:1 and most preferably of 1 :1 to 5:2.

Typically, the pulse flour employed in accordance with the present invention contains less than 25%, most preferably less than 20% of dietary fiber by weight of dry matter.

The oil content of the pulse flour preferably lies in the range of 0.3-1 2 wt.%. More preferably, the oil content is in the range of 0.5-1 0 wt.%, even more preferably in the range of 0.6-8 wt.% and most preferably in the range of 0.8-5 wt.% According to a particularly preferred embodiment, the pulse flour has the following composition, calculated on dry matter: 30-60 wt.% of starch;

1 -40 wt.% of dietary fiber;

0.5-12 wt.% of sugars;

15-35 wt.% of protein ;

0.3-1 2 wt.% of oil.

Typically, starch, dietary fiber, sugars, protein and oil together make up 90-1 00 wt.%, more preferably 95-1 00 wt.% of the dry matter contained in the pulse seed. Globulins and albumins typically represent a major part of the protein contained in the pulse flour. Accordingly, in a preferred embodiment, globulins and albumins represent at least 50 wt.%, more preferably 55-95 wt.% and most preferably 60-90 wt.% of the protein contained in the pulse flour. Emulsions of particular good quality can be obtained if the pulse flour contains globulins and albumins in a weight ratio that lies within the range of 1 0:1 to 1 :1 , or even more preferably in a weight ratio of 7:1 to 2:1 .

In accordance with another preferred embodiment the globulins legumin and vicilin together represent at least 35 wt.%, more preferably 40-75 wt.% and most preferably 45- 70 wt.% of the protein comprised in the pulse flour.

The content of globulin, albumin, legumin, vicilin, and glutelin in the pulse flour is suitably determined by the method described by Gupta & Dhillon [Gupta, R., & Dhillon, S. 1993. Characterization of seed storage proteins of Lentil {Lens culinaris M.). Annals of Biology, 9, 71 -78].

The pulse flour is advantageously obtained from a pulse selected from lentils, chickpeas, beans and combinations thereof. Even more preferably, the pulse flour is obtained from a pulse selected from lentils, chickpeas, mung beans and combinations thereof. Most preferably, the pulse flour is lentil flour.

The heat-treated pulse flour that is employed in the present method preferably has been subjected to serious heat treatment as evidenced by a lipoxygenase activity of less than 5 U/g , more preferably of less than 3 U/g and most preferably of less than 1 U/g. Also peroxidase activity in the heat-treated pulse flour has been reduced by the heat treatment that the flour has been subjected to. Typically, the heat-treated pulse flour has a peroxidase activity of less than 1 U/g, more preferably of less than 0.7 U/g and most preferably of less than 0.5 U/g.

Preferably, the heat treatment that the heat-treated pulse flour has undergone is not sufficient to remove all enzyme activity. Accordingly, it is preferred that the heat-treated pulse flour has an esterase activity of at least 0.2 U/g, more preferably of at least 0.4 U/g, most preferably of at least 0.5 U/g.

In the present method of preparing an oil-in-water emulsion the heat-treated pulse flour is preferably employed in an amount of 0.6 to 8 parts by weight, more preferably of 0.8 to 7 parts by weight and most preferably of 1 to 6 parts by weight. The heat-treated pulse flour is preferably employed in an amount of 0.6-14%, more preferably 1 -12% and most preferably 1 .5-10% by weight of the water that is contained in the oil-in-water emulsion.

In accordance with an embodiment of the present invention the heat-treated pulse flour has been heat-treated under conditions that cause most of the starch contained therein to become gelatinized. Preferably, at least 80 wt.% of the starch contained in the heat- treated flour is gelatinized.

In accordance with another embodiment the heat-treated pulse flour contains not more than a a limited amount, e.g. less than 40 wt.%, more preferably less than 20 wt.% and most preferably less than 10 wt.% of gelatinized starch. A heat-treated pulse flour containing virtually no gelatinized starch can suitably be produced by heating native pulse flour in the presence of not more than 50% water by weight of the starch that is contained in the pulse flour. More preferably, the native pulse flour is heated in the presence of less than 40% water by weight of the starch that is contained in the pulse flour for

temperatures in the range 60-80 °C and less than 25% water by weight of the starch contained in the pulse flour for temperatures in excess of 80 °C.

The heat-treated pulse flour may be produced by different heating methods. These methods may comprise heating of an aqueous suspension of native pulse flour followed by drying. Examples of suitable drying techniques that may be employed include spray drying, drum drying and moving belt drying. In case the drying yields large agglomerates of heat-treated flour particles, grinding or milling is preferably applied to produce heat- treated pulse flour having the desired particle size distribution. The heat-treated pulse flour may also be produced from native pulse flour using extrusion. Extrusion offers the advantage that little or no water needs to be used. The extrudate may be subjected to grinding or milling to produce heat-treated pulse flour having the desired particle size distribution. In accordance with a particularly preferred embodiment of the present invention, both heat-treated pulse flour and native pulse flour are employed in the preparation of the oil- in-water emulsion. The inventors have discovered that the use of a combination of heat- treated pulse flour and native pulse flour enables the preparation of O/W emulsions with a very pleasant creamy texture.

According to a preferred embodiment of the invention, the native pulse flour is employed in an amount of 0.1 to 6 parts by weight, more preferably of 0.3 to 5 parts by weight and most preferably of 0.5 to 4 parts by weight. The native pulse flour is suitably employed in an amount of 0.1 to 12%, more preferably 0.5 to 10% and most preferably 1 to 8% by weight of the water that is contained in the oil- in-water emulsion.

The native pulse flour employed in the present method preferably has a lipoxygenase activity of more than 30 U/g, most preferably of more than 40 U/g.

The peroxidase activity of the native pulse flour typically exceeds 0.8 U/g, more preferably it exceeds 1 U/g. Of the starch contained in the native pulse flour typically less than 20 wt.% , more preferably less than 10 wt.% and most preferably less than 5 wt.% of the starch is gelatinized.

In a particularly preferred embodiment of the present method a mixture of the native pulse flour and the water is heated to a temperature of more than 60 °C for more than 10 seconds, more preferably to a temperature of 70 °C for more than 10 seconds. Preferably, the heat-treated pulse flour and the native pulse flour are employed in a combined amount of 2-15%, more preferably 3-1 2% and most preferably 4-10% by weight of the water that is contained in the oil-in-water emulsion.

According to a preferred embodiment the heat-treated pulse flour and the native pulse flour are combined in a weight ratio that lies within the range of 1 :20 to 20:1 , more preferably in the range of 1 :3 to 10:1 . It is important that the pulse flour (heat-treated as well as native) employed in the present emulsion has been finely ground so that starch, protein and dietary fiber are easily released from the seed material when the flour is combined with the water. Preferably, the pulse flour (native or heat-treated) has a mass weighted average diameter of 12-200 μηι, most preferably of 15-120 μηι.

The pulse flour (native or heat-treated) preferably contains not more than 5 wt.% of flour particles having a diameter of 200 μηι or more, preferably of 1 50 μηι or more and most preferably of 120 μηι or more. The particle size distribution of pulse flour is suitably determined with the help of sieves.

The present method preferably comprises combining 10-60 parts by weight of oil, more preferably 15-50 parts by weight of oil with 40-90 parts by weight of water, more preferably 50-85 parts by weight water.

Typically, 80-100 vol.% of the oil droplets contained in the present emulsion have a diameter of less than 15 μηι, more preferably of 0.5-10 μηι.

Examples of the further ingredients that may be employed in the present method include acidulants, salts, sugar, spices, vitamins, flavouring, colouring, preservatives,

antioxidants, chelators, herbs and pieces of meat, vegetable or cheese. Preferably, these further ingredients, collectively, do not make up more than 20%, more preferably not more than 1 5% and most preferably not more than 1 0% by weight of the emulsion. According to another preferred embodiment the present method yields an emulsion containing 0.05-1 .0 wt.% of phospholipids. More preferably, phospholipids are present in the emulsion in a concentration of at least 0.1 %, more preferably of at least 0.15 wt.% and most preferably of at least 0.2 wt.%. Phospholipids may suitably be introduced into the emulsion by adding egg or an egg component. Typically, at least 0.05 parts by weight, more preferably 0.15 to 1 .0 parts by weight of egg lecithin are combined with the other ingredients in the preparation of the present emulsion. Here the term "egg lecithin" refers to phospholipids that originate from egg. Egg lecithin is preferably introduced in the emulsion by adding egg yolk.

Salt, notably NaCI and/or KCI, is preferably employed in the present method in an amount of 0.5-9% by weight of aqueous phase, more preferably of 1 .0-7.0% by weight of aqueous phase and most preferably of 1 .5-6.0% by weight of aqueous phase.

Sucrose is typically applied in the preparation of the oil-in-water emulsion in an amount of 1 -12% by weight of aqueous phase, more preferably of 2-10% by weight of aqueous phase.

According to a particularly preferred embodiment, the oil-in-water emulsion of the present invention is produced by:

• providing an aqueous dispersion containing at least 1 wt.% of pulse flour,

· adding oil to the aqueous dispersion to produce an oil-and-water mixture; and

• mixing the oil-and-water mixture to produce an oil-in-water emulsion comprising 80- 100 vol.% of oil droplets having a diameter of less than 20 μηι.

The mixing of the oil-in-water emulsion may be achieved, for instance, by homogenization in a high shear mixer (e.g. Silverson) or a rotor-stator mixer (e.g. colloid mill) or by high pressure homogenisation.

In the present method the aqueous dispersion is suitably prepared by mixing pulse flour (heat-treated pulse flour and the optional native pulse flour) with water and optionally further ingredients.

Preferably, the present method comprises the addition of an acidulant to adjust the pH of the aqueous dispersion to a pH within the range of less than 5.5, preferably to a pH of 2 to 5.5, more preferably to a pH of 3.0 to 5.0. According to a particularly preferred

embodiment, the acidulant is added, after the oil has been added to the aqueous dispersion, even more preferably after the oil-in-water emulsion has been produced by the mixing.

The acidulant employed in the present method is preferably selected from acetic acid, citric acid, lactic acid, malic acid, phosphoric acid, hydrochloric acid, glucono-delta-lactone and combinations thereof. Even more preferably, the acidulant is selected from acetic acid, citric acid and combinations thereof. Most preferably, the acidulant comprises acetic acid. In the above mentioned advantageous embodiment of the present method, the aqueous dispersion, the oil-and-water mixture or the oil-in-water emulsion are preferably heated using the following heating conditions:

• at 60-70 °C for at least 10 minutes; and/or

• at 70-80 °C for at least 2 minutes; and/or

· at 80-100 °C for at least 1 minute; and/or

• at 100-120 ^< Ό for at least 30 seconds; and/or

• at 120-150 °C for at least 10 seconds.

Depending on the heating temperature, the preferred times are as follows:

· 60-70 ^< Ό: 10-120 minutes

• 70-80 ^< Ό: 1 -80 minutes

• 80-100 ^< Ό 1 -70 minutes

• 100-120 ^< Ό: 30-1200 seconds

• 120-150 ^< Ό: 10-480 seconds

Advantageously, the present method comprises the step of heating the aqueous dispersion containing the pulse flour to gelatinize non-gelatinized starch contained therein. In case the aqueous dispersion contains native pulse flour, it is preferred to employ heating conditions that cause at least 50 wt.%, more preferably at least 79 wt.% of the starch contained in the native pulse flour to become gelatinized. After the heating of the aqueous dispersion typically 50-100 wt.%, more preferably 70-100 wt.% and most preferably 90-100 wt.% of the starch contained in the dispersion is gelatinized. Gelatinized starch is believed to enhance the emulsion stability by structuring the continuous aqueous phase of the emulsion. The extent to which the starch present in the emulsion is gelatinized can suitably be determined by cross polarised light microscopy. A particularly stable emulsion can be produced by the present method by combining the pulse flour(s) and the water and heating the resulting combination before adding the oil. Thus, in accordance with a particularly preferred embodiment, prior to the addition of oil, the combination of pulse flour(s) and water is heated to a temperature of more than 60 °C for at least 10 seconds. According to a particularly preferred embodiment of the present method the heat-treated pulse flour, the optional native pulse flour and water are combined and the combination is heated prior to the addition of oil using the

aforementioned heating conditions. In case phospholipids are added in the preparation of the present oil-in-water emulsion, it is preferred to do so after the oil-in-water emulsion has been subjected to the heat treatment. Furthermore, it is preferred to add the phospholipids after the oil-in-water emulsion has been acidified. As described in WO 01/52670, divalent metal ions, such as Ca ²⁺ and Mg ²⁺ may induce protein gelation. In order to prevent this kind of protein gelation, it is preferred that the aqueous phase of the present emulsion comprises less than 1 .0 mmol per gram of protein, more preferably less than 0.5 mmol per gram of protein of divalent metal cation selected from Ca ²⁺ , Mg ²⁺ and combinations thereof. According to another preferred embodiment the present emulsion is not in the form of a gel (as opposed to the products described in WO 01 /52670).

The emulsion of the present invention preferably contains no modified starch or modified cellulose. The term "modified starch" as used herein refers to an enzymatically or chemically modified starch. Likewise, the term "modified cellulose" as used herein refers to an enzymatically or chemically modified cellulose.

The emulsions produced by the present method typically are pourable or spoonable as opposed to solid. In case the present emulsion is non-pourable, it is preferred that the consistency of the emulsion is such that it cannot be cut into two parts that remain separate but will confluence after the cutting.

The present emulsion typically has a Stevens value at 20 °C of 35-300, more preferably of 50-250 and most preferably of 70-200. The Stevens value, expressed in grams, can be determined by using a typical mayonnaise grid in a Stevens LFRA Texture Analyzer (ex. Stevens Advanced Weighing Systems, UK) with a maximum load/measuring range of 1000 grams and by applying a penetration test of 20 mm at 1 mm/s penetration rate in a cup having a diameter of 65 mm. The mayonnaise grid comprises square openings of approximately 3x3 mm, is made up of wire with a thickness of approximately 1 mm and has a diameter of 40 mm.

The oil-in-water emulsion of the present invention preferably has a storage modulus G', measured at 20 °C, within the range of 100-3,500 Pa, most preferably in the range of 800-2,000 Pa. The viscosity of the present emulsion typically lies in the range of 100-80,000 mPa.s, more preferably in the range of 200-30,000 mPa-s at 10 s ^" and 20°C.

The G' and viscosity of the emulsion are measured using a standard protocol with the following 3 consecutive steps:

· The sample is rested for 3 minutes after the introduction into the rheometer to allow relaxation of the stresses accumulated due to the loading of the sample.

• A stress sweep as applied in which the oscillatory stress is increased from 0.1 to

1768 Pa in logarithmic steps (15 per decade). This step is terminated when the phase angle exceeds 80 °. From this step the G' (storage modulus) is taken as described below.

• A viscosity measurement is done at a shear rate of 50 s ^" for a total of 1 minute. A viscosity point is measured every 10 seconds. Typically the last point is reported. The test is carried out at 20 °C using a cone and plate rheometer. The cone used has a diameter of 4 cm and a cone angle of 2° degrees.

The storage modulus G' is the mathematical description of an object's or substance's tendency to be deformed elastically (i.e., non-permanently) when a force is applied to it. The term "storage" in storage modulus refers to the storage of the energy applied to the sample. The stored energy is recovered upon the release of the stress. The storage modulus of an oil-in-water emulsion is suitably determined by a dynamic oscillatory measurement, where the shear stress is varied (from low to high stress) in a sinusoidal manner. The resulting strain and the phase shift between the stress and strain is measured. From the amplitude of the stress and the strain and the phase angle (phase shift) the storage modulus is calculated. Herein, the G' (Pa) is taken at the plateau value at low stress (linear viscoelastic region). For these measurement a suitable state of the art rheometer is used (e.g. a TA AR2000EX, United Kingdom). The oil employed in the present method typically contains 50-1 00 wt.%, more preferably 70-100 wt.% and most preferably 90-100 wt.% of triglycerides. The oil advantageously contains a high level of unsaturated fatty acids. Typically, 40-1 00 wt.%, more preferably 50-100 wt.% and most preferably 60-100 wt.% of the fatty acids contained in the oil are unsaturated fatty acids. The melting point of the oil typically does not exceed 30 °C, more preferably it does not exceed 20 °C and most preferably it does not exceed 1 0 °C.

Examples of oils that may be employed in the present method include those which are liquid at ambient temperature like avocado, mustard, cottonseed, fish, flaxseed, grape, olive, palm, peanut, rapeseed, safflower, sesame, soybean, sunflower, mixtures thereof and the like. Examples of oils that solid at ambient temperature and suitable for use in accordance with this invention include butter fat, cocoa butter chicken fat, coconut oil, palm kernel oil mixtures thereof and the like. The present invention also encompasses the use of olein and/or stearin fractions of the aforementioned oils.

The oil content of the present emulsion preferably is in the range of 1 0 to 60 wt.%, more preferably of 12 to 55 wt.% and most preferably of 15 to 50 wt.%. The continuous aqueous phase of the emulsion preferably represents 40-90 wt.%, more preferably 45-88 wt.% and most preferably 50-85 wt.% of the emulsion.

Examples of edible oil-in-water emulsions according to the present invention include dressings, mayonnaise, soups, sauces and drinks. Preferably, the present emulsion is a dressing or a mayonnaise. Most preferably, the emulsion is a mayonnaise.

The emulsion according to the present invention typically have a shelf-life of at least 4, more preferably at least 8 weeks under ambient conditions {20 °C).

The invention is further illustrated by means of the following non-limiting examples.

EXAMPLES

Example 1

A heat-treated brown lentil flour was prepared from native brown lentil flour by pressure cooking the flour at low moisture, followed by air drying and milling. DSC and X-ray powder diffraction showed that the starch in the flour had not been gelatinized as a result of this heat treatment.

A heat-treated red lentil flour was prepared from native red lentil flour by means of extrusion. DSC and X-ray powder diffraction showed that the starch in the red lentil flour had been gelatinized during the extrusion.

Enzyme activities in the aforementioned treated lentil flours were determined. The same enzyme activities were determined in three different native pulse flours, i.e. native red lentil flour, native yellow pea flour and native black gram flour. The results are shown in Table 1 .

Table 1

Example 2

Mayonnaises were prepared on the basis of the recipe shown in Table 2.

Table 2

The mayonnaises were prepared using the following procedure: • Pulse flour (native and/or pre-treated) was added to cold water and stirred until well dispersed

• The resulting slurry was heated for 5 min at 85-90 °C while stirring

• The slurry was cooled to 30-40 ^Q C

· Sugar and salt were added at 1 ,000 rpm for 30 sec (Silverson)

• Oil was slowly added with Silverson at 7,000 rpm, moving container to help mixing of oil

• Vinegar was added (to adjust pH to 3.7) with Silverson at 7,000 rpm for 1 minute

• Egg-yolk was added with Silverson at 7,000 rpm for 1 minute

· Different flour compositions were used in the preparation of the mayonnaises as shown in Table 3.

Table 3

As described in Example 1

Syneresis, storage modulus (G') and texture of these mayonnaises were evaluated after two weeks storage at 5°C. The results are shown in Table 4.

Table 4

The method for measuring syneresis was as follows:

• 200 ml glass jars were filled with 170 gram mayonnaise • perspex tubes (inner diameter = 2cm, outer diameter = 2.5 cm, length = 4.5 cm) closed at one end by a piece of black ribbon filter paper (Whatman, Dassel,

Germany) were inserted vertically into the mayonnaise (filter paper at the bottom)

• water released into the tubes was collected at regular times using a pipette and its weight was determined

• water was put back into the tubes

The samples containing heat-treated lentil flour showed lower syneresis and improved texture (i.e. less gelling) during storage than the control sample that only contained native lentil flour.

Example 3

Example 2 was repeated except that this time the mayonnaises were prepared from native red lentil flour and/or instant extrusion cooked red lentil flour (INTIBO 1 15-5, Hanseland Ltd., Netherlands) as shown in Table 5.

Table 5

As described in Example 1

Syneresis and storage modulus (G') of these mayonnaises were evaluated after two weeks storage at 5°C. The results are shown in Table 6.

Table 6

Product G' (in Pa) Syneresis (in g)

A 2,617 0.88

B 1 ,764 0.75

C 1 ,304 0.56

D 967 0.53 Texture of the products containing the extrusion cooked lentil flour was found to be more smooth than that of the product solely containing native flour. Also, the samples containing extrusion cooked lentil flour showed less syneresis and improved texture during storage (less gelling/hardening).

Example 4

Mayonnaises were prepared on the basis of the recipe shown in Table 7.

Table 7

The extra lentil flour used was either native red lentil flour (control) or a heat-treated lentil flour. Storage modulus and syneresis of the samples so obtained was measured after 2 weeks storage at 5°C. The results are shown in Table 8. Table 8

Whole lentils cooked in salt water for 20 minutes at 100 °C to gelatinize the starch, cooled in tap water for about 5 minutes then air dried, milled, and sieved to <120 μηι. ² Aqueous slurry of lentil flour (10% w/w) was cooked at 90 °C for 30 minutes and dried on a drum dryer at 120 °C. The dried flour was ground and sieved.

Texture of the products containing heat-treated lentil flour was found to be more smooth than that of the control product solely containing native flour. Also, the samples containing heat-treated lentil flour showed improved texture and less gelling/hardening during storage compared to the control sample.