BRPI1006908A2 - method for treating a plant material containing lipid, composition and its use and food product - Google Patents

Abstract

METHOD FOR TREATING A PLANT MATERIAL THAT CONTAINS LIPID, COMPOSITION AND ITS USE AND FOOD PRODUCT. The present invention relates to the modification of lipids in plant material containing lipid, such as cereal bran for the generation of functional lipids. The present invention further relates to the preparation of compositions comprising such functional lipids as well as the use of those compositions comprising such functional lipids and other functional compounds derived from the action of enzymes that modify lipids for the preparation of a composition suitable for the preparation of bio-ethanol, as well as food products such as bread.

Description

and Invention Patent Descriptive Report for "METHOD

FOR TREATING A PLANT MATERIAL THAT CONTAINS: LIPID, COMPOSITION AND ITS USE AND FOOD PRODUCT ".

FIELD OF THE INVENTION The present invention relates to the modification of lipids in plant material containing lipid, such as cereal bran for the generation of functional lipids. The present invention also relates to the preparation of compositions that comprise such functional lipids as well as the use of those compositions that comprise functional lipids and other functional compounds derived from the action of enzymes that modify lipids for the preparation of a composition suitable for the preparation of bioethanol, as well as food products such as bread.

BACKGROUND OF THE INVENTION The use of collateral flows from the processing of fermentation residues from plant materials, such as milled cereal bran or dry soluble distillery grains (DDGS) has received little attention beyond use in animal feed.

The beneficial use of lipolytic enzymes (E.G. 3.1.1x) in applications in the food and / or animal feed industry has been known for a long time.

However, most prior art describes the use of lipolytic enzymes in flour and dough and not for side products or by-products of industrial processes. For example, in EP 0 585 988 it is claimed that the addition of lipase to the dough resulted in the improvement of the anti-aging effect. WOS94 / 04035 teaches that improved bread softness can be achieved by adding lipase to the dough without adding any additional fat / oil to the dough.

The substrate for lipases in plant material is a complex mixture of polar and non-polar lipids. Polar lipids can be divided into glycolipids and phospholipids. These lipids consist of glycerol esterified with two fatty acids and a polar group. The polar group contributes to the surface activity of these lipids.

Enzymatic cleavage of one of the fatty acids in these lipids leads to lipids with much greater surface activity. It is well known that emulsifiers, such as DATEM, with high surface activity are very functional when added to the dough.

Lipolytic enzymes hydrolyze one or more fatty acids from the lipids present in the plant material which can result in the formation of powerful emulsifying molecules.

In EP 1 193 314, the inventors found that the use of lipolytic enzymes active on glycolipids was particularly beneficial in bread making applications. Morrison et al., J. Sci. Food Agric. 1981, 32, 5679-587 describe the distribution of lipids from the soft wheat seed in the flour mill fractions. There is a need in the technique for a better use of collateral products from the processing of plant materials, such as milling cereal fluff, vegetable oil refining neutralization pastes, dry soluble distillery grains (DDGS), in which less plant material goes to low-cost applications such as livestock feed. In addition, there is a long-felt need to be able to use the bran bran in traditionally existing cereal products, with no significant impact on the appearance / structure, color or flavor of the product and for make it possible to increase the effect on health and nutrition of existing products.

OBJECTIVE OF THE INVENTION It is an objective of the present invention to provide methods for the generation of functional lipids from plant material in general and industrial side products in particular. It is an additional objective of the present invention to provide suitable methods that enable the use of compositions comprising functional lipids derived from plant material, such as bran in traditional food products, such as bread or cereal products, without significant impact on the appearance / structure, the color or the taste of the product and to make it possible to increase the | effect on health is nutritional of existing products,

SUMMARY OF THE INVENTION In a broad sense, the present invention relates to methods for the treatment of plant material containing lipids with lipid-modifying enzymes, for the generation of modified lipids, such as functional lipids from materials of plant.

In a first aspect, the present invention relates to a method for the treatment of plant material, the method comprising the step of treating a liquid suspension of at least one plant material containing lipids partially solubilized with one or more enzymes that modify the lipid. In a second aspect, the present invention relates to a composition comprising lipids and / or modified lipids, such as a functional lipid produced by a method for the treatment of lipid-containing plant material, the method comprising the step of treating a liquid suspension of at least one plant material containing partially solubilized lipids with one or more enzymes that modify the lipid.

In a third aspect, the present invention relates to the use of a composition comprising lipids and / or modified lipids, such as a functional lipid produced by a method for the treatment of lipid-containing plant material, the method comprising the step to treat a liquid suspension of at least one plant material containing lipids partially solubilized with one or more enzymes that modify the lipid, the use being for the production of a food product.

In a further aspect, the present invention relates to the use of a composition comprising lipids and / or modified lipids, such as a functional lipid produced by a method for the treatment of lipid-containing plant material, the method comprising the step to treat a liquid suspension of at least one plant material containing partially solubilized lipids with one or more enzymes that |

7 modify the lipid, the use being for the production of biovetanol. In an additional aspect, the present invention relates to a] food product obtained by using a composition comprising lipids and / or modified lipids, such as a functional lipid produced by a method for the treatment of plant material containing lipid, the method comprising the step of treating a liquid suspension of at least one plant material containing lipids partially solubilized with one or more lipid-modifying enzymes. In a further aspect, the present invention relates to bioethanol obtained by using a composition comprising modified lipids and / or lipids, such as a functional lipid produced by a method for the treatment of plant material that contains lipid, the method comprising the step of treating a liquid suspension of at least one plant material containing partially solubilized lipids with a '15 or more enzymes that modify the lipid. In a further aspect, the present invention relates to a kit in parts comprising a) a combination of enzymes comprising: one or more enzymes that modify the lipid; one or more enzymes that modify the cell wall and, optionally, one or more additional enzymes; b) instructions for use in a method according to the invention; and €) optionally, other ingredients for the preparation of a food product.

DESCRIPTION OF THE FIGURES Figure 1. Results of the cooking test. Rel. Vol. of breads versus white (%). The columns represent the volume of the bread for tests numbers 1-6 according to tables 56 6, Figure 2. Breads obtained from cooking with fractions obtained from the freezer. The numbers refer to the numbers in table 5. Figure 3. Results of the cooking test. Rel. Vol. white breads (%). The columns represent the experiments of the | s78 r cooking according to table 12. Figure 4, Breads obtained from cooking with fractions obtained from soluble bran. The numbers refer to the numbers in table 12. Figure 5. Results of the cooking test. Rel. Vol. of breads versus white (%). The columns represent the cooking party experiments according to tables 17 and 18, Figure 6. Breads obtained from cooking with fractions obtained from soluble bran. The numbers refer to the numbers in table 18. Figure 7. Breads obtained from cooking with fractions obtained from modified bran. The numbers refer to the numbers in table 24. Figure 8. Breads obtained from cooking with extracts of modified rice flour. The numbers refer to the numbers in table 30. Figure 9. Breads obtained from cooking with fractions obtained from modified bran. The numbers refer to the numbers in table 36. “7 15 DETAILED DESCRIPTION OF THE INVENTION Considerable amounts of by-products from the processing of plant materials, such as milling cereal bran, vegetable oil refining neutralization pastes, grains Dry soluble distillery (DDGS), etc., are available as raw material to generate lipid 70 deosfunctional whiskey can serve as emulsifiers in different applications such as food applications, feed applications, softeners in the production of plastic materials, etc.

Here, it was shown that the modification of the lipid fraction in, for example, wheat bran, is significantly increased if the material is coirated with other enzymes such as the enzymes that modify the cell wall and, in some modalities, also with enzymes that modify the starch. By combining these classes of enzymes, the functionalization of lipids was observed, which cannot be obtained using only lipids.

The modified lipids generated can be used, for example, in baking, by adding the soluble fraction alone or by adding the complete composition of the bran treated with the enzyme containing the lipids |

'modified that have emulsifying properties. Therefore, the compositions generated by the methods according to the present invention can be used as soluble solids. However, the compositions can also be used as a mixture of soluble and insoluble, i.e., plant material in solution, ta) as residual bran material. It should be understood that part of the lipid fraction may still be present in that residual fraction in solution.

The term "plant material containing lipid", as used here, refers to any material that comprises significant amounts of material derived from a plant that contains endogenous amounts of lipids. Suitably, 9 plant material can be obtained in 7 large quantities, contains a significant amount of lipids and can be used in industrial processes. 'In some embodiments, plant material containing lipid is a side product or by-products of industrial processes. In some embodiments, the plant material may also contain a non-plant material, such as a by-product of a fermentation, which may contain yeast cells.

In some particular embodiments, the lipid-containing plant material is cereal bran, such as, for example, wheat bran from traditional milling.

In some embodiments, an amount of at least about 100 mg, such as at least about 200 mag, such as at least about 300 mg per 100 q of the dry weight of the lipid-containing material is phospholipid.

In some embodiments, an amount of at least about 10 mg, such as at least about 20 mg, such as at least. about 30 mag per 100 g of the dry weight of the plant material containing lipid is phosphatidylinoositol (PI).

The phrase "plant material containing partially solubilized lipid", as used here, refers to plant material which contains lipids and which has been solubilized at least partially by the action |

* enzymatic or mechanical. The expression "cereal" as used here refers to the fruits of 'a plant of the Poaceae family, such a seed containing at least the bran comprising the aleurone and the starch-rich endosperm, with or without the additional presence of the pericarp, lining of the seed (alternatively called walnut) and / or germ. The term includes, but is not limited to, species such as wheat, barley, oats, spelled, rye, sorghum, corn and rice. The term “bran”, as used here, refers to a fraction of the cereal derived from milling enriched with any or all of the tissues to be selected from the aleurone, pericarp and lining of the seed M, when compared to the corresponding intact seed, The term "solubilization" as used here refers to the solubilization of plant material , like cereal bran, in the methods of a- 'cordocoma invention and is intended to include any degree of solubilization. Consequently, "solubilization" can be to obtain a 100% soluble material or it can be to obtain a degree of solubilization of less than 100%, such as less than 70%, such as in the range of 30% -60%. In some modalities, the degree of solubilization is determined on the dry mass of dry bran bran. The expression "at least partially solubilized", as used here, refers to a degree of solubilization that is greater than 1% , as high as 5%, as high as 10%. It should be understood that the action of lipid-modifying enzymes may not work optimally according to the invention, if the plant material is not soluble. to a certain degree In the specific aspects according to the present invention, in which lipid-modifying enzymes are added to work simultaneously with the treatment to obtain solubilization, such as a treatment with one or more enzymes that modify the cell wall, solubilization and the action of enzymes that modify lipid will occur at the same time.

The term "grinding fraction", as used here, refers to |

- all or part of the fractions that result from the mechanical reduction of the grain size through, for example, but not limited to cutting, crushing, crushing, breaking or grinding, with or without fractionation through, for example, but not limited to screening, selection, filtration, blowing, aspiration, centrifugal filtration, air filtration, electrostatic separation or separation by electric field.

In the context of the present invention, "functional lipids" refer to lipids that have an effect on the product, in which the functional lipid is used. In some particular embodiments, functional lipids are emulsifiers or other food improvers.

In the context of the present invention, "cell wall modifying enzyme" refers to any enzyme capable of hydrolyzing or modifying the polysaccharide matrix complex of the plant cell wall, such as] any enzyme that has activity in the "cell wall solubilization assay: 15 included here, Within that definition of" enzyme that modifies the cell wall "are included cellulases, such as cellobiohydrolase | and cellobiohydrolase | 1l, endoglycanases and beta-glycosidases and hemicellulolytic enzymes, such as xylanases.

The term "cellulases" or "cellulolytic enzymes" ”as used herein is understood to comprise cellobiohydrolases (EC 3.2.1.91)," - for example, cellobiohydrolase | and cellabiohydrolase 1, as well as endo-glycanases (EC 3.2 .1 4) and beta-glucosidases (EC 3.2.1.21).

Included within the definition of cellulases are: endoglycanases (EC 3.2.1.4) that cut the cellulose chains at random; cellobiohydrolases (EC32.1.91) that cleave the cellobiosil units of the cellulose chain endings and the beta-glucosidases (EC 3.2,1.21) that convert cellobiosis and soluble cellodextrins into glucose. Among these three categories of enzymes involved in cellulose biodegradation, cellobiohydrolases are the main enzymes for the degradation of native crystalline cellulose. The expression "cellobiohydrolase 1" is defined here as a cellulose activity 1,4-beta-cebiosidase (also referred to as exoglycanase, exo-cellobiohydrolase or 1,4-beta-cellobiohydrolase), as defined in the class of enzi- | gI78 «but EC 3.2.1.91, which catalyzes the hydrolysis of 1,4-beta-D-glycosidic bonds in cellulose and cellotetraose, by the release of cellobiosis from the unreduced Ú ends of the chains, The definition of the expression" cellobio- hydrolase | I "is identical, except that cellobiohydrolase || it attacks the diminished ends of the chains.

Cellulases can comprise a carbohydrate-binding module (CBM) that intensifies the enzyme's binding with a fiber that contains cellulose and increases the effectiveness of the active catalytic part of the enzyme. A CBM is defined as a contiguous amino acid sequence within a carbohydrate-active enzyme with a different fold that has carbohydrate-binding activity. For additional information on 7 CBMs see the CAZy internet server (Supra) or Tomme et al. (1995) in Enzymatic Degradation of Insoluble Polysaccharides (Saddler and Penner, 'eds.), Cellulose-binding domains: classification ánd properties, pp. 142-163,] 15 American Chemical Society, Washington. In a preferred embodiment, cellulases or cellulolytic enzymes can be a cellulolytic preparation as defined in U.S. application No. 60 / 941,251, which is incorporated herein by reference. In a preferred embodiment, the cellulolytic preparation comprises a polypeptide that has cellulolytic enhancing activity (GH61A), preferably that described in WO2005 / 074656. The enzyme that modifies the cell wall may additionally be a beta glycosidase, such as a beta-glycosidase derived from a strain of the genus Trichoderma, Aspergillus or Penicillium, including the fusion protein that has beta-glucosidase activity described in US application No. 60 /832,511 (Novozymes) In some embodiments, the enzyme that modifies the cell wall is a CBH II, such as cellobiohydrolase 11 from Thielavia ferrestris (CEL6A). In some embodiments, the enzyme that modifies the cell wall is a cellulase enzyme, such like that derived from Trichoderma reesei. Cellulolytic activity may, in some modalities, be derived from a fungal source; such as a strain of the Trichoderma genus, such as a strain of Trichoderma reesei; or a strain of the genus Humico- 1a, such as a strain of Humicola insolens, |

- In some embodiments, the enzyme that modifies the cell wall is a polypeptide that has the activity of cellulolytic intensification (GH61A) described in WO 2005/074656; a cellobiohydralase, such as a cellobiohydrolase 11 from Thielavia terrestris (CELGA), a beta-glucosidase (for example, the fusion protein described in US application No. 60 / 832,511) and cellulolytic enzymes, for example, derived from Trichoderma reesei In some embodiments, the enzyme that modifies the cell wall is a polypeptide that has cellulolytic enhancing activity (GH61A) described in WO 2005/074656; a beta-glycosidase (for example, the fusion protein described in U.S application: no. 60 / 832,511) and cellulosic enzymes, for example, derived from Trichoderma reesei. In some fashion, the enzyme that modifies the cell wall is a commercially available product, such as GC220 available from Genencor, A Danisco Di-Ú vision, US or CELLUCLAST? 1.5L or CELLUZYME "available from '15 Novozymes A / S, Dinamatca.

Endoglycanases (EC 3.2.1.4) catalyze endo-hydrolysis of ph | 1,4-beta-D-glucosidications in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose), lichenine, beta-14 bonds in mixed beta-1,3 glycans, such as beta-D-glycans or cereal xyloglycans or other plant material containing cellulosic parts. The authorized name is endo-1,4-beta-D-glycan 4-glycan hydrolase, but the abbreviated term endoglycanase is used in the present application. Endoglucase activity can be determined according to the procedure of Ghose, 1987, Pure and Appl. Chem, 59: 257-258, In some embodiments, endoglycanases can be derived from a strain of the genus Trichodierma, such as a strain of Trichoderma reesêi; or a strain of the genus Humicola, such as a strain of Hurmoli insolens; or a strain of Chrysosporium, preferably a strain of Ghrysosporium lucknowense.

The expression "cellobiohydrolase" means a 1,4-beta-D-glycan cellobiohydrotase (EC 3.2.1.91) that catalyzes the hydrolysis of 1,4-beta-D-glycosidic bonds in cellulose, celloligosaccharides or any polymer that |

: contains bound beta-1,4 glucose, releasing cellobiosis from the reduced or unreduced ends of the chain, Ú Examples of cellobiohydrolases are mentioned above including CBH | and CBH Il de Trichoderima reesei; Humiceola insolens and CBH II of Thielavia terrestris cellobiohydrolase (CELL6A). Cellobiohydrolase activity can be determined according to the procedures described by Lever et al., 1972, Anal.

Biochem. 47: 273-279 and by van Tilbeurgh et al., 1982, FEBS Leiters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288. The Leveretal method is suitable for assessing cellulose hydrolysis in corn straw and the method by van Tilbeurgh et al, is suitable for determining the activity of cellobic hydrolase in a fluorescent dissacaroid derivative.

The term "beta-glycosidase” means a beta-D-glycoside] glycidralase (EC 3.2.1.21), which catalyzes the hydrolysis of unreduced beta-D-] 15 glucose residues with the release of beta-D- glucose.

For the purposes of the present invention, beta-glucosidase activity is determined | nothing according to the basic procedure described by Venturi et al., 2002, J, Basic.

Microbiol. 42: 55-66, except for different conditions that were used as described here, A unit of beta-glucosidase activity is defined as 1.0 μmol p-nitrophenol produced per minute at 500C, pH 5 from p- nitrophenol-beta-D-glycopyranoside 4 mM as a substrate in 100 mM sodium citrate, TWEENº 20 0.01%. In some modalities, beta-glucosidase is of fungal origin, such as a strain of the genus Trichoderma, Aspergíllus or Penicillium.

In some embodiments, beta-glycosidase is derived from Trichoderma reesei, such as beta-glycosidase encoded by the bgal1 gene (see EP 562003). In another embodiment, beta-glycosidase is derived from Aspergillus oryzãe (produced recombinantly in Aspergillus oryzae according to WO 02/095014), Aspergillus furmigatus (produced recombinantly in Aspergillus oryzae according to Example 22 of WO 02/095014) or As- pergíllus niger (1981, J.

Appl. 3: 157-162), the terms "hemicellulolytic enzymes" or "hemicellulases", |

- as used here, refer to enzymes that can break the hemicellulose. Any hemicellulase suitable for use in the hydrolysis of hemicellulose, preferably in arabinoxylan oligosaccharides, can be used. Preferred hemicellulases include xylanases, arabinofuranosides , acetyl xylan esterase, feruloyl esterase, glucuronidases, galactanase, endo-galactanase, manases, éndo or exo arabinases, exo-galactanases, pecíinase, xyloglivanase or mixtures of two or more of these.

An example of hemicellulase suitable for use in the present invention includes Grindamy | Powerbake 930 (available from Danisco A / S, Denmark) or VISCOZYM E? (provided by Novozymes A / S, Denmark). In one embodiment, "hemicellulase is xylanase.

In one embodiment, xylanase is of microbial origin, such as fungal (eg, Trichoderma, Meripilus, Humicola, Aspergillus, Fusarium) or a bacterium (eg, Bacillus), '15 In some embodiments, the xylanase is derived from a filamentous fungus, preferably derived from an Aspergillus strain, such as Aspergillus aculeatus; or from a Humicola strain, preferably Humicula lanuginãa.

The xylanase may preferably be an endo-1,4-beta-xylanase, more preferably an endo-1,4-beta-xylanase from GH 10 or GH 11. Examples of commercial xylanases include Grindamyl H121 or Grindamyl Powerbake 930 from Danisco A / S, Denmark or SHEARZYMES and BIOFEED WHEAT "from Novezymes A / S, Denmark.

Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of non-reduced terminal alpha-L-arabinofuranoside residues in alpha-L-arabinosinosides, Galactanase (EC 3.2.1.89), endo-1,4 arabinogalactan beta-galactosidase, catalyzes the endo-hydrolysis of 1,4-D-galactoside bonds in arabinogalactans.

Pectinase (EC 3.2.1.15) catalyzes the hydrolysis of 1,4-alpha-D-galactosiduronic bonds in pectate and other galacturonans.

Xyloglycanase catalyzes the hydrolysis of xyloglycan.

The term "xylanase", as used here, refers to an enzyme that is capable of hydrolyzing the beta-1 4-glycosyl bond in non |

7 terminals of beta-D-xylanopyranosyl-1,4-beta-Dxylanopyranosyl of xylan or arabinoxylan.

Other names include 1,4-beta-D-xylan xylan hydrolase, 1,4-i beta-xylan xylan hydrolase, beta-1,4-xylan xylan hydrolase, (1-4) -beta-xylan 4-xylan hydrolase, endo-1,4-betaxylanase, endo- (1-4) -beta-xylanase, endo-beta14xylanase, endo-1,4-beta-D-xylanase, endo-1,4-xylanase, xylanase, beta-1, 4-xylanase, beta-xylanase, beta-D-xylanase.

Xylanases can be derived from a variety of organisms, including plants, fungi (for example, Aspergillus, Penicillium, Disporoirichum, Neurospora, Fusarium, Hurmicola, Trichoderma species) or bacterial species (for example, Bacillus, Aeromonas, Streptomyces species, Nocardiopsis, Thermomyces (see, for example, WOS2 / 17573, WO9S2 / 01793, WOS9S1 / 19782, 'WOSA4 / 21785). In one aspect of the invention, the xylanase used in the methods of the invention is an enzyme classified with EC 3.2.1.8. The official name is en- '15 do1,4-beta-xylanase.

The systematic name is 1,4-beta-D-xylan xylan hydrolysate.

Other names can be used, such as endo- (1-4) -beta-xylanase; (1- 4) -beta-xylan-4-xylan hydrolase; endo-1,4-xylanase; xylanase; befa-1,4-xylanase; endo-14xylanase; endo-beta-1,4-xylanase; endo1,4-beta-D-xylanase; 1 4-beta-xylan xylan hydrelase; beta-xylanase; beta-1,4-xylan xylan hydrolase; endo-1,4-beta-xylanase; beta-D-xylanase, The catalyzed reaction is the endo-hydrolysis of 1,4-beta-D-xylosidic bonds in xylans.

In one aspect of the invention, the xylanase of the invention is a xylose from Family 11 of Glycoside Hydrolase (GH). The expression "from the 11 Glycoside Hydrolase (GH) Family" means that the xylanase in question is or —can be classified in the 11 GH family.

In one aspect of the invention, the xylanase used according to the invention is a xylanase that has xylanase activity as measured in the "Xylanase assay" as described herein.

According to the CAZy website (ModO), Family 11 of glycoside 230 hydrolases can be characterized as follows; Known Activities: xylanase (EC 3.2.1.8) Mechanism: Retention |

- Nucleophile / Catalytic Base: Glu (experimental) Proton Donor Catalytic: Glu (experimental) i State of 3D Structure: Coiled: E-supercoiled Clan: GH-C As used here, "Clan C" refers to clusters of families which share a common three-dimensional fold and identical catalytic machinery (see, for example, Henrissat, B. and Bairoch, A., (1996) Bio-chem. d ,, 316, 65695-696), as used here, "Family 11" refers to a family of enzymes as established by Henrissat, B. and Bairoch, A., (1993) Biochem. J., 293, 781-788 (see also Henrissat and Davies (1997) Current Opinion in, Structural Biol. 1997, 8637-644). Common features for family members 11 include high genetic homology, a size of about kDa and a double catalytic displacement mechanism (see Tenkanen et '15 al, 1992; Wakarchuk et al., 1994). The structure of the xylanases of the 11 'family includes two large B sheets made of ribbons B and helices a.

Family 11 of the xylanases includes the following: XynA of Aspergillus niger, XynC of Aspergillus kawachii, XynA of Aspergillus tubiensis, XynA of Bacillus circulans, XynA of Bacilluspunzilus, XAynA of Bacíllus subtílis, 20 , XynC of Streptomyces lividanis, Xynll of Streptomyces therinoviolateus, XynAÃ of Thermomoanospora fusca, Xyn of Trichoderma harzianum, Xynl of Tricho- derma reesei, Xynll of Trichoderma reesei, Xyn of Trichodermaviride.

In the context of the present invention, "starch-modifying enzyme" refers to any enzyme that catalyzes the hydrolysis of a-1,3 and / or 0-1.6 glycosidic bonds to glycosides. Included within this expression are the glycoside hydrolases, typically named after the substrate on which! they act. In some embodiments according to the invention, the "starch-modifying enzyme" is selected from lactase, amylase, pululanase, isoamylase, chitinase, sucrase, maltase, neuraminidase, invertase, hyalaronidase and lysozyme.

In some embodiments, the enzyme that modifies starch is | an enzyme that debranches starch.

In one aspect of the invention, the enzyme that modifies and starch, used in accordance with the invention, is an enzyme that has starch de-branking activity as measured in the "starch de-branking activity assay" as described herein.

Enzymes that debranch starch include pullulanase (EC

3.2.1.41) and isoamylase (EC 3.2.1.68). They hydrolyze the α-1,6-D-glycosidic bonds, B-limit dextrins and pullulan. Isoamylases can be distinguished from pullulanases (EC 3.2,1.41) due to the inability of isoamylase to attack pululana and the limited action on a-limit dextrins.

By "amylase" is meant to include any amylase, such as Omo 'glycoamylases, a-amylase, B-amylases and wild-type a-amylases from Bacillus sp., Such as B. subtilis and B. licheniformes. "Amylase" should mean Ú an enzyme that is, among other things, capable of catalyzing degradation '15 doamido. Amylases are hydrolases that cleave the oD- (| -> 4) O-: dicosidic bonds of starch, Generally, a-amylases (EC 3,2,1,1; (X-D4) —4) - glycan glycanhydrolase) are defined as endo-acting enzymes that cleave aD (l— »4) O-glycosidic bonds within the molecule of starch in a random way. Exo-acting amylolytic enzymes, such as familases (EC 321.2; 0-D- (| +4) -glucan maltohydrolase) and some product-specific amylases, such as maltogenic a-amylase (EC

3.2.1.133) cleave the starch molecule from the non-reduced end of the substrate, B-Amylases, a-glycosidases (EC 3.2.1.20; aD-glycoside glucohydrolase), glycoamylase (EC 3.2.1.3; aD- (1-4) -glycan glycohydrolase), and product-specific amylases can produce glucose from starch.

By "variant a-amylase", "variant c-amylase polypeptide" and "variant enzyme" is meant an a-amylase that has been modified by substituting amino acid residues at the amino terminus of the mature o-amylase protein. As used herein, "parental enzymes", "parental sequence", "parental polypeptide", "wild-type a-amylase protein" and "parental polypeptides" mean enzymes and polypeptides from which a- variant amylase are derived. The parental enzyme can |

- be a wild-type enzyme or a cr-amylase that has been previously recombinantly engineered. The variant c-amylase may additionally include mutations in the signal sequence of the parent tr-amylase polypeptide or elsewhere in the Î ± -amylase parental polypeptide. Therefore, the α-amylase polypeptide can be a recombinantly manipulated enzyme.

In one aspect of the invention, the a-amylase used according to the invention is an o-amylase that has a-amylase activity as measured by the "u-amylase assay" described herein. In one aspect of the invention, the beta- The amylase used according to the invention is a beta-amylase that has beta-amylase activity 'as measured in the "beta-amylase assay" described herein.

The expression "pullulanase" refers to a specific type of glycanase, an amylolytic endoerzyme that degrades pullulan. It is produced as, for example, a lipoprotein anchored to the cell surface, extracellular by Gram negative bacteria of the genus Klebsiella. However, Gram-negative bacteria produce pullulanases as secreted proteins. Type pullulanases | specifically attack 0-1.6 bonds, whereas pullulanases type || they are also capable of hydrolyzing the ca1,4 bonds. It is also produced by some other bacteria and is arebacteria. Pullulanase is used as a detergent in biotechnology. Pulula nase. (EC 3.2.1.41) is also known as pululan-6-glycanhydrolase (debranching enzyme). Pullulan is observed as a chain of maltotriose units linked by q-1,6-glycosidic bonds. Pullulanase will hydrolytically cleave to pullulan (o-glycan polysaccharides).

The term "transglycosylation enzyme” refers to any enzyme that has transglycosidase activity, such as transglycoside. The term "transglycosidase" refers to any enzyme that transfers an αD-glycosyl residue to a 1,4- 4-D-glycan for the primary hydroxyl group of glucose, free or combined in a 1,4-0-D-glycan.The transglycosidase described here has an activity described as EC 2,4.1.24, according to the IUBMB enzyme nomenciatura, The systematic name for |

“Transglycosidase described here is 1,4-a-D-glycan: 1,4-1-D-glycan (D-glucose) 6- a-D-glycosyltyransferase, This enzyme may be referred to as an α-glucosidase in certain publications.

As noted above, the transglycosidase enzyme generally has an activity defined as EC 2.4.1.24 according to the JUBMB enzyme nomenclature, whose activity transfers the glycosyl residues of certain glycans to the primary hydroxyl group of glucose.

In some modalities, the enzyme may also have an activity that degrades a natural polysaccharide gum (eg polysaccharides containing egalactomannan xanthan, such as guar gum or lima bean gum), by cutting the side chains of sugar or by cleaving internal bonds to break the polysaccharide framework.

Any suitable transglycosidase enzyme finds use in the present invention (see, for example, Pazur et al., Carbohydr.

Res. 1986, 149: 137-47; and Nakamura et al., J.

Biotechnol. ] 15 53: / 75-84, 1997). In some embodiments, the transglycosidase enzyme that. finds use in the present invention is commercially available (for example, including, but not limited to, enzymes obtained from Megazyme, Wicklow, Ireland; or Danisco US Inc., Genencor Divisio, Palo Alto, CA). In some embodiments, the enzyme is a transglycosidase from Aspergillus niger produced in Trichoderma reesei cells.

In some additional embodiments, transglycosidase is a wild-type fungal transglycosidase (for example; including, but not limited to, fungal transglycosidase that has an amino acid sequence deposited in the NGBI GENBANK database as accession numbers: D45356 (GID : 2645159; As- pergillus niger, BADOSO0DE6E.1 (GID: 4031328; Aspergillus awamori), BA- AO08S125.1 (GION0S4565; Aspergillus oryzae), XPJ) JOI 210809.1 (GID: 1 15492363; Aspergillus ferreus), Aspergillus ferreus). 001271891.1 (GID: 121707620; Aspergillus clavatus), XPJ) J01266999.1 (GID: 1 19500484: Neosartorya fischeri), XP 75181 11 (GID: 70993928; Aspergillus fumigatus), XP 6596211 (GID: 67523121; GID: 67523121; ), XP 001216899.1 (GID: 115433524; Aspergillus fterreus) and XPJ) 0O1258585.1 (GID: 119473371; Neosartiorya fischerl)) or such a variant that has an amino acid sequence |

1B / 78 ° which is at least about 70% identical, at least about 80% identical, 'at least about 90% identical, at least about 95% identical or at least about 98% identical to a transglycosidase of wild type fungus.

In one aspect of the invention, the transglycosidase used according to the invention is a transglycosidase which has transglycosidase activity as measured in the "transglycosidase assay" described herein.

Assays of enzyme activity according to the invention: Cell wall solubilization assay: Preferably, the solubility of the bran is measured using the following assay. B A wheat bran suspension (0.1 M) = disodium hydrogen phosphate buffer (0.2 M), pH 5.0, is prepared to a bran concentration of 1.33% (weight / weight). From that suspension, aliquots of 750 '15 ul are transferred to eppendorph tubes under agitation. Each subs- tube. tract is preheated for 5 minutes at 40ºC. After that, 250 µl of enzyme solution is added, making the final concentration of the substrate 1%. Three dilutions (in duplicate) are made from each enzyme composition according to the invention, with increasing concentrations of enzyme (for example, 0.33; 1.0 and 3.0 µg of enzyme / bran) ) in each determination period (0, 30, 60 and 240 minutes). As a control, a heat denatured solution of the enzyme composition is used. The reaction is terminated in the determined periods, by transferring the tubes to an incubator set at 95ºC. Heat denatured samples are kept at 4ºC until all enzymatic reactions have ended. When the enzymatic reactions are finished, the Eppendorph tubes are centrifuged to obtain a clear solution. The ability of enzymes to solubilize bran is expressed as the increase in reduced end groups as determined using PAHBAH (Lever, 1972), so if the bran used contains residual starch, collateral activities such as amylase activity can interfere with the above test, the bran solubilization test should be performed only on enzymes that |

'modify the modified cell wall (which lacks amylase activity).

Alternatively, the degree of solubilization can be measured according to the following method: the degree of solubilization of a plant material, for example, cereal bran, can be determined by suspending insoluble plant material in an extraction buffer (typically 10-25% bran in buffer (weight / weight)) with no enzymes, incubating the suspension under agitation and 40º C for a controlled time (for example, 30 to 1440 minutes). After solubilization, the solubilized material is separated from the insoluble 9 material by centrifugation (20 min, 25000 x 9, room temperature). The content of: dry matter in the supernatant is determined both by lyophilization of part of the sample and by moisture analysis (Moisture Analyzer, AND ML-50, Buch & Holm, Denmark), The entire extraction buffer cannot be recovered Ú 15 using this protocol, however, it is assumed that the concentration of soluble material is the same in the recovered extraction buffer and in the non-recovered extraction buffer, because a correction is made in the extraction buffer used in total, having determined the dry matter content in the soluble fraction, knowing the amount of plant material used and the amount of extraction buffer, the degree of solubilization can be determined using the following equation.

Degree of solubilization = ((((g of dry matter / ml of recovered supernatant) x (ml of used extraction buffer)) x 100%) / g of plant material employed Ensaiode xylanase (endo-B-1,4 activity -xylanase) The samples were diluted in citric acid (0.1 M) - disodium hydrogen phosphate buffer (0.2 M), pH 5.0, to obtain approximately ODs66 = 0.7 in this assay. Three different dilutions of the sample were incubated for 5 minutes at 40ºC. At time = 5 minutes, 1 Xylazyme tablet (cross-linked, colored xylan substrate, Megazyme, Bray, Ireland) was added to the enzyme solution in a reaction volume of 1 ml, At time = 15 minutes, the reaction was finished by adding 10 m! TRIS / 2% NaOH, pH |

12. Controls were prepared using 1000 µl of buffer instead of. enzyme solution. The reaction mixture was centrifuged (1500 x 9, 10 minutes, 20ºC) and the OD of the supernatant was measured at 590 nm. A unit of xylanase (XU) is defined as the xylanase activity that increases ODss by 0025 per minute.

Α-amylase activity: α-amylases hydrolyze uD-14-glycosidic bonds and their activity can be detected as a color change rate of an iodine solution due to the hydrolysis of alpha-1,4-D bonds .

Beta-amylase activity: Beta-amylase activity can be detected as the release of maltose from the unreduced termination of a starch solution.

Transglycosidase activity: Transglycosidase catalyzes both hydrolytic and transfer reactions in incubation with a-D-glyco-oligosaccharides. The activity of. transglycosidase can be detected as the formation of isomalto-oligosaccharides such as isomaliosis, pansose and isomaliriosis after incubation with maltose or maltodextrin.

Starch de-ramification activity assay: The specific enzymes for starch a-D-1,6 glycosidic linkages currently include isoamylase (EC 3.2 1.68) and pullulanases (EC

3.2.1 41). Enzymes that act on the 0-D-1,6 glycosidic bonds of starch are also classified by their action on pullulan and their activity is measured as the specific hydrolysis of the aD-1,6 glycosidic bonds of the amidoedapululan, The expression " enzyme that modifies lipid ", as used here, refers to any enzyme that can modify a lipid.

In some preferred embodiments, the lipid-modifying enzyme is a lipolytic enzyme, such as lipase.

The term "lipolytic enzyme" as used here refers to any enzyme that hydrolyses one or more fatty acids from the lipids present in a plant material, such as cereal by-products, which can be used to |

'to result in the formation of functional lipids within the by-product of ce-: real, which provides commercial value.

The molecules that contribute to the most significant functional effects are the molecules with emulsifying characteristics, which are the products of partial hydrolysis, such as the molecules of meliso-phospholipids, smooth-glycellipids and monoglycerides.

Polar lipid hydrolysis products, such as smooth-phospholipids and smooth-glycolipids are particularly advantageous in bread production and can give equivalent functionality as emulsifiers, such as DATEM.

The substrates for lipases in cereal by-products are bran lipids, which are a complex mixture of polar and non-polar lipids.

Polar lipids can be divided into glycolipids and phospholipids.

These lipids are the builders of glycerol esterified with two fatty acids and a polar group.

The polar group contributes to the surface activity of these lipids.

The enzymatic cleavage of one of these 15 fatty acids in these lipids leads to lipids with very surface activity. bigger.

It is well known that emulsifiers, such as DATEM, with very high surface activity, are very functional when added to food.

The use of lipases (EC 3.1.1.X) in dough products can have a detrimental impact on yeast activity and / or a negative effect on bread volume.

The negative effect on bread volume is generally explained by overdosing. Overdosing can lead to a decrease in gluten 0 elasticity which results in a dough that is very firm and therefore results in reduced bread volumes.

Additionally or alternatively, such lipases can degrade the fat, oil or milk fat added to the dough, resulting in a loss of flavor in the dough and baked product.

Overdose and loss of flavor have been attributed to the accumulation of free fatty acids in the dough.

In relation to the present invention, these unwanted effects can be avoided since lipase is added to the cereal by-product such as, for example, a suspension of cereal bran which is used with or without further processing as a beneficiary of cereal. dough.

Additional processing can |

"dilution, purification of functional lipids. In addition, functional. lipids can be processed to be supplied as a liquid product or as a formulated dry product, such as a lyophilized product, Nas EP1193314, EPOS77869, WO02 / 094123, WO00 / 31758 and also in WOD1 / 39602, the use of lipolytic enzymes active in glycolipids has been reported to be particularly beneficial in the application of bread production, since the partial hydrolysis products, the smooth-glycolipids, have been found to have very high emulsification functionality, apparently resulting in a greater proportion of positive emulsification functionality compared to the harmful accumulation of free fatty acids. However, enzymes have also been found to have significant non-selective activity on triglycerides, which resulted in an unnecessary increase in free fatty acid. In addition, the application of lipases in the manufacture of bread had the addition of lipase to the dough followed by a '15 generation of emulsifier in the dough.

: The lipase can be of any origin, for example, of animal origin (such as, for example, of mammal), for example, of the pancreas (for example, bovine or porcine pancreas) or of snake venom or bee venom. Alternatively, lipase may be of microbial origin, for example, of filamentous fungus, yeast or bacteria, such as the genus or species Aspergillus, for example, A. niiger, Dictyostelium, for example, D. discoideum; Magnaporthe, for example, M. grisae, Mucor, for example, M. javanicus, M. mucedo, M, subtilissimus; Neurospora, for example, N. crassa; Rhizomucor, for example, R. pusillus; Rhizopus, for example, R, arrhizus, R japonicus, R. stolonifer, Sclerotinia, for example, S. libertiana; Triehophytan, for example, T. rubrum; Whetzelínia, for example, W. sclerotiorunt, Bacillus, for example, 8. megaterium, B. subtilis; Citrobacter, for example, CO. freundii; Enterobacter, for example, E. aerogeries, E, cloaçae; Edwardsiella, E. farda; Erwinia, for example, herbal E. Escherichia, for example, E.

col Klebsielia, for example, K. preumoniae; Proteus, for example, P. vulga- ris; It provides, for example, P. stuartii, Salmonella, for example, 8. typhimurnum; Setratia, for example, &. liquefasciens, S. marcescens; Shigella, |

7 for example, 8. flexneri, Streptomyces, for example, S. violeteoruber, Yer-: synia, for example, Y. enterocolitica. Therefore, the lipase can be fungal, for example, from the Pyrenomycetes class, such as the Fusarium genus, such as a strain of F. culmorum, E. heterosporum, F. solami, or a def strain, oxysporum. Phospholipase can also be from a strand of fungi within the genus Aspergillus, such as a strain of Aspergillus awamori, Aspergillus foetídus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzaãe.

A commercially available source of lipolytic enzymes is a microbial lipase or acyltransferase.

In some embodiments, lipase is from filamentous fungi, such as Aspergillus spp. and Fusarium spp. Lipases isolated from filamentous fungi were found to have industrially applicable characteristics and were also found to be expressed routinely in heterologous production systems, such as Aspergillus oryzae, Fusarium and yeast.

In some embodiments, the lipase is from Aspergillus turingiensis as described in EP 1433852, the patent of which is incorporated herein by reference.

In some embodiments, the lipase is from Fusarium heterosporum as described in EP1722636, the patent of which is incorporated herein by reference.

In some embodiments, the lipase is from Fusarium oxysporum as identified in EP 0 130 064 or in Hoshino et al. (1992) Biosci. Bio-tech Biochem 56: 660-664.

In some embodiments, the lipase is pig pancreatic phospholipase A2 expressed, for example, in Aspergillus niger (Cakenzyme ”, DSM).

In some embodiments, lipase is as described in EPO 8659 167, where cloning is described as the expression of a Fusari lipase- an oxysporum and its use in baking. The enzyme is described as having phospholipase activity. This enzyme is now sold by Novozymes |

"A / S (Denmark) as Lipopan Fº.

: In some embodiments, lipase is as described in WO 02/00852, which describes five lipase enzymes and their encoding polynucleotides, isolated from F. venenatum, F. sulphureum, A. berketeyanum, F.

eculmorume Ff, solani. All five enzymes are described as having triacylglycer | hydrolysis activity, phospholipase and galac-tolipase activity. Three of the enzymes have activity equivalent to that of the F. oxysporum enzyme described in EP 0 869 167: F. venenatum, F. eulmorum, F. sulphurum.

In some embodiments, the enzyme that modifies the lipid is a variant lipolytic enzyme. Variant lipolytic enzymes, with specific amino acid substitutions and fusions, have been produced, some of which have enhanced activity on polar lipids compared to wild-type parental enzymes. WO01 / 39602 describes such a variant, referred to as SP979, which is a fusion of the lipase of Thermomyces lanu- ginosus and the lipase of Fusarium oxysporum described in EP 0 869 167. This variant was found to have a significantly high rate of activity on phospholipids and glycolipids compared to triglycerides. In some embodiments, the lipid-modifying enzyme is a lipid acyltransferase.

The term "lipid acyltransferase" as used here means an enzyme that has lipase activity (generally classified as EC 3.1.1x according to the Enzyme Nomenclature Recommendations (1992) of the Nomenciature Committee of the International Union of Biochemistry and Molecular Biology), as well as it has acyltransferase activity (generally classified as EC 2.3.1.x), through which the enzyme is able to transport an acyl group from a lipid to one or more accepting substrates, such as one or more of the following : a sterol, a stanol, a carbohydrate, a protein, a protein subunit, glycerol.

In some embodiments, the lipid acyltransferase for use in the methods and / or uses of the present invention is capable of transferring an acyl group from a lipid (as defined here) to one or more of the following subs | Accepting treatments: a sterol, a stanol, a carbohydrate, a protein or its subunits or glycerol.

In some respects, the acyl receptor can be any compound comprising a hydroxy group (-OH), such as, for example, polyvalent alcohols including glycerol; sterol; stanol; carbohydrates; hydroxy acids including fruit acids, citric acid, tartaric acid, lactic acid and ascorbic acid; proteins or a subunit thereof, such as amino acids, protein hydrolysates and peptides (partially hydrolyzed protein) for example; and their mixtures and derivatives.

In some embodiments, the lipid substrate on which the lipid acyltransferase used in accordance with the present invention is one or more of the following lipids: a phospholipid, such as a lecithin, for example, phosphatidylcholine, a triglyceride, a cardiolipin , a diglyceride or a glycolipid, such as digalactosyldiglyceride (DGDG), for example: À] 15 expression lecithin when used here, includes phosphatidylcellin, phosphatidyl-. tanolamine, phosphatidylinoposit |, phosphatidylserine and phosphatidylglycerol. Preferably, in some respects, the lipid substrate on which the lipid acyltransferase acts is a phospholipid, such as lecithin, for example, phosphatidylcholine or phosphatidylinosital. In some embodiments, the lipid substrate is an alpha lipid, the which means to say that it is a lipid component of a foodstuff.

Suitably, the lipid acyltransferase used in accordance with the present invention may exhibit one or more of the following lipase activities: glycolipase activity (E.C. 3,1.1.26), triacylglycerine activity! lipase (E.C. 3.1.1.3), phospholipase A2 activity (E.C. 3.1.1.4) or phospholipase A1 activity (E.C. 3.1.1.32). The term "glycolipase activity", as used here, encompasses "galactolipase activity". Suitably, the lipid acyltransferase used in accordance with the present invention may exhibit one or more of the following activities: glycolipase activity (EC 3.1. 1.26) and / or phospholipase A1 activity (EC

3.1.1.32) and / or phospholipase A2 activity (E.O. 3.1.14), |

"Preferably, in some respects, the lipid acittransferase. Used in accordance with the present invention is capable of transferring an acyl group of a glycolipid and / or a phospholipid to a steroid | and / or an estanol to form at least one ester ester and / or a stanol ester.

Suitable sterol acyl receptors include cholesterol and phytosterols, for example, alpha-sitosterol, beta-sitosterol, stigmastero !, ergosterol, campesterol, 5,6-dihydrosterol, brassicasterol, high-spinasterol, beta-spinasterol, gamma-spinasterol, delta-spinasterol, fucosterol, dimosterol, ascostero |, serebistero |, episteral, anasterol, hipostero |, chondrilasterol, desmosterol, calinosterol, poriferastero |, clionasterol, sterol glycoside and other natural or synthetic isomeric forms and derivatives. "Preferably, in one aspect, the acyl receptor is one or more of the following: alpha-sitoster |, beta-sitosterol, stigmasterol, ergosterol, beta-sitostanol, ss-sitostane! Or campesterol |. '15 Preferably, in some respects , the lipid acyltransferase, used in accordance with the present invention, is capable of transferring an acyl group from a glycolipid and / or a phospholipid to a glycerol to form at least one diglyceride and / or a monoglyceride. of the sterols present in the plant material containing lipid can be converted to one or more stanols before or at the same time as the lipid acyltransferase is added according to the present invention. stanols can be employed, for example, the conversion can be carried out by chemical hydrogenation.The conversion can be carried out before the addition of the lipid acyltransferase according to the present invention or simultaneously the ad addition of the lipid acyltransferase according to the present invention. Enzymes suitable for converting sterol to stanols are described in WOD00 / 061771. Suitably, the present invention can be employed to produce phytostanol esters in the lipid of plant material. Phytostanol esters have adequate solubility across lipid membranes, bioavailability and increased health benefits (see, for example, |

. WOS2 / 99640). . Protocol for the determination of% acyltransferase activity: The plant material containing lipid to which a lipid acyl transferase has been added according to the present invention can | 5 will be extracted after the enzymatic reaction with CHsx: CH; OH 2: 1 and the organic phase containing the lipid material is isolated and analyzed by GLO and HPLC according to the procedure detailed below.

From the GLC and HPLC analyzes, the amount of free fatty acids and one or more sterol / stanol esters is determined; carbohydrate esters; protein esters; di-glycerides or monoglycerides.

A control of plant material containing lipid to which the enzyme has not been added is analyzed in the same way.

Calculation: From the GLC analysis is HPLC, the increase in free fatty acids Ú 15 and sterol / stanol esters and / or carbohydrate esters and / or esters. of protein and / or diglycerides and / or monoglycerides can be calculated:% fatty acid =% fatty acid (enzyme) -% fatty acid (control); Fatty acid Mv = average molecular weight of fatty acids; A = 0% sterol ester / Mv sterol ester (where A% sterol ester =% sterol ester / stanol (enzyme) -% sterol ester / stanol (control) and Mv sterol ester = average molecular weight of esters estersVestanol) - applicable where the acyl acceptor is a stereo | and / or a stanol; B = A% carbohydrate ester / Mv carbohydrate ester (where A% carbohydrate ester =% carbohydrate ester (enzyme) -% carbohydrate ester (control) is Mv carbohydrate ester = average molecular weight carbohydrate esters) - applicable where the acyl acceptor is a carbohydrate; 6 =% protein ester / Mv protein ester (where% protein deester = *% protein ester (enzyme) -% protein ester (control) is protein ester Mv = molecular weight mean of esters of protein) = applicable where the acyl acceptor is a protein; and |

, D = absolute value of diglyceride and / or monglyceride / Mv of. di / monoglyceride (where% diglyceride & / or monoglyceride =% diglyceride and / or monoglyceride (enzyme) -% diglyceride and / or monoglyceride (control) and Mv of di / monoglyceride = average molecular weight of diglice - S rideeloumonoglyceride) - applicable where the acyl receptor is glycerol, Transferase activity is calculated as a percentage of total enzyme activity:% transferase activity = A “+ B * + C * + D * x 100 A * + B * + C * + D * +% fatty acid / (Mv of fatty acid) (* - delete as appropriate). In a preferred aspect, the present invention provides a plant material containing lipid, in which the lipids have been modified to functional lipids by the action of lipolytic enzymes. This can be: used with or without the purification of functional lipids as a food ingredient.

. Suitably, the term "foodstuff", as used here, means a food in a form that is ready for consumption. Alternatively or in addition, however, the term foodstuff, as used here, may mean one or more food materials that are used in food preparation. Just as an example, the term foodstuff covers baked products produced from a dough as well as the dough used in the preparation of said baked products.

Suitably, the term "foodstuff", as used here, means a substance that is suitable for human and / or animal consumption.

In another aspect, the foodstuff according to the present invention can be animal feed. In some embodiments, the foodstuff used in accordance with the present invention is selected from one or more of the following: eggs, egg-based products, including, but not limited to. mayonnaise, salad dressings, dressings, ice cream, powdered egg, modified egg yolk |

O and products made with it; baked goods, including breads, cakes, pastry products, puff pastry, liquid pastry, muffins, donuts, cookies, toast and cookies; confectionery including chocolate, caramels, halawa, gums, including sugarless and sweetened gums, chewing gum, soft chewing gum, gum and puddings; frozen products including sorbets, preferably frozen dairy products, including ice cream and frozen milk; dairy products including cheese, butter, milk, non-dairy coffee cream, whipped cream, cream. pastry, drinks. lao- teas and yogurts; mousses, vegetable creams, meat products, including processed meat products; edible oils and fats, aerated and non-aerated creamy products, oil-in-water emulsions, e-: water-in-oil, margarine, fats and toppings including low-fat and very little fat toppings; sauces, mayonnaise, pasta, cream-based sauces, cream-based soups, beverages, emuli 15 are seasoned and pureed.

. Suitably, the foodstuff according to the present invention can be a "refined food" including cakes, pastries, confectionery, chocolates, chocolates and the like. In one aspect, the foodstuff according to the present invention can be a product of dough, such as bread, a fried product, a snack, cakes, toftas, brownies, cookies, macaroni, instant noodles, tortillas, snack items such as biscuits, graham crackers, pretzels and chips, pasta and breakfast cereals. In a further aspect, the foodstuff according to the present invention can be a plant-derived food product such as flours, premixes, oils, fats, cocoa butter, creamy coffee, sauces for salad, margarine, toppings, peanut butter, fats, soryetes, cooking oils In another aspect, 5 foodstuffs according to this invention can be a product derived from milk including butter, milk, cream, cheese such as natural, processed cheeses and imitations of cheese in a variety of ways (including crushed, chunk, sliced or grated), |

'creamy cheese, ice cream, frozen desserts, yogurt, drinks based on. yogurt, bottle butter, anhydrous fat, others. products derived from milk. The enzyme used in accordance with the present invention can improve the stability of fat in milk-based products. In another aspect, the foodstuff according to this invention can be a food product containing ingredients of animal origin. , such as processed meat products, cooking oils and fats. In a further aspect, the foodstuff according to the present invention can be a drink, a fruit, a mixture of fruits, a vegetable or a wine. In some cases, the drink may contain up to 20 q 'of added phytosterols derived from the invention. In another aspect, the foodstuff according to this invention can be an animal feed. Animal feed can be | 15 enriched with phytosterol and / or phytostanols, preferably with beta-. sitosterol / stanol, suitably; animal feed can be a poultry feed. When the food is a poultry feed, the present invention can be used to decrease the cholesterol content of the eggs produced by the birds fed with the food according to the intervention. Preferably, in one aspect, the foodstuff is selected. - swimming between one or more of the following: eggs, egg based products, including, but not limited to mayonnaise, salad dressings, pastes, ice cream, powdered egg, modified egg yolk and products made with it.

Preferably, the foodstuff according to the present invention is a foodstuff that contains water. Suitably, the foodstuff can be made up of 10-98% water, suitably 14-98%, suitably 18-98% water, suitably 20- 98%, suitably 40-98%, suitably 50-98%, suitably for 70-98%, suitably for 75-98%.

In one aspect of the invention, the functional lipid produced from the lipid-containing plant material is an emulsifier. Preferable- |

"mind, at least one emulsifier is generated in the plant material which - contains lipid.

In one aspect of the invention, at least three different emulsifiers are generated in the lipid-containing plant material.

In one aspect of the invention, at least four different emulsifiers are generated in the lipid-containing matefial.

Suitably, the emulsifier according to the present invention can be, for example, one or more of the following: a diglyceride, a monoglyceride, such as 1-monoglyceride or a lysolecithin, such as lysophosphatidylcholine or phosphatidylinositol, for example, a monoglyceride digalactosyl (DGMG). The emulsifier is preferably produced from an acyl donor lipid after removal of one or more acyl groups from said acyl donor lipid. The term lysolecithin as used herein encompasses lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylserine and lysophosphatidylglycerol. The term lysophosphatidylcholine as' used here is synonymous with the term lysolecithin and these expressions can be used alternatively, where one of the emulsifiers is a protein ester and / or a di-glyceride & / or a monaglyceride, the second emulsifier can be, for example , one or more of the following: a diglyceride, a monoglyceride, such as 1-monoglyceride, lysophosphatidylealin or a digalactosyl monoglyceride (DGMG). The second emulsifier is preferably produced from the acyl donor lipid after removal of one or more acyl groups from said acyl donor lipid.

In one embodiment, 68 functional lipids generated from the invention can be used in a process for the preparation of a foodstuff.

Functional lipids according to the present invention can be used with one or more suitable food grade enzymes.

Therefore, it is within the scope of the present invention that, in addition to the functional lipids of the invention, at least one additional enzyme is added to the foodstuff. Such additional enzymes include enzymes that |

'degrade starch such as endo- and exo-amylases, pullulanases, enzymes. debranching, hemicellulases including xylanases, cellulases, oxidoreductases, for example, glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as that which oxidizes maltose, for example, hexoseoxidase (HOX), lipases, fusfolipases , glycolipases and proteases, The plant material containing lipid treated with the lipolytic enzymes to generate functional lipids according to the present invention can be used without purification or with limited purification of the functional lipids together with one or more suitable food grade enzymes.

Therefore, it is within the scope of the present invention that, in addition to the purified or unpurified lipids of the invention, at least one at least one additional enzyme is added to the foodstuff. Such additional enzymes include enzymes that degrade starch such as endo- and exo-amylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, for example, oxidized glucose. if, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as that which oxidizes to maltose, for example, hexose oxidase (HOX), lipases, phospholipases, glycolipases and proteases.

In a preferred embodiment, the lipolytic enzyme has one or more of the following lipase activities: glycolipase activity (E.C. 3,1.1,26), triacylglycerine activity | lipase (E.C. 3.1.1.3), phospholipase A2 activity (E.C. 3.1.1.4) or phospholipase A1 activity (E.G. 3.1.1.32). Suitably, lipase enzymes are well known in the art and include, for example, the following lipases: Grindamyl Powerbake 4070 or 4080 26 (Danisco A / S), Lysomax Oil (Danisco A / S), Lipopanº F, Lipopanº Xtra, is / or LECITASE? ULTRA (Novozymes A / S, Denmark), phospholipase A2 (for example, phospholipase A2 from LIPOMODº 221 Biocatalysts, LIPOMAXº from Genencor), LIPOLASE? (Novozymes A / S, Denmark), Panomoreº (DSM Nutritional Products), the lipases described in WON3 / 97835, EP 0 977 869 or EP 1 193314, The one skilled in the art Will be able to combine ratios of lipolytic enzymes.

Traditionally, the cake industry uses boe |

'for the production of cakes and to ensure the high quality of cakes in. in terms of taste, structure, quality and aspect of the food. These cake recipients are hormonally based on lyophilized emulsifiers in a vehicle similar to starch and maltodextrin. Some beneficiaries of i5 bolot are also in gel form based on emulsifiers, sugars'; and water. These cake improvers are very important to the cake industry in order to produce a high quality cake. However, cake recipients contain emulsifiers and other "unnatural" ingredients with an "E-number". Due to consumer demand to reduce the numbers of "E-numbers", the cake industry has been researching alternative ways to produce high-quality cakes without using this type of emulsifier. 'Plant material containing lipid treated with lipolytic enzymes to generate functional lipids according to the present invention' 15 can be used as food purifiers without purification either with limited purification of functional lipids or as fully purified functional lipids.

In one aspect of the invention, the food processor is a cake processor.

In one aspect of the invention, the food processor is a bread processor.

The food processor generated in accordance with the present invention can adequately comprise one or more of the following additives: soy protein material: carotenoids, flavonoids, antioxidants and phytochemicals (especially anthocyanin, carotenoid, bioflavoine, glutathione, catechin, isoflavone, lycopene, ginsenoside, pycnogenol, alkaloid, pygeum phytosterol, sulforaphone, resveretol; grape seed extract or food containing stanol esters), vitamin (especially vitamin O, vitamin A , vitamin B3, vitamin D, vitamin E, thiamine, riboflavin, niacin, pyridoxine, cyanocobalamin, folic acid, biotin, pantothenic acid or vitamin K), minerals (especially calcium, iodine, magnesium, |

34 / T8 7 Zinc, iron, selenium, manganese, chromium, copper, cobalt, molybdenum or phosphorus. ro), fatty acids (especially gamma-linoleic acid, eicosapentae- .noic acid or docosahexaenoic acid), oil (especially borage oil, carotenoid-rich canola oil or flax seed oil), glycerol, sorbitol, amino acids (especially tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucine, isoleucine, alanine, arginine, aspartic acid, cystine, cysteine, glutamic acid, glutamine, glycine, histidine, proline, hydroxyproline, serine, taurine or tyrosine) , enzyme as defined above (especially merite bromelain, papain, amylase, cellulase or coenzyme OQ), lignin, stanol ester or favorable bacteria (especially Lacfobacíllus acidophílus, Lactobacillus bulgarícus, Lactobaciíllus bifidus, Lactobacillus facidus plantarumum or Sactobacillus fulgusicus) , insoluble and / or soluble fibers. The present invention can provide one or more of the following unexpected technical effects in egg products, particularly mayonnaise:] improved thermal stability during pasteurization; properties: improved organoleptics, improved consistency, The present invention can provide one or more of the following unexpected technical effects in dough and / or baked products: a specific improved volume of the dough or baked products (for example, bread and / or the cake); improved mass stability; an improved crust (for example, a thinner and / or more delicious bread crust), an improved crumb (for example, a more homogeneous crumb distribution and / or a finer crumb structure and / or a more softness); an improved appearance (for example, a smooth surface without bubbles or holes or substantially without bubbles or holes); reduced hardening; improved softness; improved odor; improved taste.

The present invention can provide a beneficial effect from the functional lipids since they function as highly active materials on the surface in a food without the formation of substantial amounts of free fatty acids, which reduce the food's ability to oxidize after the storage, because free fatty acids are more prone to oxidation than the corresponding fatty acid esters.

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In a further aspect, the present invention provides the use of BR a lipolytic enzyme to generate other functional compounds according to: the present invention in a plant material containing lipid, It must be understood that the action of enzymes that modify i 5 lipids, such as lipolytic enzymes on plant material containing lipids, can generate not only functional lipids, but also other functional compounds, such as with the action of a lipid transferase, in which an acyl group of a lipid is transferred for one or more other accepting substrates, such as one or more of the following: a sterol, a stanol, a carbohydrate, a protein, a protein and glycerol subunit.

In some particular modalities, the functional compounds' generated in the methods according to the present invention are functional esters. In some embodiments, both functional lipids and the other functional compounds are generated by the methods according to the price. -. feels invention.

Those functional compounds generated by the methods according to the present invention can then be used in the manufacture of a dough and / or a baked product, comprising adding the functional di-compounds to a dough and (optionally) baking the dough for making a baked product with one or more of the following: reduced viscosity of the dough; improved machinability of the dough; reduction of bubbles during the cooking of the baked product; improving the volume & / or softness of the bread; prolongation of the shelf life of the baked product and dough; improvement of the anti-aging effect of the baked product and / or pasta; improving the structure of the baked product's crust; reduction of the heterogeneity of the pore of the baked product; improving the homogeneity of the baked product pore; reduction of the average pore size of the baked product; intensification of the gluten index of the dough; improving the taste and / or odor of the baked product; perfecting the color of the baked product's crust. | In one aspect, the functional compounds generated by the |

, according to the present invention are purified or partially purified. checked. . In one aspect, the functional compounds generated by the methods according to the present invention are not purified prior to use in a humment. In one aspect, the functional compounds generated by the methods according to the present invention are formulated as a dry product.

In one aspect, 05 functional compounds are concentrated or diluted before use in a food. In another aspect of the invention, a method is provided for making noodles or a noodle dough or a noodle-based product, the method of which includes adding a functional compound according to the present invention to the noodles, noodle dough or a product “15 based on goodies,. In one aspect of the present invention, use of a functional compound according to the present invention in the manufacture of pasta or a pasta-based product is provided to enhance one or more of the yellowish color / tone characteristics, color stability, reduction of gloss, reduction of fat content, improvement of texture and bite (thickness), reduction of water activity, reduction of breakage, increase of color firmness and improvement of size maintenance during processing, In another aspect of the invention, a method for making tortilla or flour dough for tortilla is foreecido, the method of which includes adding a food processor generated according to the present invention to tortilla or dough for tortilla.

In another aspect of the invention, a method is provided for making pasta or whole grain pasta or pasta dough, the method of which includes adding a food processor generated in accordance with the present invention to the pasta or pasta dough for noodles.

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A further aspect of the present invention provides for the use of a food processor generated in accordance with the present invention in the manufacture of a tortilla or tortilla dough to improve the rolling of the tortilla, increase the flexibility of a tortilla , perfecting the anti-aging properties of tortilla and / or dough for tortilla, perfecting softness and / qu reducing the loss of flavor of the dough and / or dough for tortilla. The functionality of the food processor can be improved by combining it with emulsifiers such as DATEM. Suitably, the present invention can provide one or more of the following unexpected technical effects in a foodstuff: improved aspect, improved taste, improved stability, in particular. show improved thermal stability, improved taste, improved smoothness, improved elasticity and improved emulsification. Suitably, the present invention can provide one or more of the following unexpected technical effects in dairy products, such as ice cream: an improved taste ( preferably a more creamy taste); improved taste and improved fusion. Suitably, the present invention can provide one or more of the following unexpected technical effects on the egg or products made with egg; improved emulsion stability; ferric stability of the emulsion; improved taste; odor reduction; improved thickening properties; improved consistency Specific technical effects associated with the use of the food processor as defined here in the preparation of a food are listed in the table below: [no BR

OO EEE mechanical and increases the water absorption capacity. It increases the volume of bakery products and maintains the softness of the kernels. |

- [Care Pão de só Benefit the volume of the cake and the smooth texture 'uniform 7 4 | Cookie, cookie and cockie | Makes fat emissions more stable. And it avoids sticking to the machine. Prevents lamination of very greasy products | | Pastry and bakery Perfect the texture of fried products Noodles Prevents the adherence of the dough to the machine. Increases 6 water content and reduces loss in cooking Instant noodles Prevents noodles from sticking together Pasta Pasta conditioner prevents sticking when cooking 'Pastry cream Makes the starch paste soft and creamy and prevents dehydration Cream does not milk for | Prevents the separation of oil and water Whipped cream Provides a stable emulsion effect - ever termination 13 | Caramel, candy and nougat Improves the emulsification of melted sugar and oil. Prevents the separation of processed 14 Cam oil, salsi- | It improves the water retention capacity of sausages and ham and avoids the separation of the oily phase of pasta and purees.

In a further aspect of the present invention, the use of a lipolytic enzyme in a process for the preparation of functional lipids is provided. In another aspect of the present invention, a process for preparing a lysophospholipid, for example, lysolecithin, is provided. The process comprises treating a plant material containing lipid with the lipolytic enzyme according to the present invention.

In a further aspect of the present invention, the use of a lipolytic enzyme in a process for the preparation of a lyso-glycolipid (for example, digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG)) is provided by the treatment of a material)

"of plant containing lipid with the lipolytic enzyme according to the present invention. In some embodiments of the invention, the liquid suspension of at least part of the plant material containing partially solubilized lipid is essentially starch free.

In some embodiments of the invention, less than about 50%, such as less than about 40%, such as less than about 30%, such as less than about 20%, such as less than about - about 10%, such as less than about 6%, such as less than about 3%, such as less than 1% (weight / weight) of the liquid suspension of plant material it contains partially solubilized lipid is starch or starch-containing components, such as flour,: Consequently, in some embodiments, it is understood that enzymes are to have an enzymatic effect on plant material containing lipid which is essentially starch-free or containing: only residual starch from a previous processing step.

The present invention is not intended to cover the enzymatic treatment of compositions with additional preparations of added flour, such as in enzymatic applications in situ in bread making, Determination of galactolipase activity (glycolipase activity test): Substrate: Digalactosyldigalicerioid 0.6% (Sigma D 4651), Triten-X 100 0.4% (Sigma X-100) and CaCl; 5 mM were dissolved in HEPES 0.05 M pH7 buffer, Assay procedure: 400 µl of substrate was added to a 1.5 ml Eppendarf tube and placed in an Eppendorf Thermomixer at 37ºC for 5 minutes.

At time t = 0 min, 50 µl of enzyme solution was added.

A control with water instead of an enzyme was also analyzed.

The sample was mixed at 10100 rpm in an Eppendorf Thermomixer at 37ºC for 10 minutes.

At time t = 10 min, 6 Eppendorf tubes were placed in another mixer |

"thermal at 89ºC for 10 min. to stop the reaction.

The free fatty acid in the samples was analyzed using the NEFA € kit from WAKO GnbH. . The GLU enzyme activity at pH 7 was calculated as micro-moles of fatty acid produced per minute under the conditions of the assay.

Determination of phospholipase activity (phospholipase activity assay): Phospholipase activity was measured using two different methods that provide comparable results.

Any of these methods can be used to detect phospholipase activity according to the present invention. “PLU assay" for determining phospholipase activity 'Substrate:;' La phosphatidylcholine 95% Plant 0.6% (Avanti H441601), Triton-X 100 0.4% (Sigma X-100) and 5 mM CaCl were dissolved in buffer HE-PESOOSMpH? 7. Test procedure: 400 µl of substrate were added to a 1.5 ml Eppendorf tube and placed in an Eppendorf Thermomixer at 37ºC for 5 minutes.

At time t = 0 min., 50 µl of enzyme solution was added.

A control with water instead of an enzyme was also analyzed.

The sample was mixed at 10 * 100 rpm in an Eppendorf Thermomixer at 37ºC for 10 minutes.

At time t = 10 min., The Eppendorf tube was placed in another thermal mixer at 99ºC for 10 min. to stop the reaction.

The free fatty acid in the samples was analyzed using the NEFA C kit from WAKO GnbH.

The activity of the PLU-7 enzyme at pH 7 was calculated as fatty acid microemoles produced per minute under the conditions of the assay. "TIPU Assay" for determination of phospholipase 1 activity TIPU (Phospholipase Titration Unit) is defined as the amount of enzyme that releases 1 pumol of free fatty acid per minute under the test conditions.

Phospholipase A1 and A? catalyze the conversion of lecithin to lyso-lecithin with the release of free fatty acid from positions 1 and 2, respectively |

' mind.

The phospholipase activity can be determined by the continuous titration of the fatty acids released from lecithin during enzyme, since the. alkali consumption equals the amount of fatty acid released.

Substrate:

5 Lecithin 4%, Triton-X 100 4% is CaCl; 6 mM: 12 q of powdered lecithin (Avanti Polar Lipids X44160) and 12 g of Triton-X 100 (Merck 108643) were dispersed in approximately 200 ml! demineralized water during magnetic stirring. 3.0 ml! CaCl? 0.5 mM (p.à.

Merck 1.02382) have been added.

The volume was adjusted to 300 mL with demineralized water and the emulsion was homogenized using an Ultra Thurax.

The substrate was freshly prepared each day.

Assay procedure: an enzyme solution was prepared to give a ti- curve. between 0.06 and 0.48 ml / min. with the addition of 300 µl of enzyme.

A sample of known activity tracking is included.

The samples were dissolved in demineralized water and stirred for 15 min. at 300 rpm. . 25.00 ml of substrate had the temperature adjusted to 37ºC for 10-15 min. before the pH was adjusted to 7.0 with 0.05 M NaOH. 300 µL of enzyme solution was added and continuous titration with 0.05 M NOH was performed using a pH-Stat titrator (Phm 290, Mettler Toledo). Two activity determinations were made on each scale.

After 8 minutes, the titration is completed and the slope of the titration curve is calculated between 5 and 7 minutes.

The detection limit is 3 TIPU / ml of enzyme solution.

The phospholipase activity (TIPU / g of enzyme) was calculated as follows: Tue ma mi can, m: V, A where: a is the slope of the titration curve between 5 and 7 minutes of the reaction time (mlmin.) . N is normal for the NãaOH used (mol / 1). V1 is 6 volume in which the enzyme is dissolved (ml). |

Í m is the amount of enzyme added to V1 (g). - V2 is the volume of enzyme solution added to the substrate “(m)), Determination of triacylglyceride lipase activity: assay based on triglyceride (iributiine) as substrate (LIPU): The activity of lipase based on tributyrin is measured by according to the Food Chemical Codex, Fourth Edition, National Academic Press, 1996, p. 803, with the modifications that the sample is dissolved in deionized water instead of glycine buffer and the pH of statistical adjustment is 5.5 at the same time7. 1 LIPU is defined as the amount of enzyme that can release '1 mole of butyric acid per minute under test conditions. . In one aspect of the invention, the lipolytic enzyme used in accordance with the present invention can be obtained from a filamentous fungus. More Ú 15 preferably, the fungal lipolytic enzyme is obtained (preferably obtained). Fusarium spp. Preferably, the fungal lipolytic enzyme used in accordance with the present invention can be obtained (preferably obtained) from Fu- sarium heterosporum i Fusarium semitectum. Suitably, the fungal lipolytic enzyme used according to the present invention can be obtained (preferably obtained) from Fusarium heterosporum (CBS 782.83) or Fusari-a semitectum (IBT 9507).

Therefore; in one aspect, the fungal lipoolytic enzyme used in accordance with the present invention is preferably a filamentous fungus lipolytic enzyme, preferably a filament-sodetype-wild fungal lipolytic eryzyme.

In some of the applications mentioned here, particularly food applications, such as bakery applications, the food beneficiary generated in accordance with the present invention can be used with one or more conventional emulsifiers, including, for example, monoglycerides , esters of diacetyl tartaric acid of mono and diglycerides of fatty acids, sodium stearoyl lactylate (SSL) and lecithins.

The beneficiary of food generated by the agreement methods |

"with the present invention it is especially preferred in bread recipes with + added fat. In addition or alternatively, the food processor generated by the methods according to the present invention can be used Ú: 5 with one or more food-grade enzymes Therefore, it is within the scope of the present invention that, in addition to the lipolytic enzyme of the present invention, at least one additional enzyme can be added to the baked product and / or the dough. Such additional enzymes include enzymes that degrade starch such as endo or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, for example, glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as that which oxidizes to 'maltose, for example, hexose oxidase (HOX), lipases, phospholipases, galactic lipases and hexose oxidase, proteases and acyltransferases (such as those described in WO04 / 064987, p example).

- It is particularly preferred that the lipolytic enzyme used in accordance with the present invention is used in combination with alpha-amylases in the production of food products. In particular, amylase can give a non-maltogenic amylase, such as a polypeptide that has non-maltogenic exo-amylase activity, in particular, 14 alpha alpha maltotetrahydrolase activity (EC 321.60) (as described in WOD05 / 003339 ). Is a suitable non-maltogenic amylase available commercially as Powersoft? (made available by Danisco A / S, Denmark). Maltogenic amylases such as Novamylº (Novozymes A / S, Denmark) can also be used. In one embodiment, the combined use of alpha-amylases and the inventive foodstuff can be used in a dough and / or in the production of a baked product such as bread, cakes, donuts, donut cake or bagels . The combination of alpha-amylases and the food processor of the invention is also considered preferable for use in tortilla production methods, such as wheat and / or corn tortillas.

In another preferred modality, the food processor generates |

according to the present invention can be used in combination with - a xylanase in the production of food products.

GRINDAMYLº and PO-. WERBake 7000 are examples of commercially available xylanase enzymes. Danisco A / S levels, Other examples of xylanase enzymes can be found at VWO03 / 020923 and WOD1 / 42433. Preferably, the food processor generated in accordance with the present invention can be used in combination with a xylanase and an alpha-amylase. Suitably, the alpha-amylase can be a maltogenic or non-myaliogenic alpha-amylase (such as GRINDAMYLº or PO- WERSOft, made available by Danisco A / S) or a combination thereof.

The food processor of the invention can also preferably be used in combination with an oxidizing enzyme, such as: an enzyme that oxides Maliose (MOX), for example, hexose oxidase (HOX). Suitable methods are described in WO03 / 099016. Enzymes that oxidize maltose commercially available from Danisco A / S are - GRINDAMYL '"and SUREBake.

Optionally, an alpha-amylase, such as a non-maltogenic exo-amylase and / or a maltogenic amylase and / or an enzyme that oxidizes malitosis (MOX) in combination with the enzyme, can be used in the methods according to the present invention. for the preparation of a dough, a baked product, tortilla, cake, noodles, fried instant noodles, or a milk product such as cheese.

The food processor generated in accordance with the present invention is included in the food, typically by methods known in the art. Such methods include adding the food processor directly to the food or composition, adding the food processor in combination with a stabilizer and / or vehicle and adding a mixture comprising the food processor and a stabilizer and / or vehicle.

Stabilizers suitable for use with the present invention include, but are not limited to inorganic salts (such as NaCl, ammonium sulfate), sorbitol, emulsifiers and detergents (such as Tween 20, Tween 80, Panodan AB100 without triglycerides, polyglycerol ester, mono- |

, sorbitan oleate), oil (such as canola oil, sunflower oil and soy oil), pectin, trehalose, sorbitol and glycerol. . Vehicles suitable for use with the present invention include,. but are not limited to starch, cereal flour, milled wheat, wheat flour, NaCle citrate.

For baked goods such as bread, steamed bread and American white bread, for example, the addition of a food processor of the present invention can result in one or more of the following: increased bread volume and softness, longer life prolonged useful life and / or anti-decomposition effect, improved crumb structure, reduced pore heterogeneity, reduced average pore size, enhanced gluten index, improved flavor and / or odor and improved crust color. Advantageously. , the food processor generated in accordance with the present invention can be used to replace emulsifiers in foods such as dough and / or baked products.- The food processor generated in accordance with the present invention may have synergy with emulsifiers such as DATEM, SSL, CSL, monaglycerides, polysorbates and Tween. Therefore, the food processor generated according to the present invention can be used in combination with one or more emulsifiers, Advantageously, the use of the food processor generated in accordance with the present invention in combination with one or more emulsifiers can reduce the total amount of emulsifier used compared to the amount needed when no food processor generated according to the present invention is used.

The food processor generated according to the present invention may also have synergy with hydrocolloids, Guar, xanthan and pectin and with enzymes that oxidize maltose, such as hexose oxidase.

For donuts, donut cakes, bagels, muffins and muffins, for example, the food processor of the present invention can result in a synergistic effect when used in combination with one or more alpha-amylases, maltogenic alpha-amylase and non-maltogenic alpha-amylase.

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For cakes, sponge cake and palm cake, for example, the use of the food processor of the present invention can result in a synergistic effect when used in combination with one or more such hydrocolloids. as Guar and / or one or more emulsifiers such as DATEM.

For biscuits, for example, the use of the food processor generated in accordance with the present invention provides improved properties of rolling and handling capacity, particularly when cold (cold rolling), Advantageously, in mayonnaise and other egg-based products , for example, and using a food processor generated in accordance with the present invention could lead to improved texture, reduced mean particle size and / or reduced mean particle size distribution, improved stability thermal, better microwaves performance and / or stability, i 15 In cakes, the use of the present invention advantageously leads to improved - smoothness, volume, storage properties and improved service life.

For pasta or pasta-based products, for example, instant noodles, the food processor of the present invention Z20 can provide one or more of the following characteristics: improved yellowish color, more stable color characteristics, gloss reduction, reduction of the fat content, improvement of the texture and of the bite (thickness), reduction of the water activity, reduction of breakage, increase of the firmness of the color and improvement of the maintenance of the format during the processing.

Preferably, the food processor of the present invention can be used to reduce the fat content of a pasta or a pasta product, for example, instant noodles, In tortillas, for example, the use of a food processor generated from according to the present invention may result in one or more of the following: reduced rolling capacity of the tortilla, for example, by increasing flexibility, improved anti-decomposition properties |

Í das, improved softness and / or reduced lack of flavor. - Advantageously, the rolling capacity and / or flexibility: improved can lead to a reduced probability of shattering: of the tortilla when rolled.

The food processor generated in accordance with the present invention is particularly useful in the preparation of baked goods, such as those prepared from a dough, including breads, cakes, sweet dough products, puff pastry, liquid pastry, muffins, donuts, cookies, crackers and cookies.

The food processor generated in accordance with the present invention is particularly useful in the preparation of cereals for breakfast, such. 'like those prepared from a dough,, The food processor can also be used as an additive: for bread improvement, for example, flour dough compositions, dough additive, dough conditioners, pre -mixtures and prepara-. Similar tions conventionally added to flour and / or dough during the manufacturing processes of bread or other baked goods, to provide improved properties to bread or other baked goods. Therefore, the present invention relates to a composition for improving bread and / or a composition for improving dough which comprises a food processor generated in accordance with the present invention; and also to a dough or baked product that includes such a composition for improving bread and improving dough.

The bread improving composition and / or the dough improving composition may comprise, in addition to a fungal lipolytic enzyme according to the present invention, other substances, the substances of which are conventionally used in baking for perfecting. the properties of dough and / or baked goods. The composition for bread improvement and / or the composition |

'for the improvement of dough may comprise one or more - conventional baking agents, such as one or more of the following constituents:: powdered milk, gluten, an emulsifier, granulated fat, an oxydant, a amino acid, sugar, salt, flour or starch.

Examples of suitable emulsifiers are: monoglycerides, diacetyl tartaric acid esters of mono- or diglycerides of fatty acids, sugar esters, sodium stearoyl lactylate (SSL) and lecithins.

The bread and / or flour dough improvement composition may comprise another enzyme, such as one or more suitable food-grade enzymes, including enzymes that degrade 6 starch 7 such as endo or exoamylases, pullulanases, debranching enzymes, hemisphere -: lulases including xylanases, cellulases, oxidoreductases, for example, glycoside oxidase, pyranose oxidase, sulfhydryl oxidase or a oxidized carbohydrate such as that which oxidizes maltose, eg hexose oxidase: (HOX), lipases , phospholipases, galaciolipases and hexose oxidase, proteases and acyltransferases (such as those described in WOO04 / 064987, for example).

The term "baked product" as used here, includes a product prepared from a dough. Examples of baked products (white, light or dark) that can be advantageously produced. by the present invention include one or more of the following: bread (including white, wholegrain and rye bread), typically in the form of slices or rolls or toast, French baguette bread, pita bread, tortillas, tacos, cakes, pancakes, cookies, pasta, noodles and the like).

The dough according to the present invention can be a fermented dough or a dough to be subjected to fermentation. The dough can be fermented in various ways such as by adding sodium bicarbonate or the like or by adding a suitable yeast culture such as a Saccharomyces cerevisiae culture (bread yeast).

The dough according to the present invention can |

Í be a dough for the preparation of a dehydrated cereal product, toast, biscuit or biscuit,. Specific embodiments of the invention:: As discussed above, the present invention relates to a method for the treatment of lipid-containing plant material, the method comprising the step of treating a liquid suspension of at least one plant material containing lipid partially solubilized with one or more lipid-modifying enzymes. In some embodiments, the method further comprises a preceding or simultaneous step of treating a liquid suspension of a plant material containing lipid to obtain said plant material: partially solubilized. : In some embodiments, the method further comprises: a subsequent step of treating a liquid suspension to obtain said "solubilized plant material."

- In some embodiments, 6 treatment to obtain at least one partially solubilized plant material is a treatment with one or more enzymes that modify the cell wall. In some embodiments, the treatment to obtain at least one partially solubilized plant material is a treatment by sonication, such as ultrasonic treatment and / or extrusion, In some embodiments, the liquid suspension of plant material at least partially solubilized contains insoluble plant material, In some embodiments, the plant material is treated under the said method steps simultaneously.

In some embodiments, the plant material is treated under said steps of the method according to the present invention without removing a substantial amount of any component.

In some embodiments, the liquid suspension is treated additionally with one or more additional enzymes.

In some embodiments, the additional enzyme is one or more |

transglycosylation enzymes. z In some embodiments, the additional enzyme is a protease. : In some modalities, one or more enzymes that modify: 6 lipid is a lipolytic enzyme selected from the group consisting of: a triaciglycerol lipase, a phospholipase and a galacto-lipase.

In some modalities; one or more enzymes that modify the lipid contains two or three activities selected from the group consisting of: triacylalkyl] lipase activity, phospholipase activity and galacto-lipase activity.

In some embodiments, one or more lipid-modifying enzymes is one, two, three, four or five lipid-modifying enzymes: different. . In some embodiments, the method according to the present: invention comprises an additional step of isolating the soluble fraction. In some embodiments, one or more enzymes that modify the cell wall are selected from the group consisting of a xylanase and a cellulase, such as cellobiohydrolases, endoglycanases and beta-glycanase. In some embodiments, cellulase is selected from an endocellulase, an exocellulase, a cellobiase, an oxidative cellulase, a cellulosphosphorylase. In some embodiments, one or more additional enzymes are enzymes that modify the starch, selected from the group consisting of an alpha-amylase, a pullulanase, isoamylase and a beta- amylase.

In some embodiments, one or more transglycosylation enzymes are selected from the group consisting of enzymes of the EC enzyme class 3.2.1.20.

In some embodiments, the plant material is supplied in particles, in which the average particle size of said particulate plant material is below 3000 µm, such as below 1000 µm, as below 500 pm In some embodiments, the material of plant is a bran.

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"In some modalities, cereal bran is selected from 'wheat, barley, oats, rye, triticalêe, rice and corn.

"In some embodiments, the method according to the present: invention additionally comprises a preceding step of i) fractionating the cereal grain obtained to obtain the endosperm, bran and germ; ji) separating and distributing the endosperm, bran and germ to enable that they are treated, and iii) grind the bran. In some embodiments, the cereal bran is obtained from an industrial grinding process and subsequently ground to obtain a 1D average particle size below 500 pm, as below 400 pm, such as below 200 µm., In some embodiments, the plant material is a side product of processing plant material, such as vegetable oil refining paste, grains used for fermentation or soluble distillation dry grains (DDGS).

. In some embodiments, the composition obtained comprising the modified lipids, such as functional lipids, is further treated to inactivate the additional enzyme activity.

In some embodiments, the degree of solubilization obtained from said plant material, as determined on the dry matter versus the dry matter of plant material, is greater than 15%, such as greater than 25%, such as greater than 35%, such as greater than 40%, such as greater than 50%, such as in the range between 40% to 60%, such as in the range between 50% to 60%.

In some embodiments, the total content of modified lipids and lipids, such as functional lipids, as determined on the dry matter versus the dry matter of plant material in the soluble fraction obtained is at least about 0.05%, such as at minus about 1.0%, such as in the range of 0.05 to 5%.

In some embodiments, the method according to the present invention further comprises a step of dehydrating the obtained composition which comprises lipids and / or modified lipids, such as fungi |

national. - In some modalities, the method in accordance with this. The invention further comprises a step of spray drying the composition obtained which comprises lipids and / or modified lipids, such as functional lipids.

In some embodiments, the method according to the present invention further comprises a lyophilization step of the obtained composition which comprises lipids and / or modified lipids, such as functional lipids. In some embodiments, treatment with one or more enzymes that modify lipid generates functional lipids, such as emulsifiers. : In some modalities, treatment with one or more enzymes. but which modify lipid generates other functional compounds, such as functional sterol esters.

: In some modalities, treatment with one or more lipid-modifying enzymes generates more than 5%, such as more than 10%, such as more than 25%, such as more than 50% of conversion phosphatidylinosite] in lysophosphatidylinositol (smooth-P1).

In some embodiments, treatment with one or more lipid-modifying enzymes hydrolyzes at least 5% of the phospholipids, such as at least 10% of the phospholipids, as well as at least 20% of the phospholipids, such as at least 50% phospholipids, as well as at least 75% of phospholipids.

In some embodiments, treatment with one or more lipid-modifying enzymes hydrolyzes at least 5% of the glycolipids, such as at least. 10% of glycosipids, as well as at least 20% of glycolipids, as well as at least 50% of glycolipids, as well as at least 75% of glycolipids.

In some embodiments, treatment with a More lipid-modifying enzymes hydrolyzes at least 5% of triglycerides, as with at least 10% of triglycerides, as well as at least 20% of triglycerides.

'glycerides, such as at least 50% of triglycerides, as well as at least 75% of triglycerides. ; In some embodiments, the composition comprising the modified lipids & / or lipids, such as the functional lipids obtained in accordance with the invention method is added directly as a mixture of soluble and insoluble plant material in the production of a product alimenticio. In some embodiments, the food product according to the present invention is selected from the group consisting of bread, a breakfast cereal, pasta, biscuits, cookies, snacks and beer.

EXAMPLES 'EXAMPLE 1' Example 1. Modification in the Commercial Scale of Commercial Wheat Bran. and wheat bran lipids followed by the evaluation in baking: Bran: “Wheat bran fractions obtained from a commercial mill were used. The fractions consisted of a fraction of fine bran and a fraction of coarse bran. Before use, the coarse bran fraction was ground to obtain a smaller particle size, which will increase the specific surface of the bran, eventually increasing the efficiency of the enzymatic solubilization of the bran. The grinding was carried out in a Retch grinder to obtain an average particle size of 500 pum. However, it should be noted that a smaller particle size may be preferable, in relation to the degree of solubilization, Enzymes Table 1. Enzymes used for the modification of wheat bran Enzyme Activity - - Enzyme ID Xylanase Bacterial Xylanase Cellulase / glycanase Genencor GC220 Amylase Genencor, Spezyme Fred (4016101001) |

B4 / 78 'Enzyme Activity Enzyme ID Pululanase Genencor Optimax 1-1000 (401-05349-002). Beta-amylase Genencor Optimalt BBA (EDS 221) Fosío-galacto lipase Grindamy | Powerbake 4070 Transglycosidase Genencor TGL-500 (1600675782) Protocol: Table 2. Protocol used for bran modification Wheat bran was suspended in 50 mM NaPí, pH & (13% weight / weight) in a container / reactor with closed lid z 5 The bran suspension is heated to 100ºC with agitation and boiled for 2 min. , The sample is stirred (with the lid closed) at 50 ° C. * C and left to equilibrate in relation to temperature The enzymes are added and the reaction is continued at 50ºC for 24h (temperature and time can be further optimized) Supernatant is separated from solid residues Supernatant is boiled to inactivate additional enzymatic activity Sample is resized and stored to avoid contamination Péléte is lyophilized Supernatant is analyzed Tests: The following modifications were made to the bran (table 3) Table 3: Amount of bran, g, treated with different enzymes Grams of enzyme sample / 109 of bran Test Fo Tra e GC220 = Ú a> Transa.

Fo z 10 667 112 005 004 001 OO 005 0000007 3 10 667 112 005 DO 00) OM 0.05 - 0000017 4 10 68867 112 005 0M 00 o 005 0000172 |

'Analysis: j The soluble fraction of the bran (the supernatant) is analyzed with respect to:: Dry matter content (%): A quantitative sample of the soluble bran obtained is lyophilized. After lyophilization, the sample size is again quantified and the amount of dry matter is calculated. As a control, the buffer is included in the analysis. Evaluation of lipid modification using cooking tests: Recipient of cooking: The performance of cooking flour, flour with addition of solubilized bran was evaluated in small-scale cooking tests. (50 g of mixture and 10 9 slices) using the recipe below (table 4). i. Table 4. Recipe used to evaluate the performance of flour, flour with addition of solubilized bran and reconstituted flour with addition of - insoluble bran. Salfaçúcar is a 1: 1 (weight / weight) mixture of salt and sugar. Water is the absorption water determined by Farinographic analysis. Ingredients. Mini scale ml or g Flour so Dry yeast 1 SaltSugar 16 Water 400BU -2% Dough making and cooking The flour (or the mixture of flour and bran) and the dry ingredients are mixed for one minute, after which the water is added and mixing is continued for another five minutes.

After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were shaped into bread using a manual molder. The slices were placed on a baking tray and placed in a closed container (with a lid) and left on the table for 10 minutes. After that, the bread is fermented at 34ºC RH of |

'85% for 45 minutes and finally roasted at 230ºC for five minutes in a - Bago oven (Bago-line, Fábora, Denmark). During mass expansion, * viscosity was subjectively assessed on a scale of 1 (very viscous) to 5 (dry). The bread was cooled for 20 minutes before evaluation (weighing, volume measurement; crumb, crust and sensory evaluation). Cooking tests The cooking tests below were performed (table 5). Table 5. Experimental structure of the cooking test. 1! D refers to the composition of the flour with the addition of solubilized bran or reconstituted with insoluble bran, 68 numbers in parentheses refer to the treatment of the flour according to table 3. Flour ( g) is the amount of. flour in bread.

Bran (6) is the amount of bran used for reconstitution.

Sol bran. (Ml!) Is the amount of solubilized bran added to the flour instead of water.

Water (ml) is the amount of water added to the meal "Bran" (%) is the amount of meal, both solubilized and insoluble meal based on the weight of the flour.

TIPU refers to the Phospholipase Titration Unit as described above.

Lozimento o Farona: Foo Soo Water spout TA starts 8 0 0 2800 - 2 Bran & 150% are 2900 = 5.22 3 str o 28.00 - 4 re o o 29.00 - jo 5 OonPUca 50 o 29 , 00 - 8 Bran 5.0% a75 25 - 29.00 5.00: Results: Degree of solubilization of the bran: Based on the dry matter, the amount of bran solubilized in the tests was approximately 54%. Cooking results: |

: As can be seen in fabela 6 and figure 1, the addition of soluble fibers has little or no effect on the specific volume of the bread.

However, the combination of bran solubilization and phospholipase has a significant effect on bread volume.

Table6 Results of the cooking test.

ID refers to the composition of | flour with the addition of solubilized bran or reconstituted with insoluble bran.

Flour (9) is the amount of flour in the bread.

Bran (gq) is the amount of bran used for reconstitution.

Specific Volume (mg / ml) is the absolute specific volume of bread.

Relative volume versus control (%) is the relative volume of bread versus bread 1 (control): Baking D Specific Volume, - Relative volume mi / g versus control. k Control 3.58 100 BR 2 Bran Bran.5.0% 346 a7 Bran Bran, 5.0%,: 3 1TIPU 4.05 113 Bran Bran, 5.0%, 4 10TIPU 4.24 118 Bran Bran.5 , 0%, 5 100TIPU 497 138 6 Bran 5.0% 3.38 4 The resulting breads can be seen in figure 2, Example 2. Modification in laboratory scale of commercial wheat bran lipids sequenced by the evaluation in cooking: To evaluate the effect of modifying the lipid fraction that generates functional lipids with emulsifying properties.

Another experiment using different doses and different lipases was carried out.

Bran: Fractions of wheat bran obtained from a commercial mill were used.

The fractions consisted of a fraction of fine bran and a fraction of coarse bran. Before use, the fraction of coarse bran was ground to obtain a smaller particle size, which will increase the specific surface |

Only the bran, eventually increasing the efficiency of the solubilization en-. of the bran.

The grinding was carried out in a Retch grinder to obtain. an average particle size of 500 µm.

However, it must be observed; that a smaller particle size may be preferable, in relation to the degree of solubilization.

Enzymes: Table 7: Enzymes used for the modification of wheat bran Enzyme Activity 1D of the Enzyme Phospho-galacto lipase Grindamyl Powerbake 4070 - Lipase EDS 321 'Protocol:: Table 8. Protocol used for the modification of the bran The wheat bran was suspended in 59 mM NaPi, pH 5 (13% weight / weight) in a closed lid container / reactor The bran suspension is heated to 100ºC with stirring and boiled for 2 min.

The sample is placed under agitation (with the lid closed) at 50ºC, left to equilibrate in relation to temperature. Enzymes are added and the reaction is continued at 50ºC for 24 h (temperature and time can be optimized later). Supernatant is separated from solid residues. Supernatant is boiled to inactivate additional enzymatic activity Sample is cooled and stored to prevent contamination Pellet is lyophilized Sabrenadante is analyzed Tests: The following modifications were made to the bran (table 9) |

Ú Table 9: Amount of bran, g, treated with different enzymes: Grams of enzyme sample / 10g of. bran Test —Farelb, g Bufferg —Phosfoipass —Lipase '1 10 886.7 0.0009017 2 10 66.7 0.000172 3 10 686.7 7.28E-09 4 10 66.7 7.28E-08 66, 7 7.2BE-07 8 10 86.7 D, 00D017 7.28E-08 Analysis: The soluble fraction of the bran (the supernatant) is analyzed with re-. a: 5 Dry matter content (%): 'A quantitative sample of the soluble bran obtained is lyophilized. After lyophilization, the sample size is again quantified and a. amount of dry matter is calculated. As a control, the buffer is included in the analysis. 1D Evaluation of lipid modification using cooking tests: Cooking recipe:

The performance in cooking flour, flour with addition of solubilized modified bran was evaluated in small scale cooking tests (50 gq of mixture and 10 g slices) using the recipe below

beautiful10). Table 10. Recipe used to evaluate the performance of cooking flour, flour with added solubilized bran and reconstituted flour with added insoluble bran.

SaVaçúcar is a 1: 1 (weight / weight) mixture of salt and sugar.

To water is the water of absorption determined by the Pharyngographic analysis.

Ingredients Mini scale ml or q Flour 50 Dry yeast 1 |

"Mini scale ingredients. Salt / Sugar 16. Water 400BU -2%. Dough making and cooking The flour (or the mixture of flour and bran) and the dry ingredients are mixed for one minute, after which the water is added and mixing is continued for another five minutes.

After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were shaped into bread using a marnual molder. The slices were placed on baking sheets and placed in a closed container (with a lid) and left on the table for 10 minutes. After that, 6 bread is fermented at S4ºC RH of - 10 85% for 45 minutes and finally baked at 230ºC for a minute belt in a - Bago oven (Bago-line, Fáborg, Denmark). The bread was cooled for 20 minutes before the evaluation (weighing, measuring the volume, evaluating the crumb, the 'crust and sensory). Cooking tests The cooking tests below were performed (table 11). Table 11. Experimental structure of the cooking test. ID refers to the composition of the flour with the addition of solubilized bran, 08 numbers in parentheses refer to the treatment of the flour according to the table

9. Flour (9) is the amount of flour in the bread. Bran Extract (ml) is the amount of solubilized bran added to the flour instead of water. Water (ml) is the amount of water added to the flour. Cooking ID Pharma: ta fo TT Paste 8 o 328. 3 a = 50 29.00 - 4 Bran lipid 100 TIPU (2) to 29.00 - 5 cloth as 3 to 29.00 - |

| Cooking - Fame ID o = a. : 6 to 5 and 29.00 -. 7 Meal lipid 300 LIPU (5) 50 29.00 = 8 Meal lipid 10TIPI + 3 LIPU only 29.00 - (6) Results: Degree of bran solubilization: Based on the dry matter, the amount of bran solubilized in the testing was approximately 30%. - Cooking results:. As can be seen in table 12 and figure 3, the addition of soluble fibers has little or no effect on the specific volume of bread, however, the combination of bran solubilization with phospholipase 'has a significant effect on the volume of bread. : 10 Tabelja12. Cooking test results.

ID refers to the composition of the flour with the addition of solubilized bran, 05 numbers in parentheses refer to the treatment of the bran according to table 9, Flour (9) is the amount of flour in the bread.

Specific Volume (mgími) is the absolute specific volume of bread.

Relative volume versus control (%) is the relative volume of breads versus bread 1 (control) Baking Ta Specific Volume, Relative volume mia versus control 1 Control 3.39 100 2 Bran lipid from 3.16 to controls Lipid from bran 1D 3 O neu O) 32 7 Bran lipid 100 4 TIPU (2) 2344 101 Bran lipid 3 $ p LIPU (3) 318 o 6 Bran lipid 30 3.20 94 |

: ID YVolumeEspecific cooking, “Relative volume: 'mig versus control

The UT Meal lipid 300 '7 P UPU GS) 3.9 97 Meal lipid 8 10TIPI + 3 LIPU (6) healthy The resulting breads can be seen in figure 4.

As can be seen from the results above in experiment 2, no significant effect was obtained on cooking performance in this experiment. Compared with example 1, the structure of e- is 5 xemplo2different absence of cell wall and enzymes that modify the starch, so it can be concluded that there must be a synergistic effect between the enzymes that modify the cell wall, the starch and lipids in relation to 'generation of functional lipids from wheat bran. Example 3. Modification on a commercial scale of triple commercial bran wheat bran ellipids by cooking evaluation -2: To further evaluate the synergistic effect of modifying wheat bran with enzymes that modify the cell wall, starch and lipids , this experiment was carried out.

Bran: Fractions of wheat bran obtained from a commercial mill were used. The fractions consisted of a fraction of fine bran and a fraction of coarse bran. Before use, the coarse bran fraction was ground to obtain a smaller particle size, which will increase the specific surface of the bran, eventually increasing the efficiency of the enzymatic solubilization of the bran. The grinding was carried out in a Retch grinder. to obtain an average particle size of 500 µm. However, it should be noted that a smaller particle size may be preferable, in relation to the degree of solubilization.

Enzymes: |

'Table13: Enzymes used for the modification of wheat bran: Enzyme Activity, Enzyme ID: Xylanase Bacterial Xylanase Cellulase / Glycanase Genencor GC220: Genencor, Spezyme Fred Amilãss (4016101007) Phospho-galactose lipase Grindamyl Powerbake 4070 Protocol: Table 14. Protocol used for bran modification. The wheat bran was suspended in 50 mM NaPi, pH 5 (13% pe- - 5 s / weight) in a container / reactor with a closed lid. The bran suspension is heated to 100ºC under agitation and boiled for 2 min. .

The sample is placed under agitation (with the lid closed) at 50ºC and left to equilibrate in relation to the temperature The enzymes are added and the reaction is continued at 50ºC for 24 h (temperature and time can be optimized later) Supernatant is separated from the residues solids Supernatant is boiled to inactivate additional enzymatic activity Sample is cooled and stored to avoid contamination Pellet is lyophilized Supernatant is analyzed Tests: The following modifications were made to the bran (table 15)

|

BAfTB 'Table 15: Amount of bran, 4, treated with different enzymes: Grams of enzyme sample / 10g of bran Faso Test.

Teneão Xylanase —GCr20 Amylase Phosphalipase 1 wo 67 2 10 866.7 112 0.000172 3 10 66.7 1.12 0.05 0.000172 4 10 66.7 1.12 0.05 0.04 0.000172 66 , 7 0.05 0.000172 5 10 56.7 0.04 0.000172 7 10 66.7 0.000172 - Analysis: The soluble fraction of the bran (the supernatant) is analyzed with respect to: 7 5 Dry matter content (%): A quantitative sample of the soluble bran obtained is lyophilized. After lyophilization, the sample size is again quantified and the amount of dry matter is calculated.

As a control, the buffer is included in the analysis. 10 Evaluation of lipid modification using cooking tests: Cooking recipe:

The cooking performance of flour, flour with addition of salubilized modified bran was evaluated in small scale cooking tests (50 q of mixture and 10 g slices) using the recipe below (size

bela / 1S) Table 16. Recipe used to assess the performance of flour, flour with added solubilized bran and reconstituted flour with added insoluble bran.

Salt / sugar is a 1: 1 (weight / weight) mixture of salt and sugar.

Water is the absorption water determined by Farinographic analysis. “Ingredients - —————. Mniescala = [tt EA Flour 50 Dry yeast 1 |

B5 / T8 7 SalAçúcar 16: Water 400BU -2%. Dough making and baking: The flour (or the mixture of flour and bran) and the se-. cos are mixed for one minute, after which water is added and mixing is continued for another five minutes.

After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were shaped into bread using a manual molder. The slices were placed on baking sheets and placed in a closed container (with a lid) and left on the table for 10 minutes. After that, the bread is fermented at 34ºC RH of 10 85% for 45 minutes and finally baked at 230ºC for five minutes in a Bago oven (Bago-line, Fábora, Denmark). The bread was cooled for 20 minutes before the evaluation (weighing, measuring the volume, evaluating the crumb, the 'crust and sensory). - Cooking tests The cooking tests below were performed (table 17). Table 17, Experimental cooking test structure. ID refers to the composition of the flour with the addition of solubilized bran, the numbers in parentheses refer to the treatment of the flour according to the table

15. Flour (q) is the amount of flour in the bread. Bran Extract (ml) is the amount of solubilized bran added to the flour instead of water. Water (m)) is the amount of water added to the meal. Cooking ho. 9 is a repeat of cooking 5, however with the addition of 0.2 mg of xylan-6 0.5 mg of phospholipase / kg of flour during mixing of the dough. Cooking the Fa ra are Water, ml NS Sr Sr Sr SS DT 2 Control bran (1) so o 29 3 BSS flour, KLM1 (2) 50 29 4 BS3 bran, GC 220, KLM1 so 29 (e) 5 Fe bran MERO only 28 |

'Cooking D fo a PS Water, ml 6 Bran GC220, KLM1 (5) so 29 7 7 Bran Spezyme, KLM1 (6) 50 29: B Bran KLM1 (7) so 29 OUTER mos Results: Degree of bran solubilization: With based on dry matter, the amount of bran solubilized in the tests was, on average, approximately 30 to 54%. Results of cooking: W As can be seen in table 18 and in figure 5, the addition of soluble fibers has little or no no effect on the specific volume of the bread.

However, the combination of bran solubilization and phospholipase] has a significant effect on bread volume. . 10 Table 18 Results of the cooking test. 1D refers to the composition of the flour with the addition of solubilized bran, the numbers in parentheses refer to the treatment of the bran according to table 16. Flour (9) is the amount of flour in the bread.

Specific Volume (mgm) is the absolute specific volume of the loaves.

Relative volume versus control (%) is the relative volume of breads versus bread 1 (control) cosmarto DO SEE EEE. 1 Bran 3.88 110 2 Control bran (1) 3.08 100 3 Bran BS3, KLM1 (2) 3.14 102 4 Bran BS3, GC 220, KLM1 357 115 s) h), and 5 Bran SE ENE 3 , 84 125 6 Bran GC220, KLM1 (5) 3.57 116 7 Bran Spezyme, KLM1 (6) 3.26 106 8 Bran KLM1 (7) 3.10 101 |

'Volume Espe- Vi and relative ver-. Corimento DP. the laced Ae tip: MO Bran, BSS, .GO220, 9 Spezyme, KLM1 (+ BS3, 4.98 162 x KLM1) The resulting breads can be seen in figure 6.

As can be seen from the results above in experiment 3, a significant effect was obtained on cooking performance in this experiment by combining the bran treatment with enzymes that modify the cell wall, starch and lipids. Example 4. Laboratory scale modification of commercial trido bran and: wheat bran lipids followed by the evaluation of the modified bran during cooking:. To assess the synergistic effect of wheat bran modified with “10 enzymes that modify the cell wall, starch and lipid, the effect of the in situ generation of modified lipids that have emul-: sifying properties on the addition of wheat bran was tested. in relation to cooking performance. It is well known that the addition of bran to flour or whole grain flour, has a lower cooking potential than flour with an enamel, Bran: Fractions of wheat bran obtained from a commercial mill were used. The fractions consisted of a fraction of fine bran and a fraction of coarse bran. Before use, the coarse bran fraction was ground to obtain a smaller particle size, which will increase the specific surface of the bran, eventually increasing efficiency of the enzymatic solubilization of the bran. The grinding was carried out in a Retch grinder to obtain an average particle size of 500 µm. However, it should be noted that a smaller particle size may be preferable, in relation to the degree of solubilization.

Enzymes: '|

: Table19: Enzymes used for the modification of wheat bran Enzyme Activity Enzyme ID 'Hlanase Bacterial Xylanase Danisco - (1223449, 4010866762) Cellulase / glycanase Genencor GC220 Amylase Genencor, Spezyme Fred (4018101001) Fosfo-galacto lipase Grindam PF70. Protocol: Table 20. Protocol used for the modification of bran - Wheat bran was suspended in 50 mM NaPi, pH 5 (13% w- "5 s / weight) in a container / reactor with a closed lid. heated to 100ºC with stirring and boiled for 2 min.

The sample is placed under agitation (with the lid closed) at 50ºC and left to equilibrate in relation to the temperature The enzymes are added and the reaction is continued at 50ºC for 24 h (temperature and time can be optimized later) The modified bran is boiled to inactivate additional enzymatic activity Sample is cooled and stored to avoid contamination Tests: The following modifications were made to the bran (table 21) Table 21: Amount of bran, 9, treated with different enzymes Grams of sample of erizime / 109 of bran Test It is Torsion: Xylanase —GC220 Amylase & A sctoto = 1 10 667 0.19 005 - od 2 10 66.7 0.19 LA) 0.04 D0.00D03 Evaluation of lipid modification using cooking tests:: | i Cooking recipe: 'The cooking performance of flour and added flour. of modified bran was evaluated in small scale cooking tests. (50 q of mixture and 10 g slices) using the recipe below (table 22). Table 2z22, Recipe used to assess the performance of flour, flour with addition of modified bran.

Salaçúcar is an 11 (weight / weight) mixture of salt and sugar.

The water is the absorption water determined by the Farinographic analysis.

Mini scale ingredients. mloug, Flour 50 O Fermentaseco o - Sall / Açúcar 1,6. Water 400BU -2%: Dough making and cooking The flour and dry ingredients are mixed for one minute, after which water (or modified water and bran) is added and mixing is continued for another five minutes.

After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were molded into a mold using a manual molder. The slices were placed in crayons and placed in a closed container (with a lid) and left alone. - leave the table for 10 minutes.

After that, the bread is fermented at 34ºC 85% RH for 45 minutes and finally baked at 230ºC for five minutes in a Bago oven (Bagó-line, Fábora, Denmark). The bread was cooled for 20 minutes before the evaluation (weighing , volume measurement, crumb, crust and sensory evaluation). Cooking tests The below cooking tests were performed (table 23). |

Ú Table 23, Experimental cooking test structure. ID refers to the 'composition of the dough, the numbers in parentheses refer to the' treatment of the bran according to table 21. Flour (q) is the amount of flour in the bread. Bran Extract (mi) is the amount of solubilized bran added to the flour instead of water. Water (ml) is the amount of water added to the flour. Cooking ID Female Water, ml 1 Control (Wheat) 50 o 29 2a So o o. Enz, W Bran 3 cell wall (with insoluble) + Lipase (with 50 30 o: insol.) (2) é Results: Cooking results: 'As can be seen in table 24 and in figure 7, the addition of Modified bras had a negative effect on the specific volume of the bread. However, the addition of bran also modified with phospholipase, generating functional lipids, has a much less damaging effect on the volume of the bread compared to the addition of only bran with modi cell wall. - stayed.

Table 24, Results of the cooking test. ID refers to the composition of the meal, the numbers in parentheses refer to the treatment of the meal according to table 21. Flour (q) is the amount of flour in the bread. Specific Volume (mgml) is the absolute specific volume of the loaves. Relative volume versus control (%) is the relative volume of breads versus bread 1 (control) GCozimento D Vol E MMuliahirias 1 Control (wheat). 2.40 100> MS. stopped to go Tul (with 344 at 5 piece tos |

TIUW7B 'The resulting breads can be seen in figure 7. Ú Coma can be seen from the results above, a significant e- "achievement was obtained in the cooking performance in this experiment - by combining the bran treatment with enzymes that modify the pa - redecellular, starch and lipids before adding to the dough.

Example 5. Laboratory scale modification of rice bran and lipids from rice bran by assessing solubilized bran and modified lipids in cooking: To further assess surprising findings regarding bran solubilization combined with modification of bran. bran lipids, the effect of solubilized rice bran and modified rice bran lipids was tested in cooking tests. . Bran:. A fraction of commercial rice bran was used in the experiment “Enzymes: Table 25: Enzymes used for the modification of rice bran Enzyme Activity Enzyme ID Xylanase Bacterial Xylanase Danisco (1223449, 4010866762) Cellulase / glycanase Genencor GC220“ Genencor; Just me Fred Amilase '(4n16101007). Grindamyl Powerbake 4070 Phospho-galactose lipase Y Danisco Protocol: Table 26, Protocol used for bran modification Rice bran was suspended in 50 mM NaPj, pH 5 (13% weight / weight) in a container / reactor with a lid closed |

The bran suspension is heated to 100ºC under agitation and boiled for 2 min, * The sample is placed under agitation (with the lid closed) at - 50ºC and left to equilibrate in relation to temperature. Enzymes are added and the reaction is continued at 50 ° C for 24 h (temperature and time can be further optimized) Supernatant is separated from solid waste Supernatant is boiled to inactivate additional enzymatic activity Sample is resorced and stored to avoid contamination Tests: 'The following modifications were made to the bran of rice (table. 27). Table 27: Amount of rice bran, o, treated with different enzymes Tr “Enzyme sample / 10g bran - Taste Fo To Xilandsé GC220 Amylase acts 1 10 66.7 D, 19 0.05 D, 04 2 10 66, 7 0.19 0.05 D, 04 0.0003 Evaluation of lipid modification using cooking tests: Cooking recipe: The performance of cooking flour and flour with addition of modified bran was evaluated in small scale cooking tests ( 50 q mixture and 10 g slices) using the recipe below (table 28).

Tábela2Zs Recipe used to evaluate the performance of different dough compositions (+ £ - solubilized bran). Salaçúcar is a 1: 1 (weight / weight) mixture of salt and sugar. The water is the absorption water determined by the Farinographic analysis, Ingredients Mini scale ml or g Flour 50 Dry yeast 1 |

73MB 'Ingredients Mini scale Salt / Sugar 1.6 Water 400BU -2% s Dough making and cooking The flour and dry ingredients are mixed for one minute, after which water (or modified water and bran) is added and the mixing is continued for another five minutes.

'After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were shaped into bread using a manual molder. The slices were placed on baking sheets and placed in a closed container (with a lid) and left on the table for 10 minutes. After that, the bread is fermented at 34ºC RH of E 10 85% for 45 minutes and finally baked at 230ºC for five minutes in one. Bago oven (Baágo-line, Fábora, Denmark), The bread was cooled for 20 minutes before evaluation (weighing, volume measurement, crumb, crust and sensory evaluation). Cooking tests 18 The cooking tests below were performed (table 29), Table 29. Experimental structure of the cooking test. ID refers to the composition of the dough, the numbers in parentheses refer to the treatment of the bran according to table 27. Flour (9) is the amount of flour in the bread, Bran Extract (ral) is the amount of solubilized bran added to the flour instead of water. Water (ml) is the amount of water added to the flour. Gozimento D Flour, when not 2 Ernz. cell wall R Bran (1) 50 30 3 Enz ta RBran + 50 30 Results: Results of the cooking: |

TOUCH 'As can be seen in table 30 and in figure 6, the addition of modified soluble fibers did not have a negative effect on the specific volume of the bread, in fact it had a positive effect on the volume of the - bread However, the addition of soluble fibers also modified with phospho-lipase, generating functional lipids, has a much better effect on the bread volume compared to the addition of only modified soluble fibers, Table 30, Results of the baking fest. ID refers to the composition of the flour, the numbers in parentheses refer to the treatment of the bran according to table 27, Flour (9) is the amount of flour in the bread. Specific volume (m / ml) is the volume specific absolute of breads Relative volume versus control (9%) is 5 relative volume of breads versus bread 1 (control, le) | Chimera, a vemsemi - 1 Control (wheat) 3.40 100 2 Enz. R Bran cell wall (1) 4.16 123 3 R Bran cell wall + 4 sm 154 Lipase (2) The resulting breads before they can be seen in figure 8.

As can be seen from the results above, a significant effect was obtained in cooking performance in this experiment by the addition of soluble bran and modified lipids obtained by combining the pretreatment of rice bran with enzymes that modify the cell wall. , starch and lipids.

Example6 Modification on a laboratory scale of commercial wheat bran and wheat bran lipids using different lipases, followed by the evaluation in cooking.

To further assess the surprising findings regarding bran solubilization combined with the modification of bran lipids, other lipases other than Danisco's Phospho-galact lipase were tested, followed by the evaluation of modified lipids generated in cooking tests.

|

Fractions of wheat bran obtained from a commercial mill were used.

The fractions consisted of a fraction of fine bran and a fraction of 'coarse bran.

Before use, the coarse bran fraction was ground to obtain a smaller particle size, which will increase the specific bran surface, eventually increasing the efficiency of the enzymatic solubilization of the bran. The grinding was carried out in a Retch grinder to obtain an average particle size of 500 µm.

However, it should be noted that a smaller particle size may be preferable, in relation to the degree of solubilization.

Enzymes; : Table31: Enzymes used for the modification of wheat bran: Enzyme Activity Enzyme ID | It is - Bacterial xylanase Danisco Xylanase (1223449, 4010866762) Cellulase / glycanase Genencor GC220 Amylase Genencor, Spezyme Fred (4016101001) Phospho-galactose lipase Grindamyl Powerbake 4070 Danisco Phospho-galactose lipase Novozymes Lipopan Fase (lipopan Fam Fos-lipopan) Panaámore) Protocol: Table 32. Protocol used for bran modification The wheat bran was suspended in 50 mM NaPí, pH 5 (13% weight / weight) in a closed lid vessel / reactor The bran suspension is heated at 100ºC with stirring and boiled for 2 min.

The sample is placed under agitation (with the lid closed) at 60ºC, left to equilibrate in relation to the temperature. Enzymes are added and the reaction is continued at 50ºC for |

'24 h (temperature and time can be further optimized) Supernatant is separated from solid waste. Supernatant is boiled to inactivate additional enzymatic activity + Sample is cooled and stored to avoid contamination Tests: The following modifications were made to the bran (table 33) Table 33: Amount of bran, 9, treated with different O enzymes & ranges of sample Enzyme / 10gdefarelo - Faso Test Xylanase Buffer - GC220 Amylase - Phospholipase: TT o 67 o oo GM 2 10 86.7 0.19 0.05 0.04 Í 3 10 66.7 019 0.05 0.04 Dántsco) 0 , 00013 i ES 10 86.7 019 0.05 0.04 (Lipopan P) 5 10 66.7 p19 0.05 D, 04 (We stopped) Evaluation of lipid modification using cooking tests: Cooking performance: Performance in cooking of flour and flour with addition of solubilized modified bran (+/- modified lipids) was evaluated in - small scale cooking tests (50 g of mixture and 10 g slices) using the recipe below (table 34).

Table34 Recipe used to evaluate the performance of cooking flour, flour with the addition of solubilized bran and flour with the addition of solubilized bran and modified lipids. Salt / sugar is a 1: 1 (weight / weight) mixture of salt and sugar. Water is the absorption water determined by Farinographic analysis.

Ingredients Mini scale ml éu g Flour 50 |

T7TB] Ingredients Mini scale 'Dry yeast 1: Salt / Sugar 1.6: Water 400BU -2% Dough making and cooking The flour and dry ingredients are mixed for one minute, after which the water is added is the mixture is continued for another five minutes.

After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were shaped into bread using a manual molder. The slices were placed on baking sheets and placed in a closed container (with a lid) and left alone. on the table for 10 minutes. After that, the bread is fermented at 34ºC RH - 10 85% for 45 minutes and finally baked at 230ºC for five minutes in a Bago oven (Bago-line, Fáborg, Denmark). The bread was cooled for 20 minutes before the evaluation (weighing, volume measurement, crumb, crust and sensory evaluation). Cooking tests The cooking tests below were performed (table 35). Table 35, Experimental cooking test structure. ID refers to the composition of the flour mass, 68 numbers in parentheses refer to the treatment of the bran according to fabela 33. Farintia (9) is the amount of flour in the bread. Bran Extract (ml) is the amount of solubilized bran added to the flour through the water. Water (m)) is the amount of water added to the flour. GCozimento ID Flour, g FS = Water, ml 2 Enz, cell wall R Brán (1) so 30 3 Enz. Belular wall R Bran + 50 o Danisco (2) n + 4 Emz aan Bra so 30 |

Cooking ID Flour, bran g, ml Water, ml. 5 Enz, R Bran + ão cell wall to Panamore (4): Results: cooking results: As can be seen in table 36 and figure 9, the addition of soluble fibers had little or no effect on the specific volume of the & bread However, the combination of bran solubilization and phospholipase had a significant effect on bread volume; all bread baked with bran treated with lipases in combination with enzymes that modify the »cell wall and starch, had an increase in volume compared to the control flour.

Ú 10 Table36 Results of the cooking test. 1D refers to the composition of the dough, the numbers in parentheses refer to the treatment of the bran according to table 33. Flour (9) is the amount of flour in the bread.

Specific Volume (mgiml) is the absolute specific volume of bread.

Vol 1êl versus control (%) is 6 relative volume of breads versus bread 1 (control) Baking the Specific Volume Relative volume, mg under control 1 Control (wheat) 3.40 100 2 Enz. cell wall W Bran (1) 3,33 E 3 Eriz. cell wall W Bran + 3.38 114 Danisco (2) Enz. cell wall R Bran + 4 b 13 Lipopan F (3) 254 1 Enz. cell wall R Bran + 301 us Panamore (4) The resulting breads can be seen in figure 9. As can be seen from the results above in experiment 6, a significant effect was obtained on the performance of the cooking In this experiment by combining the bran treatment with enzymes that modify the cell wall, starch and lipids.

Claims (15)

: CLAIMS 1. Method for treating a plant material that. contains lipid, characterized by the fact that it comprises the step of treating a liquid suspension of a plant material at least partially - solubilized which contains lipid with one or more lipid-modifying enzymes. Method according to claim 1, characterized in that the liquid suspension of a plant material at least partially solubilized containing lipid is essentially free of starch. Method according to claim 1 or 2, characterized in that less than about 50%, such as less than about 40%, such as less than about 30%, such as less than about 20%, such as less than about 10%, such as less than about 6%, such as less than about 3%, such as less than about 1% (weight / weight) of liquid suspension of at least partially solubilized plant material containing lipid is starch or components containing starch, ta! like flour. Method according to any one of claims 1 to 3, characterized in that it further comprises a preceding or simultaneous step of treating a liquid suspension of plant material containing lipid to obtain said plant material at least partially solubilized, wherein said treatment for obtaining at least partially solubilized plant material is a treatment with one or more enzymes that modify the cell wall selected from the group consisting of xylanase and a cellulase, such as cellobiohydrolases, endoglycanases and beta-glycanase . Method according to any one of claims 1 to 4, characterized in that said one or more lipid-modifying enzymes is selected from the group consisting of: a triacylglycerine! lipase, a phospholipase and a galactolipase. Method according to any one of claims 1 to 5, characterized in that said plant material is cereal bran. : Method according to claim 6, characterized in that said cereal bran is obtained from an industrial milling process and additionally ground to obtain an average particle size below 500 µm, such as below 400 µm, such as below 200 pum. Method according to any one of claims 1 to 7, characterized in that said plant material is a by-product of processing plant material, such as vegetable oil refining neutralization paste, grains used for fermentation or dry soluble distillery grains (DDGS). Method according to any one of claims 1 to 8, characterized in that the obtained composition comprising modified lipids, such as functional lipids, is further treated to inactivate additional enzyme activity. Method according to any one of claims 1 to 9, characterized in that the degree of solubilization of said plant material as determined in the dry matter versus the dry matter of plant material obtained, is greater than 15%, as greater than 25%, as greater than 35%, as greater than 40%, as greater than 50%, as in the range between 40% to 60%, as in the range between 50% to 60%. Method according to any one of claims 1 to 10, characterized in that the total content of modified lipids and lipids, such as functional lipids, as determined in the dry matter versus the dry matter of plant material in the obtained soluble fraction it is at least about 0.05%, such as at least about 1.0%, such as in the range of 0.05 to 5%. Method according to any one of claims 1 to 11, characterized in that said treatment with one or more lipid-modifying enzymes converts more than 5%, such as more than 10%, such as more than 25%, such as more than 50% of It is phosphatidylinositol in lysophosphatidylinosite! (plain-PI). 13. Composition, characterized in that a method is produced by i, as defined in any one of claims 1 to 12. 14. Use of a composition as defined in claim 13, characterized by the fact that it is in the production of a food product or in the production of bioethanol. 15. Food product, characterized by the fact that it is obtained by a use, as defined in claim 14.