BRPI1006908B1 - METHOD FOR TREATMENT OF A CEREAL BRAN CONTAINING LIPIDS - Google Patents

Abstract

method for treating a lipid-containing plant material, composition and its use, and food product. The present invention relates to the modification of lipids in lipid-containing plant material 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 such compositions comprising such functional lipids and other functional compounds derived from the action of lipid-modifying enzymes for the preparation of a composition suitable for the preparation of bio-ethanol, as well as food products such as bread.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to the modification of lipids in plant material that contains 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 such compositions comprising functional lipids and other functional compounds derived from the action of lipid-modifying enzymes for the preparation of a composition suitable for the preparation of bioethanol , as well as food products such as bread.

BACKGROUND OF THE INVENTION

[0002] The use of side streams from the processing of fermentation residues from plant materials, such as bran from milling or dry distillery grains with solubles (DDGS) has received little attention beyond their use in animal feed.

[0003] The beneficial use of lipolytic enzymes (E.G. 3.1.1.x) in applications in the food and/or animal feed industry has been known for a long time.

[0004] However, most prior techniques describe 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-staling effect. WO94/04035 teaches that improved bread tenderness can be obtained by adding lipase to the dough without adding any additional fat/oil to the dough.

[0005] 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 are made up of glycerol esterified with two fatty acids and a polar group. The polar group contributes to the surface activity of these lipids.

[0006] The enzymatic cleavage of one of the fatty acids in these lipids leads to lipids with much higher surface activity. It is well known that emulsifiers such as DATEM with high surface activity are very functional when added to dough.

[0007] Lipolytic enzymes hydrolyze one or more fatty acids from the lipids present in plant material which can result in the formation of powerful emulsifying molecules.

[0008] In EP 1 193 314, the inventors found that the use of lipolytic enzymes active on glycolipids was particularly beneficial in bread-making applications.

[0009] Morrison et al., J. Sci. Food Agric. 1981, 32, 579-587 describe the distribution of soft wheat seed lipids in flour milled fractions.

[00010] There is a need in the art for better use of by-products from the processing of plant materials, such as milling cereal bran, vegetable oil refining neutralization pastes, soluble distillers dry grains (DDGS), in which less plant material goes to low-priced applications like livestock feed. Additionally, there is a long felt need to be able to use the cereal bran fraction in traditionally existing cereal products without significant impact on the appearance/structure, color or taste of the product and to make it possible to enhance the effect. on the health and nutrition of existing products.

PURPOSE OF THE INVENTION

[00011] It is an objective of the present invention to provide methods for the generation of functional lipids from plant material in general and from industrial side products in particular. It is a further 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, color or flavor of the product and to make it possible to enhance the health and nutritional effect of existing products.

SUMMARY OF THE INVENTION

[00012] In a broad aspect, the present invention relates to methods for treating lipid-containing plant material with lipid-modifying enzymes, for the generation of modified lipids, such as functional lipids from plant materials.

[00013] In a first aspect, the present invention relates to a method for treating 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.

[00014] 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 plant material containing lipid, the method comprising the step of treating a liquid suspension of at least one plant material that contains partially solubilized lipids with one or more enzymes that modify the lipid.

[00015] 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 of treating a liquid suspension of at least one plant material that contains lipids partially solubilized with one or more enzymes that modify the lipid, the use being for the production of a food product.

[00016] 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 of treating a liquid suspension of at least one plant material that contains lipids partially solubilized with one or more enzymes that modify the lipid, the use being for the production of bioethanol.

[00017] In a further 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 that contains partially solubilized lipids with one or more enzymes that modify the lipid.

[00018] In a further aspect, the present invention relates to bioethanol obtained by using 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 that contains partially solubilized lipids with one or more enzymes that modify the lipid.

[00019] 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 c) optionally other ingredients for the preparation of a food product.

DESCRIPTION OF THE FIGURES

[00020] Figure 1. Results of the cooking test. Vol. of breads versus white (%). The columns represent the bread volume for test numbers 1-6 according to tables 5 and 6.

[00021] Figure 2. Breads obtained from baking with fractions obtained from soluble bran. The numbers refer to the numbers in table 5.

[00022] Figure 3. Results of the cooking test. Vol. of breads versus white (%). The columns represent the cook test experiments according to table 12.

[00023] Figure 4. Breads obtained from baking with fractions obtained from soluble bran. The numbers refer to the numbers in table 12.

[00024] Figure 5. Results of the bake test. Vol. of breads versus white (%). Columns represent the cook test experiments according to table 17 and 18.

[00025] Figure 6. Breads obtained from baking with fractions obtained from soluble bran. The numbers refer to the numbers in table 18.

[00026] Figure 7. Breads obtained from baking with fractions obtained from modified bran. The numbers refer to the numbers in table 24.

[00027] Figure 8. Breads obtained from baking with modified rice bran extracts. The numbers refer to the numbers in table 30.

[00028] Figure 9. Breads obtained from baking with fractions obtained from modified bran. The numbers refer to the numbers in table 36.

DETAILED DESCRIPTION OF THE INVENTION

[00029] Considerable quantities of by-products from the processing of plant materials, such as milling cereal bran, vegetable oil refining neutralization pastes, soluble distillers dry grains (DDGS), etc., are available as raw material to generate functional lipids that can serve as emulsifiers in different applications such as food applications, feed applications, softeners in the production of plastic materials, etc.

[00030] Here, it was shown that the modification of the lipid fraction in, for example, wheat bran, is significantly increased if the material is co-treated with other enzymes such as enzymes that modify the cell wall and, in some modalities, also with enzymes that modify starch. By combining these classes of enzymes, the functionalization of lipids that cannot be obtained using lipases alone was observed.

[00031] The modified lipids generated can be used, for example, in baking, by adding the isolated soluble fraction or by adding the complete composition of the bran treated with the enzyme containing the modified lipids that have emulsifying properties.

[00032] Therefore, the compositions generated by the methods according to the present invention can be used as isolated solubles. However, the compositions can also be used as a mixture of solubles and insolubles, i.e. plant material in solution, such as bran waste material. It must be understood that part of the lipid fraction may still be present in this residual fraction in solution.

[00033] The term "lipid-containing plant material" as used herein refers to any material that comprises significant amounts of plant-derived material that contains endogenous amounts of lipids. Suitably the plant material can be obtained in large quantities, contains a significant amount of lipids and can be used in industrial processes.

[00034] In some embodiments, the lipid-containing plant material is a by-product or by-product of industrial processes. In some embodiments, the plant material can also contain a non-plant material, such as a by-product of a fermentation, which can contain yeast cells.

[00035] In some particular embodiments, the lipid-containing plant material is a cereal bran, such as, for example, traditional milled wheat bran.

[00036] In some embodiments, an amount of at least about 100 mg, such as at least about 200 mg, such as at least about 300 mg per 100 g of the dry weight of the lipid-containing material is phospholipid.

[00037] In some embodiments, an amount of at least about 10 mg, such as at least about 20 mg, such as at least about 30 mg per 100 g of the dry weight of the lipid-containing plant material is phosphatidylinositol (PI).

[00038] The phrase "partially solubilized lipid-containing plant material", as used herein, refers to plant material that contains lipids and has been at least partially solubilized by enzymatic or mechanical action.

[00039] The term "cereal" as used herein refers to the fruits of a plant of the Poaceae family, such seed containing at least the bran comprising the aleurone and the starch-rich endosperm, with or without the additional presence of the pericarp, coating of seed (alternatively called nut) and/or germ. The term includes, but is not limited to, species such as wheat, barley, oats, spelled, rye, sorghum, corn and rice.

[00040] The term "bran", as used herein, refers to a fraction of the cereal derived from milling enriched with any or all of the tissues to be selected among the aleurone, pericarp and seed coat, when compared to intact seed corresponding.

[00041] The term "solubilization" as used herein refers to the solubilization of plant material, such as cereal bran, in the methods according to the invention and is intended to include any degree of solubilization. Consequently, the "solubilization" can be to obtain a 100% soluble material or it can be to obtain a degree of solubilization 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 dry mass versus dry mass of bran.

[00042] The expression "at least partially solubilized", as used herein, refers to a degree of solubilization that is greater than 1%, such as as high as 5%, such as as high as 10%. It should be understood that the action of lipid modifying enzymes may not function optimally according to the invention if the plant material is not solubilized to a certain degree. In specific aspects according to the present invention, in which lipid modifying enzymes are added to function simultaneously with the treatment to achieve solubilization, such as treatment with one or more cell wall modifying enzymes, solubilization and action of the lipid-modifying enzymes will occur at the same time.

[00043] The term "milling fraction", as used herein, refers to all or part of the fractions that result from mechanically reducing grain size through, for example, but not limited to cutting, crushing, crushing, breaking or milling, with or without fractionation by, for example, but not limited to sieving, screening, filtration, blowing, aspiration, centrifugal filtration, air filtration, electrostatic separation or electric field separation.

[00044] 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.

[00045] 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 "assay of cell wall solubilization" included here. Within this definition of "cell wall modifying enzyme" are included cellulases such as cellobiohydrolase I and cellobiohydrolase II, endoglycanases and beta-glycosidases, and hemicellulolytic enzymes such as xylanases.

[00046] The term "cellulases" or "cellulolytic enzymes" as used herein is understood to comprise cellobiohydrolases (EC 3.2.1.91), for example, cellobiohydrolase I and cellobiohydrolase II, as well as endoglycanases (EC 3.2.1.41. ) and beta-glycosidases (EC 3.2.1.21).

[00047] Included within the definition of cellulases are: endoglycanases (EC 3.2.1.4) that cut the cellulose chains at random; cellobiohydrolases (EC 3.2.1.91) that cleave cellobiosil units at the ends of the cellulose chain and beta-glycosidases (EC 3.2.1.21) that convert cellobiose and soluble cellodextrins to glucose. Among these three categories of enzymes involved in cellulose biodegradation, cellobiohydrolases are the main enzymes for the degradation of native crystalline cellulose. The term "cellobiohydrolase I" is defined herein as an activity of cellulose 1,4-beta-cebiosidase (also referred to as exoglycanase, exo-cellobiohydrolase or 1,4-beta-cellobiohydrolase) as defined in the enzyme class EC 3.2.1.91, which catalyzes the hydrolysis of 1,4-beta-D-glycosidic bonds in cellulose and cellotetraose by releasing cellobiose from the unreduced ends of the chains. The definition of the term "cellobiohydrolase II activity" is identical, except that cellobiohydrolase II attacks the shortened ends of chains.

[00048] Cellulases can comprise a carbohydrate binding module (CBM) that enhances the binding of the enzyme with a fiber containing 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 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 and properties, pp. 142-163, American Chemical Society, Washington. In a preferred embodiment, the 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 having cellulolytic enhancing activity (GH61A), preferably that described in WO2005/074656. The cell wall modifying enzyme may additionally be a beta-glycosidase, such as a beta-glycosidase derived from a strain of the Trichoderma, Aspergillus or Penicillium genus, including the fusion protein that has beta-glycosidase activity described in US application no. 60/832,511 (Novozymes). In some embodiments, the cell wall modifying enzyme is a CBH II, such as Thielavia terrestris cellobiohydrolase II (CEL6A). In some embodiments, the cell wall modifying enzyme is a cellulase enzyme, such as that derived from Trichoderma reesei.

[00049] 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 Humicola, such as a strain of Humicola insolens.

[00050] 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 cellobiohydrolase, such as a cellobiohydrolase II from Thielavia terrestris (CEL6A), a beta-glycosidase (for example, the fusion protein described in US application no. 60/832,511) and cellulolytic enzymes, for example, derived from Trichoderma reesei.

[00051] 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 cellulolytic enzymes, for example, derived from Trichoderma reesei. In some embodiments, the cell wall modifying enzyme is a commercially available product, such as GC220 available from Genencor, A Danisco Division, US or CELLUCLAST® 1.5L or CELLUZYME® available from Novozymes A/S, Denmark.

[00052] Endoglycanases (EC 3.2.1.4) catalyze the endohydrolysis of 1,4-beta-D-glycosidic bonds in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glycans, such as beta-D-glycans or xyloglycans from cereal or other plant material containing cellulosic parts. The authorized name is endo-1,4-beta-D-glycan 4-glycan hydrolase, but the short term endoglycanase is used in the present application. Endoglycanase activity can be determined according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59:257-268.

[00053] In some embodiments, endoglycanases can be derived from a strain of the genus Trichoderma, such as a strain of Trichoderma reesei; or a strain of the genus Humicola, such as a strain of Humicola insolens; or a Chrysosporium strain, preferably a Chrysosporium lucknowense strain.

[00054] The term "cellobiohydrolase" means a 1,4-beta-D-glycan cellobiohydrolase (EC 3.2.1.91) that catalyzes the hydrolysis of 1,4-beta-D-glycosidic bonds in cellulose, cellooligosaccharides or any polymer that contains bound beta-1,4 glucose, releasing cellobiose from the reduced or non-reduced ends of the chain.

[00055] Examples of cellobiohydrolases are mentioned above including CBH I and CBH II from Trichoderma reesei; Humicola insolens and Thielavia terrestris cellobiohydrolase CBH II (CELL6A).

[00056] The 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 Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288. The Lever et al. is suitable for evaluating the hydrolysis of cellulose in corn straw and the method by van Tilbeurgh et al. is suitable for determining cellobiohydrolase activity in a fluorescent disaccharide derivative.

[00057] The term "beta-glucosidase" means a beta-D-glycoside glycohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis of unreduced terminal beta-D-glucose residues with the release of beta-D-glucose. For the purposes of the present invention, beta-glucosidase activity is determined according to the basic procedure described by Venturi et al., 2002, J. Basic. Microbiol. 42:55-66, except for different conditions that were employed as described here. A unit of beta-glycosidase activity is defined as 1.0 μmol of p-nitrophenol produced per minute at 500C, pH 5 from 4 mM p-nitrophenol-beta-D-glycopyranoside as substrate in 100 mM sodium citrate, TWEEN® 20 0.01%.

[00058] In some modalities, beta-glucosidase is of fungal origin, such as a strain of the genus Trichoderma, Aspergillus or Penicillium. In some embodiments, the beta-glucosidase is derived from Trichoderma reesei, as is the beta-glucosidase encoded by the bgl1 gene (see EP 562003). In another embodiment, the beta-glycosidase is derived from Aspergillus oryzae (recombinantly produced in Aspergillus oryzae according to WO 02/095014), Aspergillus fumigatus (recombinantly produced in Aspergillus oryzae according to Example 22 of WO 02/095014) or Aspergillus niger (1981, J. Appl. 3:157-163).

[00059] The terms "hemicellulolytic enzymes" or "hemicellulases" as used herein refer to enzymes that can break down hemicellulose.

[00060] Any hemicellulase suitable for use in the hydrolysis of hemicellulose, preferably into arabinoxylan oligosaccharides, can be used. Preferred hemicellulases include xylanases, arabinofuranosidases, acetyl xylan esterase, feruloyl esterase, glucuronidases, galactanase, endo-galactanase, mannases, endo or exo arabinases, exo-galactanases, pectinase, xyloglycanase or more or mixtures of the two. An example of a suitable hemicellulase for use in the present invention includes Grindamyl Powerbake 930 (available from Danisco A/S, Denmark) or VISCOZYM E® (available from Novozymes A/S, Denmark). In one embodiment, the hemicellulase is xylanase. In one embodiment, the xylanase is of microbial origin, such as fungal origin (eg, Trichoderma, Meripilus, Humicola, Aspergillus, Fusarium) or from a bacterium (eg, Bacillus). In some embodiments, the xylanase is derived from a filamentous fungus, preferably derived from a strain of Aspergillus, such as Aspergillus aculeatus; or from a strain of Humicola, preferably Humicola lanuginosa. 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 SHEARZYME® and BIOFEED WHEAT® from Novozymes A/S, Denmark.

[00061] Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminal alpha-L-arabinofuranoside residues unreduced into alpha-L-arabinosides. Galactanase (EC 3.2.1.89), arabinogalactan endo-1,4-beta-galactosidase, catalyzes the endohydrolysis of 1,4-D-galactosidic bonds in arabinogalactans.

[00062] The pectinase (EC 3.2.1.15) catalyzes the hydrolysis of 1,4-alpha-D-galactosiduronic bonds in pectate and other galacturonans.

[00063] Xyloglycanase catalyzes the hydrolysis of xyloglycan.

[00064] The term "xylanase", as used herein, refers to an enzyme that is capable of hydrolyzing the beta-1,4-glycosyl bond in non-terminal units of beta-D-xylanopyranosyl-1,4-beta-D - xylan pyranosyl of xylan or arabinoxylan. Other names include 1,4-beta-D-xylan xylan hydrolase, 1,4-beta-xylan xylan hydrolase, beta-1,4-xylan xylan hydrolase, (1-4)-beta-xylan 4-xylan hydrolase, endo -1,4-beta-xylanase, endo-(1-4)-beta-xylanase, endo-beta-1,4-xylanase, 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 (eg Aspergillus, Penicillium, Disporotrichum, Neurospora, Fusarium, Humicola, Trichoderma species) or bacterial species (eg Bacillus species, Aeromonas, Streptomyces, Nocardiopsis, Thermomyces) (see, for example, WO92/17573, WO92/01793, WO91/19782, WO94/21785).

[00065] In one aspect of the invention, the xylanase used in the methods of the invention is an enzyme classified as EC 3.2.1.8. The official name is endo-1,4-beta-xylanase. The systematic name is 1,4-beta-D-xylan xylan hydrolase. Other names can be used, such as endo-(1-4)-beta-xylanase; (1-4)-beta-xylan-4-xylan hydrolase; endo-1,4-xylanase; xylanase; beta-1,4-xylanase; endo-1,4-xylanase; endo-beta-1,4-xylanase; endo-1,4-beta-D-xylanase; 1,4-beta-xylan xylan hydrolase; beta-xylanase; beta-1,4-xylan xylan hydrolase; endo-1,4-beta-xylanase; beta-D-xylanase. The catalyzed reaction is the endohydrolysis of 1,4-beta-D-xylosidic bonds in xylans.

[00066] In one aspect of the invention, the xylanase of the invention is a Glycoside Hydrolase (GH) Family 11 xylanase. The expression "Family 11 of Glycoside Hydrolase (GH)" means that the xylanase in question is or can be classified into family 11 of GH.

[00067] In one aspect of the invention, the xylanase used according to the invention is a xylanase which possesses xylanase activity as measured in the "Xylanase assay" as described herein.

[00068] According to the CAZy website (ModO), Family 11 of glycoside hydrolases can be characterized as follows: Known Activities: xylanase (EC 3.2.1.8) Mechanism: Nucleophilic Retention/Catalytic Base: Glu (experimental) Donor of Catalytic Proton: Glu (experimental) 3D Structure State: Coiled: β-supercoiled Clan: GH-C

[00069] As used herein, "Clan C" refers to groupings of families that share a common three-dimensional folding and identical catalytic machinery (see, for example, Henrissat, B. and Bairoch, A., (1996) Biochem. J ., 316, 695-696).

[00070] As used herein, "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, &:637-644). Common features for family 11 members include high genetic homology, a size of about 20 kDa, and a double catalytic displacement mechanism (see Tenkanen et al., 1992; Wakarchuk et al., 1994). The structure of family 11 xylanases includes two large β sheets made of β strands and α helices.

[00071] The family 11 of xylanases includes the following: Aspergillus niger XynA, Aspergillus kawachii XynC, Aspergillus tubiensis XynA, Bacillus circulans XynA, Bacilluspunzilus XynA, Bacillus subtilis XynA, Neocalliniastix Strynces de patriciarum XynA, , Streptomyces lividans XynC, Streptomyces therinoviolaceus XynII, Thermomonospora fusca XynA, Trichoderma harzianum Xyn, Trichoderma reesei XynI, Trichoderma reesei XynII, Trichodermaviride Xyn.

[00072] In the context of the present invention, "enzyme that modifies starch" refers to any enzyme that catalyzes the hydrolysis of α-1,3 and/or α-1,6 glycosidic bonds in glycosides. Included within this term are 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, pullulanase, isoamylase, chitinase, sucrase, maltase, neuraminidase, invertase, hyaluronidase and lysozyme.

[00073] In some embodiments, the enzyme that modifies starch is an enzyme that debranches starch.

[00074] In one aspect of the invention, the starch modifying enzyme used according to the invention is an enzyme having starch debranching activity as measured in the "starch debranching activity assay" as described herein.

[00075] Enzymes that debranch starch include pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68). They hydrolyze α-1,6-D-glycosidic bonds, β-limit dextrins and pullulans. Isoamylases can be distinguished from pullulanases (EC 3.2.1.41) by the inability of the isoamylase to attack pullulan and by its limited action on α-limit dextrins.

By "amylase" is intended to include any amylase, such as the glycoamylases, α-amylase, β-amylases and wild type α-amylases from Bacillus sp., such as from B. subtilis and B. licheniformes. "Amylase" shall mean an enzyme that is, among other things, capable of catalyzing the degradation of starch. Amylases are hydrolases that cleave α-D-(1^4) O-glycosidic bonds in starch. Generally, α-amylases (EC 3.2.1.1; (XD-(I^4)-glycan glycanohydrolase) are defined as endo-acting enzymes that cleave α-D-(1—»4) O-glycosidic bonds within the starch molecule in a random manner. Exo-acting amylolytic enzymes, such as β-amylases (EC 3.2.1.2; α-D-(1^4)-glucan maltohydrolase) and some product-specific amylases, such as maltogenic α-amylase (EC 3.2.1.133) cleave the starch molecule from the unreduced end of the substrate, β-Amylases, α-glycosidases (EC 3.2.1.20; α-D-glycoside glucohydrolase), glucoamylase (EC 3.2. 1.3; α-D-(1—>4)-glycan glucohydrolase), and product-specific amylases can produce glucose from starch.

[00077] By "variant α-amylase", "variant α-amylase polypeptide" and "variant enzyme" is meant an α-amylase that has been modified by the substitution of amino acid residues at the amino terminus of the mature α-amylase protein . As used herein, "parent enzymes", "parent sequence", "parent polypeptide", "wild type α-amylase protein" and "parent polypeptides" mean enzymes and polypeptides from which variant α-amylase polypeptides are derived . The parent enzyme can be a wild-type enzyme or an α-amylase that has previously been recombinantly engineered. The variant α-amylase may additionally include mutations in the signal sequence of the parent α-amylase polypeptide or elsewhere in the parent α-amylase polypeptide. Therefore, the α-amylase polypeptide can be a recombinantly engineered enzyme.

[00078] In one aspect of the invention, the α-amylase used according to the invention is an α-amylase which possesses α-amylase activity as measured in the "α-amylase assay" described herein.

[00079] In one aspect of the invention, the beta-amylase used according to the invention is a beta-amylase which possesses beta-amylase activity as measured in the "beta-amylase assay" described herein.

[00080] The term "pululanase" refers to a specific type of glycanase, an amylolytic endoenzyme 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 I pullulanases specifically attack α-1,6 bonds, while type II pullulanases are also capable of hydrolyzing α-1,4 bonds. It is also produced by some other bacteria and archaebacteria. Pullulanase is used as a detergent in biotechnology. Pullulanase (EC 3.2.1.41) is also known as pullulan-6-glycanohydrolase (debranching enzyme). Pullulan is seen as a chain of maltotriose units linked by α-1,6-glycosidic bonds. Pullulanase will hydrolytically cleave pullulan (α-glycan polysaccharides).

[00081] The term "transglycosylation enzyme" refers to any enzyme that possesses transglycosidase activity, such as transglycosidase. The term "transglycosidase" refers to any enzyme that transfers an α-D-glycosyl residue in a 1,4-α-D-glycan to the primary hydroxyl group of glucose, free or combined in a 1,4-α-D -glycan. The transglycosidase described here has an activity described as EC 2.4.1.24, according to the IUBMB enzyme nomenclature. The systematic name for the transglycosidase described herein is 1,4-α-D-glycan:1,4-α-D-glycan(D-glucose) 6-α-D-glycosyltransferase. This enzyme may be referred to as an α-glucosidase in certain publications.

[00082] As noted above, the transglycosidase enzyme generally has an activity defined as EC 2.4.1.24 according to the IUBMB enzyme nomenclature, whose activity transfers the glycosyl residues of certain glycans to the primary hydroxyl group of glucose. In some embodiments, the enzyme may also have an activity that degrades a natural polysaccharide gum (for example, polysaccharides containing xanthan and galactomannan, such as guar gum or lima bean gum), by cutting the sugar side chains or by cleavage of internal bonds to break the polysaccharide backbone. 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. 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 an Aspergillus niger transglycosidase produced in Trichoderma reesei cells. In some additional embodiments, the transglycosidase is a wild-type fungal transglycosidase (for example, including but not limited to fungal transglycosidase which has an amino acid sequence deposited in the NCBI GENBANK® database as accession numbers: D45356 (GID: 2645159; Aspergillus niger), BAD06006.1 (GID:4031328; Aspergillus awamori), BAA08125.1 {G10:\054565; Aspergillus oryzae), XPJ)Ol 210809.1 (GID: 1 15492363; Aspergillus terreus), XP_001271891.1 (GID : 121707620; Aspergillus clavatus), XPJ)01266999.1 (GID: 1 19500484; Neosartorya fischeri), XP 75181 1.1 (GID:70993928; Aspergillus fumigatus), XP_659621.1 (GID:67523121; Aspergillus nidulans), XP_001:216899.1 (GID:70993928; 115433524; Aspergillus terreus) and XPJ)01258585.1 (GID: 119473371; Neosartorya fischeri)) or such a variant having an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical u at least about 98% identical to a wild-type fungal transglycosidase.

[00083] In one aspect of the invention, the transglycosidase used according to the invention is a transglycosidase which possesses transglycosidase activity as measured in the "transglycosidase assay" described herein.

[00084] Enzyme activity assays according to the invention:

Cell wall solubilization assay:

[00085] Preferably, the solubility of the bran is measured using the following test.

[00086] A suspension of wheat bran (0.1 M) - disodium hydrogenated phosphate buffer (0.2 M), pH 5.0, is prepared to a bran concentration of 1.33% (weight/weight). From this suspension, 750 µL aliquots are transferred to eppendorfh tubes under agitation. Each tube of substrate is preheated for 5 minutes at 40°C. After that, 250 µL of enzyme solution is added, making the final substrate concentration of 1%. Three dilutions (in duplicate) are made from each enzyme composition according to the invention, with increasing enzyme concentrations (eg 0.33; 1.0 and 3.0 μg enzyme/g 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 at specified times by transferring the tubes to an incubator set at 95°C. Heat denatured samples are kept at 4°C until all enzymatic reactions have ended. When the enzyme reactions are complete, the Eppendorph tubes are centrifuged to obtain a clear supernatant. The ability of enzymes to solubilize bran is expressed as the increase in reduced end groups as determined using PAHBAH (Lever, 1972).

[00087] If the bran used contains residual starch, side activities such as amylase activity, may interfere with the above assay, the bran solubilization assay should be performed only on enzymes that modify the cell wall modified (which have no activity of amylase).

[00088] Alternatively, the degree of solubilization can be measured according to the following method:

[00089] 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 and without enzymes, incubating the suspension under agitation at 40°C for a controlled time (eg, 30 to 1440 minutes). After solubilization, the solubilized material is separated from the insoluble material by centrifugation (20 min, 25000 x g, room temperature). The dry matter content of the supernatant is determined both by lyophilization of part of the sample and by moisture analysis (Moisture Analyzer, AND ML-50, Buch & Holm, Denmark). All of the extraction buffer cannot be recovered using this protocol, however it is assumed that the concentration of soluble material is the same in the recovered extraction buffer and the unrecovered extraction buffer because a correction is made to the extraction buffer used in total. Having determined the dry matter content in the soluble fraction, knowing the amount of plant material employed and the amount of extraction buffer, the degree of solubilization can be determined using the following equation. Degree of solubilization = (((g dry matter/ml supernatant recovered) x (ml extraction buffer used)) x 100%)/g plant material employed

Xylanase assay (endo-β-1,4-xylanase activity)

[00090] The samples were diluted in citric acid (0.1 M) - disodium hydrogenated phosphate buffer (0.2 M), pH 5.0, to obtain approximately OD590 = 0.7 in this assay. Three different sample dilutions were incubated for 5 minutes at 40°C. At time = 5 minutes, 1 tablet of Xylazyme (crosslinked, 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 terminated by adding 10 mL of 2% TRIS/NaOH, pH 12. Controls were prepared using 1000 µL of buffer instead of enzyme solution. The reaction mixture was centrifuged (1500 x g, 10 minutes, 20°C) and the OD of the supernatant was measured at 590 nm. A xylanase unit (XU) is defined as the xylanase activity that increases OD590 with 0.025 per minute.

[00091] α-Amylase Activity:

[00092] α-amylases hydrolyze α-D-1,4-glycosidic bonds and their activity can be detected as a rate of color change of a starch-iodine solution due to hydrolysis of alpha-1,4-D bonds.

[00093] Beta-amylase activity:

[00094] Beta-amylase activity can be detected as the release of maltose from the unreduced end of a starch solution.

[00095] Transglycosidase activity:

[00096] Transglycosidase catalyzes both hydrolytic and transfer reactions in incubation with a-D-glyco-oligosaccharides. Transglycosidase activity can be detected as the formation of isomalto-oligosaccharides such as isomaltose, pansose and isomaltriosis after incubation with maltose or maltodextrin.

[00097] Starch debranching activity test:

[00098] Specific enzymes for the α-D-1,6 glycosidic bonds of starch currently include isoamylase (EC 3.2.1.68) and pullulanases (EC 3.2.1.41). Enzymes that act on the α-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 α-D-1,6 glycosidic bonds of starch and pullulan .

[00099] The term "lipid modifying enzyme", as used herein, refers to any enzyme that can modify a lipid.

[000100] In some preferred embodiments, the lipid modifying enzyme is a lipolytic enzyme, such as lipase.

[000101] The term "lipolytic enzyme" as used herein refers to any enzyme that hydrolyzes one or more fatty acids from lipids present in a plant material, such as cereal by-products, which can result in the formation of functional lipids within the cereal by-product, which provides commercial value. The molecules that contribute to the most significant functional effects are molecules with emulsifying characteristics, which are the products of partial hydrolysis, such as lyso-phospholipid, lyso-glycolipid and monoglyceride molecules. Polar lipid hydrolysis products such as lyso-phospholipids and lyso-glycolipids are particularly advantageous in bread production and can give equivalent functionality as emulsifiers such as DATEM.

[000102] The substrates for lipases in cereal by-products are the 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. Enzymatic cleavage of one of these fatty acids in these lipids leads to lipids with a much higher surface activity. It is well known that emulsifiers such as DATEM with very high surface activity are very functional when added to food.

[000103] 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 usually explained by overdose. Overdosing can lead to a decrease in gluten elasticity which results in a dough that is too firm and therefore results in reduced volumes of bread. Additionally or alternatively, such lipases can degrade fat, oil or milk fat added to the dough, resulting in loss of flavor of the dough and baked product. Overdose and loss of flavor have been attributed to the accumulation of free fatty acids in the dough. In connection with the present invention, such unwanted effects can be avoided as lipase is added to the cereal by-product as, for example, a cereal bran suspension which is used with or without further processing as a dough improver. Further processing can be dilution, purification of functional lipids. Furthermore, functional lipids can be processed to be supplied as a liquid product or as a dry formulated product, such as a lyophilized product.

[000104] In EP1193314, EP0977869, WO02/094123, WO00/31758 and also in WO01/39602, the use of lipolytic enzymes active in glycolipids has been reported to be particularly beneficial in the application of bread production, as the products of partial hydrolysis, lyso-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, the enzymes were also found to have significant non-selective activity on triglycerides, which resulted in an unnecessary increase in free fatty acid. Additionally, the application of lipases in bread making had the addition of lipase to the dough followed by an in situ generation of emulsifier in the dough.

[000105] The lipase can be of any origin, for example, of animal origin (such as, for example, from mammal), for example, from the pancreas (for example, bovine or porcine pancreas) or from snake or snake venom. Bee. Alternatively, the lipase may be of microbial origin, e.g., from filamentous fungus, yeast, or bacteria, such as the Aspergillus genus or species, e.g., A. niger, Dictyostelium, e.g., 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; Trichophyton, for example T. rubrum; Whetzelinia, for example W. sclerotiorum; Bacillus, for example B. megaterium, B. subtilis; Citrobacter, for example C. freundii; Enterobacter, for example E. aerogenes, E. cloacae; Edwardsiella, E. tarda; Erwinia, for example, E. herbicola; Escherichia, for example, E. coli; Klebsiella, for example K. pneumoniae; Proteus, for example, P. vulgaris; Providencia, for example, P. stuartii; Salmonella, for example S. typhimurium; Serratia, for example, S. liquefasciens, S. marcescens; Shigella, for example S. flexneri; Streptomyces, e.g., S. violeceoruber, Yersinia, e.g., Y. enterocolitica. Therefore, the lipase may be fungal, for example, from the class of Pyrenomycetes, such as from the genus Fusarium, such as a strain of F. culmorum, F. heterosporum, F. solani, or a strain of F. oxysporum. The phospholipase can also be from a filamentous fungus strain within the Aspergillus genus, such as a strain of Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae.

[000106] A commercially available source of lipolytic enzymes is a microbial lipase or acyltransferase.

[000107] In some embodiments, the lipase is from filamentous fungi, such as Aspergillus spp. and Fusarium spp. Lipases isolated from filamentous fungi have been found to have industrially applicable characteristics and have also been found to be routinely expressed in heterologous production systems, such as in Aspergillus oryzae, Fusarium and yeast.

[000108] In some embodiments, the lipase is from Aspergillus turingiensis as described in EP1433852, which patent is incorporated herein by reference.

[000109] In some embodiments, the lipase is from Fusarium heterosporum as described in EP1722636, which patent is incorporated herein by reference.

[000110] In some embodiments, the lipase is from Fusarium oxysporum as identified in EP 0 130 064 or in Hoshino et al. (1992) Biosci. Biotech. Biochem. 56:660-664.

[000111] In some embodiments, the lipase is pig pancreatic phospholipase A2 expressed, for example, in Aspergillus niger (Cakenzyme®, DSM).

[000112] In some embodiments, the lipase is as described in EP0 869 167, which describes the cloning and expression of a lipase from Fusarium 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 F®.

[000113] In some embodiments, the lipase is as described in WO 02/00852, which describes five lipase enzymes and their coding polynucleotides, isolated from F. venenatum, F. sulphureum, A. berkeleyanum, F. culmorum and F. solani. All five enzymes are described as having triacylglycerol hydrolysis activity, phospholipase and galactolipase activity. Three of the enzymes have activity equivalent to the F. oxysporum enzyme described in EP 0 869 167: F. venenatum, F. culmorum, F. sulphureum.

[000114] 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 lipase from Thermomyces lanuginosus and lipase from Fusarium oxysporum described in EP 0 869 167. This variant has been found to have a significantly higher rate of activity on phospholipids and glycolipids compared to triglycerides.

[000115] In some embodiments, the enzyme that modifies the lipid is a lipid acyltransferase.

[000116] The term "lipid acyltransferase" as used herein means an enzyme that possesses lipase activity (generally classified as EC 3.1.1.x according to the Enzyme Nomenclature Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology), as well as having acyltransferase activity (generally classified as EC 2.3.1.x), whereby the enzyme is able to transfer an acyl group from a lipid to one or more acceptor substrates, such as one or more of the following: a sterol, a stanol, a carbohydrate, a protein, a protein subunit, glycerol.

[000117] 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 herein) to one or more of the following acceptor substrates: a sterol, a stanol , a carbohydrate, a protein or its subunits or glycerol.

[000118] In some aspects, the acyl receptor can be any compound that comprises 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 mixtures and derivatives thereof.

[000119] In some embodiments, the lipid substrate on which the lipid acyltransferase used according to the present invention acts, 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. The term lecithin when used herein encompasses phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidylglycerol.

[000120] Preferably, in some aspects, the lipid substrate on which the lipid acyltransferase acts is a phospholipid, such as lecithin, for example, phosphatidylcholine or phosphatidylinositol.

[000121] In some embodiments, the lipid substrate is a food lipid, which is to say that it is a lipid component of a foodstuff.

[000122] Suitably, the lipid acyltransferase used according to the present invention may exhibit one or more of the following lipase activities: glycolipase activity (EC 3.1.1.26), triacylglycerol lipase activity (EC 3.1.1.3), phospholipase activity A2 (EC3.1.1.4) or phospholipase A1 activity (EC 3.1.1.32). The term "glycolipase activity" as used herein encompasses "galactolipase activity".

[000123] Suitably, the lipid acyltransferase used according to 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 activity of phospholipase A2 (EC 3.1.1.4).

[000124] Preferably, in some aspects, the lipid acyltransferase used according to the present invention is capable of transferring an acyl group from a glycolipid and/or a phospholipid to a sterol and/or a stanol to form at least one ester of sterol and/or a stanol ester.

[000125] Suitable sterol acyl receptors include cholesterol and phytosterols, for example alpha-sitosterol, beta-sitosterol, stigmasterol, ergosterol, campesterol, 5,6-dihydrosterol, brassicasterol, alpha-spinasterol, beta-spinasterol, gamma-spinasterol, delta-spinasterol, fucosterol, dimosterol, ascosterol, serebisterol, episterol, anasterol, hyposterol, chondrilasterol, desmosterol, calinosterol, poriferasterol, clionasterol, sterol glycoside and other natural or synthetic isomeric forms and derivatives.

[000126] Preferably, in one aspect, the acyl receptor is one or more of the following: alpha-sitosterol, beta-sitosterol, stigmasterol, ergosterol, beta-sitostanol, ss-sitostanol or campesterol.

[000127] Preferably, in some aspects, the lipid acyltransferase used according to the present invention is capable of transferring an acyl group from a glycolipid and/or a phospholipid to a glycerol to form at least a diglyceride and/or a monoglyceride.

[000128] In some aspects, one or more of the sterols present in the lipid-containing plant material may be converted to one or more stanols before or at the same time as the lipid acyltransferase is added in accordance with the present invention. Any suitable method for converting sterols to 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 with the addition of the lipid acyltransferase according to the present invention. Suitable enzymes for the conversion of sterol to stanols are described in WO00/061771.

[000129] 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 eg WO92/99640).

Protocol for determining the % acyltransferase activity:

[000130] The lipid-containing plant material to which a lipid acyltransferase has been added according to the present invention can be extracted after enzymatic reaction with 2:1 CH3:CH3OH and the organic phase containing the lipid material is isolated and analyzed by GLC and HPLC according to the procedure detailed below. From the GLC and HPLC analysis, the amount of free fatty acids and one or more sterol/stanol esters is determined; carbohydrate esters; protein esters; diglycerides or monoglycerides. A control of plant material containing lipid to which the enzyme has not been added is analyzed in the same way.

[000131] Calculation:

[000132] From the GLC and HPLC analysis, the increase in free fatty acids and sterol/stanol esters and/or carbohydrate esters and/or protein esters and/or diglycerides and/or monoglycerides can be calculated:

[000133] Δ% fatty acid = % fatty acid (enzyme) - % fatty acid (control); Fatty acid Mv = average molecular weight of fatty acids;

[000134] A = Δ% sterol ester/Mv of sterol ester (where Δ% sterol ester = % sterol/stanol ester (enzyme) - % sterol/stanol ester (control) and Mv of sterol ester = average molecular weight of sterol/stanol esters) - applicable where the acyl acceptor is a sterol and/or a stanol;

[000135] B = Δ% carbohydrate ester/Mv carbohydrate ester (where Δ% carbohydrate ester = % carbohydrate ester (enzyme) - % carbohydrate ester (control) and Mv carbohydrate ester = average molecular weight of carbohydrate esters) - applicable where the acyl acceptor is a carbohydrate;

[000136] c = Δ% protein ester/Mv protein ester (where Δ% protein ester = % protein ester (enzyme) - % protein ester (control) and Mv protein ester = average molecular weight of protein esters) - applicable where the acyl acceptor is a protein; and

[000137] D = absolute value of diglyceride and/or monoglyceride/Mv of di/monoglyceride (where % diglyceride and/or monoglyceride = % diglyceride and/or monoglyceride (enzyme) - % diglyceride and/or monoglyceride (control) and Mv of di/monoglyceride = average molecular weight of diglyceride and/or monoglyceride) - applicable where the acyl receptor is glycerol.

[000138] The transferase activity is calculated as a percentage of the total enzyme activity: % transferase activity = A* + B* +C* + D* x 100 A* + B* +C* + D* + % fatty acid /(Mv of fatty acid) (* - delete as appropriate).

[000139] In a preferred aspect, the present invention provides a lipid-containing plant material, 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.

[000140] Properly, the expression "foodstuff", as used herein, means a food in a form that is ready for consumption. Alternatively or in addition, however, the term foodstuff, as used herein, can mean one or more food materials that are used in the preparation of a food. Just as an example, the expression foodstuff covers baked products produced from a flour dough as well as the flour dough used in the preparation of said baked products.

[000141] Properly, the term "foodstuff", as used herein, means a substance that is suitable for human and/or animal consumption.

[000142] In another aspect, the foodstuff according to the present invention can be an animal feed.

[000143] 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, sauces, ice cream, egg powder, modified egg yolk and products made with it; baked goods including breads, cakes, sweet dough products, puff pastries, liquid doughs, muffins, donuts, biscuits, toast and cookies; confectionery products including chocolate, caramels, halawa, gums including sugarless and sweetened gums, chewing gum, soft chewing gum, chewing 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 creamer, whipped cream, pastry cream, dairy drinks and yoghurts; mousses, whipped vegetable creams, meat products, including processed meat products; edible oils and fats, aerated and non-aerated cream products, oil-in-water emulsions, water-in-oil emulsions, margarine, fats and toppings including low-fat and very low-fat toppings; sauces, mayonnaise, pastes, cream-based sauces, cream-based soups, beverages, flavored emulsions and purees.

[000144] Suitably, the foodstuff according to the present invention can be a "refined food" including cakes, pastries, confectionery, chocolates, candies and the like.

[000145] In one aspect, the foodstuff according to the present invention can be a dough product, such as bread, a fried product, a snack, cakes, pies, brownies, cookies, noodles, instant noodles, tortillas , snack items such as crackers, graham crackers, pretzels and chips, pasta and breakfast cereal.

[000146] 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, salad dressings, margarine, toppings, peanut butter, shortenings, ice cream, cooking oils.

[000147] In another aspect, the foodstuff according to the present invention may be a milk-derived product including butter, milk, cream, cheese such as natural cheeses, processed and cheese imitations in a variety of forms (including crushed, block, sliced or grated), cream cheese, ice cream, frozen desserts, yoghurt, yoghurt drinks, bottled butter, anhydrous fat, other dairy products. The enzyme used in accordance with the present invention can improve fat stability in milk-based products.

[000148] In another aspect, the foodstuff according to the present invention can be a food product that contains ingredients of animal origin, such as processed meat products, cooking oils and fats.

[000149] In a further aspect, the foodstuff according to the present invention can be a beverage, a fruit, a fruit mixture, a vegetable or a wine. In some cases, the beverage may contain up to 20 g/l of added phytosterols derived from the invention.

[000150] In another aspect, the foodstuff according to the present invention can be an animal feed. The animal feed can be fortified with phytosterol and/or phytostanols, preferably with beta-sitosterol/stanol. Suitably the animal feed can be a poultry feed. When the food is a poultry feed, the present invention can be used to lower the cholesterol content of eggs produced by birds fed the food according to the invention.

[000151] Preferably, in one aspect, the foodstuff is selected from one or more of the following: eggs, egg-based products, including but not limited to mayonnaise, salad dressings, pastes, ice cream, egg powder, egg yolks. modified egg and products made with it.

[000152] Preferably, the foodstuff according to the present invention is a foodstuff containing water. Suitably the foodstuff can be constituted by 1098% water, suitably 14-98%, suitably 1898% water, suitably 20-98%, suitably 4098%, suitably 50-98%, suitably 70 -98%, appropriately by 75-98%.

[000153] In one aspect of the invention, the functional lipid produced from the lipid-containing plant material is an emulsifier. Preferably, at least one emulsifier is generated in the lipid-containing plant material.

[000154] In one aspect of the invention, at least three different emulsifiers are generated in the lipid-containing plant material.

[000155] In one aspect of the invention, at least four different emulsifiers are generated in the lipid-containing material.

[000156] Suitably, the emulsifier according to the present invention may 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 digalactosyl monoglyceride (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 expression lysophosphatidylcholine as used herein is synonymous with the expression lysolecithin and these expressions can be used alternatively.

[000157] Where one of the emulsifiers is a protein ester and/or a diglyceride and/or a monoglyceride, the second emulsifier can be, for example, one or more of the following: a diglyceride, a monoglyceride, such as 1-monoglyceride, lysophosphatidylcholine 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.

[000158] In one embodiment, the functional lipids generated from the invention can be used in a process for the preparation of a foodstuff.

[000159] The 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 starch degrading enzymes such as endo- and exo-amylases, pullulanases, debranching enzymes, hemicellulases including the xylanases, cellulases, oxidoreductases, e.g. glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as that which oxidizes maltose, for example, hexose oxidase (HOX), lipases, phospholipases, glycolipases and proteases.

[000160] The lipid-containing plant material 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 starch degrading enzymes such as endo- and exo-amylases, pullulanases, debranching enzymes, hemicellulases including the xylanases, cellulases, oxidoreductases, e.g. glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as that which oxidizes maltose, for example, hexose oxidase (HOX), lipases, phospholipases, glycolipases and proteases.

[000161] In a preferred embodiment, the lipolytic enzyme has one or more of the following lipase activities: glycolipase activity (EC 3.1.1.26), triacylglycerol lipase activity (EC 3.1.1.3), phospholipase A2 activity (EC 3.1. 1.4) or phospholipase A1 activity (EC 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 (Danisco A/S), Lysomax Oil (Danisco A/S), Lipopan® F, Lipopan® Xtra, and /or LECITASE® ULTRA (Novozymes A/S, Denmark), phospholipase A2 (eg phospholipase A2 from LIPOMOD® 22L from Biocatalysts, LIPOMAX® from Genencor), LIPOLASE® (Novozymes A/S, Denmark), Panomore® (DSM Nutritional Products), the lipases described in WO03/97835, EP 0 977 869 or EP 1 193 314. One skilled in the art will be able to combine proportions of lipolytic enzymes.

[000162] Traditionally, the cake industry uses cake processors for the production of cakes and to ensure the high quality of the cakes in terms of flavor, structure, quality and appearance of the food. These cake improvers are typically based on freeze-dried emulsifiers in a carrier similar to starch and maltodextrin. Some cake improvers are also in gel form based on emulsifiers, sugars and water. These cake processors are very important to the cake industry in order to produce a high quality cake. However, cake improvers contain emulsifiers and other "unnatural" ingredients with an "E-number". Due to consumer demand to reduce the numbers of "Enumbers", the cake industry has been researching alternative ways to produce high quality cakes without using these types of emulsifiers.

[000163] The lipid-containing plant material treated with lipolytic enzymes to generate functional lipids according to the present invention can be used as food improvers without purification or with limited purification of the functional lipids or as fully purified functional lipids.

[000164] In one aspect of the invention, the food processor is a cake processor.

[000165] In one aspect of the invention, the food processor is a bread improver.

[000166] The food processor generated according to the present invention may suitably comprise one or more of the following additives:

[000167] soy protein material; carotenoids, flavonoids, anti-oxidants and phytochemicals (especially anthocyanin, carotenoid, bioflavonoid, glutathione, catechin, isoflavone, lycopene, ginsenoside, pycnogenol, alkaloid, pygeum phytosterol, contain sulphorafone, resveremente deu keu extract, stanol esters), vitamin (especially vitamin C, 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, zinc, iron, selenium, manganese, chromium, copper, cobalt, molybdenum or phosphorus), fatty acids (especially gamma-linoleic acid, eicosapentaenoic acid or docosahexaenoic acid), oil (especially borage oil, rich canola oil in carotenoid or flaxseed 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 bromelain, papain, amylase, cellulase or coenzyme Q), lignin, stanol ester or favorable bacteria (especially Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus bifidus, Lactobacillus plantarum or Streptococcus faecium), folic acid, insoluble and/or soluble fibers.

[000168] The present invention can provide one or more of the following unexpected technical effects in egg products, particularly mayonnaise: improved thermal stability during pasteurization; improved organoleptic properties, improved consistency.

[000169] The present invention can provide one or more of the following unexpected technical effects on dough and/or baked goods: an improved specific volume of the dough or baked goods (e.g., bread and/or cake); improved dough stability; an improved crust (eg a thinner and/or crunchier bread crust), an improved crumb (eg a more even crumb distribution and/or a finer crumb structure and/or a softer crumb) ; an improved appearance (for example, a smooth surface with no bubbles or holes or substantially no bubbles or holes); reduced hardening; improved softness; improved odor; improved flavor.

[000170] The present invention can provide a beneficial effect from functional lipids as 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 storage , because free fatty acids are more prone to oxidation than the corresponding fatty acid esters.

[000171] In a further aspect, the present invention provides the use of a lipolytic enzyme to generate other functional compounds according to the present invention in a lipid-containing plant material.

[000172] It should be understood that the action of lipid-modifying enzymes, such as lipolytic enzymes on lipid-containing plant material can generate not only functional lipids, but also other functional compounds, such as with the action of a lipid transferase , wherein an acyl group of a lipid is transferred to one or more other acceptor substrates, such as one or more of the following: a sterol, a stanol, a carbohydrate, a protein, a protein subunit and glycerol.

[000173] In some particular embodiments, the functional compounds generated in the methods according to the present invention are functional esters.

[000174] In some embodiments, both functional lipids and other functional compounds are generated by the methods according to the present invention.

[000175] These 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 said functional compounds to a dough and (optionally) baking the dough to make a baked product with one or more of the following: reduced dough viscosity; improved machinability of the dough; reduction of bubbles during cooking of the baked product; improvement in bread volume and/or tenderness; extending the shelf life of the baked product and/or pasta; improvement of the anti-hardening effect of the baked product and/or the dough; improvement of the crust structure of the baked product; reduced pore heterogeneity of the baked product; improving the homogeneity of the pore of the baked product; reduction in the average pore size of the baked product; enhancement of the gluten index of the mass; improving the taste and/or odor of the baked product; perfecting the color of the crust of the baked product.

[000176] In one aspect, functional compounds generated by the methods according to the present invention are purified or partially purified.

[000177] In one aspect, functional compounds generated by the methods according to the present invention are not purified prior to use in a food.

[000178] In one aspect, functional compounds generated by the methods according to the present invention are formulated as a dry product.

[000179] In one aspect, functional compounds are concentrated or diluted prior to use in a food.

[000180] In another aspect of the invention, a method is provided for making noodles or a noodle dough or a product based on noodles, which method comprises adding a functional compound according to the present invention to the noodles, noodle dough or a product based on noodles.

[000181] In one aspect of the present invention, the use of a functional compound according to the present invention in the manufacture of noodles or a noodle-based product is provided to improve one or more of the yellowish color/tone, stability of the color, reduced shine, reduced fat content, improved texture and bite (thickness), reduced water activity, reduced breakage, increased color fastness, and improved size maintenance during processing.

[000182] In another aspect of the invention, a method for making tortilla or tortilla dough is provided, which method comprises adding a food improver generated in accordance with the present invention to tortilla or tortilla dough.

[000183] In another aspect of the invention, a method is provided for making whole grain noodles or noodles or pasta dough for noodles, which method comprises adding a food improver generated in accordance with the present invention to pasta or dough for noodles. noodle.

[000184] A further aspect of the present invention provides the use of a food processor generated in accordance with the present invention in the manufacture of a tortilla or tortilla batter to improve tortilla curl, increase flexibility of a tortilla, improve the anti-hardening properties of the tortilla and/or tortilla dough, improve the tenderness and/or reduce the loss of flavor of the tortilla and/or tortilla dough.

[000185] The functionality of the food processor can be improved by combining it with emulsifiers such as DATEM.

[000186] Suitably, the present invention can provide one or more of the following unexpected technical effects in a foodstuff: improved appearance, improved taste, improved stability, in particular improved thermal stability, improved flavor, improved tenderness, improved elasticity and improved emulsification .

[000187] 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 creamier taste); improved flavor and improved melting.

[000188] Suitably, the present invention can provide one or more of the following unexpected technical effects on egg or egg products: improved emulsion stability; thermal stability of the emulsion; improved flavor; odor reduction; improved thickening properties; improved consistency

[000189] Specific technical effects associated with the use of the food processor as defined herein in the preparation of a food are listed in the table below:

[000190] 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.

[000191] In another aspect of the present invention, a process is provided for the preparation of a lysophospholipid, for example, lysolecithin, which process comprises treating a lipid-containing plant material with the lipolytic enzyme according to the present invention.

[000192] In a further aspect of the present invention, the use of a lipolytic enzyme in a process for the preparation of a lysoglycolipid (e.g., digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG)) by treating a lipid-containing plant material with the lipolytic enzyme according to the present invention.

[000193] In some embodiments of the invention, the liquid suspension of at least part of the plant material that contains partially solubilized lipid is essentially free of starch.

[000194] 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 10%, such as less than about 6%, such as less than about 3%, such as less than about 1% (weight/weight) of the plant material liquid suspension containing Partially solubilized lipid is starch or starch-containing components such as flour.

[000195] Consequently, in some embodiments, it is understood that enzymes are to have an enzymatic effect on lipid-containing plant material that is essentially free of starch or that contains only residual starch from a previous processing step. The present invention is not intended to encompass enzymatic treatment of compositions with additional preparations of added flour, such as in in situ enzymatic applications in bread making.

Determination of galactolipase activity (glycolipase activity assay):

[000196] Substrate:

[000197] Digalactosyldiglyceride 0.6% (Sigma D 4651), Triton-X 100 0.4% (Sigma X-100) and 5 mM CaCl 2 were dissolved in 0.05 M HEPES buffer pH 7.

[000198] Test procedure:

[000199] 400 μL of substrate were added to a 1.5 mL Eppendorf tube and placed in an Eppendorf Thermomixer at 37°C for 5 minutes. At time t=0 min, 50 µL of enzyme solution was added. A control with water instead of enzyme was also analyzed. The sample was mixed at 10*100 rpm in an Eppendorf Thermomixer at 37°C for 10 minutes. At time t=10 min, the Eppendorf tube was placed in another thermal mixer at 99°C for 10 min. to stop the reaction. Free fatty acid in the samples was analyzed using the NEFA C kit from WAKO GnbH.

[000200] The GLU enzyme activity at pH 7 was calculated as micromoles of fatty acid produced per minute under the assay conditions.

Determination of phospholipase activity (phospholipase activity assay):

[000201] Phospholipase activity was measured using two different methods that provide comparable results. Any of these methods can be used to determine phospholipase activity in accordance with the present invention.

"PLU Assay" for Determination of Phospholipase Activity

[000202] Substrate:

[000203] L-α phosphatidylcholine 95% Plant 0.6% (Avanti #441601), Triton-X 100 0.4% (Sigma X-100) and 5 mM CaCl 2 were dissolved in 0.05 M HEPES buffer pH 7.

[000204] Test procedure:

[000205] 400 μL of substrate were added to a 1.5 mL Eppendorf tube and placed in an Eppendorf Thermomixer at 37°C for 5 minutes. At time t=0 min., 50 µL of enzyme solution was added. A control with water instead of enzyme was also analyzed. The sample was mixed at 10*100 rpm in an Eppendorf Thermomixer at 37°C for 10 minutes. At time t=10 min., the Eppendorf tube was placed in another thermal mixer at 99°C for 10 min. to stop the reaction. Free fatty acid in the samples was analyzed using the NEFA C kit from WAKO GnbH.

[000206] The PLU-7 enzyme activity at pH 7 was calculated as micromoles of fatty acid produced per minute under the assay conditions.

"TIPU Assay" for determination of phospholipase activity

[000207] 1 TIPU (Phospholipase Titration Unit) is defined as the amount of enzyme that releases 1 µmol of free fatty acid per minute under the test conditions.

[000208] Phospholipase A1 and A2 catalyze the conversion of lecithin into lysolecithin with the release of free fatty acid from positions 1 and 2, respectively. Phospholipase activity can be determined by continuous titration of the fatty acids released from lecithin during enzymatic, as the alkali consumption equals the amount of fatty acid released.

[000209] Substrate:

[000210] 4% Lecithin, 4% Triton-X 100 and 6 mM CaCl 2 : 12 g of powdered lecithin (Avanti Polar Lipids #44160) and 12 g of Triton-X 100 (Merck 108643) were dispersed in approximately 200 ml of demineralized water during magnetic stirring. 3.0 ml of 0.6 mM CaCl 2 (p.a. Merck 1.02382) was 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. Test procedure:

[000211] an enzyme solution was prepared to give a titration curve between 0.06 and 0.18 ml/min. with the addition of 300 µL of enzyme. A control sample of known activity is included. The samples were dissolved in demineralized water and shaken for 15 min. at 300 rpm. 25.00 ml of substrate had the temperature adjusted to 37°C 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 NaOH 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 enzyme solution.

[000212] The phospholipase activity (TIPU/g enzyme) was calculated as follows:

[000213] where:

[000214] α is the slope of the titration curve between 5 and 7 minutes of the reaction time (mL/min.).

[000215] N is the normality of the NaOH used (mol/l).

[000216] V1 is the volume in which the enzyme is dissolved (mL).

[000217] m is the amount of enzyme added to V1 (g).

[000218] V2 is the volume of enzyme solution added to the substrate (mL).

Determination of triacylglyceride lipase activity: assay based on triglyceride (tributyrin) as substrate (LIPU):

[000219] Tributyrin-based lipase activity is measured 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 statistical adjustment pH is 5.5 instead of 7.

[000220] 1 LIPU is defined as the amount of enzyme that can release 1 mole of butyric acid per minute under the test conditions.

[000221] In one aspect of the invention, the lipolytic enzyme used in accordance with the present invention can be obtained from a filamentous fungus. More preferably, the fungal lipolytic enzyme is obtained (preferably obtained) from Fusarium spp. Preferably, the fungal lipolytic enzyme used according to the present invention can be obtained (preferably obtained) from Fusarium heterosporum or 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 Fusarium semitectum (IBT 9507).

[000222] Therefore, in one aspect, the fungal lipolytic enzyme used in accordance with the present invention is preferably a filamentous fungus lipolytic enzyme, preferably a wild-type filamentous fungus lipolytic enzyme.

[000223] In some of the applications mentioned here, particularly food applications, such as bakery applications, the food improver generated according to the present invention can be used with one or more conventional emulsifiers, including, for example, monoglycerides, diacetyl tartaric acid esters of mono and diglycerides of fatty acids, sodium stearoyl lactylate (SSL) and lecithins.

[000224] The food improver generated by the methods according to the present invention is especially preferred in bread recipes with added fat.

[000225] Additionally or alternatively, the food improver generated by the methods 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 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 starch degrading enzymes such as endo or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g. glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as that which oxidizes maltose, for example, hexose oxidase (HOX), lipases, phospholipases, galactolipases and hexose oxidase, proteases and acyltransferases (such as those described in WO04/064987, for example).

[000226] It is particularly preferred that the lipolytic enzyme used according to the present invention is used in combination with alpha-amylases in the production of food products. In particular, the amylase can give a non-maltogenic amylase, such as a polypeptide that possesses non-maltogenic exo-amylase activity, in particular, 1,4-alpha-maltotetrahydrolase glycan activity (EC 3.2.1.60) (as described in WO05/003339). A suitable non-maltogenic amylase is commercially available as Powersoft® (available from Danisco A/S, Denmark). Maltogenic amylases such as Novamyl® (Novozymes A/S, Denmark) can also be used. In one embodiment, the combined use of alpha-amylases and the food improver of the invention can be used in a dough and/or in the production of a baked product such as bread, cakes, donuts, cake donuts or bagels. The combination of alpha-amylases and the food improver of the invention is also considered to be preferable for use in tortilla production methods such as wheat and/or corn tortillas.

[000227] In another preferred embodiment, the food improver generated according to the present invention can be used in combination with a xylanase in the production of food products. GRINDAMYL® and POWERBake 7000 are examples of xylanase enzymes commercially available from Danisco A/S. Other examples of xylanase enzymes can be found in WO03/020923 and WO01/42433.

[000228] Preferably, the food improver generated according to 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-maltogenic alpha-amylase (such as GRINDAMYL® or POWERSoft, available from Danisco A/S) or a combination thereof.

[000229] The food improver of the invention can also preferably be used in combination with an oxidizing enzyme, such as an enzyme that oxidizes maltose (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.

[000230] Optionally, an alpha-amylase, such as a non-maltogenic exo-amylase and/or a maltogenic amylase and/or an enzyme that oxidizes maltose (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, instant noodles/fried snacks or a milk-derived product such as cheese.

[000231] The food processor generated according to the present invention is included in the food, typically, by methods known in the art. Such methods include adding the food improver directly to the food or composition, adding the food improver in combination with a stabilizer and/or vehicle, and adding a mixture comprising the food improver and a stabilizer and/or vehicle.

[000232] Suitable stabilizers 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, ester of polyglycerol, sorbitan monooleate), oil (such as canola oil, sunflower oil and soybean oil), pectin, trehalose, sorbitol and glycerol.

Suitable carriers for use with the present invention include, but are not limited to starch, cereal flour, ground wheat, wheat flour, NaCl and citrate.

[000234] For baked goods such as bread, steamed bread and American white loaf bread, for example, the addition of a food improver of the present invention may result in one or more of the following: increased bread volume and tenderness, extended shelf life and/or anti-decomposition effect, improved crumb structure, reduced pore heterogeneity, reduced average pore size, enhanced gluten content, improved taste and/or odor, and improved crust color.

[000235] Advantageously, the food processor generated according to the present invention can be used to replace emulsifiers in foods, such as dough and/or baked goods.

[000236] The food processor generated according to the present invention can have synergy with emulsifiers such as DATEM, SSL, CSL, monoglycerides, polysorbates and Tween. Therefore, the food improver generated in accordance with the present invention can be used in combination with one or more emulsifiers. Advantageously, the use of the food improver 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 improver generated in accordance with the present invention is used.

[000237] The food processor generated according to the present invention can also have synergy with hydrocolloids, Guar, xanthan and pectin and with enzymes that oxidize maltose, such as hexose oxidase.

[000238] For donuts, donut cake, bagels, muffins and muffins, for example, the use of the food improver 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.

[000239] For cakes, sponge cake and palm cake, for example, the use of the food improver of the present invention can result in a synergistic effect when used in combination with one or more hydrocolloids such as Guar and/or one or more emulsifiers such as DATEM.

[000240] For biscuits, for example, the use of the food processor generated according to the present invention confers improved winding and handling capacity properties, particularly when cold (cold winding).

[000241] Advantageously, in mayonnaise and other egg-based products, for example, the use of a food processor generated according to the present invention can lead to improved texture, reduced mean particle size and/or reduced distribution of average particle size, improved thermal stability, better microwave performance and/or stability.

[000242] In cakes, the use of the present invention advantageously leads to improved softness, volume, storage properties and improved shelf life.

[000243] For noodles or noodle-based products, for example, instant noodles, the food improver of the present invention can provide one or more of the following characteristics: improved yellowish color/tone, more stable color characteristics, reduction in brightness, reduction fat content, improved texture and bite (thickness), reduced water activity, reduced breakage, increased color firmness, and improved shape maintenance during processing.

[000244] Preferably, the food improver of the present invention can be used to reduce the fat content of a noodle or a noodle product, for example instant noodles.

[000245] In tortillas, for example, the use of a food processor generated in accordance with the present invention can result in one or more of the following: reduced tortilla curling capacity, for example, by increased flexibility, improved anti-decomposition properties , improved tenderness and/or reduced lack of flavor.

[000246] Advantageously, the improved rollability and/or flexibility can lead to a reduced probability of the tortilla shattering when rolled.

[000247] The food processor generated according to the present invention is particularly useful in the preparation of baked products, such as those prepared from a dough, including breads, cakes, sweet dough products, puff pastries, liquid doughs , muffins, donuts, cookies, crackers and cookies.

[000248] The food processor generated according to the present invention is particularly useful in the preparation of cereals for breakfast, such as those prepared from a flour dough.

[000249] The food improver can also be used as an additive for improving bread, for example dough compositions, dough additive, dough conditioners, premixes and similar preparations conventionally added to flour and/or to dough during the manufacturing processes of bread or other baked goods to provide improved properties to bread or other baked goods.

[000250] Therefore, the present invention relates to a composition for improving bread and/or a composition for improving dough that comprises a food improver generated according to the present invention; and also to a dough or baked product which comprises such a composition for improving the bread and/or improving the dough.

[000251] The composition for improving bread and/or composition for improving dough may comprise, in addition to a fungal lipolytic enzyme according to the present invention, other substances, which substances are conventionally used in baking for improve the properties of the dough and/or baked products.

[000252] The composition for improving bread and/or composition for improving dough may comprise one or more conventional baking agents, such as one or more of the following constituents:

[000253] Powdered milk, gluten, an emulsifier, granulated fat, an oxidant, an amino acid, sugar, salt, flour or starch.

[000254] 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.

[000255] The composition for improving bread and/or dough may comprise another enzyme, such as one or more suitable food grade enzymes, including starch degrading enzymes such as endo or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g. glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as that which oxidizes maltose, e.g., hexose oxidase (HOX), lipases, phospholipases, galactolipases and hexose oxidase, proteases and acyltransferases (such as those described in WO04/064987, for example).

[000256] The term "baked product" as used herein includes a product prepared from a dough. Examples of baked goods (of the white, light or dark type) that may advantageously be produced by the present invention include one or more of the following: bread (including white, wholemeal and rye bread), typically in the form of slices or rolls or toast, French baguette bread, pita bread, tortillas, tacos, cakes, pancakes, cookies, noodles, noodles and the like).

[000257] 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 (bread yeast) culture.

[000258] The dough according to the present invention can be a dough for the preparation of a dehydrated cereal product, toast, biscuit or biscuit.

[000259] Specific modalities of the invention:

[000260] As discussed above, the present invention relates to a method for treating lipid-containing plant material, the method comprising the step of treating a liquid suspension of at least one partially solubilized lipid-containing plant material with one or more lipid-modifying enzymes.

[000261] In some embodiments, the method further comprises a preceding or simultaneous step of treating a liquid suspension of a lipid-containing plant material to obtain said partially solubilized plant material.

[000262] In some embodiments, the method further comprises a subsequent step of treating a liquid suspension to obtain said solubilized plant material.

[000263] In some embodiments, the treatment to obtain at least a partially solubilized plant material is a treatment with one or more enzymes that modify the cell wall.

[000264] In some embodiments, the treatment to obtain at least one partially solubilized plant material is a sonication treatment, such as ultrasonic treatment and/or extrusion.

[000265] In some embodiments, the liquid suspension of at least partially solubilized plant material contains insoluble plant material.

[000266] In some embodiments, the plant material is treated under said method steps simultaneously.

[000267] In some embodiments, the plant material is treated under said method steps according to the present invention without removing a substantial amount of any component.

[000268] In some embodiments, the liquid suspension is further treated with one or more additional enzymes.

[000269] In some embodiments, the additional enzyme is one or more transglycosylation enzymes.

[000270] In some embodiments, the additional enzyme is a protease.

[000271] In some embodiments, one or more enzymes that modify the lipid is a lipolytic enzyme selected from the group consisting of: a triacylglycerol lipase, a phospholipase and a galactolipase.

[000272] In some embodiments, one or more enzymes that modify the lipid contains two or three activities selected from the group consisting of: triacylglycerol lipase activity, phospholipase activity and galacto-lipase activity.

[000273] In some embodiments, one or more lipid-modifying enzymes is one, two, three, four or five different lipid-modifying enzymes.

[000274] In some embodiments, the method according to the present invention comprises an additional step of isolating the soluble fraction.

[000275] 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.

[000276] In some embodiments, cellulase is selected from an endocellulase, an exocellulase, a cellobiase, an oxidative cellulase, a cellulose phosphorylase.

[000277] In some embodiments, one or more additional enzymes are starch-modifying enzymes, selected from the group consisting of an alpha-amylase, a pullulanase, isoamylase and a beta-amylase.

[000278] 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.

[000279] In some embodiments, the plant material is provided in particles, in which the average particle size of said particulate plant material is below 3000 µm, such as below 1000 µm, such as below 500 µm.

[000280] In some embodiments, the plant material is a cereal bran.

[000281] In some modalities, the cereal bran is selected from wheat, barley, oats, rye, triticale, rice and corn.

[000282] In some embodiments, the method according to the present invention further comprises a preceding step of i) fractionating the cereal grain obtained to obtain the endosperm, bran and germ; ii) separate and distribute the endosperm, bran and germ to enable them to be treated; and iii) grinding the bran.

[000283] In some embodiments, cereal bran is obtained from an industrial milling process and further ground to obtain an average particle size below 500 μm, such as below 400 μm, such as below 200 μm.

[000284] In some embodiments, plant material is a by-product of processing plant material, such as neutralizing paste from the refining of vegetable oils, grains used for fermentation, or dry distillers grains with solubles (DDGS).

[000285] In some embodiments, the composition obtained comprising the modified lipids, such as functional lipids, are further treated to inactivate additional enzymatic activity.

[000286] In some embodiments, the degree of solubilization obtained from said plant material, as determined on dry matter versus 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%.

[000287] In some embodiments, the total content of lipids and modified lipids, such as functional lipids, as determined on dry matter versus plant material dry matter in the soluble fraction obtained is at least about 0.05%, such as at least about 1.0%, such as in the range between 0.05 to 5%.

[000288] In some embodiments, the method according to the present invention further comprises a step of dehydrating the composition obtained which comprises lipids and/or modified lipids, such as functional lipids.

[000289] In some embodiments, the method according to the present invention further comprises a step of spray drying the composition obtained which comprises lipids and/or modified lipids, such as functional lipids.

[000290] In some embodiments, the method according to the present invention further comprises a step of lyophilization of the composition obtained which comprises lipids and/or modified lipids, such as functional lipids.

[000291] In some modalities, treatment with one or more lipid-modifying enzymes generates functional lipids, such as emulsifiers.

[000292] In some embodiments, treatment with one or more lipid-modifying enzymes generates other functional compounds, such as functional sterol esters.

[000293] In some embodiments, 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% conversion of phosphatidylinositol to lysophosphatidylinositol (lyso-PI).

[000294] 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, such as at least 20% of the phospholipids, such as at least 50% of the phospholipids, such as at least 75% of phospholipids.

[000295] In some embodiments, treatment with one or more lipid-modifying enzymes hydrolyzes at least 5% of the glycolipids, such as at least 10% of the glycolipids, such as at least 20% of the glycolipids, such as at least 50% of the glycolipids, such as at least 75% of glycolipids.

[000296] In some embodiments, treatment with one or more lipid-modifying enzymes hydrolyzes at least 5% of the triglycerides, such as at least 10% of the triglycerides, such as at least 20% of the triglycerides, such as at least 50% of the triglycerides, such as at least 75% of triglycerides.

[000297] In some embodiments, the composition comprising the lipids and/or modified lipids, such as the functional lipids obtained in the method according to the invention is added directly as a mixture of soluble and insoluble plant material in the production of a product food.

[000298] In some embodiments, the food product according to the present invention is selected from the group consisting of bread, a breakfast cereal, pasta, cookies, cookies, snacks and beer.

EXAMPLES EXAMPLE 1

[000299] Example 1. Laboratory-Scale Modification of Commercial wheat bran and wheat bran lipids followed by evaluation in bakery:

[000300] Bran:

[000301] Wheat bran fractions obtained from a commercial mill were used. The fractions consisted of a fine bran fraction and a coarse bran fraction. 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. Grinding was performed 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, with regard to the degree of solubilization.

[000302] Protocol: Table 2. Protocol used for bran modification

[000303] Wheat bran was suspended in 50 mM NaPi, pH 5 (13% w/w) in a closed-lid vessel/reactor

[000304] The bran suspension is heated to 100°C under stirring and boiled for 2 min. The sample is placed under agitation (with the lid closed) at 50 °C and allowed to equilibrate to the 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 further enzymatic activity Sample is cooled and stored to avoid contamination Pellet is lyophilized Supernatant is analyzed

[000305] Tests: The following modifications were made to the bran (table 3) Table 3: Amount of bran, g, treated with different enzymes

[000306] Analysis:

[000307] The soluble fraction of the bran (the supernatant) is analyzed with respect to:

[000308] Dry matter content (%):

[000309] 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.

[000310] Evaluation of lipid modification using cooking tests:

[000311] Cooking recipe:

[000312] The cooking performance of flour, flour with added solubilized bran was evaluated in small-scale cooking tests (50 g of mix and 10 g slices) using the recipe below (table 4).Table 4. Recipe used for the evaluation of the performance of flour, flour with addition of solubilized bran and reconstituted flour with addition of insoluble bran. Salt/sugar is a 1:1 (weight/weight) mixture of salt and sugar. Water is the absorption water determined by Farinographic analysis.

Pasta making and baking

[000313] The flour (or flour and bran mixture) and dry ingredients are mixed for one minute, after which water is added and mixing is continued for another five minutes.

[000314] After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were molded into a loaf shape using a hand 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. Thereafter, the bread is fermented at 34°C 85% RH for 45 minutes and finally baked at 230°C for five minutes in a Bago oven (Bago-line, Fâborg, Denmark). During dough expansion, viscosity was subjectively rated on a scale of 1 (very viscous) to 5 (dry).

[000315] The bread was cooled for 20 minutes before evaluation (weighing, volume measurement, crumb, crust and sensory evaluation).

Cooking tests

[000316] The cooking tests below were performed (table 5). Table 5. Experimental structure of the cooking test. ID refers to the composition of the flour either with the addition of solubilized bran or reconstituted with insoluble bran, the numbers in parentheses refer to the flour treatment according to table 3. Flour (g) is the amount of flour in the bread. Bran (g) 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 flour "Bran" (%) is the amount of bran, both solubilized and insoluble bran based on the weight of the flour. TIPU refers to the Phospholipase Titration Unit as described above.

[000317] Results:

[000318] Degree of solubilization of the bran:

[000319] Based on dry matter, the amount of bran solubilized in the tests was approximately 54%.

[000320] Cooking results:

[000321] As can be seen in table 6 and figure 1, the addition of soluble fiber has little or no effect on the specific volume of the bread. However, the combination of bran solubilization with phospholipase has a significant effect on bread volume.Table 6. Baking test results. ID refers to the composition of the flour either with the addition of solubilized bran or reconstituted with insoluble bran. Flour (g) is the amount of flour in the bread. Bran (g) is the amount of bran used for reconstitution. Specific Volume (mg/mL) is the absolute specific volume of the breads. Relative volume versus control (%) is the relative volume of bread versus bread 1 (control)

[000322] The resulting loaves can be seen in figure 2.

[000323] Example 2. Laboratory scale modification of commercial wheat bran lipids followed by cooking evaluation:

[000324] To assess 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.

[000325] Bran:

[000326] Wheat bran fractions obtained from a commercial mill were used. The fractions consisted of a fine bran fraction and a coarse bran fraction. 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. Grinding was performed 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, with regard to the degree of solubilization.

[000327] Enzymes: Table 7: Enzymes used for the modification of wheat bran

[000328] Protocol: Table 8. Protocol used for bran modification Wheat bran was suspended in 50 mM NaPi, pH 5 (13% wt/wt) in a closed-lid vessel/reactor

[000329] The bran suspension is heated to 100°C under stirring and boiled for 2 min. The sample is placed under stirring (with the lid closed) at 50°C and allowed to equilibrate to the 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 further enzymatic activity Sample is cooled and stored to avoid contamination Pellet is lyophilized Supernatant is analyzed

[000330] Tests: The following modifications were made to the bran (table 9) Table 9: Amount of bran, g, treated with different enzymes

[000331] Analysis:

[000332] The soluble fraction of the bran (the supernatant) is analyzed with respect to:

[000333] Dry matter content (%):

[000334] 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.

[000335] Evaluation of lipid modification using cooking tests:

[000336] Cooking recipe:

[000337] The baking performance of the flour, flour with added solubilized modified bran was evaluated in small-scale baking tests (50 g of mix and 10 g slices) using the recipe below (table 10). Table 10. Recipe used to evaluate the cooking performance of flour, flour with addition of solubilized bran and reconstituted flour with addition of insoluble bran. Salt/sugar is a 1:1 (weight/weight) mixture of salt and sugar. Water is the absorption water determined by Farinographic analysis.

Pasta making and baking

[000338] The flour (or a mixture of flour and bran) and dry ingredients are mixed for one minute, after which water is added and mixing is continued for another five minutes.

[000339] After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were molded into a loaf shape using a hand 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. Thereafter, the bread is fermented at 34°C 85% RH for 45 minutes and finally baked at 230°C for five minutes in a Bago oven (Bago-line, Fâborg, Denmark). The bread was cooled for 20 minutes before evaluation (weighing, volume measurement, crumb, crust and sensory evaluation).

Cooking tests

[000340] 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, the numbers in parentheses refer to the flour treatment according to table 9. Flour (g) 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.

[000341] Results:

[000342] Degree of solubilization of the bran:

[000343] Based on dry matter, the amount of bran solubilized in the tests was approximately 30%.

[000344] Cooking results:

[000345] As can be seen in table 12 and figure 3, the addition of soluble fiber has little or no effect on the specific volume of the bread. However, the combination of bran solubilization with phospholipase has a significant effect on bread volume.Table 12. Results of the bake test. ID refers to the composition of the flour with the addition of solubilized bran, the numbers in parentheses refer to the bran treatment according to table 9. Flour (g) is the amount of flour in the bread. Specific Volume (mg/mL) is the absolute specific volume of the breads. Relative volume versus control (%) is the relative volume of bread versus bread 1 (control)

[000346] The resulting loaves can be seen in figure 4.

[000347] As can be seen from the above results in experiment 2, no significant effect was obtained on cooking performance in this experiment. Compared to example 1, the structure of example 2 differed in the absence of cell wall and starch modifying enzymes. So, it can be concluded that there must be a synergistic effect between cell wall modifying enzymes, starch and lipids in relation to the generation of functional lipids from wheat bran.

Example 3. Laboratory-Scale Modification of Commercial Wheat Bran and Wheat Bran Lipids by Cooking Evaluation -2:

[000348] 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.

[000349] Bran:

[000350] Wheat bran fractions obtained from a commercial mill were used. The fractions consisted of a fine bran fraction and a coarse bran fraction. 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. Grinding was performed 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, with regard to the degree of solubilization.

[000351] Enzymes: Table13: Enzymes used to modify wheat bran

[000352] Protocol: Table 14. Protocol used for bran modification Wheat bran was suspended in 50 mM NaPi, pH 5 (13% w/w) in a closed-lid vessel/reactor

[000353] The bran suspension is heated to 100°C under stirring and boiled for 2 min. The sample is placed under stirring (with the lid closed) at 50°C and allowed to equilibrate to the 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 further enzymatic activity Sample is cooled and stored to avoid contamination Pellet is lyophilized Supernatant is analyzed

[000354] Tests: The following modifications were made to the bran (table 15) Table 15: Amount of bran, g, treated with different enzymes

[000355] Analysis:

[000356] The soluble fraction of the bran (the supernatant) is analyzed with respect to:

[000357] Dry matter content (%):

[000358] 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.

[000359] Evaluation of lipid modification using cooking tests:

[000360] Cooking recipe:

[000361] The baking performance of the flour, flour with added solubilized modified bran was evaluated in small-scale baking tests (50 g of mix and 10 g slices) using the recipe below (table 16). Table 16. Recipe used to evaluate the performance of flour, flour with addition of solubilized bran and reconstituted flour with addition of insoluble bran. Salt/sugar is a 1:1 (weight/weight) mixture of salt and sugar. Water is the absorption water determined by Farinographic analysis.

Pasta making and baking

[000362] The flour (or a mixture of flour and bran) and dry ingredients are mixed for one minute, after which water is added and mixing is continued for another five minutes.

[000363] After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were molded into a loaf shape using a hand 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. Thereafter, the bread is fermented at 34°C 85% RH for 45 minutes and finally baked at 230°C for five minutes in a Bago oven (Bago-line, Fâborg, Denmark). The bread was cooled for 20 minutes before evaluation (weighing, volume measurement, crumb, crust and sensory evaluation).

Cooking tests

[000364] The cooking tests below were performed (table 17). Table 17. Experimental structure of the cooking test. ID refers to the composition of the flour with the addition of solubilized bran, the numbers in parentheses refer to the flour treatment according to table 15. Flour (g) 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 no. 9 is a repeat of baking 5, however with the addition of 0.2 mg xylanase and 0.5 mg phospholipase/kg flour during dough mixing.

[000365] Results:

[000366] Degree of solubilization of the bran:

[000367] Based on dry matter, the amount of bran solubilized in the tests was, on average, approximately 30 to 54%. Cooking Results:

[000368] As can be seen in table 18 and in figure 5, the addition of soluble fiber has little or no effect on the specific volume of the bread. However, the combination of bran solubilization with phospholipase has a significant effect on bread volume.Table 18. Baking test results. ID refers to the composition of the flour with the addition of solubilized bran, the numbers in parentheses refer to the bran treatment according to table 15. Flour (g) is the amount of flour in the bread. Specific Volume (mg/mL) is the absolute specific volume of the breads. Relative volume versus control (%) is the relative volume of bread versus bread 1 (control)

[000369] The resulting loaves can be seen in figure 6.

[000370] As can be seen from the above results in experiment 3, a significant effect was obtained on cooking performance in this experiment by combining the treatment of the bran with cell wall modifying enzymes, starch and lipids.

[000371] Example 4. Laboratory scale modification of commercial wheat bran and wheat bran lipids followed by evaluation of modified bran in cooking:

[000372] To evaluate the synergistic effect of modified wheat bran with enzymes that modify the cell wall, starch and lipid, the effect of in situ generation of modified lipids that have emulsifying properties on the addition of wheat bran in relation to 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 endosperm.

[000373] Bran:

[000374] Wheat bran fractions obtained from a commercial mill were used. The fractions consisted of a fine bran fraction and a coarse bran fraction. 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. Grinding was performed 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, with regard to the degree of solubilization.

[000375] Enzymes:Table 19: Enzymes used to modify wheat bran

[000376] Protocol: Table 20. Protocol used for bran modificationWheat bran was suspended in 50 mM NaPi, pH 5 (13% wt/wt) in a closed-lid vessel/reactor

[000377] The bran suspension is heated to 100°C under stirring and boiled for 2 min. The sample is placed under stirring (with the lid closed) at 50°C and allowed to equilibrate to the temperature. Enzymes are added and the reaction is continued at 50°C for 24 h (temperature and time can be further optimized) modified bran is boiled to inactivate further enzymatic activity Sample is cooled and stored to avoid contamination Testing: The following modifications were made to the bran (table 21) Table 21: Amount of bran, g, treated with different enzymes

[000378] Evaluation of lipid modification using cooking tests:

[000379] Cooking recipe:

[000380] The baking performance of flour and flour with addition of modified bran was evaluated in small-scale baking tests (50 g of mix and 10 g slices) using the recipe below (table 22). Table 22. Recipe used to evaluate the performance of flour, flour with the addition of modified bran. Salt/sugar is a 1:1 (weight/weight) mixture of salt and sugar. Water is the absorption water determined by Farinographic analysis.

Pasta making and baking

[000381] The flour and dry ingredients are mixed for one minute, after which water (or water and modified bran) is added and mixing is continued for another five minutes.

[000382] After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were molded into a loaf shape using a hand 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. Thereafter, the bread is fermented at 34°C RH 85% for 45 minutes and finally baked at 230°C for five minutes in a Bago oven (Bagoline, Fâborg, Denmark). The bread was cooled for 20 minutes before evaluation (weighing, volume measurement, crumb, crust and sensory evaluation).

Cooking tests

[000383] The cooking tests below were performed (table 23). Table 23. Experimental structure of the cooking test. ID refers to dough composition, numbers in parentheses refer to bran treatment according to table 21. Flour (g) 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.

[000384] Results:

Cooking Results:

[000385] As can be seen in table 24 and figure 7, the addition of modified fibers 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 detrimental effect on bread volume compared to the addition of bran with modified cell wall alone.Table 24. Baking test results. ID refers to flour composition, numbers in parentheses refer to bran treatment according to table 21. Flour (g) is the amount of flour in the bread. Specific Volume (mg/mL) is the absolute specific volume of the breads. Relative volume versus control (%) is the relative volume of bread versus bread 1 (control)

[000386] The resulting loaves can be seen in figure 7.

[000387] As can be seen from the above results, a significant effect was obtained on cooking performance in this experiment by combining the treatment of the bran with cell wall modifying enzymes, starch and lipids prior to addition to the dough.

[000388] Example 5. Laboratory-scale modification of rice bran and rice bran lipids by evaluating the solubilized bran and modified bran lipids in cooking:

[000389] To further evaluate the surprising findings regarding the solubilization of bran combined with the modification of the bran lipids, the effect of solubilized rice bran and modified rice bran lipids in cooking tests was tested.

[000390] Bran:

[000391] A commercial rice bran fraction was used in the experiment.

[000392] Enzymes:Table 25: Enzymes used for modification of rice bran

[000393] Protocol: Table 26. Protocol used for bran modification Rice bran was suspended in 50 mM NaPi, pH 5 (13% wt/wt) in a closed-lid vessel/reactor

[000394] The bran suspension is heated to 100°C under stirring and boiled for 2 min. The sample is placed under stirring (with the lid closed) at 50°C and allowed to equilibrate to the 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 further enzymatic activity Sample is cooled and stored to avoid contamination

[000395] Tests: The following modifications were made to rice bran (table 27) Table 27: Amount of rice bran, g, treated with different enzymes

[000396] Evaluation of lipid modification using cooking tests:

[000397] Cooking recipe:

[000398] The baking performance of flour and flour with addition of modified bran was evaluated in small-scale baking tests (50 g of mix and 10 g slices) using the recipe below (table 28).Table 28. Recipe used to evaluate the performance of different dough compositions (+/- solubilized bran). Salt/sugar is a 1:1 (weight/weight) mixture of salt and sugar. Water is the absorption water determined by Farinographic analysis.

Pasta making and baking

[000399] The flour and dry ingredients are mixed for one minute, after which water (or water and modified bran) is added and mixing is continued for another five minutes.

[000400] After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were molded into a loaf shape using a hand 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. Thereafter, the bread is fermented at 34°C 85% RH for 45 minutes and finally baked at 230°C for five minutes in a Bago oven (Bago-line, Fâborg, Denmark). The bread was cooled for 20 minutes before evaluation (weighing, volume measurement, crumb, crust and sensory evaluation).

Cooking tests

[000401] The cooking tests below were performed (table 29). Table 29. Experimental structure of the cooking test. ID refers to dough composition, numbers in parentheses refer to bran treatment according to table 27. Flour (g) 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.

[000402] Results:

[000403] Cooking results:

[000404] As can be seen in table 30 and figure 8, the addition of modified soluble fiber did not have a negative effect on the specific bread volume, in fact it had a positive effect on the bread volume. However, the addition of soluble fibers also modified with phospholipase, generating functional lipids, has a much better effect on bread volume compared to adding only soluble fibers modified.Table 30. Baking test results. ID refers to flour composition, numbers in parentheses refer to bran treatment according to table 27. Flour (g) is the amount of flour in the bread. Specific Volume (mg/mL) is the absolute specific volume of the breads. Relative volume versus control (%) is the relative volume of bread versus bread 1 (control)

[000405] The resulting breads can be seen in figure 8.

[000406] As can be seen from the above results, a significant effect was obtained on cooking performance in this experiment by adding soluble bran and modified lipids obtained by combining pretreatment of rice bran with cell wall modifying enzymes, starch and lipids.

Example 6. Laboratory scale modification of commercial wheat bran and wheat bran lipids using different lipases followed by cooking evaluation.

[000407] To further evaluate the surprising findings regarding the solubilization of bran combined with the modification of the bran lipids, other lipases other than Danisco's Phospho-galacto lipase were tested, followed by the evaluation of the modified lipids generated in cooking tests.

[000408] Bran:

[000409] Wheat bran fractions obtained from a commercial mill were used. The fractions consisted of a fine bran fraction and a coarse bran fraction. 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. Grinding was performed 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, with regard to the degree of solubilization.

[000410] Enzymes:Table 31: Enzymes used to modify wheat bran

[000411] Protocol: Table 32. Protocol used for bran modification Wheat bran was suspended in 50 mM NaPi, pH 5 (13% wt/wt) in a closed-lid vessel/reactor

[000412] The bran suspension is heated to 100°C under stirring and boiled for 2 min. The sample is placed under stirring (with the lid closed) at 50°C and allowed to equilibrate to the 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 further enzymatic activity Sample is cooled and stored to avoid contamination

[000413] Tests: The following modifications were made to the bran (table 33) Table 33: Amount of bran, g, treated with different enzymes

[000414] Evaluation of lipid modification using cooking tests:

[000415] Cooking recipe:

[000416] The cooking performance of flour and flour with addition of solubilized modified bran (+/- modified lipids) was evaluated in small-scale cooking tests (50 g of mix and 10 g slices) using the recipe below ( table 34). Table 34. Recipe used to evaluate performance in cooking flour, flour with added solubilized bran and flour with added 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.

Pasta making and baking

[000417] The flour and dry ingredients are mixed for one minute, after which water is added and mixing is continued for another five minutes.

[000418] After mixing, four blocks of dough were weighed, each containing 10 grams of flour. These were molded into a loaf shape using a hand 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. Thereafter, the bread is fermented at 34°C 85% RH for 45 minutes and finally baked at 230°C for five minutes in a Bago oven (Bago-line, Fâborg, Denmark). The bread was cooled for 20 minutes before evaluation (weighing, volume measurement, crumb, crust and sensory evaluation).

Cooking tests

[000419] The cooking tests below were performed (table 35). Table 35. Experimental structure of the cooking test. ID refers to dough composition, numbers in parentheses refer to bran treatment according to table 33. Flour (g) is the amount of flour in the bread. Bran Extract (mL) is the amount of solubilized bran added to flour instead of water. Water (mL) is the amount of water added to the flour.

[000420] Results:

[000421] Cooking results:

[000422] As can be seen in table 36 and figure 9, the addition of soluble fiber had little or no effect on the specific volume of the bread. However, the combination of bran solubilization with phospholipase had a significant effect on bread volume; all bread baked with bran treated with lipases in combination with cell wall modifying enzymes and starch had an increase in volume compared to the control flour. Table 36. Cooking test results. ID refers to dough composition, numbers in parentheses refer to bran treatment according to table 33. Flour (g) is the amount of flour in the bread. Specific Volume (mg/mL) is the absolute specific volume of the breads. Rel rel versus control (%) is the relative volume of bread versus bread 1 (control)

[000423] The resulting loaves can be seen in figure 9.

[000424] As can be seen from the above results in experiment 6, a significant effect was obtained on cooking performance in this experiment by combining the treatment of the bran with cell wall modifying enzymes, starch and lipids.

Claims (25)

1. Method for the treatment of a lipid-containing cereal bran, characterized in that it comprises: (a) treating a liquid suspension of a lipid-containing cereal bran with one or more enzymes to obtain a solubilized cereal bran having a grade of solubilization greater than 1% in terms of dry matter basis; wherein said lipid-containing cereal bran comprises from 1% to 20% (weight/weight) of starch, and (b) treating the liquid solubilized cereal bran suspension containing lipids with one or more exogenous phospholipase, wherein said treatments yield a treated composition comprising 0.05%, in terms of dry matter basis, of a solubilized fraction of cereal bran functional lipids, such as lyso-phospholipid, lyso-glycolipid, monoglyceride or DATEM emulsifying molecules. 2. Method according to claim 1, characterized in that 1% to 10% (weight/weight) of the liquid suspension of a cereal bran containing lipids is starch. 3. Method according to claim 1, characterized in that steps (a) and (b) are performed simultaneously. 4. Method according to claim 1 or 3, characterized in that said one or more enzymes used to obtain the solubilized cereal bran comprises one or more cell wall modifying enzymes. 5. Method according to claim 3, characterized in that said liquid cereal bran suspension containing lipids also contains insoluble cereal bran. 6. Method according to claim 1 or 3, characterized in that said one or more enzymes used to obtain the solubilized cereal bran comprises one or more protease enzymes. 7. Method according to claim 1, characterized in that said cereal bran is further treated with one or more lipolytic enzymes, in addition to treatment with phospholipase. 8. Method according to claim 1, characterized in that it further comprises a step of isolating the soluble fraction. 9. Method according to claim 4, characterized in that said one or more cell wall modifying enzymes are selected from the group consisting of a xylanase and a cellulase. 10. Method according to claim 9, characterized in that said cellulase is selected from an endo-cellulase, an exo-cellulase, a cellobiase, an oxidative cellulase and a cellulose phosphorylase. 11. Method according to claim 1 or 2, characterized in that said one or more enzymes is a starch modifying enzyme selected from the group consisting of an alpha-amylase, a pullulanase, an isoamylase and a beta- amylase. 12. Method according to any one of claims 1 to 11, characterized in that the lipid-containing cereal bran is supplied in particles, in which the average particle size of said particulate cereal bran is less than 3000 μm. 13. Method according to claim 1, characterized in that the cereal bran is selected from wheat, barley, oats, rye, triticale, rice and corn. 14. Method according to claim 1, characterized in that it further comprises the previous steps of (i) fractionating a cereal grain to obtain endosperm, bran and germ; (ii) separate and distribute the endosperm, bran and germ to allow them to be treated; and (iii) grinding the bran. 15. Method according to claim 1, characterized in that said cereal bran is obtained from an industrial milling process and subsequently ground to obtain an average particle size below 500 µm. 16. Method according to claim 1, characterized in that said cereal bran containing lipids is a side stream of cereal bran processing. 17. Method according to claim 1, characterized in that said composition comprising functional lipids, such as emulsifying molecules of lyso-phospholipids, lyso-glycolipids, monoglyceride or DATEM, is further treated to inactivate additional enzymatic activity . 18. Method according to claim 1, characterized in that the degree of solubilization of said cereal bran containing lipids obtained before said treatment with phospholipase is greater than 15% in terms of dry matter basis. 19. Method according to claim 1, characterized in that the total content of solubilized functional lipids, such as emulsifying molecules of lyso-phospholipids, lyso-glycolipids, monoglyceride or DATEM, obtained is in the range of 0.055% in terms of dry matter basis. 20. Method according to claim 1, characterized in that said method further comprises a step of drying the composition comprising the solubilized fraction of functional lipids, such as emulsifying molecules of lyso-phospholipids, lyso-glycolipids, monoglyceride or DATEM. 21. Method according to claim 1, characterized in that said method further comprises a step of spray drying the composition comprising the solubilized fraction of functional lipids, such as lyso-phospholipid emulsifying molecules, lyso- glycolipids, monoglyceride or DATEM. 22. Method according to claim 1, characterized in that said method further comprises a lyophilization step of the composition comprising the solubilized fraction of functional lipids, such as emulsifying molecules of lyso-phospholipids, lyso-glycolipids, monoglyceride or DATEM. 23. Method according to claim 1, characterized in that said treatment with phospholipase generates functional lipids comprising emulsifiers, such as lyso-phospholipid, lyso-glycolipid, monoglyceride or DATEM molecules. 24. Method according to claim 1, characterized in that said treatment with phospholipase also generates functional sterol esters. 25. Method according to claim 1, characterized in that said treatment with phospholipase converts more than 5% of phosphotidylinositol present in cereal bran into lysophosphatidylinositol.

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