IMPROVED FIBER-WASHING IN CORN WET-MILLING

Abstract

A method for increasing starch yield and/or gluten yield from corn kernels in a wet milling process, comprising contacting a fiber rich fraction of ground kernels, with an effective amount of SO.sub.2, and an effective amount of one or more hydrolytic enzymes, wherein at least one of said hydrolytic enzymes is selected from xylanase and/or cellulase enzymes, during a fiber-washing step.

Claims

1. A method for increasing starch yield and/or gluten yield from corn kernels in a wet milling process, comprising contacting a fiber rich fraction of ground kernels, with an effective amount of SO.sub.2, and an effective amount of one or more hydrolytic enzymes, wherein at least one of said hydrolytic enzymes is selected from xylanase and/or cellulase enzymes, during a fiber-washing step.

2. The method according to claim 1, wherein the amount of starch and/or gluten released from fiber during the wet milling process is increased compared to a process where no SO.sub.2 is present/added.

3. The method according to claim 2, comprising the steps of: a) soaking the corn kernels in water to produce soaked kernels; b) grinding the soaked kernels to produce ground kernels; c) separating germs from the ground kernels to produce a corn kernel mass comprising fiber, starch and gluten; d) subjecting the resultant corn kernel mass comprising fiber to a fiber washing procedure, thereby separating starch, gluten, and fiber; wherein at least a xylanase and/or cellulase enzyme(s) and an effective amount of SO.sub.2 is present/added before or during step d).

4. The method according to claim 3, wherein SO.sub.2 is present/added during fiber wash step d) in amounts of at least 400 ppm, at least 450 ppm, at least 500 ppm, at least 600 ppm, at least 700 ppm, or at least 800 ppm.

5. The method according to claim 3, wherein SO.sub.2 is present/added during fiber wash in amounts in a range from 400-3000 ppm, 500-2000 ppm, 600-1500 ppm, or 600-1200 ppm.

6. The method according to claim 3, wherein the xylanase is selected from the group consisting of a GH5 xylanase, GH30 xylanase, a GH10 xylanase, a GH11 xylanase, a GH8 xylanase, and a combination thereof.

7. The method according to claim 3, wherein the hydrolytic enzymes comprise one or more cellulases.

8. The method according to claim 7, wherein the cellulase(s) comprise(s) one or more enzymes selected from the group consisting of an endoglucanase (EG), and a cellobiohydrolase (CBH).

9. The method according to claim 8, wherein the cellulase(s) comprise(s) one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase I, a cellobiohydrolase II, and a combination thereof.

10. The method according to claim 3, wherein the hydrolytic enzymes further comprise an arabinofuranosidase.

11. The method according to claim 10, wherein the arabinofuranosidase is selected from the group consisting of a GH43 polypeptide, a GH62 polypeptide, and a GH51 polypeptide.

12. The method according to claim 3, wherein said fiber washing procedure comprises a fiber washing system comprising a space (V)/tank configured to provide a total retention time in the fiber washing system of at least 35 minutes and less than 48 hours.

13. The method according to claim 12, wherein the incubation time in said space (V)/tank configured into the fiber washing system is between 5 minutes and 48 hours, between 35 minutes and 24 hours, between 35 minutes and hours, between 35 minutes and 6 hours, between 35 minutes and 5 hours, between 35 minutes and 4 hours, between 35 minutes and 3 hours, between 35 minutes and 2 hours, between 45 minutes and 48 hours, between 45 minutes and 24 hours, between 45 minutes and 12 hours, between 45 minutes and 6 hours, between 45 minutes and 5 hours, between 45 minutes and 4 hours, between 45 minutes and 3 hours, between 45 minutes and 2 hours.

14. The method according to claim 13, wherein the incubation temperature in said space (V)/tank configured into the fiber washing system is between 25° C. and 95° C., between 25 and 90° C., between 25 and 85° C., between 25 and 80° C., between 25 and 75° C., between 25 and 70° C., between 25 and 65° C., between 25 and 60° C., between 25 and 55° C., between 25 and 53° C., between 25 and 52° C., between 30 and 90° C., between 30 and 85° C., between 30 and 80° C., between 30 and 75° C., between 30 and 70° C., between 30 and 65° C., between 30 and 60° C., between 30 and 55° C., between 30 and 53° C., between 30 and 52° C., between 35 and 90° C., between 35 and 85° C., between 35 and 80° C., between 35 and 75° C., between 35 and 70° C., between 35 and 65° C., between 35 and 60° C., between 35 and 55° C., between 35 and 53° C., between 35 and 52° C., between 39 and 90° C., between 39 and 85° C., between 39 and 80° C., between 39 and 75° C., between 39 and 70° C., between 39 and 65° C., between 39 and 60° C., between 39 and 55° C., between 39 and 53° C., between 39 and 52° C., or between 46 and 52° C.

15. The method according to claim 3, wherein the level of SO.sub.2 present/added during fiber wash results in the same extraction yield of starch and gluten while reducing the required contact time between hydrolytic enzyme and ground corn kernel mass compared to a method where SO.sub.2 levels are below 400 ppm.

16. The method according to claim 3, wherein the one or more hydrolytic enzymes is expressed in a organism with a Trichoderma reesei cellulase background.

17. The method according to any of the preceding claims, wherein the one or more hydrolytic enzymes are purified.

18. The method according to any of the preceding claims, wherein the one or more hydrolytic enzymes is/are in a liquid composition.

19. The method according to any of the preceding claims, wherein the one or more hydrolytic enzymes is/are in a solid composition.

20. The method according to any of the preceding claims, wherein the effective amount of one or more hydrolytic enzymes admixed/contacted with one or more fractions of said ground corn kernel mass, is between 0.005-0.5 kg enzyme protein/metric tonne corn kernels entering the wet milling process.

21. The method according to any of the preceding claims, wherein the source of SO.sub.2 is sodium metabisulfite (Na.sub.2S.sub.2O.sub.5), and/or addition of SO.sub.2 gas.

22-30. (canceled)

Description

DETAILED DESCRIPTION

[0072] It is an object of the present invention to provide a method that improves starch and gluten yield from a corn wet milling process.

[0073] Particularly, it is an object of the present invention to provide a method for improving the starch and/or gluten yields that can be obtained from corn kernels in a wet milling process, by treating the fiber fraction with a hydrolytic enzyme composition, preferably during a fiber washing procedure. The inventors of the present invention has surprisingly found that the enzymatic treatment of corn fiber in the presence of at least a xylanase and/or cellulase and an effective amount of SO.sub.2, increases the release of bound starch and gluten from fiber and thus improve the starch and/or gluten yields that can be obtained.

[0074] The Wet Milling Process:

[0075] Corn kernels are wet milled in order to open up the kernels and separate the kernels into its four main constituents: starch, germ, fiber and gluten.

[0076] The wet milling process can vary significantly from mill to mill, however conventional wet milling usually comprises the following steps:

[0077] 1. Steeping

[0078] 2. Grinding

[0079] 3. Separation into streams comprising: [0080] i) germ; ii) fiber, iii) starch and gluten

[0081] 4. Fiber washing, pressing and drying

[0082] 5. Starch/gluten separation, and

[0083] 6. Starch washing.

[0084] Steeping, Grinding and Germ Separation

[0085] Corn kernels are softened by soaking in water for between about 30 minutes to about 48 hours, preferably 30 minutes to about 15 hours, such as about 1 hour to about 6 hours at a temperature of about 50° C., such as between about 45° C. to 60° C. During steeping, the kernels absorb water, increasing their moisture levels from 15 percent to 45 percent and more than doubling in size. The optional addition of e.g. 0.1 percent sulphur dioxide (SO.sub.2) and/or NaHSO.sub.3 to the water prevents excessive bacteria growth in the warm environment. As the corn swells and softens, the mild acidity of the steep water begins to loosen the gluten bonds within the corn and release the starch. After the corn kernels are steeped they are cracked open by grinding to release the germ. The germ contains corn oil. The germ is separated from the heavier density mixture of starch, gluten and fiber (corn kernel mass comprising fiber, starch and gluten) essentially by “floating” the germ segment free of the other substances under closely controlled conditions. This method serves to eliminate any adverse effect of traces of corn oil in later processing steps. Subsequently the germ may be dried and oil extracted.

[0086] The corn kernel mass comprising fiber, starch and gluten are subsequently separated into fiber, starch, and gluten fractions, e.g., in a fiber-washing step.

[0087] Fiber Washing, Pressing and Drying

[0088] To get maximum starch and gluten recovery, while keeping any fiber in the final product to an absolute minimum, it is necessary to wash the free starch and gluten from the fiber during processing. The free starch and gluten is separated from fiber during screening (washing) and collected as mill starch. The remaining fiber is then pressed to decrease the water content.

[0089] Starch Gluten Separation

[0090] The starch-gluten suspension as well as additional starch gluten released from the fiber-washing step, called mill starch, is separated into starch and gluten. Gluten has a low density compared to starch. By passing mill starch through a centrifuge, the gluten is readily spun out.

[0091] Starch Washing

[0092] The starch slurry from the starch separation step contains some insoluble protein and much of solubles. They have to be removed before a top quality starch (high purity starch) can be made. The starch, with just one or two percent protein remaining, is diluted, washed 8 to 14 times, re-diluted and washed again in hydro-clones to remove the last trace of protein and produce high quality starch, typically more than 99.5% pure.

[0093] Products of wet milling: Wet milling can be used to produce, without limitation, corn steep liquor, corn gluten feed, germ, corn oil, corn gluten meal, corn starch, modified corn starch, syrups such as corn syrup, and corn ethanol.

[0094] An aspect of the present disclosure is to provide a method to increase the total starch yield and/or gluten yield that can be obtained from corn kernels in a wet milling process, the method comprising: Admixing corn kernels or a fraction of the corn kernels with an enzyme composition comprising an effective amount of one or more hydrolytic enzymes, wherein at least one of said hydrolytic enzymes is selected from the group consisting of a xylanase polypeptide, and/or cellulase polypeptide or a combination thereof.

[0095] Some of the starch and/or gluten in corn kernels or fractions of corn kernels, may be bound to the fiber fraction and never released during the wet milling process. However, addition of hydrolytic enzymes, which may include any catalytic protein that can use water to break down substrates present in corn kernels, may release some of the bound starch and/or gluten and thus increase the total yield of starch and/or gluten in the wet milling process.

[0096] The present inventors have surprisingly found that the effect of adding hydrolytic enzymes, such as cellulases and xylanases, to the fiber rich fraction of the ground kernel mass, particularly in a fiber-washing step can be boosted at elevated levels of SO.sub.2.

[0097] In a first aspect the present invention therefore relates to a method for increasing starch yield and/or gluten yield from corn kernels in a wet milling process, the method comprising contacting ground corn kernels or a fraction of the ground kernels, particularly a fiber rich fraction, with an effective amount of SO.sub.2, and an effective amount of one or more hydrolytic enzymes, wherein at least one of said hydrolytic enzymes is selected from xylanase and/or cellulase enzymes. Preferably, the contact is performed during a fiber-washing step.

[0098] In one embodiment, the method of the present invention leads to an increase in the amount of starch and/or gluten released from fiber during the wet milling process compared to a process where no SO.sub.2 is present/added.

[0099] The specific procedure and the equipment used in the wet milling process can vary, but the main principles of the process remains the same (see description on wet milling process).

[0100] In one particular embodiment, the method of the invention comprise the steps of: [0101] a) soaking the corn kernels in water to produce soaked kernels; [0102] b) grinding the soaked kernels to produce ground kernels; [0103] c) separating germs from the ground kernels to produce a corn kernel mass comprising fiber, starch and gluten; and [0104] d) subjecting the resultant corn kernel mass comprising fiber to a fiber washing procedure, thereby separating starch, gluten, and fiber;

[0105] wherein at least a xylanase and/or cellulase and an effective amount of SO.sub.2 is present/added before or during step d).

[0106] To get maximum starch and gluten recovery, while keeping any fiber in the final product to an absolute minimum, it is necessary to wash the free starch and gluten from the fiber fraction during processing. The fiber is collected, slurried and screened, typically after soaking, grinding and separation of germs from the corn kernels, to reclaim any residual starch or gluten in the corn kernel mass. This process is herein referred to as the fiber washing procedure.

[0107] In a preferred embodiment, said corn kernels or a fiber rich fraction of said corn kernels is admixed with said one or more hydrolytic enzymes during the step of subjecting the corn kernel mass to a fiber washing procedure.

[0108] According to the invention, in order to maximize the effect of the hydrolytic enzymes during the fiber washing step, a boosting effect is observed when an effective amount of SO.sub.2 is present during the fiber wash. SO.sub.2 is often added in the step of soaking the kernels, however, in the downstream steps, such as in the fiber-washing step, SO.sub.2 levels will have dropped below the levels claimed according to the present method.

[0109] In one embodiment SO.sub.2 is present/added during fiber wash in amounts of at least 400 ppm, at least 450 ppm, at least 500 ppm, at least 600 ppm, at least 700 ppm, at least 800 ppm.

[0110] In another embodiment SO2 is present/added during fiber wash in amounts in a range from 400-3000 ppm, 500-2000 ppm, 600-1500 ppm, such as 600-1200 ppm.

[0111] The specific equipment used in the fiber washing procedure may vary, but the main principle of the process remains the same. WO2018/053220 describes a fiber-washing system including a dedicated enzyme incubation space/tank. Based on this disclosure and the general knowledge of the skilled person it will be possible to design a fiber-washing system resulting in sufficient incubation time for the hydrolytic enzymes to work. In one embodiment, said corn kernels or a fraction of said corn kernels, e.g., a fiber rich fraction, is allowed to react with said one or more hydrolytic enzymes for at least 15 minutes, such as at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes or at least 120 minutes.

[0112] In one embodiment, said fiber washing procedure comprise the use of a fiber washing system optimized for introduction of one or more hydrolytic enzymes, wherein the fiber washing system comprise a space (V) configured to provide a total reaction time in the fiber washing system (retention time) of at least 35 minutes, such as at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes or at least 120 minutes and less than 48 hours, such as less than 40 hours, less than 36 hours, less than 30 hours, less than 24 hours, less than 20 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours. In one embodiment the total retention time in the fiber washing system is between 35 minutes and 48 hours such as between 35 minutes and 24 hours, 35 minutes and 12 hours, 35 minutes and 6 hours, 35 minutes and 5 hours, 35 minutes and 4 hours, 35 minutes and 3 hours, 35 minutes and 2 hours, 45 minutes and 48 hours, 45 minutes and 24 hours, 45 minutes and hours, 45 minutes and 6 hours, 45 minutes and 5 hours, 45 minutes and 4 hours, 45 minutes and 3 hours, 45 minutes and 2 hours 1-48 hours, 1-24 hours, 1-12 hours, 1-6 hours, 1-5 hours, 1-4 hours, 1-3 hours, 1-2 hours.

[0113] In one embodiment, the fiber washing system comprises: [0114] a plurality of screen units (S1 . . . S4) being fluidly connected in a counter current washing configuration; each screen unit being configured for separating a stream of corn kernel mass and liquid into two fractions: a first fraction (s) and a second fraction (f), said second fraction (f) containing a higher amount measured in wt % fiber than the first fraction (s); [0115] a space (V) arranged in the system and being fluidly connected to receive said first fraction (s), said second fraction (f), or a mixed first and second fraction (s,f), preferably only a second fraction (f), and configured to provide an incubation time for one or both fractions received in the space; and outletting the thereby incubated one or both fractions to a downstream screen unit (S4),

[0116] wherein the system is configured for [0117] inletting corn kernel mass and liquid to the most upstream screen unit (S1) [0118] outletting the first fraction (s1) from the most upstream screen unit (S1) as a product stream containing starch, [0119] inletting process water, preferably arranged for inletting process water to a most downstream screen unit (S4), [0120] outletting the second fraction (f4) from most downstream screen unit (S4) as a washed corn kernel mass containing a lower amount of starch and gluten than the original corn kernel mass. [0121] introducing hydrolytic enzymes into the system.

[0122] In one embodiment, the incubation time in said space (V) configured into the fiber washing system is at least 5 minutes such as at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes or at least 120 minutes and less than 48 hours, such as less than 40 hours, less than 36 hours, less than 30 hours, less than 24 hours, less than 20 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours.

[0123] In one embodiment the incubation time in said space (V) is between 35 minutes and 48 hours such as between 35 minutes and 24 hours, 35 minutes and hours, 35 minutes and 6 hours, 35 minutes and 5 hours, 35 minutes and 4 hours, 35 minutes and 3 hours, 35 minutes and 2 hours, 45 minutes and 48 hours, 45 minutes and 24 hours, 45 minutes and 12 hours, 45 minutes and 6 hours, 45 minutes and 5 hours, 45 minutes and 4 hours, 45 minutes and 3 hours, 45 minutes and 2 hours 1-48 hours, 1-24 hours, 1-12 hours, 1-6 hours, 1-5 hours, 1-4 hours, 1-3 hours, 1-2 hours.

[0124] In one embodiment, the incubation temperature in said space (V) is between 25 and 95° C., such as between 25 and 90° C., 25 and 85° C., 25 and 80° C., 25 and 75° C., 25 and 70° C., 25 and 65° C., 25 and 60° C., 25 and 55° C., 25 and 53° C., 25 and 52° C., 30 and 90° C., 30 and 85° C., 30 and 80° C., 30 and 75° C., 30 and 70° C., 30 and 65° C., 30 and 60° C., 30 and 55° C., 30 and 53° C., 30 and 52° C., 35 and 90° C., 35 and 85° C., 35 and 80° C., 35 and 75° C., 35 and 70° C., 35 and 65° C., 35 and 60° C., 35 and 55° C., 35 and 53° C., 35 and 52° C., 39 and 90° C., 39 and 85° C., 39 and 80° C., 39 and 75° C., 39 and 70° C., 39 and 65° C., 39 and 60° C., 39 and 55° C., 39 and 53° C., 39 and 52° C., such as 46 and 52° C.

[0125] Further, the dimension of the space (in m.sup.3) is preferably configured to provide an incubation time of at least at least 5 minutes, such as at least 10 minutes, at least 15 minutes, at least 20 minutes at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes.

[0126] The space (V) designated for incubation preferably has a volume of at least 30 m.sup.3, at least 40 m.sup.3, at least 50 m.sup.3, at least 60 m.sup.3, at least 70, m.sup.3, at least 80, m.sup.3, at least 90, m.sup.3, at least 100 m.sup.3, at least 110 m.sup.3, at least 120 m.sup.3, at least 130 m.sup.3, at least 140 m.sup.3, at least 150 m.sup.3, at least 160 m.sup.3, at least 170 m.sup.3, at least 180 m.sup.3, at least 190 m.sup.3, at least 200 m.sup.3, at least 210 m.sup.3, at least 220 m.sup.3, at least 230 m.sup.3, at least 240 m.sup.3, at least 250 m.sup.3, at least 260 m.sup.3, at least 270 m.sup.3, at least 280 m.sup.3, at least 290 m.sup.3, at least 300 m.sup.3, at least 400 m.sup.3, or at least 500 m.sup.3. The incubation time may also be in more than one space V with a total or combined volume of at least 100 m.sup.3, at least 110 m.sup.3, at least 120 m.sup.3, at least 130 m.sup.3, at least 140 m.sup.3, at least 150 m.sup.3, at least 160 m.sup.3, at least 170 m.sup.3, at least 180 m.sup.3, at least 190 m.sup.3, at least 200 m.sup.3, at least 210 m.sup.3, at least 220 m.sup.3, at least 230 m.sup.3, at least 240 m.sup.3, at least 250 m.sup.3, at least 260 m.sup.3, at least 270 m.sup.3, at least 280 m.sup.3, at least 290 m.sup.3, at least 300 m.sup.3, at least 400 m.sup.3, at least 500 m.sup.3.

[0127] During the incubation time, it is preferred that the fluid received in the space V is not screened. Thus, the fluid leaving the space V has the same composition, e.g. of starch and fiber, as the fluid received in the space V, although it preferably contains a higher proportion of starch that has been released from the fibers.

[0128] To assure intimate contact between the enzymes and the fiber, it may be preferred to configure the space V for agitation of matter contained in said space V, such as by comprising a rotor or impeller.

[0129] It is preferred to arrange the space V downstream of the most upstream screen unit S1 and upstream of said most downstream screen unit S4; in particular, the space V is arranged to feed fluid into the second most downstream screen unit S3.

[0130] Hydrolytic Enzymes Suitable for the Method of the Invention

[0131] In one embodiment, hydrolytic enzymes suitable for use in the method of the invention comprise one or more enzymes selected form the group consisting of: cellulases (EC 3.2.1.4), xylanases (EC 3.2.1.8) arabinofuranosidases (EC 3.2.1.55 (Non-reducing end alpha-L-arabinofuranosidases); EC 3.2.1.185 (Non-reducing end beta-L-arabinofuranosidases) cellobiohydrolase I (EC 3.2.1.150), cellobiohydrolase II (E.C. 3.2.1.91), cellobiosidase (E.C. 3.2.1.176), beta-glucosidase (E.C. 3.2.1.21), beta-xylosidases (EC 3.2.1.37) and proteases (E.C 3.4).

[0132] In one embodiment the xylanase is selected from the group consisting of a GH5 polypeptide, GH30 polypeptide, a GH10 polypeptide, a GH11 polypeptide, a GH8 polypeptide or a combination thereof.

[0133] In another embodiment the hydrolytic enzymes comprise one or more cellulases. The cellulases may be selected from at least the group consisting of an endoglucanase (EG), and a cellobiohydrolase (CBH). More particularly, the cellulase(s) comprise(s) one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase I, a cellobiohydrolase II, or a combination thereof.

[0134] In one embodiment the hydrolytic enzymes further comprise an arabinofuranosidase. The arabinofuranosidase may be selected from the group consisting of a GH43 polypeptide, a GH62 polypeptide, GH51 polypeptide. Particularly a GH62 polypeptide.

[0135] In one embodiment, the one or more hydrolytic enzymes is expressed in an organism with a cellulase background, such as Trichoderma reesei. According to these embodiments the xylanase and or arabinofuranosidase polypeptides defined according to the invention is/are expressed together with endogenous cellulases from Trichoderma.

[0136] In one embodiment, the enzyme composition comprising one or more hydrolytic enzymes may comprise cellulases expressed in Trichoderma reesei and other hydrolotic enzymes which are added to the enzyme composition in a purified or semi-purified form.

[0137] In one embodiment, the one or more hydrolytic enzymes are purified. The purified enzymes may be used in an enzyme composition as described in other embodiments of the present invention.

[0138] In one embodiment, the one or more hydrolytic enzymes is/are in a liquid composition. The composition may be homogenous or heterogeneous.

[0139] In one embodiment, the one or more hydrolytic enzymes is/are in a solid composition.

[0140] In one embodiment, the effective amount of one or more hydrolytic enzymes admixed with one or more fractions of said corn kernel mass, is between 0.005-0.5 kg enzyme protein (EP)/metric tonne (MT) corn kernels entering the wet milling process, such as between 0.010-0.5 kg EP/MT corn kernel, such as between 0.05-0.5 kg/MT corn kernel or 0.075-0.5 kg/MT or 0.1-0.5 kg/MT corn kernel or 0.005-0.4 kg/MT corn kernel or 0.01-0.4 kg/MT corn kernel or 0.05-0.4 kg/MT corn kernel or 0.075-0.4 kg/MT corn kernel or 0.1-0.4 kg/MT corn kernel or 0.005-0.3 kg/MT corn kernel or 0.01-0.3 kg/MT corn kernel or 0.05-0.3 kg/MT corn kernel or 0.075-0.3 kg/MT or 0.1-0.3 kg/MT corn kernel or 0.005-0.2 kg/MT corn kernel or 0.010-0.2 kg/MT corn kernel or 0.05-0.2 kg/MT corn kernel or 0.075-0.2 kg/MT or 0.1-0.2 kg/MT corn kernel or such as 0.075-0.10 kg/MT corn kernel or 0.075-0.11 kg/MT corn kernel.

[0141] In preferred embodiments the enzyme composition comprises cellulase obtained from a culture of Trichoderma reesei, such as a culture of Trichoderma reesei ATCC 26921. Suitable cellulases are available; e.g. from Novozymes A/S under the commercial name Celluclast®.

[0142] Polypeptides Having Xylanase Activity

[0143] Xylanases are suitable to be applied in the method according to the invention. The xylanase polypeptide may be selected from family GH5, GH10, GH30, GH11, and GH8.

[0144] More specific embodiments relates to the method according to the invention, wherein the GH5 xylanase enzyme is selected from the group consisting of: [0145] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1; [0146] (b) a variant of the mature polypeptide of SEQ ID NO: 1 comprising a substitution, deletion, and/or insertion at one or more positions; and [0147] (c) a fragment of the polypeptide of (a), or (b) that has xylanase activity.

[0148] The mature polypeptide is in one embodiment amino acids 25 to 551 of SEQ ID NO: 1.

[0149] Another specific embodiment relates to the method according to the invention, wherein the GH10 xylanase is selected from the group consisting of: [0150] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2; [0151] (b) a variant of the mature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion, and/or insertion at one or more positions; and [0152] (c) a fragment of the polypeptide of (a), or (b) that has xylanase activity.

[0153] The mature polypeptide is in one embodiment amino acids 21 to 405 of SEQ ID NO: 2.

[0154] Another specific embodiment relates to the method according to the invention, wherein the GH10 xylanase is selected from the group consisting of: [0155] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 4; [0156] (b) a variant of the mature polypeptide of SEQ ID NO: 4 comprising a substitution, deletion, and/or insertion at one or more positions; and [0157] (c) a fragment of the polypeptide of (a), or (b) that has xylanase activity.

[0158] The mature polypeptide is in one embodiment amino acids 20 to 319 of SEQ ID NO: 4.

[0159] Polypeptides Having Arabinofuranosidase Activity

[0160] Another specific embodiment relates to the method according to the invention, wherein the GH62 arabinofuranosidase is selected from the group consisting of: [0161] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 3; [0162] (b) a variant of the mature polypeptide of SEQ ID NO: 3 comprising a substitution, deletion, and/or insertion at one or more positions; and [0163] (c) a fragment of the polypeptide of (a), or (b) that has xylanase activity.

[0164] The mature polypeptide is in one embodiment amino acids 17 to 325 of SEQ ID NO: 3.

[0165] Sources of Polypeptides Having Xylanase Activity

[0166] A polypeptide having xylanase activity may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0167] The polypeptide may be a bacterial polypeptide. For example, the polypeptide may be a Gram-positive bacterial polypeptide such as a Bacillus, Chryseobacterium, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having pectin lyase activity, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.

[0168] In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide.

[0169] In another aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide.

[0170] In another aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide.

[0171] The polypeptide may be a fungal polypeptide. For example, the polypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, e.g., Aspergillus niger, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, e.g., Talaromyces leycettanus, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.

[0172] Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0173] The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

[0174] Enzyme Compositions

[0175] An enzyme compositions for use in the method according to the invention may comprise a xylanase polypeptide as the major enzymatic component, e.g., a mono-component composition. Alternatively, the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of cellobiohydrolase, cellulase, endoglucanase, and/or arabinofuranosidase.

[0176] The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art.

[0177] The invention is further disclosed in the following numbered embodiments.

[0178] Embodiment 1. A method for increasing starch yield and/or gluten yield from corn kernels in a wet milling process, comprising contacting a fiber rich fraction of ground kernels, with an effective amount of SO.sub.2, and an effective amount of one or more hydrolytic enzymes, wherein at least one of said hydrolytic enzymes is selected from xylanase and/or cellulase enzymes, during a fiber-washing step.

[0179] Embodiment 2. The method according to embodiment 1, wherein the amount of starch and/or gluten released from fiber during the wet milling process is increased compared to a process where no SO.sub.2 is present/added.

[0180] Embodiment 3. The method according to any of the preceding embodiments, comprising the steps of: [0181] a) soaking the corn kernels in water to produce soaked kernels; [0182] b) grinding the soaked kernels to produce ground kernels; [0183] c) separating germs from the ground kernels to produce a corn kernel mass comprising fiber, starch and gluten; [0184] d) subjecting the resultant corn kernel mass comprising fiber to a fiber washing procedure, thereby separating starch, gluten, and fiber;

[0185] wherein at least a xylanase and/or cellulase enzyme(s) and an effective amount of SO.sub.2 is present/added before or during step d).

[0186] Embodiment 4. The method of any of embodiments 1-3, wherein SO.sub.2 is present/added during fiber wash step (d) in amounts of at least 400 ppm, at least 450 ppm, at least 500 ppm, at least 600 ppm, at least 700 ppm, at least 800 ppm.

[0187] Embodiment 5. The method of any of the embodiments 1-4, wherein SO.sub.2 is present/added during fiber wash in amounts in a range from 400-3000 ppm, 500-2000 ppm, 600-1500 ppm, such as 600-1200 ppm.

[0188] Embodiment 6. The method of any of the preceding embodiments, wherein the xylanase is selected from the group consisting of a GH5 polypeptide, GH30 polypeptide, a GH10 polypeptide, a GH11 polypeptide, a GH8 polypeptide or a combination thereof.

[0189] Embodiment 7. The method of any of the preceding embodiments, wherein the hydrolytic enzymes comprise one or more cellulases, particularly cellulases obtained from Trichoderma, more particularly from Trichoderma reesei.

[0190] Embodiment 8. The method of embodiment 7, wherein the cellulase(s) comprise(s) one or more enzymes selected from the group consisting of an endoglucanase (EG), and a cellobiohydrolase (CBH).

[0191] Embodiment 9. The method of embodiment 8, wherein the cellulase(s) comprise(s) one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase I, a cellobiohydrolase II, or a combination thereof.

[0192] Embodiment 10. The method of any of the preceding embodiments, wherein the hydrolytic enzymes further comprise an arabinofuranosidase.

[0193] Embodiment 11 The method of embodiment 10, wherein the arabinofurasnosidase is selected from the group consisting of a GH43 polypeptide, a GH62 polypeptide, and a GH51 polypeptide.

[0194] Embodiment 12. The method according to any of embodiments 3-9, wherein said fiber washing procedure comprises the use of a fiber washing system comprising a space (V)/tank configured to provide a total retention time in the fiber washing system of at least 35 minutes and less than 48 hours.

[0195] Embodiment 13. The method according to any of the preceding embodiments, wherein the incubation time in said space (V)/tank configured into the fiber washing system is at least 5 minutes and less than 48 hours, such as between 35 minutes and 24 hours, 35 minutes and hours, 35 minutes and 6 hours, 35 minutes and 5 hours, 35 minutes and 4 hours, 35 minutes and 3 hours, 35 minutes and 2 hours, 45 minutes and 48 hours, 45 minutes and 24 hours, 45 minutes and 12 hours, 45 minutes and 6 hours, 45 minutes and 5 hours, 45 minutes and 4 hours, 45 minutes and 3 hours, 45 minutes and 2 hours.

[0196] Embodiment 14. The method according to any of the preceding embodiments, wherein the incubation temperature is between 25° C. and 95° C., such as between 25 and 90° C., 25 and 85° C., 25 and 80° C., 25 and 75° C., 25 and 70° C., 25 and 65° C., 25 and 60° C., 25 and 55° C., 25 and 53° C., 25 and 52° C., 30 and 90° C., 30 and 85° C., 30 and 80° C., 30 and 75° C., 30 and 70° C., 30 and 65° C., 30 and 60° C., 30 and 55° C., 30 and 53° C., 30 and 52° C., 35 and 90° C., 35 and 85° C., 35 and 80° C., 35 and 75° C., 35 and 70° C., 35 and 65° C., 35 and 60° C., 35 and 55° C., 35 and 53° C., 35 and 52° C., 39 and 90° C., 39 and 85° C., 39 and 80° C., 39 and 75° C., 39 and 70° C., 39 and 65° C., 39 and 60° C., 39 and 55° C., 39 and 53° C., 39 and 52° C., preferably 46 and 52° C.

[0197] Embodiment 15. The method according to any of the preceding embodiments, wherein the level of SO.sub.2 present/added during fiber wash results in the same extraction yield of starch and gluten while reducing the required contact time between hydrolytic enzyme and ground corn kernel mass compared to a method where SO.sub.2 levels are below 400 ppm.

[0198] Embodiment 16. The method according to any of the preceding embodiments, wherein the one or more hydrolytic enzymes is expressed in an organism with a cellulase background, such as Trichoderma reesei.

[0199] Embodiment 17. The method according to any of the preceding embodiments, wherein the one or more hydrolytic enzymes are purified.

[0200] Embodiment 18. The method according to any of the preceding embodiments, wherein the one or more hydrolytic enzymes is/are in a liquid composition.

[0201] Embodiment 19. The method according to any of the preceding embodiments, wherein the one or more hydrolytic enzymes is/are in a solid composition.

[0202] Embodiment 20. The method according to any of the preceding embodiments, wherein the effective amount of one or more hydrolytic enzymes admixed/contacted with one or more fractions of said ground corn kernel mass, is between 0.005-0.5 kg enzyme protein/metric tonne corn kernels entering the wet milling process.

[0203] Embodiment 21. The method according to any of the preceding embodiments, wherein the source of SO.sub.2 is sodium metabisulfite (Na.sub.2S.sub.2O.sub.5), and/or addition of SO.sub.2 gas.

[0204] Embodiment 22. The method according to any of the preceding embodiments wherein the xylanase is selected from the group consisting of: [0205] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1; [0206] (b) a variant of the mature polypeptide of SEQ ID NO: 1 comprising a substitution, deletion, and/or insertion at one or more positions; and [0207] (c) a fragment of the polypeptide of (a), or (b) that has xylanase activity.

[0208] Embodiment 23. The method of embodiment 22, wherein the mature polypeptide is amino acids 1 to 551 of SEQ ID NO: 1.

[0209] Embodiment 24. The method according to any of the preceding embodiments wherein the xylanase is selected from the group consisting of: [0210] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2; [0211] (b) a variant of the mature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion, and/or insertion at one or more positions; and [0212] (c) a fragment of the polypeptide of (a), or (b) that has xylanase activity.

[0213] Embodiment 25. The method of embodiment 24, wherein the mature polypeptide is amino acids 21 to 405 of SEQ ID NO: 2.

[0214] Embodiment 26. The method of any of the preceding embodiments, wherein the arabinofuranosidase is selected from the group consisting of: [0215] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 3; [0216] (b) a variant of the mature polypeptide of SEQ ID NO: 3 comprising a substitution, deletion, and/or insertion at one or more positions; and [0217] (c) a fragment of the polypeptide of (a), or (b) that has xylanase activity.

[0218] Embodiment 27. The method of embodiment 26, wherein the mature polypeptide is amino acids 17 to 325 of SEQ ID NO: 3.

[0219] Embodiment 28. The method according to any of the preceding embodiments wherein the xylanase is selected from the group consisting of: [0220] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 4; [0221] (b) a variant of the mature polypeptide of SEQ ID NO: 4 comprising a substitution, deletion, and/or insertion at one or more positions; and [0222] (c) a fragment of the polypeptide of (a), or (b) that has xylanase activity.

[0223] Embodiment 29. The method of embodiment 28, wherein the mature polypeptide is amino acids 20 to 319 of SEQ ID NO: 4.

[0224] Embodiment 30. The method according to any of the preceding embodiments, wherein the celullases are derived from Trichoderma reesei.

[0225] The invention is further illustrated by the following examples.

EXAMPLES

[0226] Enzymes:

[0227] GH5 Xylanase A: GH5 xylanase derived from Chryseobacterium sp-10696 and disclosed as SEQ ID NO: 1

[0228] Cellulase A: A whole cellulase derived from Trichoderma reesei. This cellulase composition will comprise all cellulase activities expressed in T. reesei; e.g., endoglucanases, and cellobiohydrolases.

Examples 1

[0229] 10-g fiber assay is performed with 5% fiber dry substance incubating at pH4.0, 50° C. for 150 minutes at dose of 200 μg or 300 μg enzyme protein per gram fiber dry substance, using a blend including Cellulase A and GH5 Xylanase A, in combination with either 400 ppm or 800 ppm hydrogen sulfite (HSO.sub.3.sup.−). Blend consists of 8% of GH5 Xylanase A, and 92% of Cellulase A based on enzyme protein. Hydrogen sulfite is generated by adding sodium metabisulfite (Na.sub.2S.sub.2O.sub.5) into the buffer following the reaction of Na.sub.2S.sub.2O.sub.5+H.sub.2O->2Na.sup.++2HSO.sub.3.sup.−. For comparison, blend containing 92% Cellulase A and 8% GH5 Xylanase A only (no SO.sub.2) at both low dose (200 μg EP/g-ds fiber) and high dose (300 μg EP/g-ds fiber) were included. The corn fiber with 17.77% residual starch and 9.88% residual protein was used as substrate in the fiber assay. Release of starch+gluten (dry substance) as well as individual protein from corn fiber at the specified treatment below was measured.

TABLE-US-00001 TABLE 1 Starch and gluten yield with and without enzymatic treatment Dose (μg enzyme Starch + Individual protein/g-ds Gluten Protein Treatments Fiber) Recovered Recovered No Enzyme 0 10.05% 0.65% Cellulase A + GH5 Xylanase A 200 12.20% 1.02% Cellulase A + GH5 Xylanase 200 14.45% 1.60% A + HSO.sub.3.sup.− (400 ppm) Cellulase A + GH5 Xylanase 200 15.00% 1.74% A + HSO.sub.3.sup.− (800 ppm) Cellulase A + GH5 Xylanase A 300 14.65% 1.48% Cellulase A + GH5 Xylanase 300 15.35% 1.82% A + HSO.sub.3.sup.− (400 ppm)

[0230] Therefore, the addition of Hydrogen sulfite on top of Cellulase A+GH5 Xylanase A can significantly increase the yield of starch+gluten as well as protein in corn wet-milling process.

Example 2

[0231] A 10-g fiber assay generally includes incubating wet fiber samples obtained from wet-milling plant, in the presence of enzymes, at conditions relevant to the process (pH 4, temp around 50° C.) and over a time period of between 1 to 4 hrs. After incubation the fiber is transferred and pressed over a 75 micron screen where the filtrates consisting mainly of the separated starch and gluten are then collected. A number of washes are done over the screen, and the washing are collected together with the initial filtrate. The collected filtrates are allowed to sit overnight letting the insoluble settle to the bottom of the flask. The bulk of the supernatant is aspirated via vacuum and the rest of the insolubles are then centrifuged in 50 ml conical tubes and the supernatant is decanted leaving a wet insoluble pellet. The wet insoluble pellet is lyophilized o/n to complete dryness. This insoluble dry mass is weighed to determine % insoluble yield and then analyzed for total nitrogen content (protein) via Leco analysis.

[0232] This 10-g fiber assay was performed with 6.4% fiber dry substance incubating at pH 4.0, 48° C. for 240 minutes at dose of 5000 μg enzyme protein per gram fiber dry substance, using a blend including Cellulase A and GH5 Xylanase A. The blend consists of 10% of GH5 Xylanase A, and 90% of Cellulase A based on enzyme protein. This blend was tested both in as-is substrate and in substrate sheared (homogenized) for 3 minutes in a blender. The homogenized substrate was treated with the enzyme blend with and without 1000 ppm SO2 (added from a 20× dilution of 1.48 g Na metabisulfate dissolved in 50 ml water) and a no enzyme treatment. The as-is substrate was treated with the enzyme blend both with and without 1000 ppm SO2 while two no enzyme treatments were performed with and without 1000 ppm SO2. The weights of insoluble mass (starch and gluten) and insoluble protein (gluten) released by the specified treatments are given below.

TABLE-US-00002 TABLE 2 Results Insoluble % CGM insolubles % Protein % increase over of starting of starting respective no Treatement fiber fiber ezyme control homogenized no enzyme 37.1% 1.9% Xylanase A 38.3% 3.3% 0.23% Xylanase A + 39.7% 4.0% 0.35% SO2 as is no enzyme 29.6% 1.2% no enzyme + 29.5% 1.5% 0.04% SO2 Xylanase A 36.5% 3.4% 0.36% Xylanase A + 36.9% 3.7% 0.42% SO2