WO2002000911A1

WO2002000911A1 - Starch gluten separation process

Info

Publication number: WO2002000911A1
Application number: PCT/DK2001/000426
Authority: WO
Inventors: Hans Sejr Olsen; Bjarne Rønfeldt Nielsen
Original assignee: Novozymes A/S
Priority date: 2000-06-29
Filing date: 2001-06-19
Publication date: 2002-01-03
Also published as: AU2001265825A1

Abstract

The present invention relates to an improved process of starch gluten separation, comprising subjecting mill starch to an acidic protease.

Description

STARCH GLUTEN SEPARATION PROCESS

FIELD OF THE INVENTION

The present invention relates to an improved starch gluten separation process constituting one of the steps in, e.g., the wet milling process used to process corn kernels and other crop kernels into high quality starch suitable for the conversion of starch into mono- di-, oligosaccharides, ethanol, sweeteners etc.

Further the invention also relates to an enzyme compo- sition comprising an acidic protein degrading activities for use in a starch gluten separation process.

BACKGROUND OF THE INVENTION

Before starch - being an important constituent in the kernels of most crops, such as corn, wheat, rice, sorghum bean, barley or fruit hulls - can be used for conversion of starch into saccharides, such as dextrose, fructose; alcohols, such as ethanol; and sweeteners, the starch must be made available and treated in an manner to provide a high purity starch. If starch contains more than 0.5% impurities, including proteins, it is not suitable as starting material for starch conversion processes. To provide such pure starch product staring out from the kernels of crops the kernels are often milled, as will be described further below.

The Composition of Corn Kernels Corn kernels, such as the yellow dent corn kernel, have an outer covering referred to as the "Pericarp" that protects the germ in the kernels. It resists water and water vapour and is undesirable to insects and microorganisms.

The only area of the kernels not covered by the "Peri- carp" is the "Tip Cap", which is the attachment point of the kernel to the cob.

The "Germ" is the only living part of the corn kernel. It contains the essential genetic information, enzymes, vitamins, and minerals for the kernel to grow into a corn plant. About 25 percent of the germ is corn oil. The endosperm covered surrounded by the germ comprises about 82 percent of the kernel dry weight and is the source of energy (starch) and protein for the germinating seed. There are two types of endosperm, soft and hard. In the hard endosperm, starch is packed tightly together. In the soft endosperm, the starch is loose.

Wet milling

Wet milling is often used for separating corn kernels into its four basic components: starch, germ, fiber and protein.

Typically wet milling processes comprise four basic steps. First the kernels are steeped for 30 to 48 hours to begin breaking the starch and protein bonds. The next step in the process involves a coarse grind to separate the germ from the rest of the kernel. The remaining slurry consisting of fiber, starch and protein is finely ground and screened to separate the fiber from the starch and protein. The starch is separated from the remaining slurry in hydrocyclones . The starch then can be converted to syrup or alcohol.

Dry Milling

In dry milling processes crop kernels, in particular corn kernels, are grinded in substantially dry state, without pre- soaking the kernels to separate the kernels into its major constituents: starch, germ, fiber and protein.

Today enzymes are not commonly for wet or dry milling of crop kernels. However, the use of enzymes has been suggested for the steeping step of wet milling processes. The commercial enzyme product Steepzyme® (available from Novozymes A/S) have been shown suitable for the first step in wet milling processes, i.e., the steeping step where corn kernels are soaked in water.

Accordingly, the object of the invention is to provide an improved starch gluten separation process. BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows a corn wet milling process,

Figure 2 shows the processes used in a corn steeping plant in a schematic form.

Figure 3 shows a dry milling process including starch and protein recovery process. In the case of corn the feed product is similar to corn gluten and the protein is similar to corn gluten meal.

DETAILED DESCRIPTION OF THE INVENTION The object of the present invention is to provide an improved starch gluten separation process.

When using the term "kernels" it is intended to include kernels from corn (maize) , rice, barley, sorghu bean, or fruit hulls, or wheat. In the context of the present invention, the term "enriched" is intended to indicate that the enzyme activity in question of the enzyme preparation has been increased, e.g., with an enrichment factor of at least 1.1, conveniently due to addition of a recombinant mono-component enzyme.

The Milling Process

The kernels are milled in order to open up the structure and to allow further processing. Two processes are used: wet or dry milling. In dry milling processes the whole kernels are milled and used in the remaining part of the process. Wet milling gives a very good separation of germ and meal (starch granules and protein) .

Dry Milling Dry milling processes are well known in the art. The term "dry milling" is in the context of the invention meant to include all such processes where the kernels are grinded in dry state. Dry milling may for instance be carried out as follows: Dry kernels are first cleaned to remove chaff and other external vegetable matter. The hulls of the cleaned dry kernels are intentionally broken to facilitate subsequent milling, and passed through an impact-degerminating mill to loosen up the kernels germ. The discharge from the degerminating mill, comprising germ, fibre (hull) and endosperm (which is the raw material for the starch recovery process) , is sifted into fractions according to particle size. The sifted fractions are subjected to suction using air aspirators, which separates the hull fiber. The dehulled discharge from the air aspirators, comprising germ and endosperm, is passed over vibrating gravity tables to separate the germ from the endosperm. The germ is collected from the gravity tables and, if desired, sent to oil expelling station.

Wet milling

Degradation of the kernels of corn (see also Fig. 1 and Fig. 2) and other crop kernels into starch suitable for conversion of starch into mono-, di-, oligo saccharides, ethanol, sweeteners etc. consists of four main steps:

1. Steeping and germ separation,

2. Fiber washing and drying,

3. Starch gluten separation,

4. Starch washing.

1. Steeping and germ separation

Corn kernels are softened by soaking in water for between 30 and 48 hours at a temperature about 50°C. During steeping, the kernels absorb water, increasing their moisture levels from 15 percent to 45 percent and more than doubling in size. The addition of 0.1% sulfur dioxide (S0₂) and/or NaHSU3 to the water prevents excessive bacteria growth in the warm environment. As the corn swells and softens, the mild acidity of the steepwater begins to loosen the gluten bonds within the corn and release the starch. After the corn kernels are steeped they are cracked open to release the germ. The germ contains the valuable corn oil. The germ is separated from the heavier density mixture of starch, hulls and fiber 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 . 2. Fiber washing and drying

To get maximum starch recovery, while keeping any fiber in the final product to an absolute minimum, it is necessary to wash the free starch from the fiber during processing. The fiber is collected, slurried and screened to reclaim any residual starch or protein.

3. Starch separation

The starch-gluten suspension 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.

4. Starch washing.

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 hydroclones to remove the last trace of protein and produce high quality starch, typically more than 99.5 percent pure . Process Of The Invention

The inventors of the present invention have surprisingly found that selected enzyme activities and combinations thereof may be used to improve starch gluten separation processes step. Advantages may be that the yield of the final starch product and/or quality of the final starch product is increased. In the first aspect the invention relates to a process of separating starch and gluten from mill starch, comprising subjecting the mill starch to an effective amount of acidic protease activity. A process may comprise the following steps: a) crop kernels are steeped by soaking in water; b) kernels are cracked open to release germ; c) the germ is removed to provide a starch fraction; d) the starch fraction obtained in step c) is freed from fibers to provide mill starch starch-gluten suspen^¬ sion) ; e) the mill starch obtained in step d) is subjected to an effective amount of acidic protease to provide a starch and a protein slurry; f) washing the starch slurry obtained from step e) g) recover starch. The steeping step may be carried out at a temperature between 40 and 60°C, preferably around 50°C and/or in the pres^¬ ence of 0.01-1%, preferably 0.05-0.3%, especially 0.1% SO2 and/or NaHS0₃.

In a preferred embodiment the mill starch is further sub^¬ jected to an effective amount of xylanase activity.

In another aspect the invention relates to a process of separating starch and gluten of milled endoperm (grits), com- prising subjecting the milled endosperm (grits) to an effective amount of acidic protease activity.

The process may comprise the following steps: i) dry milling of kernels into endosperm grits; ii) subjecting the milled kernels to an effective amount of acidic protease activity to provide a starch and a gluten slurry; iii) washing the starch slurry obtained in step ii) . In a preferred embodiment of the invention the dry milled kernels (the grits) obtained in step i) are grinded in wet state until the kernels have an average particles diameter of below 450 micro meters, preferably below 200 micro meters, especially below 100 micro meters and then subjected to step ii) . In a preferred embodiment the milled endosperm (grits) are further subjected to an effective amount of xylanase activity.

In a further embodiment also an effective amount of cellu- lase and/or arabinofuranosidase is added.

In a preferred embodiment of the invention the milled kernels are subjected to an effective amount of acidic protease activity to provide a starch and a gluten slurry is carried out in the presence of 0.01-1%, preferably 0.05-0.3%, especially 0.1% S0₂ and/or NaHS0₃.

Acidic Proteases

Suitable acidic proteases include fungal and bacterial proteases, i.e., proteases characterized by the ability to hy- drolyze proteins under acidic conditions below pH 7. Suitable acid fungal proteases include fungal proteases derived from Aspergill us, Mucor, Rhizopus, Candida , Coriolus, Endothia, Enthomophtra , Irpex, Penicilli um, Sclerotiυm and To- rulopsis . Especially contemplated are proteases derived from Aspergillus niger (see, e . g. , Koaze et al . , (1964) , Agr. Biol . Chem. Japan, 28, 216) , Aspergill us sai toi (see, e . g. , Yoshida,

(1954) J. Agr. Chem. Soc . Japan, 28, 66) , Aspergillus awamori

(Hayashida et al . , (1977) Agric. Biol. Chem., 42(5), 927-933,

Aspergill us aculeatus (WO 95/02044), or Aspergillus oryzae, and acidic proteases from Mucor pusillus or Mucor miehei . In an embodiment the acidic protease is a protease clom- plex from A. oryzae sold under the tradename Flavourzyme® (from Novozymes A/S) or an aspartic protease from Rhizomucor miehei or Spezyme® FAN or GC 106 from Genencor Int.

In a preferred embodiment the process of the invention is carried out in the presence of the acidic Protease I derived from A. aculeatus CBS 101.43 in an effective amount.

The kernels are subjected to a composition of the invention as described so that the mil starch is subjected to from from 4,000-20,000 HUT/100 g DS kernels acidic protease, pref- erably 5,000-10,000 HUT/100 g DS kernels, especially from 6,000-16,500 HUT/100 g DS kernels. In a preferred embodiment the acidic protease is an as- partic protease, such as an aspartic protease derived from a strain of Aspergill us, in particular A. aculeatus, especially A. acul eatus CBS 101.43.

Preferred acidic proteases are aspartic proteases, which retain activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF, or EDTA. Protease I derived from A. aculeatus CBS 101.43 is such acidic protease.

Xylanase

In a preferred embodiment of the invention an effective amount of a xylanase activity is also present or added during treatment of the milled kernels. The xylanase activity may be derived from any suitable organism, including fungal and bacterial organisms, such as

Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium and Trichoderma .

Examples of suitable xylanases include xylanases derived from H. insolens (WO 92/17573; Aspergillus tubigensis (WO

92/01793); A. niger (Shei et al., 1985, Biotech, and Bioeng.

Vol. XXVII, pp. 533-538, and Fournier et al., 1985, Biotech.

Bioe g. Vol. XXVII, pp. 539-546; WO 91/19782 and EP 463 706); A. aculeatus (WO 94/21785) . In a specific embodiment the xylanase is Xylanase I, II, or

III disclosed in WO 94/21785.

Contemplated commercially available xylanase include

Shearzyme®, Biofeed wheat® (from Novozymes A/S) and Spezyme® CP

(from Genencor Int.) . The xylanase may be added in an amount of 1-100 FXU per 100 g DS kernels, preferably 5-90 FXU per 100 g DS kernels, especially 10-80 FXU per 100 g DS kernels.

Cellulases

In another preferred embodiment an effective amount of a cellulase activity is also present or added during treatment of the milled kernels. The cellulase may be of microbial origin, such as derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusari um) . Specific examples of cellu- lases include the endo-glucanase (endo-glucanase I) obtainable from H. insolens and further defined by the amino acid sequence of fig. 14 in WO 91/17244 and the 43 kD H. insolens endogluca- nase described in WO 91/17243.

Commercially available cellulase which may be used include Celluclast®, Celluzyme® (available from Novozymes A/S), Spezyme® CP (available from Genencor Int.) and Rohament® 7069 W (available from Rohm, Germany) .

The cellulase may be added in an amount of 1-1,000 NCU per 100 g DS kernels, preferably 170-900 NCU per 100 g DS kernels, especially 200-800 NCU per 100 g DS kernels.

Arabinofuranosidase

In an even further embodiment of the process of the invention an effective amount of an arabinofurasidase activity is also present or added during treatment of the milled kernels. Examples of contemplated arabinofuranosidases include A. niger alpha-L-arabinofuranosidase A and B disclosed in WO 97/42301; the Aspergill us sp. arabinofuranosidase disclosed in EP 871,745; the Aspergillus niger Kl alpha-L- arabinofuranosidase disclosed in DD 143925.

Other enzyme activities

According to the invention an effective amount of one or more of the following activities may also be present or added during treatment of the kernels: endoglucanase, beta-glucanase, pentosanase, pectinase, arabinanase, xyloglucanase activity, or mixtures thereof.

In an embodiment of the invention the enzyme acitivities added during the process of the invention is derived from the enzyme product Steepzyme® further enriched with one or more of the following activities: xylanase, cellulase, arabinosidase, endoglucanase, beta-glucanase, pentosanase, pectinase, arabinanase, xyloglucanase and/or acidic protease activity.

It is believed that the acidic protease facilitates the separation of the protein from the starch. Thereby the starch is washed out more easily in the subsequent step.

Composition of the invention

The invention also relates to an enzyme composition. For starch gluten separation the composition may comprise an acidic activity only or a combination of an acidic protease activity and one or more enzyme activities.

In this aspect the invention relates to a composition suitable for starch gluten separation comprising one or more of the following enzyme activities: endoglucanase, beta-glucanase, xy- lanase, cellulase, pentosanase, pectinase, arabinofurasidase, arabinanase, xyloglucanase and/or acidic protease activity.

In a preferred embodiment the composition comprises an acidic protease activity. The composition may further comprise endoglucanase, beta-glucanase, xylanase, cellulase, pentosanase, pectinase, arabinofurasidase, arabinanase, xyloglucanase and/or cellulase activity.

In a preferred embodiment the composition further comprises a xylanase. The composition may also comprise an arabinofurasidase and/or a cellulase activity. In an embodiment the composition of the invention is the enzyme product Steepzyme® further enriched with xylanase activity and/or cellulase activity and/or arabinofuranosidase activity and/or an acidic protease activity.

In an embodiment the composition of the invention compris- ing more that 3740 HUT/g and/or more that 45 FXU/g and/or more that 1694 NCU/g.

The enzyme composition of the invention may in an embodiment comprise a mono-component Protease I from A. aculeatus CBS 101.43. It may further comprise Xylanase II from A. aculeatus CBS 101.43 (WO 94/21785). Use of a Composition of the invention

A composition of the invention may be used for the starch gluten separation step.

MATERIALS & METHODS Enzymes :

Steepzyme®: multi activity enzyme complex derived from A. aculeatus 101.43 (is available from Novozymes A/S on request)

Shearzyme®: A. aculeatus CBS 101.43 xylanase II disclosed in WO 94/21785 (is available from Novozymes A/S)

Flavourzye®: multi proteolytic activity enzyme complex derived from A. oryzae (is available from Novozymes A/S)

Protease I: acidic protease from Aspergillus aculeatus CBS 101.43 (disclosed in WO 95/02044).

METHODS

Determination of protease HUT activity:

The HUT activity was determined according to the AF92/2 method published by Novozymes A/S, Denmark. 1 HUT is the amount of enzyme which, at 40°C and pH 4.7 over 30 minutes forms a hydrolysate from digesting denatured hemoglobin equivalent in absorbancy at 275 nm to a solution of 1.10 μg/ml tyrosine in 0.006 N HC1 which absorbancy is 0.0084. The denatured hemoglobin substrate is digested by the enzyme in a 0.5 M acetate buffer at the given conditions. Undigested hemoglobin is precipitated with trichloroacetic acid and the absorbance at 275 nm is measured of the hydrolysate in the supernatant.

Determination of xylanase activity (FXU)

The endo-xylanase activity is determined by an assay, in which the xylanase sample is incubated with a remazol-xylan substrate (4-O-methyl-D-glucurono-D-xylan dyed with Remazol

Brilliant Blue R, Fluka) , pH 6.0. The incubation is performed at 50°C for 30 in. The background of non-degraded dyed substrate is precipitated by ethanol. The remaining blue colour in the supernatant is determined spectrophotometrically at 585 nm and is proportional to the endoxylanase activity.

The endoxylanase activity of the sample is determined rela- tively to an enzyme standard.

The assay is further described in the publication AF 293.6/1- GB, available upon request from Novozymes A/S, Denmark.

Determination of Endo-Glucanase Units (ECU) The ECU (endocellulose unit) is determined relatively to an enzyme standard.

Endocellulase decomposes carboxylmethylcellulose, CMC. The resulting reduction in viscosity is determined by a CMC- vibration Viscosimeter (e.g. MIVI 3000 available from Sofraser, France) . The prepared substrate solution contain 35 g/1 CMC (Blanose Aqualon) in 0.1 M phosphate buffer at pH 7.5. The enzyme sample to be analyzed is determined is dissolved in the same buffer. 0.15 ml standard enzyme solution or the unknown enzyme sample is placed in 10 ml test tubes. 5 ml CMC-substrate solution, preheated to 40°C, is added. The joint solution is mixed thoroughly, incubated for 30 minutes and placed in the viscometer .

The method is further described in AF302/1-GB available from Novozymes A/S upon request.

Determination of endo-glucanase activity (EGU)

The fermentation broths are analyzed by vibration viscosimetry on CMC at pH 6.0. More specifically, a substrate solution containing 34.0 g/1 CMC (Blanose Aqualon) in 0.1 M phosphate buffer, pH 6.0 is prepared. The enzyme sample to be analyzed is dissolved in the same buffer. 14 ml substrate solution and 0.5 ml enzyme solution are mixed and transferred to a vibration viscosimeter (e.g. MIVI 3000 available from Sofraser, France) thermostated at 40°C. Endoglucanase unit (EGU) is determined as the ratio between the viscosity of the sample and the viscosity of a standard enzyme solution. Cellulytic Activity

The cellulytic activity is determined with carboxymethyl cellulose (CMC) as substrate.

One Novo Cellulase Unit (NCU) is defined as the amount of enzyme which, under standard conditions (i.e. at pH 4.80; 0.1 M acetate buffer; 10 g/1 Hercules CMC type 7 LFD as substrate; an incubation temp, of 40.0°C; an incubation time of 20 in; and an enzyme concentration of approximately 0.041 NCU/ l) forms an amount of reducing carbohydrates equivalent to 1 micro mol glu- cose per minute. A folder AF 187,2/1 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Arabinofuranosidase assay

The synthetic substrate p-nitrophenyl alpha-L- arabinofuranoside (SIGMA) is used as substate. Following cleavage of the enzyme, the p-nitrophenyl molecule is liberated and the development in yellow colour can be measured by visible spectrometty at 405 nm.

Stock solution: 1 g/ml p-nitrophenyl alpha-L- arabinofuranoside in DMSO.

Substrate solution: 0.2 mg/ml p-nitrophenyl alpha-L- arabinofuranoside diluted in 50 mM Sodium acetate, pH 4.5.

Procedure: 100 microlitre enzyme and 100 microlitre is mixed in a 96-well plate and the development of yellow colour due to the enzymatic reaction is measured from 0 to 15 minutes at 405 nm. The slope of the time dependent OD405 curve is di- rectly proportional to the amount of alpha- arabinofuranosidase . Example 1

A description of the results of the work with enzymatic separation of starch and gluten

Using Yellow Corn Meal (YCM) for extraction of starch and protein (called gluten) the use of enzymes on an aqueous slurry to release the main components of the flour as complete as possible was tested.

In laboratory trials hydrolysis trials using the enzymes were performed at pH = 4.5, T = 50°C, at a substrate concen- tration of 25 g YCM/100 g reaction mixture. The time dependent hydrolytic effects were demonstrated by measurements of the increase of soluble dry matter (refractometer) , and the increase of osmolality (using an osmometer) as well as the phase separation of the reaction mixture after centrifugation. Even though YCM itself contained hydrolytic enzymes a separation of the starch and gluten within 24 hours could not be obtained without addition of enzymes.

The overall most efficient cell wall degrading enzyme was shown to be Steepzyme but a high dosage had to be used to ob- tain the separation.

Supplementation of Steezyme's protease activity using Flavourzyme or Protease I improve the separation of starch and gluten compared to the effect of Steepzyme alone.

Combining Steepzyme and Flavourzyme so that the overall protease dosage was approximately 18000 HUT/100 g YCM resulted in an almost complete separation of starch and gluten after 24 hours of reaction time.

Using Protease I instead of Flavourzyme the protease activity dosage of only 3600 HUT/g secured an almost complete separation under the same conditions.

Claims

1. A process of separating starch and gluten from mill starch, comprising subjecting the mill starch to an effec- tive amount of an acidic protease activity.

2. A process comprising the steps of: a) crop kernels are steeped by soaking in water; b) kernels are cracked open to release germ; c) the germ is removed to provide a starch fraction; d) the starch fraction obtained in step c) is freed from fibers to provide mill starch starch-gluten suspension; e) the mill starch obtained in step d) is subjected to an effective amount of acidic protease to provide a starch and a protein slurry; f) washing the starch slurry obtained from step e) g) recovering starch.

3. The process of claims 1 or 2, wherein the mill starch is further subjected to an effective amount of a xylanase activity.

4. A process of separating starch and gluten of milled endoper (grits), comprising subjecting the milled endosperm (grits) to an effective amount of an acidic protease activity.

5. A process comprising the steps of: j) dry milling of kernels into endosperm grits; ii) subjecting the milled kernels to an effective amount of acidic protease activity to provide a starch and a gluten slurry; iii) washing the starch slurry obtained in step ii) .

6. The process of claims 4 or 5, wherein the dry milled kernels (the grits) obtained in step i) are grinded in wet state until the kernels have an average particles diameter of below 450 micro meters, preferably below 200 micro me- ters, especially below 100 micro meters and then subjected to step ii) .

7. The process of claims 4-6, wherein the milled endosperm (grits) are further subjected to an effective amount of a xylanase activity.

8. The process of claims 1-7, wherein xylanase is added in an amount of 1-100 FXU per 100 g DS kernels, preferably 5-90 FXU per 100 g kernels, especially 10 to 80 FXU per 100 g DS kernels.

9. The process of claims 1-8, wherein also an effective amount of a cellulase is added.

10. The process of claim 1-9, wherein a cellulase is added in an amount of 1-1000 NCU per 100 g DS kernels, preferably 170-900 NCU per 100 g DS kernels, especially 200 to 800 NCU per 100 g DS kernels.

11. The process of claims 1-10, wherein also an effective amount of an arabinofuranosidase is added.

12. The process of claims 1-11, wherein the acidic protease is added in an amount of 1-10,000 HUT/100 g kernels, prefera- bly 300-8,000 HUT/100 g kernels, especially 3,000-6,000 HUT/ 100 g kernels.

13. The process of claim 1-12, wherein further one or more of the following enzyme activities are added: endoglucanase, beta-glucanase, pentosanase, pectinase, arabinanase, and/or xyloglucanase, or mixtures thereof.

14, A process of claims 1-13, wherein steeping of kernels is carried out at a temperature between 40 and 60°C, preferably around 50°C.

15. The process of claims 1-14, wherein the steeping step is carried out in the presence of 0.01-1%, preferably 0.05- 0.3%, especially 0.1% S0₂ and/or NaHS0₃.

16. The process of claim 1-14, wherein the subjection of the milled kernels to an effective amount of an acidic protease activity to provide a starch and a gluten slurry in performed in the presence of 0.01-1%, preferably 0.05- 0.3%, especially 0.1% S0₂ and/or NaHS0₃.

17. he process of claims 1-16, wherein the crop kernels are from corn (maize) , rice, barley, sorghum bean, or fruit hulls, or wheat.

18. The process of claims 1-17, wherein the xylanase, and/or the cellulase, and/or the arabinofurasidase and/or the acidic protease is derived from the genus Aspergill us, preferably A. aculeatus, especially A. aculea tus CBS

101.43.

19. The process of claims 1-18, wherein the enzyme activities is derived from Steepzyme® enriched with one or more of the following activities: xylanase, cellulase, arabinosidase, endoglucanase, beta-glucanase, pentosanase, pectinase, arabinanase, xyloglucanase and/or acidic protease activity.

20. The process of claims 1-20, wherein the kernels are subjected to the Steepzyme® enzyme activities enriched to provide a total HUT/100 g DS kernels from 4,000-20,000 HUT/100 g DS kernels acidic protease, preferably 5,000- 10,000 HUT/lOOg, especially from 6,000-16,500 HUT/g DS kernels .

21, The process of claims 1-21, wherein the acidic protease is an aspartic protease, such as an aspartic protease derived from a strain of Aspergillus, in particular A. aculeatus, especially A. aculeatus CBD 101.43.

22. The process of claims 1-22, wherein the aspartic protease retains activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF, and EDTA.

23. The process of claims 1-22, wherein the acidic protease is protease I derived from A. aculeatus CBS 101.43.

24. A composition comprising one or more of the following enzyme ^• activities : endoglucanase, beta-glucanase, xylanase, cellulase, pentosanase, pectinase, arabinofurasidase arabinanase, xyloglucanase and/or acidic protease activity.

25. The composition of claim 24 comprising a xylanase and an acidic protease activity.

26. he composition of claim 25, wherein the composition further comprises arabinofurasidase and/or cellulase activ- ity.

27. The composition of claim 24-26, wherein the^' composition is Steepzyme® enriched with a cellulase and/or a xylanase and/or an arabinofuranosidase and/or an acidic protease.

28. The composition of claim 24-27, comprising more that 3740 HUT/g.

29. The composition of claim 24-28, comprising more that 45 FXU/g.

30. The composition of claim 24-29, comprising more that 1694 NCU/g.

31. Use of a composition of claims 24-30, for separating mill starch into starch and gluten slurries.

32. Use of a composition of claims 24-30 for separating milled kernels into starch and gluten slurries.

33. Use of a composition of claims 24-30 in a process of claims 1-23.

34. Use of claims 31-33, for separation of corn or sorghum mill starch.