GB2402600A - Removing enzymes on grain - Google Patents

Abstract

A cereal flour that is substantially free of an exogenous enzyme is prepared from grain that has been treated to remove enzymes produced by bacteria or fungus. The treatment may be washing, heating or a mechanical treatment, e.g. polishing. The amount of enzyme on the grain may be tested by milling the grain, extracting the flour in water and measuring the amount of enzyme in the extraction.

Description

FLOUR

FIELD OF THE PRESENT INVENTION

The present invention relates to a cereal flour. In particular, a cereal flour which is substantially free of an exogenous enzyme.

The present invention also relates to a dough prepared from a cereal flour which is substantially free of an exogenous enzyme. In particular a refrigerated dough.

Furthermore, the present invention relates to a process of preparing the dough.

The present invention further relates to a dough product, such as bread, prepared from a cereal flour which is substantially free of an exogenous enzyme. Furthermore, the present invention relates to a process of preparing the baked product.

The present invention also relates to a method of determining activity and/or amount of an exogenous enzyme on a grain.

BACKGROUND TO THE INVENTION

Typically, doughs such as refrigerated doughs comprise flour and water. In the refrigerated dough, the wheat flour contains 2 - 5 % arabinoxylan (AX) (Fincher and Stone, 1986, Cell walls and their components in cereal grain processing. pp 207-295.

In: Advances in cereal science and technology. Vol. VIII. Pomeranz, Y. ed. Am. Assoc. Cereal. Chem., St. Paul. Minnesota, USA) originating from the endosperm cell walls. Arabinoxylan is a complex non-starch polymer that has a unique capacity to bind water. Girhammer (1992, Water-soluble nonstarch polysaccharides from cereals.

PhD dissertation, Lund, Sweden) reports that arabinoxylans are capable of binding water in amounts of up to about 10 times their own weight.

Refrigerated dough is a rapidly increasing bread market. In this respect, pre-made dough, prepared by industrial bakeries, can be stored for a long time, and fresh baked bread can be produced very easily and rapidly by the end consumer. The whole concept of refrigerated dough, fits well to the developing demands of the consumers viz: being able to have fresh baked bread independent of opening hours, time to bakery, etc. However, there is a problem with doughs such as refrigerated doughs. In this regard, refrigerated doughs are known to exhibit syruping. Syruping is a consequence of the deleterious breakdown of arabinoxylan, and hence a decrease in water holding capacity in the dough. Otherwise expressed, syruping is a lack of water holding capacity as a function of time. It is believed that the breakdown of arabinoxylan is due to the presence of xylanases in the flour.

Xylanases (EC 3.2.1.32) break down arabinoxylan. Xylanase inhibitors have been shown to reduce or prevent the enzymatic degradation of arabinoxylan.

Wheat endogenous xylanase inhibitors (Sibbesen and Sorensen, 2000; Gebruers et al., 2001; Goesaert et al., 2001; Gebruers, 2002; Gebruers et al. 2002a; Gebruers et al. 2002b; Flatman et al., 2002) have been the topic of much attention in the past few years. Flatman et al., (2002) show that microbial xylanases have variable sensitivity to xylanase inhibitors, some are very sensitive, others are not.

The prior art teaches that it is possible to reduce -or even eliminate syruping in dough using for example, hydrocolloids (see, for example, USA-5792499 which describes the addition of xylan to dough) or enzyme inhibitors (see, for example, p8144 which describes the addition of xylanase inhibitors to dough).

Noots et al (1998, Critical Reviews in Microbiology 25:121-153) teach that barley grains carry microbial populations and discusses the microflora of barley grain. In addition, Noots et al teach that fungi isolated from barley grain can degrade arabinoxylan. Furthermore, Noots et al teach the use of barley grain in malt for brewing and that the types of microbes present on the grain influence the quality of malt and beer. However, Noots et al do not teach nor suggest the decontamination of wheat kernels nor that microbial xylanase contamination influences flour properties.

Dorfer (2001, Getreide Mehl und Brat 55:154-157) examines the activity of pentosan reducing enzymes, such as endo-,B-xylanase and a-amylase, on the texture of doughs made from rye flour. Dorfer (2001) teaches that the viscosity is reduced in a dough composition comprising high xylanase activity. In addition, Dorfer (2001) teaches that fungal enzymes are problematical. However, Dorfer does not teach nor suggest the decontamination of wheat kernels nor that decontaminated kernels produce a dough with an improved viscosity.

SUMMARY ASPECTS OF THE PRESENT INVENTION

Several attempts have been made to correlate the level of endogenous wheat xylanase inhibitor to the level of endogenous wheat xylanase. However, all these attempts have failed. Furthermore, attempts to predict the performance of a flour batch based on the level of endogenous xylanase activity have not been successful. This led to the idea that the xylanase activity in flour may not all be originating from wheat but that some of the xylanase activity may be of microbial origin. However, it has been extremely difficult to characterize microbial xylanase activity in wheat flour, due to the level of endogenous xylanase inhibitor activity and the sensitivity of microbial xylanases to these inhibitors.

The present invention is based on the surprising finding that the activity of an exogenous enzyme present on the surface of a grain can influence the properties of a cereal flour from the milled grain. Additionally, the present invention is based on the unexpected finding that even a low reduction in the amount and/or activity of an exogenous enzyme from said grain results in an improved cereal flour. Such improvements in the cereal flour include an improved viscosity of a dough comprising the cereal flour and an improved water holding capacity as a function of time.

In one aspect, the present invention provides a cereal flour wherein said cereal flour is substantially free of an exogenous enzyme.

In another aspect, the present invention provides a dough prepared from a cereal flour wherein said cereal flour is substantially free of an exogenous enzyme.

According to another aspect of the present invention, there is provided a dough product prepared from a cereal flour wherein said cereal flour is substantially free of an exogenous enzyme or prepared from a dough wherein said dough is prepared from a cereal flour which is substantially free of an exogenous enzyme.

In a further aspect, the present invention provides a process of preparing a dough comprising the steps of admixing a cereal flour, wherein said cereal flour is substantially free of an exogenous enzyme, with one or more other dough ingredients.

According to one aspect of the present invention, there is provided a process of preparing a dough comprising the steps of milling a grain into a cereal flour and admixing the cereal flour, wherein said cereal flour is substantially free of an exogenous enzyme, with one or more other dough ingredients; wherein said grain for milling is substantially free of exogenous enzyme.

The present invention provides, in a further aspect thereof, a process of preparing a dough comprising the steps of i) treating a grain to substantially remove an exogenous enzyme ii) milling the treated grain into a cereal flour iii) admixing the cereal flour with an aqueous solution, preferably water.

In another aspect, the present invention provides a method of determining a reduction in activity andlor amount of an exogenous enzyme on a grain comprising the steps of i) optionally treating the grain, ii) milling the grain into a cereal flour iii) extracting the cereal flour in an aqueous solution, preferably water, iv) measuring the activity and/or amount of the exogenous enzyme of the cereal flour extraction v) comparing the activity andlor amount of the exogenous enzyme of an untreated grain with the activity and/or amount of the exogenous enzyme of a treated grain.

In a further aspect, the present invention provides the use of a treatment procedure for the removal of an exogenous enzyme from a grain.

The present invention provides, in a further aspect thereof, the use of a treatment procedure in the preparation of a cereal flour wherein said treatment procedure reduces the amount andlor activity of an exogenous enzyme in said cereal flour, preferably wherein said cereal flour is substantially free of said exogenous enzyme.

In another aspect, the present invention provides the use of a cereal flour that is substantially free of an exogenous enzyme in the preparation of a dough, preferably a refrigerated dough.

In another aspect, the present invention provides cell wall components, of a milled grain, of improved quality wherein the quality of said cell wall components is improved by a reduction in the activity andlor amount of an exogenous enzyme on a gram.

In a further aspect, the present invention provides a process of preparing cell wall components, of a milled grain, of improved quality wherein said process comprises the steps of i) treating a grain to substantially remove an exogenous enzyme ii) milling the treated grain.

The present invention provides, in a further aspect thereof, the use of a treatment procedure in the preparation of cell wall components, of a milled grain, of improved quality wherein said treatment procedure reduces the amount and/or activity of an exogenous enzyme on said milled grain, preferably wherein said milled grain is substantially free of said exogenous enzyme.

DETAILED ASPECTS OF THE PRESENT INVENTION

Preferably, the exogenous enzyme as described herein is a xylanase (EC 3. 2.1.8).

The term "exogenous enzyme" as used herein refers to an enzyme which is produced by at least one microbial species, such as a bacteria or a fungus or an algae. The term "endogenous enzyme" refers to an enzyme produced by a cereal grain.

Preferably, the cereal flour as described herein is one or more of the following: wheat flour, rye flour, oat flour, corn flour and barley flour.

In a preferred embodiment, the grain as described herein is one or more of the following: wheat, rye, oats, corn and barley.

In a preferred embodiment of the present invention, the grain is milled.

In a preferred aspect, the cereal flour as described herein is a flour andlor a whole meal flour.

Preferably, the exogenous enzyme as described herein is an enzyme present on the surface of a grain. More preferably, the exogenous enzyme as described herein is an enzyme present on the surface of a grain before said grain is subjected to a treatment procedure, such as a decontamination-procedure. In a highly preferred embodiment, the exogenous enzyme of the present invention is an enzyme present on the surface of a grain before said grain is treated and milled into a cereal flour.

According to the present invention, the grain can be treated to substantially remove an exogenous enzyme. Here, the treatment may be any suitable treatment - such as washing. In some cases, we refer to the treatment as a "decontamination" step.

As used herein, the terms "decontamination", "decontaminating", "decontaminated" "decontamination treatment" and "decontamination procedure" refer to the removal of an exogenous enzyme on a grain.

Treatment as described herein includes, but is not limited to, the following procedures or treatments: chemical surface disinfection, mechanical treatment such as polishing, pearling or shelling the kernels, washing with an aqueous solution, or heat treatment.

As used herein the terms "non-decontamination", "non-decontaminating" and "non- decontaminated" refer to grain which has not been treated for the removal of an exogenous enzyme on a grain, i.e. the control grain.

Suitably, the grain as described herein is treated by washing said grain in an aqueous solution such as water.

In one aspect of the present invention, the dough is, or is processed as, or is processed into a refrigerated dough.

As used herein, the term "refrigerated dough" refers to a dough which may be stored below room temperature (for example, at about 5 C) for a period of time.

In one aspect, the dough as described herein is one or more of the following: a bread dough, a cookie dough, a pastry dough, a pasta dough and a cake dough.

Preferably the dough, as described herein, is then baked.

Preferably the dough product as described herein is one or more of the following: a bread, a cookie, a pastry, a pasta and a cake.

In a highly preferred embodiment, the dough product as described herein is a bread.

The dough may be prepared by mixing the cereal flour as described herein with other dough ingredients before an aqueous solution is added. Alternatively, the dough may be prepared by mixing the cereal flour as described herein and the aqueous solution before the other dough ingredients are added. Alternatively, the dough may be prepared by mixing the aqueous solution with the other dough ingredients before the cereal flour as described herein is added. Combinations of these process steps are also encompassed by the present invention.

In a preferred aspect, the other dough ingredients are one or more of the following: salt, sugar, fruit, fruit extracts or parts thereof, yeast, chemical leavener(s), spice(s), antioxidant(s), colourant(s), stabilier(s) , flavouring(s), egg(s) or extracts thereof, dairy product(s), emulsifier(s), inhibitor(s) of the exogenous enzyme(s), enzymes such as amylase(s), protease(s), xylanase(s), oxidase(s), lipase(s), phospholipase(s), glycolipase(s), emulsifier(s), bread improver(s), xylanase inhibitor(s), enzymatic inhibitor(s) of the exogenous enzyme(s) etc. Preferably, the inhibitor of the exogenous enzyme as described herein is a xylanase inhibitor.

In one aspect, the xylanase inhibitor may be obtainable from suitable cereals, such as wheat, rye etc. Preferably, the xylanase inhibitor is a wheat endogenous xylanase inhibitor.

Preferably, the exogenous enzyme is removed from the surface of a grain. More preferably, the exogenous enzyme is removed from the surface of said grain by a treatment (e.g. decontamination) procedure - such as washing said grain with an aqueous solution, preferably water. In a highly preferred embodiment, the exogenous enzyme is removed from the surface of said grain by a treatment procedure, before said grain is milled into the cereal flour.

As used herein the phrase "cell wall components" refers to fibre and/or dietary fibre of grain and milled grain.

As used herein the term "cell wall components, of a milled grain, of improved quality" includes reference to a reduction in the decrease in the molecular weight of said cell wall components when said grain is milled.

SOME ADVANTAGES

One advantage of the present invention is that the amount and/or activity of an exogenous enzyme present on a grain is reduced.

A further advantage of the present invention is that the amount and/or activity of an exogenous enzyme present in a cereal flour is reduced.

A further advantage of the present invention is that the amount and/or activity of an exogenous enzyme present in a dough is reduced.

A further advantage of the present invention is that the effect of exogenous enzyme of the properties of a dough is reduced.

Advantageously, a decrease in the molecular weight of the cell wall components in a milled grain as described herein is reduced.

Advantageously, the amount of arabinoxylan degradation in a milled grain as described herein is reduced.

A further advantage of the present invention is that the amount of arabinoxylan degradation in a dough and/or a refrigerated dough is reduced.

Another advantage of the present invention is that the water retention of a dough and/or a refrigerated dough is enhanced and/or increased and the amount of syruping in a dough andlor refrigerated dough is reduced. s

Another advantage of the dough and/or refrigerated dough of the present invention is that the reduction in the exogenous enzyme in the cereal flour improves the viscosity of dough.

Advantageously the amount andlor activity of an exogenous enzyme activity present on the surface of a grain of the present invention can be determined.

Another advantage is that once the exogenous enzyme is removed, addition of an inhibitor (if desired) of the exogenous enzyme to the flour can be dosed more 1 5 accurately.

CEREAL FLOUR

As used herein, the phrase "cereal flour is substantially free of exogenous enzyme" means that the amount of the exogenous enzyme andlor activity of the exogenous enzyme in a cereal flour is reduced after the grain, before milling, has been treated (for example, by washing the grain in an aqueous solution) when compared to a milled grain which has not been treated (untreated grain)). For example, the amount of the exogenous enzyme andlor the activity of the exogenous enzyme in the cereal flour of the treated grain compared to the cereal flour of the untreated grain is reduced by at least 5%, 10%, 15% or 20%%. More preferably, the reduction is by at least 40%, 50%, 60%, 70%, 75%, 80%, 85%. In a more preferred embodiment, the reduction is by at least 90%.

The term "cereal flour" as used herein is a synonym for the finely-ground meal of a cereal grain. The term "grain", as used herein, is synonymous with the term "kernel".

As used herein, the phrase "grain for milling is substantially free of exogenous enzyme" means that the amount of the exogenous enzyme andlor the activity of the exogenous enzyme in a grain is reduced after the grain has been treated (for example, by washing the grain in an aqueous solution) when compared to a grain which has not been treated. For example, the amount of the exogenous enzyme and/or the activity of the exogenous enzyme in the treated grain compared to the untreated grain is reduced by at least 5%, 10%, 15% or 20%. More preferably, the reduction is by at least 40%, 50%, 60%, 70%, 75%, 80%, 85%. In a more preferred embodiment, the reduction is by at least 90%.

XYLANASES

Xylanases have been used in bakery for several years.

Xylanases are inter alla capable of catalysing the depolymerisation of arabinoxylan which may be present in cereal (e.g. wheat) - e.g. an enzyme that is inter alla capable of catalysing the solubilisation of water unextractable arabinoxylan (WU-AX) and catalysing the depolymerisation of water extractable arabinoxylan (WE-AX) which may be present in cereal (e. g. wheat).

In this regard, it is known that cereal flour (e.g. wheat flour) contains arabinoxylan originating from the endosperm cell walls. The amount of arabinoxylan in the flour differs depending on the origin of the flour for example, see Rouau et al, Journal of Cereal Science (1994), 19, 259272 Effect of an Enzyme Preparation Containing Pentosanases on the Breadmaking Quality of Flour in Relation to Changes in Pentosan Properties; Pincher and Stone, (1986) Advances in Cereal Technology, Vol. VIII (Y Pomeranz, Ed.) AACC, St Paul, Minnesota, 207-295; and Meuser and Suckow (1986), Chemistry and Physics of Baking (J.M.V. Blanchard, P J Frasier and T Gillard, Eds.) Royal Society of Chemistry, London, 42-61. Typically the amount of arabinoxylan can vary from 2-5% ((w/w) based on flour dry weight).

Fincher and Stone (1986, pp 207-295. In: Advances in cereal science and technology.

Vol. VIII. Pomeranz, Y. ed. Am. Assoc. Cereal. Chem., St. Paul. Minnesota, USA) report 70% of the polysaccharides in the endosperm cell wall are arabinoxylan. A characteristic feature of arabinoxylan is its ability to bind water. Part of the arabinoxylan is WU-AX and part is WE-AX. Experimental results have shown a positive correlation between degradation of WU-AX to high molecular weight (HMW) water soluble polymers and bread volume.

During the production of a dough product, it is known that using a xylanase at a proper dosage may result in a more stable dough system (which will typically comprise salt, flour, yeast and water) and a better volume of, for example, raised bread.

In this respect, a good xylanase for increasing bread volume should solubilise WU-AX giving an increased viscosity in the dough liquid without further degradation of WE AX into xylose oligomers. This degradation of WU-AX into low molecular weight (LMW) WE-AX is believed to be detrimental for the dough properties and may give rise to stickiness (Rouau et al and McCleary (1986) International Journal of Biological Macro Molecules, 8, 349-354).

US-A-5306633 discloses a xylanase obtained from a Bacillus subtilis strain.

Apparently, this xylanase may improve the consistency and increase the volume of bread and baked goods containing the same.

To date, fungal xylanases have been typically used in baking. For example, J Maat et al. (Xylans and Xylanases, edited by J Visser et al, 349-360, Xylanases and their application in bakery) teach a p-1,4-xylanase produced by an Aspergillus niger var.

awamori strain. According to these authors, the fungal xylanase is effective in increasing the specific volume of breads, without giving rise to a negative side effect on dough handling (stickiness of the dough) as can be observed with xylanases derived from other fungal or from bacterial sources.

Typically, to date, the dosages of xylanases added to the dough have been carefully calculated based on the amounts of the endogenous xylanases and endogenous xylanase inhibitors. Such calculations, however, do not take into account the activities of exogenous enzymes that may be present on the grain surface. Therefore, reducing the activity and/or amount of an exogenous enzyme such as xylanase will assist in improving the accuracy of such calculations.

In a preferred aspect of the present invention the reduction of the activity andlor amount of the exogenous enzyme, such as xylanase, can reduce or prevent the excessive enzymatic degradation of arabinoxylan present in cereal flour. For example, the reduction of the activity andlor amount of the exogenous enzyme may reduce the enzymatic degradation of arabinoxylan in the cereal flour of the treated grain compared to the cereal flour of the untreated grain by at least 5%. More preferably, the reduction is by at least 10%, 20% or 30%. In a more preferred embodiment, the reduction is by at least 40%, 50% or 75%.

An assay for determining endo--1,4-xylanase activity is presented below.

XYLANASE INHIBITORS

As indicated above, in a preferred aspect of the present invention the exogenous enzyme inhibitor that can reduce or prevent the enzymatic degradation of arabinoxylan present in the cereal flour is preferably a xylanase inhibitor.

The xylanase inhibitor may be any suitable xylanase inhibitor. A suitable assay for screening for suitable xylanase inhibitors is presented in a later section.

By way of example, the xylanase inhibitor may be the inhibitor described in WO-A 98/49278 andlor WO 01/98474 andlor the xylanase inhibitor described by Rouau, X. and Surget, A. (1998), McLauchlan, R., et al. (1999) and/or the xylanase inhibitor described in UK patent application number 9828599.2 (filed 23 December 1998), UK id patent application number 9907805.7 (filed 6 April 1999) and UK patent application number 9908645.6 (filed 15 April 1999) and WO01/52657.

DOUGH PRODUCT s

The present invention provides a process for preparing a foodstuffespecially a baked product from a refrigerated dough. Typical dough (such as baked) products in accordance with the present invention include bread - such as loaves, rolls, buns, pizza bases etc. - pretzels, tortillas, cakes, cookies, biscuits, crackers etc.

EXAMPLES

The present invention may now be described, by way of example only, in which reference may be made to the following figure: Figure 1. Microbial xylanase activity on the surface of wheat kernels.

METHODS

Cleaning of grain grams of cereal kernels are washed with 50 ml McIlvaine's buffer, by gently stirring for 5 minutes. Hereafter, the buffer is separated from the kernels by filtering on a glass filter. Kernels are washed further using tap water to remove buffer and remaining enzyme activity from the surface. The cleaned grain samples (hereafter referred to as washed) are dried at 40 C in an oven until water content is approx. 8 - 9 %. Control samples of the same cereal are dried under same conditions (hereafter referred to as control).

Milling to obtain whole meal flour Cereal samples (washed and control) are processed into whole meal flour (hereafter I referred to as WM) using an IKA mill (IKA Analysis Mill, A 10). 1 Milling to obtain milling fractionation To obtain industrially comparable milling fractions, cereal samples (washed and control) are milled using a Brabender mill (Brabender Quadrumat Senior mill). Before milling, the samples are conditioned to 12. 5% water content. Bran, shorts and flour fractions are obtained. I Cereal flour extraction 5-gram cereal flour (WM or flour) is extracted in 15 ml of water, by stirring. ; Depending on the further analysis, different extraction times and temperatures are used. For extraction of endogenous xylanase activity and of crude endogenous xylanase inhibitor preparations, an extraction time of 10 minutes at 20 C is used. For extraction of soluble flour components, extraction times of O. 2, 10 and 50 minutes at C are used. After extraction, the slurry in all cases is cooled to approximately 0 C and the solid phase is separated from soluble phase by centrifugation (10 minutes, 10000 x8, 4 C). The soluble phase is kept at 0 C or frozen until further analysis.

Xylanase activity measurement, TXU Xylanase activities are determined as follows. In short, samples are diluted in citric acid (0.1 M) - di-sodiumhydrogen phosphate (0.2 M) buffer, pH 5.0, to obtain approximately 0D = 0. 7 in the final assay. Three dilutions of the sample and an internal standard with a defined activity are thermostated for 5 minutes at 40 C. At time = 5 minutes, 1 Xylazyme tab (crosslinked, dyed xylan substrate) is added to the enzyme solution. At time = 15 minutes (or in some cases longer, depending on the xylanase activity present in the sample) the reaction is terminated by adding 10 ml of 2% TRIS/NaOH, pH 12. The reaction mixture is centrifuged and the OD of the supernatant is measured at 590 nm. Taking into account the dilutions and the reaction time, the activity (TXU, Total-Xylanase-Units) of the sample can be calculated relative to the standard.

Xylanase inhibitor preparation Cereal flour was extracted as mentioned under "Cereal flour extraction" above, to produce a crude preparation of endogenous cereal Xylanase inhibitor. The extract is analysed according to the "Xylanase inhibitor activity determination" described below.

Inhibitor activity is determined to 126 XIU/ml.

Xylanase inhibitor activity determination To quantify the concentration of the endogenous Xylanase inhibitor the below assay is used.

1 XIU (Xylanase Inhibitor Unit) is deft ned as the amount of inhibitor that decreases I TXUto 0.5 TXU under the conditions described below.

The Xylanase used in this assay is preferably a Bacillus subtilis XynA (Accession no. P18429).

250,u1 Xylanase solution (containing 17 TXU/ml), 1001 Xylanase inhibitor solution and McIlvaine's buffer, pH 5, to reach a reaction volume of 10001 is pre-incubated for minutes at 40 C. At time = 5 minutes, 1 Xylazyme tablet is added to the reaction mixture. At time = 15 minutes the reaction is terminated by addition of 10 ml 2% TRIS/NaOH, pH 12. The solution is filtered and the absorbance of the supernatant is measured at 590 nm. By choosing several different concentrations of inhibitor in the above assay, it is possible to create a plot of OD versus inhibitor concentration. Using the slope (a) and intercept (b) from this plot and the concentration of the Xylanase it is possible to calculate the amount of XIU in a given inhibitor solution (equation 1).

Equation 1 XIU = ((bl2)1-a)lx x = TXU in assay (approx. 3 TXU/ml assay volume) Xylanase inhibiffon analysis s Varying amounts of a xylanase inhibitor fraction, 250 pi xylanase solution (containing 17 TXU microbialxylanase/ml, if possible, if not a longer incubation time is used) and buffer (0.1 M citric acid - 0.2M all-sodium hydrogen phosphate buffer, pH 5.0) to a final volume of 1000 pi are mixed. The mixture is thermostated for 5 minutes at 40 C.

At time = 5 minutes one Xylazyme tab is added. At time = 15 minutes (or longer if xylanase concentration is low) the reaction is terminated by adding 10 ml 2% TRIS.

The reaction mixture is centrifuged (3500 g, 10 minutes, room temperature) and the I supernatant is measured at 590 nm. The inhibition is calculated as residual activity compared to the blank. The blank is prepared in the same way, except that the inhibitor is substituted with 100 Ill buffer (0.1 M citric acid - 0.2 M all-sodium hydrogen phosphate buffer, pH 5.0).

Viscosity determination of flour extracts The viscosity of the cereal flour extract is measured using a Brookfield cone and plate viscosimeter (Brookfield model DV-III, Programmable Rheometer). The samples are kept on ice, to avoid any further xylanase activity in the sample. Measurements are done at 10 C.

MATERIALS

Wheat samples Thirteen commercial wheat samples collected from commercial millers and feed producers were used in the examples described below. The wheat samples and their origin are summarised in table 1.

Table 1. Wheat samples used with information on variety, origin and comments.

Sample No. Variety | Country Comment 1 Tarso Sweden Winter wheat Northern spring USA Spring wheat Durum Russia Winter wheat Tarso Sweden Winter wheat Durum Spain 6 KV Sweden Spring wheat KV Sweden Spring wheat Ritmo Denmark Winter wheat Solist Denmark Yacht Denmark 11 Ritmo Denmark Winter wheat 12 Ure Denmark 13 Kris Denmark EXAMPLE 1 - Variation In Xylanase Activity On The Surface Of Wheat By analysing the xylanase activity in the wash water obtained from washing the wheat samples mentioned in table 1, different levels of activity was found. Due to the nature of the methodology used, the activity found in the wash water was expected to be of microbial origin. The process used did not damage the pericarp of the wheat kernels thus, no extraction of endogenous wheat xylanase activity can have occurred.

Table 2. Microbial xylanase activity on the surface of wheat kernels. For reference to wheat batch see table 1.

Wheat Batch TXU/kg st. dev.

1 24259 2 65238 3 60439 4 23592 55124 6 25638 7 880279 8 712 9 5512 3499 11 482353 12 12623 13 31077 The results from table 2 are illustrated in figure 1.

EXAMPLE 2 - Variation In Xylanase Activity In Whole Meal Flour Samples The variation seen in table 2 (more than 10 fold) can not be accounted for by the variation in endogenous wheat xylanase activity. It was therefore of interest to study the effect of washing the grain, before milling, on the level of endogenous wheat xylanase activity. Analysing xylanase activity in the flour instead of the activity on the surface brings another variable into consideration, the endogenous wheat xylanase inhibitor. Milling the grain makes the inhibitor extractable. The xylanase activity determined in the control WM may be expected to be higher than the activity found in the washed WH, because of the removal of activity during the washing step. However, as can be seen from table 3, there is not much difference in the xylanase activities found in the washed and control flours. This may be due to inhibition of the microbial xylanase activity found on the surface of the wheat by the endogenous wheat xylanase inhibitor.

Table 3. Xylanase activity extractable from control and washed WMflour. Data represent average of a replica, analysis error is expressed as standard deviation (qt.

dev.).

Wheat batch Control st dev Washed st dev 1 166 4 134, 3 2 656 6 346 13 3 446 4 245 16 4 260 11 152 47 152 1 104 15 6 259 9 163 9 7 424 103 315 21 8 493 34 276 9 9 447 13 171 35 359 20 226 41 11 925 33 832 18 12 241 4 133 30 13 589 16 363 35 EXAMPLE 3 - Differences In The Sensitivity Of Exogenous Xylanases Towards Endogenous Xylanase Inhibitors To study the nature of the xylanase activities found on the surface of the wheat samples, the sensitivity of xylanase populations to endogenous xylanase inhibitors was evaluated. As can be seen from table 4, the xylanase populations found on the surface of the wheat differ significantly in their sensitivity towards the xylanase inhibitor.

Some of the xylanases populations are highly sensitive others are much less sensitive.

The difference in inhibitor sensitivity between xylanase populations indicates different xylanase populations and thus different microbial populations.

Table 4. Sensitivity of Placate activity extractedfrom wheat grain surface towards endogenous wheat xylanase inhibitors. Relative residual xylanase activity (a/) is illustrated as a function of xylanase inhibitor activity (MU).

Xylanase inhibitor addition, XIU/ml Sample No. Variety 0 2,5 5 10 Tarso 100 44 32 26 Northern 2 spring 100 67 56 44 Durum 100 56 44 35 Tarso 100 81 76 67 Durum 100 44 35 23 6 KV 100 66 55 45 7 KV 100 59 45 35 8 Ritmo 100 88 82 73 Solist 100 n.d. 88 78 Yacht 100 47 40 26 11 Ritmo 100 73 66 53 12 Ure 100 40 30 22 13 Kris 100 43 33 24 EXAMPLE 4 - Higher Levels Of Viscosity In Flour Slurries From Flour With A Reduced Level Of Exogenous Xylanase Compared To Control Flour To evaluate the impact of the xylanase population found on the surface of the wheat, viscosity was evaluated in flour slurries of the selected washed and control WM flours

(table 5).

Surprisingly, the difference in viscosity between the flour slurries produced from the washed and control flours differed significantly. The difference may both be observed as the initial viscosity and the change in viscosity as a function of stirring time. In all cases, the flour slurries produced from the control WM flours (having different levels of microbial xylanase activity) had a lower initial viscosity. Furthermore, only moderate viscosity increases may be detected in the slurries produced from the control WM flour, compared to the slurries produced from washed WM flour. The absolute; viscosity of the slurries vary, due to different composition in damaged starch etc., but the impact of the microbial xylanase activity from the surface is clear for all flour - the viscosity is lowered due to depolymerisation of arabinoxylan. Furthermore, the endogenous wheat xylanases may be more specific for the water unextractable arabinoxylan, since a net viscosity increase is seen from all washed WM flour.

Alternatively, the endogenous wheat xylanase may have a low activity, a higher activity might depolymerise the AX population.

Table 5. Effect of removing the surface microbial xylanase activity of the wheat kernels on the viscosity of WMflour slurries. Slurries stirredfor 2, 10 and 50 minutes.

Sample nomenclature: 5Wis identical to WHflour obtainedirom washed wheat 5; 5C is identical to WMflour obtainedirom control wheat 5, etc. Tim e, mint 5 W S C 7 W 7 C W C 9 W 9 C 11 W 11 C. 2 2,5 5,0 3,6 3,9 3,4 3,3 3,0 l 3,5 2,7 4,6 4,2 2,7 5,2 3,3 2,9 5,0 2,8 6,0 4,6 3,2 6,0 3,8 5,8 The surface microbial xylanase contamination has an effect on the viscosity of the flour slurries. Removing the microbial xylanase contamination from the wheat before processing the wheat into whole meal flour prevents a significant drop in flour slurry viscosity. Interestingly, the absolute level of xylanase found on the surface does not correlate with the changes seen in the flour slurry viscosity.

Sample No 7 (or wheat 7) has a high level of xylanase activity on the surface of the grain whereas sample No 8 (or wheat 8) has a low level of xylanase activity on the surface of the grain. However, as can be seen, both flours respond to the removal of surface xylanase activity, by increased viscosity. Actually, the wheat having the lowest xylanase activity on the surface (wheat 8) seems to respond the most. As mentioned earlier, the system is very complex, because the two flours also contain different levels of endogenous xylanase inhibitor. Wheat 7 actually has more than the double inhibitor activity than wheat 8. Furthermore, as already indicated in table 4, the xylanase population differs between wheat batches, the xylanase population on the surface is inhibited to different extends.

All the data presented above, indicate that there are different levels of microbial contaminations (and populations) on the surface of wheat. This has been demonstrated before. What is new is that this microbial contamination of the wheat produces enzyme activities and/or amounts of enzyme (i.e. an exogenous enzyme) that have a significant influence on the technological properties of the flour that can be obtained from this wheat. However, the above data are all from whole meal flour, including the outer layers of the kernels, the bran fraction. This means that an enzyme activity on the surface of the kernel will end up in the flour. However, most flour used in the production of bread is only based on the endosperm of the kernels, the bran fraction is excluded. To test if this has any influence on our findings, milling fractions were produced.

EXAMPLE 5 - Reduced Exogenous Xylanase Activity In Milling Fractions And Increased Viscosity Of Flour Slurries Of Flour From Washed Grain A wheat batch mixture having high xylanase activity on the surface (hereafter referred to as high) and a batch having low activity on the surface (hereafter referred to as low) was produced. The xylanase activities in the two wheat batches are listed in table 6.

Table 6. Surface xylanase activity (TXU/kg) on wheat batches produced to represent a high and low level of activity.

Wheat batch TXU/kg High 919 Low 281 The wheat batches were split in two, one part was washed as described above and the other part left as a control. Thus, four fractions were obtained, a washed and a control fraction of the wheat batch with high xylanase activity on the surface (hereafter referred to as high wash and high control, respectively) and a washed and control fraction of the wheat batch having low xylanase activity on the surface (hereafter referred to as low wash and low control, respectively).

To evaluate if the impact of different xylanase populations is limited to WM flour the experiments were repeated with flour fractions obtained through milling fractionation of the wheat samples.

Table 7. Effect of removing (washing) the surface microbial xylanase activity of High wheat kernels on the viscosity of WMflour slurries andflour obtained by milling fractionation of wheat. Compared to control samples. In both cases the xylanase level in the control was high. Slurries were stirredfor 2, 10 and 50 minutes and viscosity (cP) determined at 10 C.

Time, minutes WH - wash WH - control Flour - wash Flour - control 2 3,1 1 2,70 4,49 4,17 3,68 3,26 4,35 4,25 5,12 3,92 5,63 4,29 Table 8. Effect of removing (washing) the surface microbial xylanase activity of Low wheat kernels on the viscosity of WMflour slurries andflour obtained by milling fractionation of wheat. Compared to control samples. In both cases the xylanase level in the control was low. Slurries were stirredfor 2, 10 and 50 minutes and viscosity 5(cP) determined at 10 C.

Time, minutes WH - wash WH - control Flour - wash Flour - control 3,82 3,44 6,46 6,45 4,05 4,16 6,89 6,59 4,44 4,96 7,21 7,34 1 As can be seen from tables 7 and 8, the absolute viscosity (cP) differs between flour slurries produced from whole meal flour and flour. The higher absolute viscosity obtained from flour is probably due to other constituents than arabinoxylan, e.g. damaged starch. Furthermore, it can be seen that in both flours produced (whole meal and flour) from wheat having a high xylanase activity on the surface, the importance of removing the surface xylanase activity is highly significant. Removing the surface xylanase by washing before processing the wheat into flour, results in a higher viscosity of the flour slurries. Since this can be seen for both whole meal flour and flour, it can be concluded that the microbial xylanase on the surface of the kernel ends up in the flour fraction after milling fractionation of the wheat.

Compared to the significant results obtained from the wheat high in microbial xylanase activity, the results obtained from the wheat low in microbial xylanase activity are less consistent. However, some of the results indicate a higher flour slurry viscosity in the I washed samples. The less significant results obtained from the wheat low in microbial xylanase activity are of course expected. Analysis of the TAXI-like xylanase inhibitor level in the two wheat samples, however, explained the difference in "functionality" of the microbial xylanase levels in the two wheat batches. The high xylanase wheat batch only contained approximately half the inhibitor concentration of the low xylanase wheat batch, so in addition to the high xylanase activity in this wheat batch, the counteracting inhibitor level was low (table 9).

Table 9. TAM-like xylanase inhibitor level in whole meal (WM)flour orflour obtained from wheat high or low microbial xylanase activity, before (control) or after removal of microbial xylanase activity.

Flour XIU/g High flour - washed 287 High flour- control 343 High WM washed 313 High WM - control 309 Low flour- washed 457 Low flour- control 495 Low WM - washed 622 Low WM - control 607

CONCLUSION

Based on the findings in these examples, it can be concluded that different levels of microbial xylanase activity can be isolated from the surface of commercial wheat samples used for production of commercial flour or for feed. The activity found on the surface is probably directly correlated to the environmental conditions under which the wheat was grown, harvested and stored. The higher the humidity during growth, harvest and storage, the higher the microbial enzyme activity on the surface of the cereal can be expected. Furthermore, from the data shown here, it can be concluded that microbial xylanase activity has an impact on the technological properties of the flour produced from this wheat. Finally it can be concluded that the microbial xylanase activity is not excluded from the flour during the milling fractionation of wheat, even though the pericarp or bran is excluded from this fraction. Due to the mechanical treatment during the milling process, microbial xylanase activity will as most other small particles end up in the flour.

Some of the variations in flour performance/quality seen from batch to batch and from year to year may be due to different levels of microbial load and thus, different enzyme activities.

A direct impact of these finding is in the production of refrigerated dough. The presence of microbial xylanase activity is not detectable in the flour, however, it has an impact on the arabinoxylan population in the dough and hence on the water holding capacity of the dough. High microbial xylanase activity will no doubt cause syruping.

The influence of microbial activity may also influence other parameters such as flour falling number, flour stability, dough consistency, etc. One way of utilising the findings described here is to remove the microbial contamination from wheat before processing. Such a cleaning may be facilitated through washing of the grains, mechanical treatment combined with wind sieving or even pearling.

Another way to utilise the finding is to ensure that the right cereal batches are processed for the right application. This may be done by simple analysis of the microbial enzyme activity on the cereals, before processing. Thus, it will be possible to obtain flour batches low in microbial enzyme activity for special applications as e.g. refrigerated dough.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

REFERENCES

Flatman, R., McLaughlan, W.R., Juge, N., Furniss, C., Berrin, J-G., Hughes, R.K., Manzanares, P., Ladbury, J.E., O'Brien, R. and Williamson, G. (2002). Biochem. J. 365 (3), 773-781.

Gebruers, K., Debyser, W. Goesaert, H., Proost, P., Van Damme, J., Delcour, J.A.

Triticum aestivum L. endoxylanase inhibitor (TAXI) consists of two inhibitors, TAXI I and TAXI II, with different specificities, 2001, Biochemical Journal, 353, 239-244.

Gebruers, K. Endoxylanase inhibitors in wheat (Triticum aestivum L.): isolation, characterization and use for endoxylanase purification, 2002, PhD dissertation, K. U. Leuven.

Gebruers, K., Goesaert, H., Brijs, K., Courtin, C.M., and Delcour, J.A. Purification of TAXI-like endoxylanase inhibitors from wheat (Triticum aestivum L.) whole meal using cation exchange and affinity chromatography with immobilized Bacillus subtilis endoxylanase, 2002a, Journal of Enzyme Inhibition and Medicinal Chemistry, 17, 61- 68.

Gebruers, K., Courtin, C.M., Goesaert, H., Van Campenhout, S. and Delcour, J.A.

Endoxylanase inhibition activity in different European wheat cultivars and milling fractions, 2002b, Cereal Chemistry, 79 (5), 613-616.

Goesaert, H., Debyser, W., Gebruers, K., Proost, P., Van Damme, J., and Delcour, J.A.

Purification and partial characterization of an endoxylanase inhibitor from barley, 2001, Cereal Chemistry, 78(4), 453-457.

Sibbesen, O. and S0rensen, J. F. Proteins, 2000, Patent application WO 00/39289, Danisco A/S.

Sibbesen, O. and Soerensen, J.F. 2001, Enzyme. Patent application WO 01/66711, Danisco A/S

Claims (1)

1) A cereal flour wherein said cereal flour is substantially free of an exogenous enzyme.

2) A cereal flour according to claim 1 wherein said exogenous enzyme is a xylanase.

3) A cereal flour according to any one of claims I to 2 wherein said cereal flour is a milled grain of one or more of the following grains: wheat, barley, oats, corn and rye.

4) A cereal flour according to any one of the preceding claims wherein said exogenous enzyme is an enzyme present on the surface of a grain before said grain is treated and milled into cereal flour.

5) A cereal flour according to claim 4 wherein said grain is treated by washing said grain with an aqueous solution.

6) A cereal flour according to claim 5 wherein said aqueous solution is water.

7) A dough prepared from the cereal flour according to any one of claims 1 to 6.

8) A dough according to claim 7 wherein said dough is a refrigerated dough.

9) A dough product prepared from the cereal flour according to any one of claims 1 to 6 or the dough according to claim 7 or claim 8.

10) A dough product according to claim 9 wherein said product is bread.

1 1) A process of preparing a dough comprising the steps of admixing the cereal flour according to any one of claims 1 to 6 with one or more other dough ingredients.

12) Process according to claim 11 wherein said dough is or is processed as a refrigerated dough.

13) A process of preparing a dough comprising the steps of milling a grain into a cereal flour and admixing the cereal flour according to any one of claims 1 to 6 with one or more other dough ingredients; wherein said grain for milling is substantially free of exogenous enzyme.

14) Process according to claim 13 wherein said dough is or is process into a refrigerated dough.

15) Process according to any one of claims 13 to 14 wherein said dough is then baked.

16) A process of preparing a dough comprising the steps of i) treating a grain to substantially remove an exogenous enzyme ii) milling the treated grain into a cereal flour iii) admixing the cereal flour with an aqueous solution, preferably water.

17) Process according to claim 16 wherein said exogenous enzyme is a xylanase.

18) Process according to any one of claims 16 to 17 wherein said grain is one or more of the following: wheat, rye, oats, corn and barley.

19) Process according to any one of claims 16 to 18 wherein said exogenous enzyme is present on the surface of the grain before said grain is treated and milled into cereal flour.

20) Process according to any one of claims 16 to 19 wherein said dough is or is processed into a refrigerated dough.

21) Process according to any one of claims 16 to 20 wherein said dough is then baked.

22) Process according to any one of claims 16 to 21 wherein said grain is treated by washing said grain with an aqueous solution, preferably water.

23) A method of determining a reduction in activity and/or amount of an exogenous enzyme on a grain comprising the steps of i) optionally treating the grain, ii) milling the grain into a cereal flour iii) extracting the cereal flour in an aqueous solution, preferably water, iv) measuring the activity and/or amount of the exogenous enzyme of the cereal flour extraction v) comparing the activity and/or amount of the exogenous enzyme of an untreated grain with the activity and/or amount of the exogenous enzyme of a treated grain.

24) A method according to claim 23 wherein said exogenous enzyme is a xylanase.

25) A method according to any one of claims 23 to 24 wherein said grain is one or more of the following: wheat, barley, oats, corn and rye.

26) A method according to any one of claims 23 to 25 wherein said exogenous enzyme is removed from the surface of said grain by treatment before said grain is milled into the cereal flour.

27) A method according to any one of claims 23 to 26 wherein said optional treatment of a grain is by washing the grain with an aqueous solution, preferably water.

28) Use of a treatment procedure for the removal of an exogenous enzyme from a grain.

29) Use according to claim 28 wherein said exogenous enzyme is a xylanase.

30) Use according to any one of claims 28 to 29 wherein said grain is one or more of the following: wheat, barley, oats, corn and rye.

31) Use according to any one of claim 28 to 30 wherein said exogenous enzyme is S removed from the surface of the grain by a treatment procedure comprising washing the grain with an aqueous solution, preferably water.

32) Use of a treatment procedure in the preparation of a cereal flour wherein said treatment procedure reduces the amount and/or activity of an exogenous enzyme in said cereal flour, preferably wherein said cereal flour is substantially free of said exogenous enzyme. I 33) Use according to claim 32 wherein said exogenous enzyme is a xylanase.

34) Use according to any one of claims 32 to 33 wherein said grain is one or more of the following: wheat, barley, oats, corn and rye.

35) Use according to any one of claims 32 to 34 wherein said exogenous enzyme is removed from the surface of a grain by the treatment procedure before said grain is I milled into cereal flour.

36) Use according to any one of claims 33 to 35 wherein said treatment procedure comprises washing said grain with an aqueous solution, preferably water.

37) Use of a cereal flour that is substantially free of an exogenous enzyme in the I preparation of a dough, preferably a refrigerated dough.

38) A cereal flour according to claim 37 wherein said exogenous enzyme is a xylanase. I 39) A cereal flour according to any one of claims 37 to 38 wherein said cereal flour is a milled grain of one or more of the following grains: wheat, barley, oats, corn and rye.

40) A cereal flour according to any one of claims 37 to 39 wherein said exogenous enzyme is an enzyme present on the surface of a grain before said grain is treated and milled into the cereal flour.

41) A cereal flour according to claim 40 wherein said grain is treated by washing said i grain with an aqueous solution, preferably water.