METHOD FOR PREPARING FOOD PRODUCTS COMPRISING RYE

Abstract

The present invention relates to a method for the preparation of a food product comprising rye, which comprises the steps of preparing a primary food mixture; adding to said primary food mixture a composition comprising at least one glycoside hydrolase family 10 (GH10) enzyme; and processing said primary food mixture to produce said food product comprising rye. The invention further provides GH10 enzymes, compositions comprising said enzymes and the use of said enzymes and said composition in preparing food products.

Claims

1. A method for the preparation of a food product comprising rye, said rye comprising arabinoxylan, and said method comprising the steps of: preparing a primary food mixture; adding to said primary food mixture a composition comprising at least one glycoside hydrolase family 10 (GH10) enzyme; and processing said primary food mixture to produce said food product comprising rye; wherein the addition of said at least one GH10 enzyme results in dough improvement.

2. The method of claim 1, wherein said at least one GH10 enzyme has hemicellulolytic activity, preferably xylanolytic activity.

3. The method according to claim 1, wherein said processing means treating of said primary food mixture with heat, such as baking, steaming, cooking or otherwise heating.

4. The method according to claim 1, wherein said composition comprising at least one GH10 enzyme is added during the mixing and/or blending of the primary food mixture.

5. The method according to claim 1, wherein said composition comprising at least one GH 10 enzyme further comprises another agent, which is selected from the group consisting of other enzymes, hydrocolloids, emulsifiers, oxidants, fats and lipids, flavors, (poly)saccharides, proteins, salts and acids, leavening agents, milk and cheese products or a mixture thereof.

6. The method according to claim 1, wherein said at least one GH10 enzyme is added to said primary food mixture in a form selected from the group consisting of a cell extract, a cell-free extract, a partially purified protein and a purified protein.

7. The method according to claim 1, wherein said composition comprising at least one GH10 enzyme further comprises an enzyme carrier and optionally a stabilizer and/or a preservative and/or another agent selected from extenders, fillers, binders, flavour maskers, bitter blockers and activity enhancers.

8. The method according to claim 1, wherein said at least one GH10 enzyme is isolated from a microorganism.

9. The method according to claim 1, wherein said at least one GH10 enzyme is a recombinant enzyme.

10. The method according to claim 1, claims, wherein dough improvement means improving dough processing such that dough stability is increased, dough resistance to extension is reduced, stickiness is reduced and/or improving the quality of final food products, such that the final food products show a less compact structure, an increased softness, a volume increase, a homogeneous pore distribution and/or a softer crumb structure.

11. The method of claim 1, wherein dough improvement means a dough stability increase within the range of 115% and 225% , a reduction of dough resistance to extension within the range of 9% and 30%, a reduction of dough stickiness within the range of 8% and 18%, a reduction of crumb hardness within the range of 18% and 49% and/or a volume increase within the range of 108% and 122%, when compared to dough processed without the GH 10 enzyme of the present invention.

12. Use of a GH10 enzyme in the production of food products comprising arabinoxylan.

13. The method according to claim 1, wherein said GH10 enzyme comprises or consists of a polypeptide which has at least 75% amino acid sequence identity to a polypeptide selected from SEQ ID NO: 1 -6 and which shows hemicellulolytic activity.

14. A GH10 enzyme with hemicellulolytic activity, which comprises or consists of a polypeptide which has at least 75% amino acid sequence identity to the polypeptide according to SEQ ID NOs 4 to 6, with the proviso that the GH10 enzyme is not the polypeptide of SEQ ID NO: 1, 2 or the polypeptide of SEQ ID NO: 3.

15. A nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding the GH10 enzyme as claimed in claim 14.

16. An expression vector comprising the nucleic acid molecule as claimed in claim 15.

17. A host cell comprising the nucleic acid of claim 15, wherein said host cell expresses the GH10 enzyme.

18. A method of producing a GH10 enzyme, the method comprising culturing a host cell according to claim 17 under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.

Description

[0186] The present invention is further described by the following figures and examples that should not be construed as limiting the scope of the invention.

[0187] Herein,

[0188] FIG. 1 shows the relative enzyme activity increase (%) of SEQ ID NOs 4 to 6 enzymes in relation to the enzyme activity of the wildtype enzyme of SEQ ID NO 1. Surprisingly, the enzyme activity of the SEQ ID NOs 4 to 6 enzymes increases 4-8 fold in comparison to enzyme activity of the SEQ ID NO 1 enzyme. Enzyme activity: the release of micromol reducing sugars from arabinoxylan per mg of enzyme per minute was determined with the DNSA assay. Enzyme activity was measured in triplicates.

[0189] FIG. 2 shows an SDS-PAGE of recombinantly produced enzymes of SEQ ID NOs 1, 4, 5 and 6, respectively. PageRuler Prestained Protein Ladder 10 to 180 kDa (#26616, ThermoFischer Scientific) was used as protein standard.

[0190] FIG. 3 shows the relative enzyme activity of GH10 enzymes of SEQ ID NOs 2 and 3 compared to the GH family 11 enzymes SEQ ID NO 7 and Pentopan Mono BG*. The activity of the SEQ ID NO 6 xylanase was set to 100%. Enzyme activity: the release of micromol reducing sugars from arabinoxylan per mg of enzyme per minute was determined with the DNSA assay. Enzyme activity was measured in triplicates.

[0191] FIG. 4 shows the results of using GH10 enzymes SEQ ID NOs 2, 3 and 6, compared to the GH family 11 enzymes of SEQ ID NO 7 and Pentopan Mono BG*, on the bread volume in a method of baking rye bread. The enzyme concentration used was 0.1 ppm each. Baking and volume estimations were made in triplicates. The standard bread volume w/o enzyme addition was set to 100%. Means with different letters are significantly different (One-Way ANOVA followed by Tukey test, p<0.05, n=3).

[0192] FIG. 5 shows a photograph of rye bread, baked at a pH of 4.3 with 0.13 ppm GH10 enzyme of SEQ ID NO 6 (right), compared to the standard w/o addition (left) of a GH10 enzyme

[0193] FIG. 6 shows the crumb improving effect of using 0.13 ppm GH10 enzyme of SEQ ID NO 6, compared to the standard w/o addition of a GH10 enzyme, by reducing the crumb hardness in a method of baking rye bread at a pH of 4.3. Hardness calculations were made in duplicates.

[0194] FIG. 7 shows the impact of GH10 and GH11 Xylanases on the stability of rye dough samples in a torque-measuring Z-kneader. Means with different letters are significantly different (One-Way ANOVA followed by Tukey test, p<0.05, n=4).

[0195] FIG. 8 shows the impact of GH10 Xylanase with the amino acid sequence of SEQ ID NO:

[0196] 6 of different xylanase quantities 0.0 ppm (.circle-solid.), 0.01ppm () 0.1 ppm (), on the stability on rye-wheat doughs at a ratio of 70% rye and 30% wheat (A) and 10% rye and 90% wheat (B) dough samples in a torque-measuring Z-kneader. Means with different letters are significantly different (One Way ANOVA followed by Turkey test p<0.05, n=2).

[0197] FIG. 9 shows the impact of different xylanase quantities 0.0 ppm (.circle-solid.), 0.01ppm () 0.1 ppm (), on the stickiness of rye-wheat (ratio of rye (R) to wheat (W) 30:70%) dough samples determined by using the Ta.XT plus Texture Analyzer (Stable Micro Systems Ltd., Godalming, UK) equipped with a Chen-Hoseney Dough Stickiness Rig. Means with different letters are significantly different (One-Way ANOVA followed by Tukey test, p<0.05, n=2).

[0198] FIG. 10 shows the impact of different xylanase quantities 0.0 ppm (.circle-solid.), 0.01ppm () 0.1 ppm (), on the resistance to extension R.sup.K.sub.max of rye-wheat (ratio of rye (R) to wheat (W) A) 10:90%, B) 30:70%, and C) 70:30%) dough samples determined by using the Ta.XT plus Texture Analyzer (Stable Micro Systems Ltd., Godalming, UK) equipped with a SMS/Kieffer Extensibility Rig. Means with different letters are significantly different (One-Way ANOVA followed by Tukey test, p<0.05, n=2).

EXAMPLE 1

Cloning of SEQ ID NOs 14-19 Encoding Xylanases

Materials

[0199] Chemicals used as buffers and substrates were commercial products of at least reagent grade. Escherichia coli DH1OB was used for routine cloning and E. coli BL21 Star (DE3) for expression of Clostridium thermocellum DSM1237 (also known as Ruminiclostridium thermocellum DSM1237) and Herbivorax saccincola DSM101079 xylanase genes of SEQ ID NO 14 an SEQ ID NOs 16-19 respectively. Synthetic DNA for cloning was received from Integrated DNA Technologies (Leuven, Belgium). Expression of Fusarium verticilloides xylanase of SEQ ID NO 15, Pichia pastoris X33 (ThermoFisher) was used. Aeromonas punctata GH10 xylanase of SEQ ID NO 3 was purchased from Megazyme (E-XYNAP, Megazyme, Bray, Ireland) and Pentopan Mono BG* was purchased from Sigma-Aldrich (Sigma Aldrich, St. Louis, Mo., USA)

DNA Modification

[0200] Preparation of chromosomal and plasmid DNA, endonuclease digestion, and ligation were carried out by standard procedures (Sambrook J and Russell D W. 2001).

Cloning of Genes Encoding GH10 and GH11 Xylanases

[0201] The genes with the nucleic acid sequences of SEQ ID NOs: 14, 16 to 18 encoding GH10 xylanases were amplified from chromosomal DNA from C. thermocellum DSM1237 in accordance with manufacturer's instructions (Phusion High-Fidelity DNA Polymerase, F530S, ThermoFisher Scientific) using primer set of SEQ ID NOs: 8 and 9 and SEQ ID NOs: 10 and 9, primer set SEQ ID NOs 8 and 11 and primer set SEQ ID Nos 10 and 11, respectively. With the SEQ ID NO 4 to 6 enzymes, the effect of different protein module deletions on enzyme activity and function was investigated. The cellulose binding domain was deleted in the SEQ ID NO 4 enzyme. The dockerin module was deleted in the SEQ ID NO 5 enzyme and both modules were deleted in in the SEQ ID NO 6 enzyme. All PCR products were subsequent cloned by Gibson assembly (NEB, Cat. Nr. E2611S) in NdeI/XhoI digested pET24c(+) vector (Novagen, MerckMillipore) and sequenced by Eurofins to confirm the correct sequence.

[0202] The DNA coding for sequence SEQ ID NO 15 of Fusarium verticilloides GH10 xylanase was deduced from the amino acid sequence disclosed in WO2014019219A1. DNA was synthesized by Integrated DNA Technologies, inserted in Sa/I/EcoRI digested pICZa A (EasySelect Pichia Expression Kit) using Gibson assembly (NEB, Cat. Nr. E2611S) and sequenced by Eurofins to confirm the correct sequence.

[0203] The coding sequence SEQ ID NO 19 of GH11 xylanase from Herbivorax saccincola was amplified from the respective chromosomal DNA of strain DSM101079 employing primer pair of SEQ ID NOs: 12 and 13. A new cellulose degrading bacterial strain named Herbivorax saccincola was isolated from a 20 I fermenter operated with cow manure and fed with maize silage at 55 C. (Koeck et al. 2016). For genome sequencing of the strain Herbivorax saccincola, a total of 4 microgram genomic DNA was used to construct an 8-k mate-pair sequencing library (Nextera Mate Pair Sample Preparation Kit, Illumina Inc.), which was sequenced applying the paired-end protocol on an Illumina MiSeq system. Analysis and interpretation of the Herbivorax saccincola genome sequence within GenDB and by means of the Carbohydrate-active-enzyme database dbCAN (Yin et al., 2012) revealed more than 100 genes predicted to encode enzymes that mainly belong to different families of Glycoside Hydrolases (GH) and Carbohydrate-Binding Modules (CBM). One of the enzymes (SEQ ID NO 7) was identified as glycoside hydrolase family 11 member.

Construction of Pichia Pastoris X33 Recombinant Strains Expressing the SEQ ID NO 15 Xylanase.

[0204] To transform P. pastoris strain X33 pICZalpha A containing the nucleic acid of SEQ ID NO 15 was linearized with SacI and used to transform electro competent P. pastoris cells (Lin-Cereghino et al., 2005). Clones growing on antibiotics containing YPDS Agar (1% (wt/vol) yeast extract, 2% (wt/vol) peptone and 2% (w/v) dextrose, 1M sorbitol and 1.5% (wt/vol) agar) were screened for insertion of the nucleic acid of SEQ ID NO 15 using colony PCR with primers provided in the EasySelect Pichia Expression Kit. Positive clones were selected for production of SEQ ID NO 2 enzyme.

EXAMPLE 2

Protein Production of the Enzymes of SEQ ID NOs 1-7

Growth of Cells

[0205] Fed-batch fermentations of recombinant E. coli strains harbouring the GH10 Xylanase genes from C. thermocellum ATCC27405/DSM1237 of SEQ ID NOs 14, 16-18 and Herbivorax saccincola DSM101079 xylanase gene SEQ ID NO 19 were carried out in a 10 L Uni-Vessel controlled and equipped with a Biostat B Twin DCU (Sartorius AG, Gttingen, Germany). Temperature, pH, foam, turbidity, weight and dissolved oxygen were monitored online during fermentation. The dissolved oxygen (DO %) was set to 25% (vol/vol) and maintained with increasing agitation and constant air flow. The formation of foam was controlled by the addition of Antifoam 206 (Sigma Aldrich, St. Louis, Mo., USA). A pH of 6.9 was maintained by addition of a 25% (vol/vol) ammonium hydroxide solution and 25% (vol/vol) HPO4 solution. E. coli strains were cultivated in Riesenberg medium (Korz et al., 1995) at the 10 L scale, the feeding solution consists of 1021 g/L glycerol, 20 g/L MgSO.sub.4.7 H.sub.2O, 13 mg/L EDTA, 4 mg/L CoCl.sub.2. 6 H.sub.2O, 23.5 mg/L MnCl.sub.2.4 H.sub.2O, 2.5 mg/L CuCl.sub.2.2 H.sub.2O, 5 mg/L H.sub.3BO.sub.3, 4 mg/L Na.sub.2MoO.sub.42 H2O, 16 mg/L Zn(CH.sub.3COO).sub.2.2 H.sub.2O, 40 mg/L Fe(III)citrate (Korz et al., 1995). After the consumption of the initial carbohydrate substrate, growth rate was controlled according to EQUATION 1, whereby ms, is the mass flow of substrate (g h.sup.1), .sub.set the desired specific growth rate (h.sup.1), Y.sub.X/S the biomass/substrate yield coefficient (g g.sup.1), m the specific maintenance coefficient (g g.sup.1 h.sup.), V the cultivation volume (L), and X the biomass concentration (g L.sup.1):

[00001]$\begin{array}{cc}{m}_S=\left(\frac{{}_{s.Math.e.Math.t}}{Y_{X/S}}+m\right).Math.V\left(t\right).Math.X\left(t\right).Math.e^{{}_{\mathrm{set}}\left(t-t_F\right)}& \mathrm{EQUATION}.Math..Math.1\end{array}$

[0206] The inoculation procedure was the following: Based on a cryo-stock, a fresh agar plate containing adequate antibiotics was prepared. With one colony an Erlenmeyer flask containing 30 mL Lysogeny Broth (Sambrook et al. 1989) was inoculated and incubated for 12 to 15 h at 30 C. 30 mL of this first preculture was used to inoculate 500 mL of the fermentation medium in a 5 L Erlenmeyer flask and incubated for further 14 h. The 10 L fermenter was filled with 6 L fermentation medium and inoculated with 500 mL of the second preculture. Kanamycin was added at 50 g/mL. Protein production was induced by changing the glycerol feed to lactose feed. Cells were harvested after 48 h by centrifugation for 1 h at 9000 rpm and 22 C. Portions of 300 g cells were solved in 3 L lysis buffer (50 mM MOPS pH 7.3, 0.1 M NaCl, 20 mM imidazol). Cell lysis was achieved by ultrasonic treatment in a ultrasonic flow through chamber. Cell debris was separated by centrifugation (9000 rpm, 22 C.). Supernatant was clarified from residual cells or debris by tangential filtration applying a 0.2 m filter cassette and three volumes washing with lysis buffer. The enzyme solution was concentrated employing tangential filtration with a 30 kDa filter cassette followed by dialysis with three volumes lysis buffer. GH10 xylanases were purified by immobilized metal ion affinity chromatography (IMAC). Pure enzymes were eluted with elution buffer containing 50 mM MOPS, pH 7.3, 0.25 M imidazole, 0.1 M NaCl, and 20 mM CaCl.sub.2.

[0207] Another possibility for producing variants of the SEQ ID NO 1 enzyme, such as the enzymes of SEQ ID NOs 4-6 of the invention is to transform a competent B. subtilis strain with an appropriate vector comprising the mutated DNA and cultivating the recombinant strain in accordance with Park et al., 1991.

[0208] Production of Fusarium verticilloides xylanase of SEQ ID NO 2 was performed in P. pastoris X33 by inoculating a preculture harboring SEQ ID NO 15 genomic insertion in 5 mL YPD broth (1% (wt/vol) Yeast extract, 2% (w/v) peptone and 2% (wt/vol) dextrose) in a 50 mL Erlenmeyer flask from a single colony grown on YPD agar (1% (wt/vol) yeast extract, 2% (wt/vol) peptone and 2% (wt/vol) dextrose, 1.5% (wt/vol) agar) and incubating the preculture for 6 h at 30 C. in an orbital shaker at 180 rpm. One ml of preculture was used to inoculate 300 ml expression culture in BMD 1% medium (0.2M potassium phosphate buffer pH6, 13.4 g/L Yeast Nitrogen Base, 0.4 mg/I Biotin, 1.1% (wt/vol) glucose) in a baffled 5 l Erlenmeyer flask and incubated for 64 h at 22 C. in an orbital shaker at 180 pm. Expression was induced by addition of 240 ml BMM2 medium (0.2M Potassium Phosphate buffer pH 6, 13.4 g/L Yeast Nitrogen Base, 0.4 mg/I biotin, 1% (vol/vol) methanol) and transferred to 28 C. The expression culture was fed every 24 h with 9% (vol/vol) BMM 10 medium (0.2M Potassium Phosphate buffer pH 6, 13.4 g/L Yeast Nitrogen Base, 0.4 mg/I Biotin, 5% (vol/vol) methanol) and 1% (vol/vol) pure methanol. The culture supernatant was used to purify the enzyme by immobilized metal ion affinity chromatography (IMAC) as described above.

EXAMPLE 3

Electrophoretic Methods

[0209] Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was performed in accordance with Laemmli (1970). Proteins were resuspended in denaturating buffer and heated for 15 min at 95 C. The PageRuler Prestained Protein Ladder 10 to 180 kDa (#26616, ThermoFischer Scientific) was used as molecular weight standard. The proteins were stained with Coomassie brilliant blue R-250 (Weber and Osbourne, 1969).

EXAMPLE 4

Protein Quantification

[0210] The protein amount was determined by using Pierce BCA Protein Assay Kit (#23225, ThermoFischer Scientific) according to the instructions of the manufacturer.

EXAMPLE 5

Activity Test, Arabinoxylan Degradation

Xylanase Activity

[0211] For the purpose of the present invention, any of the commercially available xylanase activity measurement kits is suitable to determine xylanase activity. One suitable way of measuring the xylanase activity is as follows:

[0212] Xylanase activity measurement was performed at the temperature and pH optimum of the respective enzymes of SEQ ID NOs 1, 4, 5, 6 and 7 at 60 C., pH 5.8 and SEQ ID NOs 2 and Pentopan Mono BG* at 50 C., pH 5.8 for 1 h with a final concentration of 0.86% (wt/vol) arabinoxylan (wheat arabinoxylan, medium viscosity, Megazyme, Ireland), reaction buffer (100 mM MOPS, pH 6.5, 50 mM NaCl, 10 mM CaCl.sub.2) and the appropriate amount of enzyme. Quantification of reducing sugars was performed as described by Wood and Bhat (1988) using 3,5-dinitrosalicylic acid (DNSA). The amount of liberated reducing sugar ends was determined based on a calibration curve with glucose. One unit (U) is defined as the amount of enzyme required to liberate one pmole reducing sugar equivalents in one minute. All assays were performed at least in triplicates.

EXAMPLE 6

Characterization of the Enzymes of SEQ ID NOs 4-6 in Comparison to the Enzyme of SEQ ID NO 1

[0213] The Clostridium thermocellum GH10 xylanases (SEQ ID NOs: 1, 4-6) were produced, purified and quantified as described in Examples 1-4. Xylanase activity was determined as described in Example 5 with the exception that every enzyme was measured using a temperature and pH range in order to determine pH optimum (pH range) and temperature optimum (temperature range). The specific activity of the enzymes of SEQ ID NOs 1 and 4 to 6 were calculated as U/mg. The results were calculated as ratio to the wildtype enzyme SEQ ID NO 1. FIG. 2 shows the size of the enzymes of SEQ ID Nos: 4 to 6 compared to the wildtype protein of SEQ ID NO 1. All variants of SEQ ID NOs 4 to 6 are between 12 and 36% smaller compared to the wildtype protein. However, surprisingly the specific enzyme activities of the variants of SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6 are at least 4 fold higher (FIG. 1), which does not correspond to the size changes only (FIG. 2). Surprisingly, the deletion of protein domains as shown in the variants of SEQ ID NOs: 4 to 6 leads to a significant increase in enzyme activity. The individual domain deletions in the enzymes of SEQ ID NO 4 and 5 results in an approximately each 4 fold activity increase. The deletions of both domains in the enzyme of SEQ ID NO 6 leads to an even further specific enzyme activity increase which is not reflected by the size reduction of the enzyme.

EXAMPLE 7

Comparison GH11 and GH10 Enzyme Activity

[0214] The GH11 enzyme of SEQ ID NO 7 and the GH10 enzymes of SEQ ID NOs 2 and 6 were cloned and produced as described in Examples 1 and 2. The respective protein concentration including the purchased GH11 enzyme Pentopan Mono BG* was determined as described in Example 4. Specific enzyme activity for SEQ ID NO 3 was provided by the manufacturer (Megazyme, Ireland). The specific enzyme activity calculated according to example 5 revealed very high specific enzyme activities of both GH11 enzymes, the enzyme of SEQ ID NO 7 and Pentopan Mono BG* in comparison to a rather low specific enzyme activities of GH10 enzymes of SEQ ID NOs 2, 3 and 6. In FIG. 3 the results are shown as relative enzyme activity. The activity of the SEQ ID NO 6 xylanase was set to 100%.

EXAMPLE 8: Baking Experiments Using GH10 and GH11 Enzymes

[0215] Baking experiments were performed to determine the effect of GH11 and GH10 enzymes on bread volume comprising rye flour (100%). In each case, 0.1 ppm enzyme in respect to rye flour was added in baking experiments according to the following recipe:

Baking Recipe 1

[0216]

TABLE-US-00002 Rye flour 120 g (Type 1150, Rosenmhle) Instant Dry Yeast (species 1% (based on flour amount) Saccharomyces cerevisiae) Salt 2% (based on flour amount) Sugar 2% (based on flour amount) Water 57% (based on flour amount)

[0217] The enzymes of SEQ ID NOs 2, 3, 6 and 7 as well as Pentopan Mono BG* were used, each 0.1 ppm based on rye flour.

Procedure

[0218] 1. Scaling of ingredients, addition of flour, yeast, salt, sugar and enzyme

[0219] 2. Enzyme was dosed 0.1 ppm in relation to flour; excl. the negative control (no enzyme)

[0220] 3. Temperature adjustment of water in order to reach a dough temperature of 30 C. after mixing, scaling and addition of water into mixer bowl

[0221] 4. Mixing: 1 min with an hand mixer (Philips) at full speed

[0222] 5. The dough is given 30 min at 30 C. in an oven.

[0223] 6. Mixing: 1 min with a hand mixer (Philips) at full speed

[0224] 7. The dough is given 45 min at 30 C. in an oven.

[0225] 8. Mixing: 1 min with an hand mixer (Philips) at full speed

[0226] 9. Dough was divided in equally heavy portions and molded to rolls

[0227] 10. The molded roll is given 10 min bench-time

[0228] 11. The rolls are transferred to a covered baking sheet.

[0229] 12. The rolls are baked for 25 min (220 C. heated from the top and the bottom)

[0230] 13. The rolls are allowed to cool down

[0231] 14. The rolls are evaluated

Volume Estimation

[0232] The volume of baked rolls and bread was measured by rapeseed displacement. A suitable beaker (three times height, two times wide and two times length compared to the biggest test sample) was filled with rapeseeds until the beaker was completely filled. Surplus rapeseeds were removed with a plate, which completely covered the beaker hole. The test roll was placed on top on the rapeseeds in the middle of the beaker and was covered by the plate. By applying steady moderate pressure on the plate, the roll was plunged into the rapeseeds. Displaced rapeseeds were collected and measured in a measuring glass. Displaced ml of rapeseed were considered as rolls volume. For every baking experiment volume calculations were run in triplicates. Several xylanases belonging to families 10 and 11 obtained from a variety of species were tested. FIG. 4 summarizes the baking experiments of five xylanases, two GH11 family representatives and three GH10 xylanases obtained from different bacteria or fungi. The same amount of the individual enzymes was used for the baking trials. The GH10 enzymes are characterized by a low specific activity on arabinoxylan, however these enzymes demonstrate the best increase in bread volume. As shown in Example 7, representatives of GH11 enzymes show a much higher specific enzyme activity compared to GH10 enzyme. However, it was surprisingly discovered that, using the same amounts of enzymes in baking experiments, GH11 enzymes failed to increase the bread volume.

EXAMPLE 9

Effects of the Enzyme of SEQ ID NO 6 on pH Adjusted Rye Bread

[0233] Following a different baking recipe (2), including pH adjustment, the effects of the enzyme of SEQ ID NO 6 on rye bread volume and hardness were tested.

Baking Recipe 2

[0234]

TABLE-US-00003 Rye flour 100% Type 997 Instant Dry Yeast 1.8% (species: S. cerevisiae) (based on flour amount) Salt 1.8% (based on flour amount) Water 70% (based on flour amount) Enzyme: SEQ ID NO 6, 0.13 mg/kg flour (Control experiments w/o enzyme) (based on flour amount) Lactate 0.0-2.0 ml for pH adjustment (4.3, 4.7, 5.1 and 5.9) based on flour amount

Procedure

[0235] 1. Scaling of ingredients, addition of flour, yeast, salt and enzyme

[0236] 2. Temperature adjustment of water in order to reach a dough temperature of 30 C. after mixing, scaling and addition of water into mixer bowl

[0237] 4. Kneading: 3 min with a Z-kneader at 63 rpm

[0238] 5. Dough resting occurred for 10 min at 30 C. in a proofing chamber

[0239] 6. Each 200 g breads are molded and transferred into baking tins

[0240] 7. Proofing occurred 75 min at 30 C. in a proofing chamber (90% relative humidity).

[0241] 8. The breads are baked for 5 min (230 C. from the top 200 C. from the bottom) and 0.5 I steam

[0242] 9. The breads are baked for 7 min (200 C. from the top, 200 C. from the bottom)

[0243] 10. The breads are baked for 7 min (200 C. from the top, 200 C. from the bottom)

[0244] 11. The breads are chilled and stored at room temperature

[0245] 12. The breads are evaluated

Measurement of Crumb Structure

[0246] Crumb Hardness was analyzed by means of a texture analyzer (TVT 300 XP, TexVol Instruments AB, Viken, Sweden) according to the AACC International method 2011.

[0247] The baking experiments were done in duplicates and demonstrated a volume increase in pH adjusted rye bread as shown in FIG. 5. Compared to a baking control experiment w/o enzyme, the crumb structure was significantly improved. FIG. 6 shows that using the enzyme of SEQ ID NO 6 reduced the bread crumb hardness significantly.

EXAMPLE 10

Effects of the Enzymes with the Amino Acid Sequences of SEQ ID NO: 2 and SEQ ID NO: 6 on Rye-Wheat Bread in Different Flour Ratios

[0248] Following a different baking recipe 3, including pH adjustment, the effects of the enzymes with the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6 at 0.3 ppm on rye-what bread volume and crumb hardness were tested.

Baking Recipe 3

[0249] Rye flour Type 997: 70, 50 and 30% [0250] Wheat Flour Radboud: 30, 50 and 70% [0251] Salt: 1.8% [0252] Compressed Yeast: 3.5% [0253] Ascorbic acid: 25 ppm [0254] Citric acid: 0.3% [0255] Calcium propionate: 0.1% [0256] Water: 65%

Procedure

[0257] 1. Scaling of ingredients, addition of flour, yeast, salt and 0.3 ppm enzyme

[0258] 2. Temperature adjustment of water in order to reach a dough temperature of 30 C. after mixing, scaling and addition of water into mixer bowl

[0259] 4. Kneading: 3 min with a Z-kneader at 63 rpm

[0260] 5. Dough resting occurred for 15 min at 33 C. in a proofing chamber (80% relative humidity).

[0261] 6. Each 750 g breads are molded and transferred into baking tins.

[0262] 7. Proofing occurred 30 min at 33 C. in a proofing chamber (80% relative humidity).

[0263] 8. The breads are baked for 30 min at 225 C.

[0264] 9. The breads are chilled and stored at room temperature

[0265] 10. The breads are evaluated

[0266] Specific volume increase in percent is defined as specific volume of bread with enzyme (ml/g) divided by specific volume of the standard (ml/g) multiplied with 100.

TABLE-US-00004 TABLE 1 Specific volume increase of rye-wheat bread and crumb hardness after 1 day and 6 days after baking supplemented with xylanases Specific Crumb hardness Rye Wheat Volume after baking [N] Enzyme flour flour increase [%] Day 1 Day 6 standard 70 30 100 1604 2795 SEQ ID NO: 6 70 30 110 1307 2131 SEQ ID NO: 2 70 30 118 1010 1406 standard 50 50 100 1237 1965 SEQ ID NO: 6 50 50 114 730 1239 SEQ ID NO: 2 50 50 122 661 1490 standard 30 70 100 868 1481 SEQ ID NO: 6 30 70 110 690 1107 SEQ ID NO: 2 30 70 115 714 1381

[0267] Surprisingly both xylanases of SEQ ID NO: 2 and SEQ ID NO: 6 were able to increase the specific volume of rye-wheat bread and reduce the crumb hardness significantly after 1 and 6 days after baking when compared to bread without enzyme supplementation.

EXAMPLE 11

Effect of the Enzyme of SEQ ID NO: 6 on Rye-Wheat Dough in Different Flour Ratios

[0268] The effect of enzymes of SEQ ID NO: 2, SEQ ID NO: 6 and Pentopan mono BG on different dough qualities was investigated. Dough was prepared according to dough recipe 3 or 4 and analyzed as described in the following:

Dough Recipe 3

[0269]

TABLE-US-00005 Rye flour 100% Type 1150 Instant Dry Yeast 1.8% (Species: S. cerevisiae) (based on flour amount) Salt 1.8% (based on flour amount) Water 70% (based on flour amount) Enzyme: SEQ ID NO: 2, SEQ ID NO: 6 or Pentopan mono BG was added in concentrations w/o enzyme of 0.17 ppm (based on flour amount)

Dough Recipe 4

[0270]

TABLE-US-00006 Rye flour 10 and 70% Type 1150 Wheat flour 90 and 70% Type 550 Instant Dry Yeast 1.8% (Species: S. cerevisiae) (based on flour amount) Salt 1.8% (based on flour amount) Water 57% (based on flour amount)

[0271] In accordance with the American Association of Cereal Chemists (AACC) method 54-21.02 a torque measuring Z-kneader (doughLAB; Perten Instruments, Germany) was applied to prepare rye-wheat dough in ratios of 100:0%, 70:30%, and 10:90%, using commercial rye flour type 1150 and wheat flour type 550 (Rosenmhle, Landshut, Germany). To 100 parts flour mixture (moisture content corrected to 14.00 g/100 g flour) 70.0 parts de-mineralized water and 0.17 ppm of the xylanase of SEQ ID NO: 2, SEQ ID NO: 6 and Pentopan mono BG were added. Subsequently, kneading was performed for 153 s at 63 rpm and 30 C.

[0272] To 100 parts flour mixture (moisture content corrected to 14.00 g/100 g flour) 57.0 parts de-mineralized water and 0.01 ppm or 0.01 ppm of xylanase of SEQ ID NO: 2 were added to rye-wheat flour mixtures. Subsequently, kneading was performed for 153 s at 63 rpm and 30 C.

Methods

1. Kneading Properties Determined by a Z-Kneader

[0273] In accordance to the mentioned AACC method 54-21.02 a torque measuring Z-kneader (doughLAB; Perten Instruments, Germany) was used to determine the dough stability, which describes the period of time between exceeding the 500 FU-line and the first drop under 500 FU and provides information about dough processability. All measurements were done at least by double identification.

[0274] As shown in FIG. 7, addition of SEQ ID NO: 2 or SEQ ID NO: 6 xylanase cause an increase of dough stability during kneading in rye dough, while Pentopan mono a commercial GH11 xylanase does not. This enables an improved processability on dough with rye only. Using different quantities of the xylanase of SEQ ID NO: 2 with different ratios of rye-wheat flour (70:30% (A), or 10:90% (B)) also improved dough stability during kneading as shown in FIG. 8. Increasing dough stability during kneading improves process reliability and processability of rye wheat doughs during (industrial) breadmaking.

2. Dough Stickiness by Chen-Hoseney Dough Stickiness Cell

[0275] Analysis of dough stickiness was carried out by using the Ta.XT plus Texture Analyzer (Stable Micro Systems Ltd., Godalming, UK) equipped with a Dough Stickiness Rig as described by Chen and Hoseney (1995). The stickiness (g) of the dough was measured of a piece of dough extruded through a grid to a cylinder in polished Plexiglas (diameter 25 mm). Dough was placed into the cell after kneading and rested for 5 min at room temperature. The dough sample is compressed by the cylinder at a speed of 0.5 mm/s, an applied force of 40 g, and a contact time of 0.10 s; then the cylinder goes back to 4 mm at speed of 10 mm/s. All the measurements were done by double identification.

[0276] As shown in FIG. 9, by increasing the SEQ ID NO: 6 xylanase quantity, the dough stickiness gets significantly reduced, which indicates an improved dough handling and machinability on dough with a rye ratio of 30%.

3. Elongation Properties by SMS/Kieffer Extensibility Rig

[0277] Uniaxial elongation was analyzed using the SMS/Kieffer Extensibility Rig for the Ta.XT plus Texture Analyzer (Stable Micro Systems Ltd., Godalming, UK). For this purpose, each dough sample was placed into the Kieffer sample plate. After 40 minutes equilibration time at 30 C. the maximum peak force (resistance to extension (R.sub.Kmax)) of ten strands of each dough sample were recorded. The test settings were: pre-test speed: 2.00 mm/s; test speed: 3.3 mm/s; post-test speed: 10.0 mm/s; distance: 75 mm; trigger force: 5.0 g. All the measurements were done by double identification.

[0278] As shown in FIG. 10, by increasing the SEQ ID NO 6 xylanase quantity, the dough resistance to extension gets significantly reduced, which improves loosening of dough during fermentation and enables higher bread volumes on dough with all tested rye ratios of 10% (A), 30% (B) and 70% (C) rye flour.

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