Field

The present invention relates to a process for making a baked good using protein arginine deiminase, a premix for a baked good including protein arginine deiminase, and a process for improving a baked good by using protein arginine deiminase.

Background

In the baking industry, e.g. in industrial dough, batter, and bread making, processing aids are commonly used to improve properties of a dough, a batter, and/or a baked good. Dough and/or batter properties that may be improved comprise stability, gas retaining capability, elasticity, extensibility, moldability etcetera. Properties of the baked goods that may be improved comprise loaf volume, crust crispiness, oven spring, crumb texture, crumb structure, crumb softness, crumb firmness, flavour, relative staleness and shelf life.

Processing aids, such as chemical additives and enzymes are added to flour and I or dough to improve the properties of a dough or a baked good.

Loosening or softening of doughs and batters can increase air incorporation which can increase volume of the baked good. However, such loosening or softening can decrease production efficiency by making the doughs and/or batters unstable, sticky, and/or hard to handle. This concern for production efficiency is why processing aids such as the use of protease enzymes has been limited. Even though such proteases can break down gluten protein in the doughs and batters and loosen those doughs and batters, the results can be unpredictable and thus their use is limited.

Therefore, there is a need for processing aids that can loosen or soften doughs and batters to improve the properties of baked goods.

Summary

The present invention relates to methods of making baked goods including the steps of contacting a dough with a protein arginine deiminase to form a treated dough and baking the treated dough to form the baked good.

The present invention also relates to a baked good or a dough wherein the baked good or dough has 1% to10% less arginine as a result of a protein arginine deiminase treatment.

The present invention also relates to a premix comprising a protein arginine deiminase, and a bulking agent. The present invention also relates to a method of increasing a volume of a baked good comprising the steps of contacting a dough with a protein arginine deiminase and baking.

The present invention also relates to use of protein arginine deiminase for increasing volume in a baked good.

The present invention further relates to the use of protein arginine deiminase for improving crumb softness of a baked good.

Definitions

The term “baked good” refers to a baked food good prepared from a dough or a batter.

Examples of baked goods, whether of a white, brown, or whole-grain such as wholemeal or whole-wheat type, include bread, typically in the form of loaves or rolls, French baguette-type bread, pastries, croissants, brioche, panettone, pasta, noodles (boiled or (stir- )fried), pita bread and other flat breads, tortillas, tacos, cakes, pancakes, cookies in particular biscuits, doughnuts, including yeasted doughnuts, bagels, pie crusts, steamed bread, crisp bread, brownies, sheet cakes, snack foods (e.g., pretzels, tortilla chips, fabricated snacks, fabricated potato crisps). Baked goods are typically made by baking a dough at a suitable temperature for making the baked good such as a temperature between 100 °C and 300 °C. A baked good as disclosed herein may be a white, a brown, a whole-meal or a whole-wheat bread.

The term “dough” is defined herein as a mixture of flour and other ingredients. Usually, dough is firm enough to knead or roll. The dough may be fresh, frozen, prepared or parbaked. Dough is usually made from basic dough ingredients including (cereal) flour, such as wheat flour or rice flour, water and optionally salt. For leavened products, primarily baker’s yeast is used, and optionally chemical leavening compounds can be used, such as a combination of an acid (generating compound) and bicarbonate. Cereals from which flour can be made include maize, rice, wheat, barley, sorghum, millet, oats, rye, triticale, buckwheat, quinoa, spelt, einkorn, emmer, durum and kamut. The term dough herein also includes a batter. The term “batter” is a semi-liquid mixture, being thin enough to drop or pour from a spoon, of one or more flours combined with liquids such as water, milk or eggs used to prepare various foods, including cake.

The term “pre-mix” is to be understood in its conventional meaning, i.e. as a mix of baking agents, generally including bulking agents and/or standardization agents such as flour, starch, maltodextrin and / or salt, which may be used not only in industrial bread-baking plants/facilities, but also in retail bakeries. A pre-mix comprises a protein arginine deiminase as disclosed herein. A pre-mix may contain additives as mentioned herein.

Additives are in most cases added in powder form. Suitable additives include oxidants (including ascorbic acid, bromate and azodicarbonamide (ADA), reducing agents (including L- cysteine), emulsifiers (including without limitation mono- and diglycerides, monoglycerides such as glycerol monostearate (GMS), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE) and diacetyl tartaric acid esters of mono- and diglycerides (DATEM), propylene glycol monostearate (PGMS), lecithin), gums (including guar gum, pectin, gellan gum, and xanthan gum), flavours, acids (including citric acid, propionic acid), starches, modified starches, humectants (including glycerol) and preservatives.

Detailed Description

The invention provides a method of making a baked good comprising the steps of contacting a dough with a protein arginine deiminase to form a treated dough and baking the treated dough to form the baked good and wherein said dough is a mixture of flour and other ingredients. Suitable “other ingredients” will depend on the desired specific baked good. The skilled person is very well capable of determining said other ingredients. Salt and water are examples of “other ingredients”. Other, non-limiting, examples of “other ingredients” can be found in the experimental part herein.

The term protein arginine deiminase and peptidyl arginine deiminase (PAD) are used interchangeably herein. Protein or peptidyl arginine deiminases belong to a family of enzymes (EC 3.5.3.15) which convert peptide or protein bound arginine into peptide or protein bound citrulline. This process is called deimination or citrullination. In the reaction from arginine to citrulline, one of the terminal nitrogen atoms of the arginine side chain is replaced by an oxygen. The reaction uses one water molecule and yields ammonia as a side product (http://en.wikipedia.org/wiki/Citrullination). Whereas arginine is positively charged at a neutral pH, citrulline is uncharged.

Peptidyl arginine deiminase (PAD) may be derived from any suitable origin, for instance from mammalian or microbial origin. PAD’S used in the present invention are advantageously derived from a microbial source, i.e. the PAD used in a process of the invention is a microbial PAD. For instance, PAD’S may be derived from fungal origin such as from Fusarium sp. such as Fusarium graminearum, Chaetomium globosum, Phaesphaeria nodorum or from bacterial origin such as from the bacteria Streptomyces, eg Streptomyces scabies, Streptomyces clavuligeres. Peptidyl arginine deiminases are for instance known from W02008/000714. W02008/000714 discloses a PAD derived from Fusarium graminearum, as well as a process for producing said PAD enzyme.

A peptidyl arginine deiminase may be a pure or purified peptidyl arginine deiminase. A pure of purified peptidyl arginine deiminase is an enzyme that may be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 80% pure, at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% pure for instance as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art.

In some embodiments, the protein arginine deiminase may be a peptidylarginine deiminase enzyme (EC 3.5.3.15) from Gibberella zeae (also called Fusarium graminearum) as described in W008000714.

The used PAD enzyme may be added as an enzyme preparation. It will be understood that the enzyme preparation further comprises carriers, stabilizers and/or diluents which will not interfere with the intended purpose of the PAD enzyme and still be regarded as isolated. The PAD enzyme may also be in a substantially purified form, in which case it will generally comprise the enzyme in a preparation in which more than 70%, e.g. more than 80%, 90%, 95%, 98% or 99% of the proteins in the preparation is PAD enzyme.

The PAD enzyme in an enzyme preparation is provided in a form such that the PAD enzyme is outside their natural cellular environment. Thus, the PAD enzyme may be substantially isolated or purified, as discussed above, or in a cell in which they do not occur in nature, for example a cell of other fungal species, animals, plants or bacteria.

Preferably, the used PAD is not endogenous yeast PAD.

In some embodiments, doughs and baked goods where PAD is used in the composition show a reduction in arginine content of from 1 % to 10% as compared to doughs and baked goods without PAD. In some embodiments, the doughs and baked goods treated with PAD show an arginine reduction from 1 %, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, or 9.5% to 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% as compared to doughs and baked goods without PAD. Preferably, said dough is not a sourdough. Preferably, said baked good is a baked good which is not prepared from sourdough.

Amino acid analyses such as high-performance liquid chromatography (HPLC) methods can be used to measure this reduction in arginine. One such method of measuring this reduction in arginine is described in Example 3. A dough composition may be a solid or fluid composition. In some embodiments, a dough composition may comprise one or more components selected from the group consisting of a milk powder, a gluten, a granulated fat, an additional enzyme, an amino acid, a salt, an oxidant, a reducing agent, an emulsifier, a lecithin, a sodium stearoyl lactylate, a calcium stearoyl lactylate, one or more polyglycerol esters of fatty acids and one or more diacetyl tartaric acid esters of mono- and diglycerides, a hydrocolloid, a flavour, an acid, a starch, a modified starch, a humectant and a preservative. In some embodiments, a dough composition can be a mixture of (cereal) flour, water and optionally salt. In some embodiments, a dough is firm enough to knead or roll. The dough may be fresh, frozen, prepared or parbaked. For leavened products primarily baker's yeast is used and optionally chemical leavening compounds can be used, such as a combination of an acid (generating compound) and bicarbonate. The term dough herein also includes a batter. A batter is a semi-liquid mixture, being thin enough to drop or pour from a spoon, of one or more flours combined with liquids such as water, milk or eggs used to prepare various foods, including cake.

In some embodiments, a premix composition comprises a protein arginine deiminase, and a bulking agent and/or standardization agent. A pre-mix as defined herein is a mix of baking agents, generally including bulking agents and/or standardization agents such as flour, starch, maltodextrin and/or salt, which may be used not only in industrial bread-baking plants/facilities, but also in retail bakeries. In some embodiments, a bulking agent and/or standardization agent can include a flour, a starch, a maltodextrin, a salt, or combinations thereof.

A dough, baked good, or pre-mix composition as disclosed herein may comprise one or more further enzyme(s) such as an phospholipase, an amylase such as an alpha-amylase, for example a fungal alpha-amylase (which may be useful for providing sugars fermentable by yeast), a beta-amylase; a maltogenic amylase, a glucanotransferase; a peptidase in particular, an exopeptidase (which may be useful in flavour enhancement); a transglutaminase; a cellulase; a hemicellulase, in particular a pentosanase such as xylanase (which may be useful for the partial hydrolysis of pentosans, more specifically arabinoxylan, which increases the extensibility of the dough); protease (which may be useful for gluten weakening in particular when using hard wheat flour); a protein disulfide isomerase, e.g., a protein disulfide isomerase as disclosed in WO 95/00636; a glycosyltransferase; a peroxidase (which may be useful for improving the dough consistency); a laccase; an oxidase, such as an hexose oxidase, a glucose oxidase, aldose oxidase, pyranose oxidase; a lipoxygenase; L-amino acid oxidase (which may be useful in improving dough consistency) and / or an asparaginase. In some embodiments including a phospholipase, the term “phospholipase” refers to enzymes that hydrolyze the ester bonds of phospholipids. Phospholipases differ in their specificity according to the position of the bond attacked in the phospholipid molecule. Phospholipase A1 (PLA1) removes the 1 -position fatty acid to produce free fatty acid and 1- lyso-2-acylphospholipid. Phospholipase A2 (PLA2) removes the 2-position fatty acid to produce free fatty acid and 1-acyl-2-lysophospholipid. PLA1 and PI.A2 enzymes can be intra- or extracellular, membrane-bound or soluble. Intracellular PI.A2 is found in almost every mammalian cell. Patatins are another type of phospholipase, thought to work as a PLA (see for example, Hirschberg HJ, et al., (2001), Eur J Biochem 268(19):5037-44). [general to specific]. Phospholipids can comprise phosphatidic acid (PA), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), and phosphatidylcholine (PC).

A polypeptide having phospholipase A1 activity as used herein is a phospholipase A1 according to the enzyme classification E.C. 3.1.1.32. Phospholipase A1 is an enzyme that cleaves a phospholipid at the SN1 position forming a lysophospholipid and a fatty acid. The wording “phospholipase A1” and a “polypeptide having phospholipase A1 activity” is used interchangeably herein. A polypeptide having phospholipase A1 activity as disclosed herein does not have phospholipase A2 activity.

A phospholipase A1 may be derived from any suitable organism, for instance from fungi. Suitable fungi include filamentous fungi, such as Aspergillus, Talaromyces, Trichoderma, and yeast such as Pichia, Saccharomyces, for instance Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Talaromyces emersonii, Pichia pastoris, Saccharomyces cerevisiae. A polypeptide having phosphoplipase A1 activity may be derived from Aspergillus niger.

A phospholipase A1 used in a process as disclosed herein may be a natural occurring polypeptide or a variant polypeptide.

A phospholipase A1 may also be a pure or an isolated phospholipase A1 , i.e. a polypeptide having phospholipase A1 activity that is removed from at least one component eg. other polypeptide material with which it is naturally associated.

A phospholipase A2 (PLA2) releases fatty acids from the second carbon group of glycerol and belongs to enzyme classification EC 3.1.1.4.

The phospholipase is typically added as an enzyme preparation.

The present invention also relates to a process for preparing a dough comprising adding a protein arginine deiminase as disclosed herein or a composition containing a protein arginine deiminase as disclosed herein which has been added to the dough. A process for preparing a dough is known to a person skilled in the art and usually comprises mixing dough ingredients such as flour, water, and optionally sugar, yeast, leavening compounds, and salt. A process for preparing a dough as disclosed herein comprises adding a protein arginine deiminase to the dough, for instance during preparation of the dough, for instance during mixing other dough ingredients. A suitable amount of a protein arginine deiminase is added to the dough. A suitable amount of a protein arginine deiminase may for instance be expressed in enzyme weight percent by weight of protein in flour or in units (U) per kilogram (kg) of flour.

A suitable amount of a protein arginine deiminase may be from 0.05% to 0.4%, preferably 0.05% to 0.20% or 0.05% to 0.10% w/w by protein weight of flour in the dough. As used herein, “protein weight of flour” is understood to be the weight percent of protein in a flour. The amount of protein present in a flour will be provided by a miller and said amount can be determined by multiple methods. For example, there are standardized official methods to determine protein content in flour - AACC International Method 46-10 — Crude Protein. The Kjeldahl procedure AACC International Method 46-30 — Crude Protein Combustion nitrogen analysis (CNA). In some embodiments, the amount of protein arginine deiminase is from 0.05%, 0.06%, 0.07%, 0.08%, or 0.09% to 0.06%, 0.07%, 0.08%, 0.09%, or 0.10% w/w by protein weight of flour.

A suitable amount of protein arginine deiminase can also be from 500 U/kg to 4000 U/kg, preferably 500 U/kg of flour to 2000 U/kg flour or 500 U/kg of flour to 1 ,000 U/kg flour. In some embodiments, the amount of protein arginine deiminase can be from 500 U/kg, 550 U/kg, 600 U/kg, 650 U/kg, 700 U/kg, 750 U/kg, 800 U/kg, 850 U/kg, 900 U/kg, or 950 U/kg to 550 U/kg, 600 U/kg, 650 U/kg, 700 U/kg, 750 U/kg, 800 U/kg, 850 U/kg, 900 U/kg, 950 U/kg, or 1000 U/kg. As used herein, one unit (U) of peptidyl arginine deiminase is expressed as 1 pmol of citrulline formed I min I mg of protein as measured by measuring the formation of citrulline residues in a-N-Benzoyl-L-arginine-ethyl ester (BAEE). The incubation mixture contained 100mM tris-HCI buffer (pH 7.5), 5 mM CaCI2, 10 mM DTT, 10 mM BAEE in a final volume of 700 pl. Incubation was performed at 55°C for 30 min, and the reaction was stopped by adding 10OpI 8 N HCIO4. Citrulline was determined by colorimetry according to the method of Guthdhrlein and Knappe, (1968) Anal. Biochem. 26, 188.

The present invention also relates to a dough comprising a protein arginine deiminase as disclosed herein or a composition including a protein arginine deiminase as disclosed herein. Surprisingly, it was found that a dough or a baked good made with protein arginine deiminase as disclosed herein has an improved property as compared to a dough or baked good prepared without a protein arginine deiminase. In particular, the improved property was found when the dough was made with 0.05% to 0.10% of a protein arginine deiminase (w/w by protein weight of the flour). The improved property was also found when the dough was made with 500 to 1 ,000 U/kg flour of a protein arginine deiminase. An improved property may be an improved bread/baked good volume.

Yet another improved property may be a more relaxed dough. Moreover, a method according to the invention is a more robust method when compared to other methods for obtaining an improved property (for example by using a protease). Yet another advantage is that overdose of the PAD does not result in negative effects (compared to proteases which do result in negative effects when overdosed).

In some embodiments, PAD is used in amounts that do not decrease the stability of the dough. As used herein, the term “dough stability” refers to rheological properties as preferably measured using a farinograph according to the method described in the Examples below. Unstable dough has a tendency to collapse before or during the baking process resulting in a loss of volume. Surprisingly, and especially when compared to proteases, adding PAD to dough samples did not result in excess dough softening. Thus, the baked goods including PAD show increased volume without the adverse side effect of excessive dough softening.

The term “increased volume of the baked good” is preferably measured as the volume of a given loaf of bread or baked good as determined by an automated bread volume analyser (e.g. BVM-3, TexVol Instruments AB, Viken, Sweden), using ultrasound or laser detection as known in the art and compared to the volume of a reference baked good. In case the volume is increased, the property is improved. Alternatively, the height of the baked good after baking in the same size tin is an indication of the baked good volume. In case the height of the baked good has increased, the volume of the baked good has increased.

Whether or not a property of a dough or a baked good is improved is determined by comparing the obtained result with an otherwise identically obtained dough or baked good which does not comprise PAD.

Other methods of measuring the volume of baked goods can include American Association of Cereal Chemists International (AACCI) Method 10-05.01 which is a volume displacement method where an item to be measured is placed in a graduated, calibrated container which is then filled with rapeseed and the volume of the item is measured by reading the level of rapeseed as shown on the container. For such displacement methods involving comparisons of baked goods of differing weights, a specific volume can be calculated by dividing the volume measured by the weight of the baked good sample in grams (cm ³ /g).

Still other volume measuring methods include laser topography (scanning) as described in AACCI Approved Method 10-14.01 , image analysis, and ultrasound detection. The present invention also relates to a process for preparing a baked good comprising baking a dough as disclosed herein. A person skilled in the art knows how to prepare a baked good, and such preparation usually comprises baking a dough in an oven at a suitable temperature to prepare the baked good. Suitable temperatures for preparing a baked good are for instance between 100°C and 300°C.

The present invention also relates to the use of a protein arginine deiminase as disclosed herein to increase the volume of a bread or baked good made from the dough. Accordingly, disclosed herein is a method for improving bread/baked good volume, using a protein arginine deiminase as disclosed herein. The use of a protein arginine deiminase as disclosed herein to increase the volume of a baked good as disclosed herein is relative to the use of a reference baked good that does not include a protein arginine deiminase. Accordingly, disclosed herein is a method for improving the volume of a baked good using a protein arginine deiminase as disclosed herein.

The present invention also relates to a process for increasing the volume of a baked good, comprising

- preparing a dough comprising a protein arginine deiminase as disclosed herein and

- baking the dough, wherein the volume of the baked good is increased as compared to the volume of a baked good prepared from a dough without a protein arginine deiminase (PAD). The dough is a mixture of flour and other ingredients. Preferably, said PAD is added as an enzyme preparation. The dough may further include a(n) (enzyme preparation comprising a) phospholipase.

A dough or a baked good disclosed herein may comprise any suitable cereal flour, for instance wholemeal flour, or a mixture of different flours. Cereal flour may also comprise bran, grains and I or seeds. Cereals include maize, rice, wheat, barley, sorghum, millet, oats, rye, triticale, buckwheat, quinoa, spelt, einkorn, emmer, durum and kamut. Wholemeal flour, also referred to as whole-wheat flour, is flour made from the entire wheat kernel or grain including the outer part. Flour may comprise a free fatty acid content of between 0.01 to 0.8 w/w%, for instance 0.05 to 0.6 w/w%, for instance a free fatty acid content of between 0.1 to 0.5 w/w% or between 0.14 to 0.4 w/w%. During storage the content of free fatty acids in flour usually increases. Free fatty acids may for instance be linoleic acid (C18: 2), palmitic acid (C16: 0), oleic acid (C18: 1 ), linolenic acid (C18:3) (see for instance MacMurray and Morrison, J.Sci. Food Agr. 21 :520-528 (1970). Free fatty acids in flour may be determined by methods known to a person skilled in the art, for instance as disclosed in Fierens et al, J. of Cereal Science 65 (2015), p. 81-87. The present invention also relates to the use of protein arginine deiminase for increasing volume in a baked good. The invention also provides use of protein arginine deiminase for improving crumb softness of a baked good, preferably improving crumb softness after 1 -5 days of storage of said baked good at room temperature. Preferably, PAD is added (to the flour or dough) as an enzyme preparation. The definitions and embodiments as provided above equally apply to this part of the invention.

The invention will be explained in more detail in the following example, which are not limiting the invention.

Examples

The PAD enzyme used in the herein described examples is described and claimed in W02008/000714.

Example 1: Effect of PAD on dough rheology

Materials & Methods

For each test sample, a Brabender farinograph with an automatic water dosing system was used to assess dough softness. For each sample, 300 grams kolibri flour*** was mixed with 58% water and yeast and the enzyme treatment was pipetted into the mixing chamber. Doughs were removed from the mixing chamber and left to rest for 10 minutes in a humidity cabinet before being evaluated by a trained assessor.

Results

Table 1 - Farinograph and dough evaluation

*The protease was Bakezyme® Protease GBW, from DSM, The Netherlands

** PAD dose was weight percent based on the protein weight of flour (assumed and later confirmed to be 10%) *** Kolibri flour sourced from Dossche Mills

As shown in Table 1 , adding protease enzyme to a dough results in an overly soft and sticky dough. This is shown in the low stability reading, the high degree of softening, the low farinograph quality number and confirmed by the dough evaluation. In contrast, adding PAD resulted in a dough with very similar properties to the blank. This testing confirms that addition of PAD does not result in making the dough difficult to process.

Example 2 - Effect of PAD dose on dough handling and bread volume/texture Materials & Methods

Table 2 - Bread Compositions

Table 3 - Test Procedure

Table 4 - Process

Extensibility and Elasticity Test Method:

1 . Dough is distributed and rounded to small dough balls of 51 g using a Rotamat (at size 3 and revolutions at 11).

2. Balls are proofed 25 min at 28C 186% humidity.

3. Temperature of press are set at 0C.

4. Press for 3 seconds at size nr 7 (approx. 2,5 mm)

5. After pressing each dough measured in fourfold at two point across the dough

6. After resting each dough is measured in fourfold again at two points across the dough

7. Retraction is calculated by = (1 -((original size-size after retraction)/original size))*100% Results

Table 5 - Extensibility and Elasticity of Doughs weight of flour) show increased extensibility and decreased elasticity. It is thus expected that these doughs, while slightly stickier, were more developed and would provide an improved baked good with better structure to provide increased volume.

Bread Volume Test Method:

Volume analysis are performed by using the laser volumeter from TexVol instrument (Perten). Crumb firmness and Resilience Test Method:

Crumb firmness (or hardness; both being the opposite of softness) and crumb resilience of bread slices were measured using the Texture Analyzer and were also evaluated empirically by the skilled test baker.

To evaluate these crumb properties, breads were cut into slices with a thickness of 2.5 cm, and the firmness was measured using a Texture Analyzer TA-XT Plus (Stable Micro Systems), with the following settings: compression test mode, pre-test speed of 3 mm/s, test 25 speed of 1 mm/s, distance of 5 mm, hold time of 10 s and trigger force of 5 g. The crumb firmness is the maximum peak force during compression, recorded in gram. The crumb resilience is the ratio of the force at the end of the 10 s hold time to the maximum force. Crumb resilience is expressed as percentage.

Table 6 - Baked Bread Evaluation

These results show that adding PAD to doughs result in improved baked breads with higher volume and softer crumb texture.

Example 3 - Amino acid analysis in dough and bread treated with PAD

Materials & Methods

Doughs and breads were prepared as in Example 2.

Amino Acid Analysis Method: Amino acid analysis was carried out according to the Pico Tag method specified in the operator manual of the Amino Acid Analysis System of Waters (Milford MA, USA). To that end, samples were dried and directly derivatized using phenylisothiocyanate. The position of citrulline and ornithine in the chromatogram was established by test runs in which the pure compounds (Sigma) were derivatized and subjected to chromatography. The quantification of the amino acids was done using the HPLC method. Incubation mixture containing proteins or peptides which were used as substrate for the PAD enzyme were first acid hydrolysed according to the operators manual of the Amino Acid Analysis System of Waters, then derivatized, separated and quantified by HPLC.

Results

Table 7 - Amino Acid Analysis

These results show a decrease in arginine of 5-7% in the doughs and breads with effective levels of PAD (effective levels being those that showed improvements from Example 2).

Example 4 - Effect of enzyme combinations on bread volume in breads with regular flour Materials & Methods

Table 8 - Bread Compositions (amounts in grams except for enzymes and ascorbic acid amounts which are in ppm)

Each test composition was prepared as a bread according to the process in Example 2.

Results

Table 9 - Bread Volume

*The test method as described in Table 6 was also used here.

These results show the clear benefit of increased bread volume when PAD is used in the dough and thus the baked good (bread) when regular flour is involved. Additionally, these results show a further enhanced bread volume benefit with PAD is combined with phospholipase. Example 5 - Effect of enzyme combinations on bread volume in breads with strong flour

Materials & Methods

Table 10 - Bread Compositions (amounts in grams except for enzymes and ascorbic acid which are in ppm)

Each test composition was prepared as a bread according to the process in Example 2.

Results

Table 11 - Bread Volume* *The test method as described in Table 6 was also used here. While results in strong flour are less marked than those in regular flour, these results still show the clear benefit of increased bread volume when PAD is used in the dough and thus the baked good (bread) when strong flour is involved. Here again, these results show a further enhanced bread volume benefit with PAD is combined with phospholipase.

Example 6 - Mini bread Materials & Methods

Prototype Mini-breads were made using Thermomixes® (Vorwerk).

This process comprises:

Weighing flour and other ingredients

Transfer of all ingredients into a Thermomix®

Mixing 3 minutes using Thermomix® bread dough program

Divide dough in pieces of 120 gram and put through dough rounder (Gefra Conical Rounder)

Shape the dough pieces uniformly in a Bertrand Moulders machine and transfer to appropriately sized baking moulds.

Proving time 60 minutes in climate chamber 32 degrees Celsius and 85% humidity Bake-off 25 minutes 220 degrees Celsius

We used Mini breads to use small amounts of PAD to test viable functionality. Volume was tested and positive effects previously seen were again observed. Surprisingly, it was observed that upon keeping the breads longer (more than 3 days) the PAD breads showed better crumb softness than blank. Subsequently this effect was tested for PAD in mini breads and compared against Bakezyme Master and Blank.

Table 12 - Bread Compositions

Freshness test is done with the Texture Analyser analysis (on day 1 , 5 and 7). TA measurements were performed using a Texture Analyser (Stable Micro Systems) equipped with a 25 mm Radiused Cylinder Probe. Two breads were used for the measurements per recipe. For each measurement a fresh slice of the center of the bread was analyzed. The probe (SMSP25 this is a smaller probe than used for the big breads) reached the sample with a speed of 3 mm/s, compressed 5 mm of the bread crumb in the center of the slice with a speed of 1 mm/s and held the compression for 10 s, then returned to the original position with a speed of 5 mm/s. Trigger Force was 5 g. The firmness of the crumb is defined as the maximum force (F1) required for the slice compression. The averaged firmness data points were then depicted against the storage time in days, to follow the effect of staling on the texture of the bread crumb.

Volume was determined by measurement including (not shown) bread pictures (TexVol Instruments)

Results

The volume measurements showed that the addition of Bakezyme Master alone does not result in volume increase when compared to the blank. The addition of 0.1 % PAD together with 25 ppm Bakezyme Master resulted in a volume increase of 24% compared to the blank.

The TA measurements showed that on all days, the Bakezyme Master and/or PAD treated breads are significantly softer compared to the blank. The 0.5% PAD treated bread shows on all days comparable softness to the 25 ppm Bakezyme Master treated bread. The 0.1 % PAD + 25 ppm Bakezyme Master bread shows a softness which is approximately in between the softness of the blank and 25 ppm Bakezyme Master (or 0.5% PAD).

Example 7 - Big bread - freshness effect

A regular big bread experimental set up was used to see if the mini bread results (see previous example) could be transferred to a bigger bread matrix and if we would still see similar results regarding volume and softness. This was indeed the case (experimental data not shown). As a follow-up, we determined if we could still observe the freshness effect in breads wherein bread improver rich tin was used. We observed negligible impact on volume; however, we continued to observe softness effect.

Materials & Methods

Breads were made via a regular bread make process and were baked in a deck oven (Wachtel Piccolo).

Table 13 - Bread Compositions

The conditions were identical to the conditions in Table 3.

Measurements performed on the produced breads: Freshness test is done with the Texture Analyser analysis (on day 1 , 5 and 7). TA measurements were performed using a Texture Analyser (Stable Micro Systems) equipped with a P/36R 36mm Radiused Cylinder Probe. Two breads were used for the measurements per recipe. For each measurement a fresh slice of the center of the bread was analyzed. The probe reached the sample with a speed of 3 mm/s, compressed 5 mm of the bread crumb in the center of the slice with a speed of 1 mm/s and held the compression for 10 s, then returned to the original position with a speed of 5 mm/s. Trigger Force was 5 g. The firmness of the crumb is defined as the maximum force (F1) required for the slice compression. The averaged firmness data points were then depicted against the storage time in days, to follow the effect of staling on the texture of the bread crumb.

Volume was determined by measurement including bread pictures (TexVol Instruments)

Results

Volumes were measured in duplicate with a laser volumeter. No big differences were detected for all different PAD dosages compared to the blank.

The texture analysis hardness results show that on day 1 , all PAD dosages result in a bread which is softer in comparison to the blank. On day 5, PAD dosages up to and including 0.2% show a softer crumb compared to the blank: approximately 16% softer for the 0.2% PAD dosage and 11% for the 0.1 % PAD dosage.

Hence, the softness effects seen are not directly related to increase in volume and this implies a freshness effect that stands on its own.

Example 8 - Dough softness as determined by using a Farinograph

We used Farinograph to see if PAD has an effect on dough softness before baking. A softer dough without an overdose effect (like typically seen in proteases) could make doughs better processable and machinable.

Materials & Methods

Table 14 - Bread Compositions

* For the farinograph the addition of water is corrected by the amount of PAD enzyme to maintaining a dough consistency of 500 BU.

** 5 separate blanks were included in this example Farinograph analysis

Dough properties determination was performed using a Brabender farinograph. The dough is prepared according to the following procedure:

1. Calibration: the automatic water dosing system is calibrated according to the instruction manual 2. Programming: Rotating speed is determined for 63 min, Temperature is at 30°C, Mixing time is up to 10 min (moisture content is set at 14%). After leaving the dough to rest for 30 min in the mixing chamber the torque is measured for another 10 min.

3. Mixing Kolibri flour (300g), yeast, salt and basic white tin is transferred into the mixing chamber. Addition of water and enzyme after 30 s of mixing. Water absorption was recorded as the amount of water required to centre the farinograph curve on the 500 BU line Results

The intention was to differentiate between Basic Tin Improver (bread improver used in this example) effect on dough and differentiating said effect from the effect of PAD on the dough. Clear effect was observed of tin improver on the dough development. The dough with tin improver (sample 1) was less soft after proofing. (7% softer than sample 2). Subsequently, effect of PAD addition was tested on dough development. All tested PAD concentrations show a softer dough after the proofing step compared to the blank with 0.2% PAD addition (sample 4) leading to a dough softness that is 13% softer than the sample with tin improver (sample 1).