WO2013092731A1

WO2013092731A1 - Method for making a dough with a glutamyl endopeptidase

Info

Publication number: WO2013092731A1
Application number: PCT/EP2012/076178
Authority: WO
Inventors: Neil Carr; José MASTENBROEK; Luppo Edens
Original assignee: Dsm Ip Assets B.V.
Priority date: 2011-12-21
Filing date: 2012-12-19
Publication date: 2013-06-27

Abstract

The present invention relates to a method for the relaxation of a dough in the process of making a bakery product. The method comprises using a protease which has high specificity for glutamyl residues as the only proteolytic component in the dough to achieve relaxation. The method precludes extended proteolysis and the dough can be satisfactorily used industrially for preparing baked products such as bread, biscuits and pasta products. The method is particularly suitable for reworked doughs.

Description

METHOD FOR MAKING A DOUGH WITH A GLUTAMYL ENDOPEPTIDASE

Field of the invention

The present invention relates to a method for preparing a dough. In particular to a method for preparing a dough by using proteases, in particular glutamyl specific proteases.

Background of the invention

It is common to use dough relaxants in the baking industry. In particular, additives such as I- cysteine or yeast derivatives rich in glutathione find common use in order to increase the extensibility of the gluten proteins in the dough. Such dough relaxants are especially useful for the manufacture of bread rolls, pizzas, and tortillas so that products can be formed optimally. Dough relaxation is also critical within wider bakery applications when these use flour sources that are overly strong in terms of the gluten protein.

A particularly important dough relaxant is sodium metabisulfite (SMS) which is used commonly during the manufacture of biscuits. Sodium metabisulfite reduces the gluten protein to increase extensibility of the dough, such that the dough can be sheeted more satisfactorily without shrinkage. A disadvantage of SMS is that it may be an allergen to certain people.

Proteases are used as alternatives to SMS and other dough relaxants. In particular, where an exceptional degree of extensibility is required, such as in biscuit making, proteases can be used to assist in the breakdown of gluten in hard doughs. Commonly used commercial endoproteases include papain, bacillolysin, and thermolysin, and subtilisin. However, these commercial preparations are perceived as leading to excessive digestion of gluten, and are therefore not favoured by, for example, the biscuit industry. The main drawback is that their action is progressive. In other words, the breakdown continues while the dough stands. If this breakdown is achieved by mechanical or chemical means, there is no further reaction after the required degree is obtained. The amount of gluten softening produced by a reductant, such as SMS, is dependent only on the amount added, while enzyme effects depend both on the amount added, temperature and the length of time allowed for protease action. Progressive digestion is perceived as problematic, especially if reworked dough is used, since dough characteristics may change with different standing times, which influences the quality and commercial value of the end product.

Brief description of the Figures

Figure 1. Proteolytic specificity of protease derived from Bacillus as determined upon incubation with ZAAX-pNA with Xaa representing the amino acid residue (in the one-letter code) preceding pNA.

Figure 2. Proteolytic specificity of protease derived from Staphylococcus as determined upon incubation with ZAAX-pNA with Xaa representing the amino acid residue (in the one- letter code) preceding pNA.

Detailed description of the invention

The present invention relates to a method for relaxing dough in the process of making a bakery product The method comprises using a protease which has high specificity for glutamyl residues as the only proteolytic component in the dough and as a dough relaxant. One advantage of the method according to the invention is that the method according to the invention allows for improved doughs. Without wishing to be bound by any theory, the inventor(s) believe that using the method according to the invention improved doughs are obtained because excessive digestion of the gluten in the dough is prevented and a more controlled breakdown of gluten is achieved. The improved doughs have improved texture and structure compared to a reference dough in which no protease was used which has high specificity for glutamyl residues as the only proteolytic component and as a dough relaxant. This is in particular of importance when a reworked dough, viz. a dough that has been mixed and processed and added back to a fresh batch of dough, is involved.

In the present context, a protease which has high specificity for glutamyl residues is also referred to as a glutamate-specific protease, glutamyl endoprotease or glutamic acid specific protease. In the present context, the term 'protease' is interchangeably used with the term 'endoprotease' and the term 'endopeptidase' and refers to an enzyme which catalyzes the cleavage of peptide bonds in other proteins or polypeptides, wherein the peptide bonds are distinct from the amino or carboxy termini of the substrate protein or polypeptide. It may belong to any of the groups into which endoproteases are commonly classified, viz. serine proteases, aspartic proteases, cysteine proteases and metalloproteases, which refer to their catalytic mechanism.

The protease which is used in the method according to the invention may be any type of protease, be it a neutral protease, an acidic protease or an alkaline protease. Preferably, it is an alkaline protease, which refers to a protease which has its pH optimum between about pH 8 and 12.

The protease which is used in the method according to the invention may be obtainable from any organism, be it a plant, animal or microorganism, such as a bacterium, fungus, yeast or virus. In one embodiment it is obtainable from a prokaryotic cell, e.g. a Gram-negative or Gram-positive bacterium. Suitable bacteria include Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Moraxella, Neisseria, Staphylococcus, Streptomyces or Thermoactinomyces. Preferably, it is obtainable from a bacterial cell, in particular from a Staphylococcus, Streptomyces, Thermoactinomyces or a Bacillus, more in particular from an S. aureus, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. intermedius, B. puntis, B. megaterium, B. halodurans or B. pumilus.

In another embodiment, the protease is obtainable from a eukaryotic cell. Preferably, the eukaryotic cell is a mammalian, insect, plant, fungal, or algal cell. Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, PerC6 cells, and hybridomas. Preferred insect cells include e.g. Sf9 and Sf21 cells and derivatives thereof. More preferably, the eukaryotic cell is a fungal cell, i.e. a yeast cell, such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. More preferably from Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris, or a filamentous fungal cell . Most preferably, the eukaryotic cell is a filamentous fungal cell. Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al , In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). Filamentous fungal strains include strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma. Preferred filamentous fungal cells belong to a species of an Aspergillus, Chrysosporium, Penicillium, Talaromyces, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Myceliophtora thermophila, Fusarium oxysporum, Trichoderma reesei or Penicillium chrysogenum. The protease may be obtained by any means available in the art, including genetic engineering techniques, such as cloning, mutation and nucleotide or protein synthesis.

The protease may be comprised in a preparation comprising or consisting of the protease. The protease preparation may comprise between 0.001 % and 100% w/w of the protease based on total protein. Preferably, the preparation comprises between 1 % and 70% w/w of the protease based on total protein. In one embodiment, the preparation comprises between 1 % and 50% w/w of the protease based on total protein. In yet another embodiment, the preparation comprises between 1 % and 30% w/w of the protease based on total protein.

The protease may be the major enzymatic component in the preparation. Alternatively, the protease preparation may comprise several enzymatic activities, as long as they do not interfere with the benefits offered by the method according to the invention. This means that excessive relaxation of the dough, most likely due to excessive digestion of the gluten, must be prevented. Therefore, the protease is preferably the only proteolytic component, in any case the only functioning proteolytic component, present in the preparation.

The protease which is used in the method according to the invention has high specificity towards glutamyl residues in a polypeptide chain. In the present context, an enzyme is said to have 'specificity' for a certain amino acid when under given circumstances the peptide bond at the carboxy or amino terminal site of the certain amino acid is cleaved with a preference substantially in excess of other peptide bonds. In the present context, 'high specificity' refers to a preference for cleavage at a peptide bond of a certain amino acid, whereby the preference is in excess at least three fold , preferably at least four fold , preferably at least five fold , preferably at least six fold relative to any other peptide bond. More preferably, the preference at least seven fold, at least eight fold, at least nine fold or at least ten fold in excess to any other peptide bonds. Glutamyl endopeptidases (EC 3.4.21.19) have high specificity for cleaving at the carboxy terminal side of glutamyl residues. Suitable glutamyl endopeptidases which may be used in the method according to the present invention are glutamyl endopeptidases which cleave at the glutamyl residue with a preference in excess of at least five fold, preferably at least six fold, at least seven fold, at least eight fold, at least nine fold, at least ten fold, relative to any other amino acids. Some endoprotease have broad specificity and hydrolyse peptide bonds of, for example, (all) hydrophobic residues. Although they may have certain specificity for glutamyl residues, they will not have high specificity for glutamyl residues, and therefore such proteases should not be used in the method according to the invention, because this may lead to excessive relaxation of the dough, most likely due to excessive digestion of the gluten. On the other hand, if a protease has high specificity for glutamyl residues, and (low) specificity for peptide bonds adjacent to other amino acids residues, it may still be used in the method according to the invention as long as it does not lead to excessive relaxation of the dough, most likely due to excessive digestion of the gluten. One unit (U) of the glutamyl endopeptidolytic activity is defined as the amount of enzyme required for the production of 1 μηηοΙ p-nitroanilide per minute

(ε₄₀5 = 9650 mol^"1cm^"1) using as the substrate ZAAE-p-nitroanilide (1 ,0 millimol/liter) in a buffer containing 100 millimol/liter (mM) MES buffer of pH 6.5, running the enzymatic reaction for a period of 10 min. at 37°C.

In an embodiment of the method according to the invention, the method comprises contacting the dough with a protease which has a high specificity for glutamyl residues, wherein said high specificity is defined as a preference for a glutamyl peptide bond of at least three fold, preferably at least four fold, preferably at least five fold, preferably at least six fold, preferably at least seven fold, preferably at least eight fold, relative to a peptide bond of any other amino acid residue as determined via incubating 1 ,25 mM (millimol/l) solutions of ZAAX-p-nitroanilide comprising 10 microgram/ml, preferably purified, protease in a buffer containing 100 mM MES buffer of pH 6.5 at 37°C for a period of 30 min and after these 30 minutes measuring the optical density at 405 nm, using 1 ,25 mM (millimol/l) solutions of ZAAX-p-nitroanilide in the buffer containing 100 mM MES buffer of pH 6.5 at 37°C without protease as a reference, wherein X represents either A, I, L, V, S, G, P, Q, E, R, D, K, N, Y, H, F, C or T. A suitable apparatus to carry out the optical density measurement of this embodiment is a Tecan Genios MTP reader (Salzburg, Vienna).

In a further aspect of this embodiment X represents either A, I, L, V, S, G, P, Q, E, R, D, K, N, Y, H or F. The one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Accordingly, A stands for Alanine, I for Isoleucine, L for Leucine, V for Valine, S for Serine, G for Glycine, P for Proline, Q for Glutamine, E for Glutamic acid, R for Arginine , D for Aspartic acid , K for Lysine, N for Asparagine, Y for Tyrosine, H for Histidine , F for Phenylalanine^ for Cysteine, T for Threonine.

In the present context, "dough relaxation" refers to reduced elasticity within the dough. Dough relaxation can be measured by a number of established techniques that characterise the rheology of dough, for example by oscillatory rheometry, or by stress-relaxation tests (see Zaidel et al, J. Applied Sciences 10:2478-2480, 2010) or, more satisfactorily, through the evaluation of the dough during processing such that the dough exhibits a reduced tendency to contract after lamination and an increased tendency for extensional flow during handling and proof. In the present context, the term 'gluten' refers to the protein composite found in wheat and related grains, such as rye and barley. Gluten is an important constituent of the flour used for making doughs and gives cohesiveness to dough. The gluten may be any gluten, be it from wheat, rye or barley. In a preferred embodiment, the gluten is wheat gluten. Wheat gluten is formed from the proteins gliadin and glutenin. In the present invention, the term 'dough' refers to an elastic, pliable protein network mixture that minimally comprises a flour, or meal and a liquid, such as milk or water, which is typically used to prepare a food product. A typical dough may comprise, in addition to the flour, or meal and the liquid, such as milk or water, one or more of the following ingredients: grain, yeast, sponge, salt, shortening, sugar, yeast nutrients, dough conditioners and preservatives. The dough may be any dough, in particular a dough for baked goods. The term 'baked good' refers to baked or to be baked food products, including bread (e.g. rye, wheat, oat, potato, white, whole grain products, mixed flours, loaves, twists, buns, rolls, pitas, matzos, focaccia, melba toast, zwieback, croutons, soft pretzels, soft and hard bread, bread sticks), such as yeast leavened and chemically-leavened bread, white bread, variety breads, rolls, muffins, cakes, danish, croissants, bagels, cookies, in particular biscuits, confectionery coatings, crackers, doughnuts and other sweet pastry goods, pie and pizza crusts, pretzels, pita and other flat breads, tortillas, pasta products, par-baked products and refrigerated and frozen dough products. The dough and the glutamyl endoprotease may be brought into contact by any suitable way. Typically, this is done by adding the glutamyl endoprotease to the flour which is used to prepare the dough. The dough comprising the glutamyl endoprotease may be kneaded and rested for at least 5 min, at least 10 min, at least 30 min, at least 45 min, at least 60 min, at least 75 min, at least 90 min, at least 120 min, without negative effects to the characteristics of the dough or of the end product. The protease may be added before kneading or during kneading. The method of the invention is particularly advantageous for doughs which are reworked.

The skilled person will understand that the amount of glutamate specific protease to be added to the flour for the dough will vary depending on the type of flour used, the desired degree of digestion and the duration of the digestion. The amount of peptidase added will typically range from about 1 unit of enzyme per kg flour to about 2000 units of enzyme per kilogram of flour. Preferably, 50 - 2000 units of enzyme per kg flour is used, more preferably, 100-1000 units of enzyme per kg flour is used. One unit (U) of the glutamyl endopeptidolytic activity is defined as the amount of enzyme required for the production of 1 μηηοΙ p- nitroanilide per minute (ε₄₀5 = 9650 modern^"1) using the substrate ZAAE-p-nitroanilide (1 mM) in a buffer containing 100 mM MES buffer/pH 6.5. The enzymic reaction is run at 37°C over a period of 30 min.

The skilled person will understand that the duration of the digestion will vary, depending on the circumstances and the application. Generally speaking, the duration of the digestion may range from a few minutes to many hours, such as, from about 5 or 10 minutes to about 1 hour or 2 hours. The digestion will end when all the peptide substrates for the protease have been converted. Alternatively, the peptidase may be inactivated actively, for example by heating the dough. In both cases, excessive relaxation of the dough, probably due to excessive digestion of the gluten, will be prevented.

In another aspect, the present invention relates to the use of a protease for the relaxation of a dough. All the embodiments which were mentioned above, with all the preferences mentioned above, also apply to this aspect of the invention.

In yet another aspect, the present invention relates to a dough obtained by a method according to the invention. The dough may be used in baking, to prepare a baked or bakery product, such as a bread product (e.g. rye, wheat, oat, potato, white, whole grain products, mixed flours, loaves, twists, buns, rolls, pitas, matzos, focaccia, melba toast, zwieback, croutons, soft pretzels, soft and hard bread, bread sticks), such as yeast leavened and chemically-leavened bread, white bread, variety breads, rolls, muffins, cakes, danish, croissants, bagels, cookies, in particular biscuits, confectionery coatings, crackers, doughnuts and other sweet pastry goods, pie and pizza crusts, pretzels, pita and other flat breads, tortillas, pasta products, par-baked products and refrigerated and frozen dough products. More preferably, the dough is a biscuit dough. Baked products obtained by using a dough prepared according to the method according to the invention are also encompassed by the present invention. All the embodiments which were mentioned above, with all the preferences mentioned above, also apply to doughs and baked products according to the invention.

EXAMPLES

Example 1 Glutamyl endopeptidase assay

The bacterial glutamyl endopeptidases (EC 3.4.21 .19) may be divided into three groups according to the source organisms and sequence relationships: a Staphylococcal group, a Bacillus group and a Streptomyces group (Handbook of Proteolytic Enzymes, A.J. Barrett, N.D. Rawlings and J.F. Woessner eds, Academic Press). In the present experiments the cleavage preferences of the glutamyl endopeptidases are exemplified by enzyme samples obtained from either Staphylococcus aureus (Sigma) or from Bacillus (over expressed and isolated essentially as described by Matsumoto et al.(J. Ferment. Bioeng.1995, 79,23-27)).

One unit (U) of the glutamyl endopeptidolytic activity was defined as the amount of enzyme required for the production of 1 μηηοΙ p-nitroanilide per minute (ε₄₀5 = 9650 modern^"1) using as the substrate ZAAE-p-nitroanilide (1 ,0 millimol/liter (mM)); Z re p rese n ti n g benzyloxycarbonyl and A and E using the one-letter code for amino acids) in a buffer containing 100 mM MES buffer of pH 6.5. Liberation of pNA by each preparation was followed at 405 nm.

The two enzyme preparations were determined to have the activity listed in Table 1 . Table 1. Activity of glutamyl endopeptidases

^* Activity expressed with respect to enzyme preparation

Example 2: Cleavage specificity

Substrates based on ZAAX-p- nitroanilinenitroanilide (1 ,25 mM; Z representing benzyloxycarbonyl and A the one-letter amino acid code for alanine), where X= A, I, L, V, S, G, P, Q, E, R, D, K, N, Y, H, F (again in the one-letter code for amino acid residues), were incubated with glutamyl endopeptidase derived from Staphylococcus and Bacillus, as specified in Example 1 (at a glutamyl endopeptidase concentration of 10 g/ml), using 0.1 M MES buffer at pH 6.5, and incubating mixtures for 30 min at 37°C. As a reference 1 ,25 mM (millimol/l) solutions of ZAAX-p-nitroanilide in the buffer containing 100 mM MES buffer of pH 6.5 at 37°C without protease were used. A Tecan Genios MTP reader (Salzburg, Vienna) was used for the optical density measurements.

Results are shown in Figures 1 and 2. The enzyme preparations in both cases were shown to have high specificity towards peptide cleavage at the glutamate residue, with a preference substantially in excess of ten-fold relative to the other amino acid residues.

Example 3: Comparison of dough rheology made with different formulations

In this Example, the dough rheology of dough made with three different formulations was compared. One formulation contained sodium metabisulfite (SMS); another contained, glutamyl endopeptidase isolated from Staphylococcus aureus; and a formulation with a commercial bacillolysin (Bakezyme® Protease GBW from DSM, The Netherlands) with broad specificity, viz. which specificity for peptide bonds involving hydrophobic residues, such as leucine, isoleucine, phenylalanine and valine.

Doughs were prepared according to the formulation of Table 2a/b, using a Farinograph mixing bowl set at 40°C, and mixing 12 minutes beyond peak torque. Dough pieces were then held at 40°C allowing periodic rheological measurement to be made by means of a stress-relaxation test using a texture analyser fitted with a 45mm probe (Microstable TAXT2); the probe was compressed into a dough piece (15g) and compressed by 50% measuring the decay in force from its maximum value over a period of 25s. Results are provided in Table 3.

Table 2a. Base recipe

Table 2b. Recipe variants with dose-range for sodium metabisulfite and glutamyl endopeptidase (additions with respect to flour weight)

Sodium Bakezyme S. aureus

metabisulfite Protease GBW Glutamyl

(%) endopeptidase

(ppm)

(U/kg)

Mix 1 0 0 0

Mix 2.1 0.0125 0 0

Mix 2.2 0.025 0 0

Mix 2.3 0.05 0 0

Mix 2.4 0.1 0 0

Mix 3.1 0 25 0

Mix 3.2 0 50 0

Mix 3.3 0 100 0

Mix 4.1 0 0 100

Mix 4.2 0 0 200 Table 3. Stress relaxation values for doughs incubated at 40°C using formulations including sodium metabisulfite, Bakezyme® Protease GBW and glutamyl endopeptidase

Stress relaxation values (g/s)

15min 30min 60min 120min

Mix 1 21 .92 19.95 20.36 19.33

Mix 2.1 21 .81 25.85 20.12 25.85

Mix 2.2 30.17 27.69 29.15 28.76

Mix 2.3 37.53 36.30 35.17 37.23

Mix 2.4 No analysis possible

Mix 3.1 41 .4 42.28 50.35 56.95

Mix 3.2 No analysis possible

Mix 3.3

Mix 4.1 23.43 22.37 23.21 23.84

Mix 4.2 27.76 25.85 26.53 29.37

It was seen that sodium metabisulfite produced dough that was more extensible, yielding higher stress-relaxation values, in accordance with the dose used, until the dough was no longer suitable for testing because it was too fluid to handle. The general pattern showed that sodium metabisulfite had reduced the protein of dough by the time of the first measurement (i.e. 15 min after the end of mixing), with there being no discernible pattern of further relaxation with the later measurements, which is consistent with the qualities attributed to this material in biscuit-making.

The influence of Bakezyme® Protease GBW, a commercialised enzyme that brings broad cleavage specificity, contrasted with sodium metabisulfite. This enzyme at its lowest inclusion level was seen to lead to progressive relaxation of dough over time, while the higher inclusion levels gave dough that was too fluid to measure through this approach. This shows that, while this protease does relax dough, it does not do this in a satisfactorily controlled way as a consequence of the qualities of dough changing markedly with time and dose level. This is a characteristic of proteases now available to the biscuit industry and for this reason these do not form a satisfactory alternative to sodium metabisulfite The glutamyl endopeptidase, however, was shown to act in a manner that was comparable to sodium metabisulfite; specifically it was shown to lead to mild relaxation only, comparable to 0.0125 - 0.025% sodium metabisulfite, which did not change measurably over the incubation period nor over a two fold increase in the usage level of the enzyme. This shows that glutamyl endopeptidase can be used practically in biscuit dough where variations in processing time and reworking are known to occur.

Accordingly, it can be seen that the glutamyl endopeptidase brings substantial advantage over enzymes of broad cleavage specificity and forms an alternative to sodium metabisulfite.

Example 4: Semi-sweet biscuits made with formulations containing sodium metabisulfite and glutamyl endopeptidase isolated from Bacillus

Sodium metabisulfite and glutamyl endopeptidase were tested in a semi-sweet biscuit recipe as given in Table 4a/b. Dough was mixed in a Morton mixer to a temperature of 40±2°C and rested at 40°C for 10 minutes. A portion of the dough was then sheeted using a Rondo pastry brake; Rondo settings of 25, 15, 10, 5, 3, 2, and 1 .5 were used respectively for seven passes, turning the dough piece 90 degrees between the third and fourth pass and resting the dough for approximately 10 seconds between each pass, in order to achieve dough pieces of satisfactory thickness. A circular cutter was used then to form 24 biscuits which were baked in a Spooner travelling oven for 5 seconds in a first section and 30 seconds in a second section (first section set at 180°C and the second section was set at 245°C).

Measurements were taken of weight, both of the dough after cutting and the biscuit, as well as the dimensions of the biscuit in terms of height (in terms of stacking), and maximum and minimum length; results are given in Table 5. Separately judgements were also made on biscuit colour and texture.

It was seen that sodium metabisulfite led to less mass within the cut dough pieces which, in turn, led to less mass within the biscuit. This result was expected as a consequence of the dough being more relaxed and extensible such that it became thinner during sheeting. This effect was also seen directly by the measurement of stack height that was seen to lessen with the use of sodium metabisulfite. Further evidence for a more relaxed and extensible dough was provided by the distortion away from the circular form in which the dough had been cut; that is dough with unacceptable extensibility contracts to a greater along the direction of sheeting such that the dough and, in turn the baked biscuit, loses the circular form in which it has been cut and forms an oval shape instead. This can be seen from the ratio of the minimum length of the biscuit to its maximum length. Ideally this ratio should be one and, with the use of sodium metabisulfite, biscuits were measured to become closer to this ideal ratio.

Table 4a. Base recipe

Ingredient % (Flour basis)

Flour (biscuit) 100.00

Sugar (pulverised) 20.83

Fat (biscuit) 16.07

Glucose syrup 1 .31

Salt 1 .20

Sodium acid pyrophosphate 0.10

Sodium bicarbonate 0.50

Ammonium bicarbonate 1 .07

Water 18

Table 4b. Recipe variants with dose-range for sodium metabisulfite and glutamyl endopeptidase (additions with respect to flour weight)

Sodium Glutamyl

metabisulfite endopeptidase

(%)

(U/kg)

Mix 1 0 0

Mix 2.1 0.01 0

Mix 2.2 0.02 0

Mix 2.3 0.04 0

Mix 3.1 0 190

Mix 3.2 0 380

Mix 3.3 0 760

Mix 3.4 0 1520

Table 5. Weights of cut dough and biscuits (average of 24 pieces) and dimensions of ten aligned biscuits made with formulations including sodium metabisulfite and glutamyl endopeptidase.

With regard to formulation including the glutamyl endopeptidase, two key influences were observed: firstly, in absolute terms, the glutamyl endopeptidase brought closely similar influence to sodium metabisulfite on the properties of the dough and biscuits as can be seen by comparing the weights of the cut dough and those of the biscuits and, additionally, by the dimensions taken of the biscuits; secondly, and more importantly, the scale of influence remained of the same magnitude in spite of the enzyme being used over an 8 fold dose level.

Accordingly this shows that glutamyl endopeptidase can fully replace sodium metabisulfite by giving both similar relaxation properties with respect to the dough and similar process- tolerance, because extended proteolysis is precluded, such that the dough can be handled satisfactorily within industrial processes. Other judgements taken on colour and textural characteristics also showed that the protease affected biscuit qualities in a comparable manner to sodium metabisulfite.

Claims

1 . Method for the relaxation of a dough in the process of making a bakery product, the method comprising using a protease which has a high specificity for glutamyl residues as the only proteolytic component in the dough and as a dough relaxant.

2. Method according to claim 1 , wherein the dough is a rework dough.

3. Method according to claim 1 or 2 , wherein the dough is a biscuit dough.

4. Method according to claims 1 -3, wherein the protease is an alkaline protease.

5. Method according to any one of claims 1 -4, wherein the protease is obtainable from a bacterial, yeast or fungal cell.

6. Method according to claim 5, wherein the protease is obtainable from an Aspergillus, a Bacillus, a Staphylococcus, a Streptomyces or Kluyveromyces cell.

7. Method according to any one of claims 1 -6, wherein the bakery product is selected from bread, rolls, muffins, cakes, cookies, biscuits, confectionery coatings, crackers, sweet pastry goods, pie and pizza crusts, pretzels, flat breads, tortillas, pasta products, and refrigerated and frozen dough products.

8. A dough for making a bakery product prepared by the method according to any one of claims 1 -7.

9. Use of a dough according to claim 8, wherein the bakery product is selected from bread, rolls, muffins, cakes, cookies, biscuits, confectionery coatings, crackers, sweet pastry goods, pie and pizza crusts, pretzels, flat breads, tortillas, pasta products, and refrigerated and frozen dough products.

10. A bakery product prepared by using a dough according to claim 8.