WO2005117595A1

WO2005117595A1 - Method to improve flavour of baked cereal products

Info

Publication number: WO2005117595A1
Application number: PCT/EP2005/052533
Authority: WO
Inventors: Luppo Edens; Jan Dirk Rene Hille
Original assignee: Dsm Ip Assets B.V.
Priority date: 2004-06-04
Filing date: 2005-06-02
Publication date: 2005-12-15
Also published as: AR049071A1

Abstract

The present invention describes the use of a proline-specific endoprotease to improve the flavour of baked cereal products. The invention also relates to novel compositions comprising a proline-specific endoprotease and an exopeptidase and dough comprising such composition suitable for improving flavour in baked cereal products, a method for improving flavour in baked products, and to baked products having an improved flavour.

Description

METHOD TO IMPROVE FLAVOUR OF BAKED CEREAL PRODUCTS

The present invention relates to the use of a composition comprising a proline- specific protease for the improvement of the flavour of baked cereal products. The invention also relates to novel compositions and dough suitable for improving flavour in baked cereal products, a method for improving flavour in baked products, and to baked products having an improved flavour. The flavour of freshly baked cereal products, for example bread, is generally appreciated. According to Jackel (1969, Baker's Dig. 43 (5), 24-28) bread flavour results from four categories of factors: ingredients, fermentation, degradations, and thermal reactions. Ingredients used in bread making, mainly (wheat or rye) flour, have peculiar aromatic characteristics, but they need to undergo several changes in order to produce a complete flavour. Fermentation of sugars and amino acids by yeast leads to a large number of volatile compounds that are supposed to be responsible for distinctive characteristics associated to bread flavour. Mechanical and enzymatic degradations are necessary to eliminate the starchy residual taste of flour, which can be partly obtained by partial starch gelatinisation and by adequate relaxation of gluten. Thermal reactions, including caramellization and non-enzymatic browning during baking, promote crust flavour and colour. Although bread flavour is composed of a large number of components the pleasant aroma of wheat bread crust is mainly determined by the formation of roasty smelling compounds during the baking process (Grosch & Schieberle (1997), Cereal Chem. 74, 91- 97). Praline is considered as one of the precursors of bread flavour. Proline is an amino (in fact imino) acid occurring in gluten forming proteins present in wheat flour. After baking the bread, the fresh bread aroma disappears rapidly while off-flavours appear. In order to improve and stabilize the desired fresh bread flavour supplementation with both amino acids and combinations of amino acids with sugars have been applied (Fadel & Hegazy (1997) Die Nahrung 4, 386 - 394). This resulted in an improvement of the crust colour and fresh bread flavour while the freshness of the flavour was maintained during a storage period of up to three days. However, addition of free amino acids to the dough recipe (apart from L-cysteine) is in many countries not permitted. Wheat flour contains several proteins such as gluten that is rich in the amino acids proline, glutamine and glycine. Up to now, it has been practically impossible to liberate specifically proline as a single amino acid from gluten, due to the fact that all known proteases are in general not able to cleave protein next to a proline residue. Instead, most known proteases cleave gluten in such a way that the gluten network is destroyed which results in baked products with very bad structural properties. In the case of bread, the dough looses its gas holding capacity, which results in very low volumes of the bread baked therefrom. We have now surprisingly found that the flavour of baked cereal products can be improved significantly by using a composition that comprises a proline-specific endoprotease or by using a composition that comprises a combination of said endoprotease with an exoprotease, whereby the structural properties of the dough and baked products are not impaired. A peptide or oligopeptide is defined herein as a chain of at least two amino acids that are linked through peptide bonds. The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires. A "polypeptide" is defined herein for chains containing more than 30 amino acid residues (Fϋlop,V., Szeltner,Z. and Polgar, L (1998) Cell 94, 61 -170). All (oligo)peptide and polypeptide formulas or sequences herein are written from left to right in the direction from amino-terminus to carboxy-terminus, in accordance with common practice. 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). An endoprotease is defined herein as an enzyme that hydrolyses peptide bonds in a polypeptide in an endo-fasion and belongs to the group EC 3.4. The endoproteases are divided into sub-subclasses on the basis of catalytic mechanism and specificity is used only to identify individual enzymes within the groups. These are the sub-subclasses of serine endoproteases (EC 3.4.21), cysteine endoproteases (EC 3.4.22), aspartic endoproteases

(EC 3.4.23), metalloendoproteases (EC 3.4.24) and threonine endoproteases (EC 3.4.25). A proline-specific endoprotease is defined herein as an endoprotease which has a preference to cleave a peptide bond adjacent to a proline residue in a peptide chain, either at the C-terminal side of the proline residue or at the N-terminal side of the proline residue. Cleavage at the C-terminal side results in a peptide chain with a C-termiήal proline. Likewise, cleavage at the N-terminal side results in a peptide chain with a N-terminal proline. Cleavage between two adjacent proline residues results in two peptides with a C- terminal and N-terminal proline residue respectively. In a first aspect, the invention provides the use of a composition for the improvement of the flavour of a baked cereal product characterized in that the composition comprises a proline-specific endoprotease. Suitable proline-specific endoproteases for the invention can be endoproteases, which yield either an amino terminal proline residue or a carboxy terminal proline residue. It was surprisingly found, that the use of proline-specific endoproteases according to the invention in effective amounts results in significant flavour improvement in the dough during normal length of proofing while maintaining the structural properties, such as the gas-retaining capacity, of the dough. Preferably, a proline-specific endoprotease is used that can cleave the gluten proteins in the dough in the native form, i.e. without requiring the prior formation of smaller (poly)peptides. The specificity of a proline-specific endoprotease can be determined by methods that are generally known to the person skilled in the art. For example, the activity of proline- specific endoproteases that cleave at the carboxy-terminal side of proline, can be measured by using the synthetic peptide Z-Gly-Pro-pNA (Z = benzyloxycarbonyl) as a substrate while monitoring the formation of the yellow coloured para-nitroanilide (pNA). The activity of proline-specific endoproteases that cleave at the amino-terminal side of the proline residue can be' identified using for example the synthetic substrate FA-Pro-Ala-Ala (FA = 3-(2-furylacryloyl)) and whereby the carboxy-terminus of the substrate preferentially is blocked by methods known in the art. The activity of proline-specific endoproteases that cleave in between two adjacent proline residues can be identified using for example an internally quenched fluorogenic substrate such as HOO-E(EDANS)PPPPK(DABCYL)NH2 according to the method described by Matayoshi et al (1990), Science 247, 954-958. In this substrate, the fluorescent donor (EDANS) is linked to the side chain carboxyl group of the N-terminal glutamic acid and the fluorescent quenching acceptor (DABCYL) is linked to the side chain amino group of the C-terminal lysine. An additional advantage of the use of such a proline-specific endoprotease is that flavour improvement in the bread is enabled in situ, thereby avoiding the need to add for instance a protein hydrolysates, enriched in amino acids (e.g. proline), to the bread in order to improve its flavour. A further advantage of the use of such a proline-specific endoprotease is that it can be used for improving flavour in bread types whereby the flavour has to be developed in a relatively short time, for example for bread prepared from frozen dough. Preferably proline-specific endoproteases are used having their optimum proline- specific proteolytic activity at or below ^* pH 7.0. More preferably, proline-specific endoproteases having a maximum proline-specific proteolytic activity at or below pH 6.0 are used. Most preferably, proline-specific endoproteases, having a proline-specific

5 proteolytic activity around the pH of the dough, are used. An "isolated" or "purified" polypeptide or protein is defined herein as a polypeptide or protein, which is isolated from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have beeno purified to some extend by any suitable technique known in the art. The proline-specific endoprotease suitable for the use and method according to the invention may be derived from animals, plants or microorganisms such as bacteria, yeasts and fungi. Preferably, the proline-specific endoprotease is derived from fungi, in particular from the genus Aspergillus, most preferably Aspergillus niger. The latter organism is widely5 known as a food grade microorganism. Although proline-specific endoproteases are known in the art, their use for the improvement of the flavour of cereal, baked products has neither been described nor suggested in the prior art. The proline-specific endoprotease may be isolated via methods known in the art. Fo instance, proline-specific endoprotease may be isolated from culture broths obtainedo from (large scale) fermentation processes wherein the proline-specific endoprotease- producing microorganism, such as Aspergillus niger, is grown. In a preferred embodiment, the proline-specific endoprotease is isolated from a microbial host that is genetically engineered in order to over-express a gene encoding the proline-specific endoprotease. Suitable hosts known in the art are bacteria (i.e. of the genus Bacillus, Escherichia5 etceteras), yeasts (i.e. of the genus Saccharomyces or Kluyveromyes) or fungi (i.e. from the genus Aspergillus, such as Aspergillus niger, Aspergillus ory∑ea and others known in the art). Most preferred is the over-expression of the gene encoding an Aspergillus niger proline-specific endoprotease by an engineering Aspergillus niger host. Examples of suitable fungal proline-specific endoproteases are those disclosed ino WO02/45524. Surprisingly, we have found that the proline-specific endoproteases described in WO02/45524 can hydrolyse intact proteins, larger polypeptides as well as smaller (oligo)peptides. Most preferred is the specific proline-specific endoprotease referred to in claims 1-5, 11 and 13 of WO02/45524. The proline-specific endoprotease from Aspergillus niger as described in WO02/45524 is specifically preferred as endoprotease for the use according to this invention, to be used in the compositions according to the invention and for use in the methods according to the invention. This proline-specific endoprotease has the additional advantage that it is very active under the pH conditions usually present in dough, being below pH 6.5, more specifically around pH 5.5. Although WO02/45524 discloses that proline-specific endoproteases can be used for anti-staling of bread, the use of these proline-specific endoproteases to improve the flavour of baked cereal products has neither been disclosed nor suggested in WO02/45524, nor that the proline-specific endoproteases would be capable of cleaving a gluten protein without substantially destroying the gluten structure. In a second aspect, the invention provides the use of a composition for the improvement of the flavour of a baked cereal product characterized in that the composition comprises a proline-specific endoprotease described hereinbefore in combination with an exopeptidase. An exopeptidase is defined herein as an enzyme that hydrolyses peptide bonds in peptide chains in an exo-fasion and as such belongs to the group EC 3.4. The exopeptidase act only near the ends of polypeptide chains, and those acting at a free N- terminus liberate a single amino-acid residue (aminopeptidases, EC 3.4.11), or a dipeptide or a tripeptide (dipeptidyl-peptidases and tripeptidyl-peptidases, EC 3.4.14). The exopeptidases acting at a free C-terminus liberate a single residue (carboxypeptidases, EC 3.4.16-18) or a dipeptide (peptidyl-dipeptidases, EC 3.4.15). The carboxypeptidases are allocated to four groups on the basis of catalytic mechanism: the serine-type carboxypeptidases (EC 3.4.16), the metallocarboxypeptidases (EC 3.4.17) and the cysteine-type carboxypeptidases (EC 3.4.18). In a preferred embodiment, the exopeptidase is capable of cleaving off a proline residue from either the N-terminal or the C-terminal side of the peptide chain. Preferably, the exopeptidase has preference, more preferably a high preference, for a proline residue compared to other amino acids. Preferably the exopeptidase can cleave off proline residues with an efficiency that is comparable or higher than the efficiency by which Carboxypeptidase-Y (CPD-Y) can liberate the proline residue from a synthetic Z-Ala-Pro- OH substrate (Dal Degan et al. Appl. Environ. Microbiol., July 1992, p 2144-2152). More preferably the exoprotease is able to release proline from a synthetic poly-proline substrate, such as Z-Pro-Pro-OH. In case the proline-specific endoprotease yields a peptide chain with a carboxy- terminal proline residue, the exopeptidase is preferably a carboxypeptidase, more preferably a carboxypeptidase liberating a single amino acid from the peptide chain (EC 3.4.16-18). Furthermore, it is preferred that the carboxypeptidase has a high preference for a carboxy-terminal proline residue. Suitable examples are the carboxypeptidase CPD-Y (as referred to above) or the proline-specific carboxypeptidases obtainable from Xanthomonas or Escherichia. Preferably, carboxypeptidases obtained from Xanthomonas citrii or Xanthomonas campestris or Escherichia coli are used. In case the proline-specific endoprotease yields a peptide chain with an amino- terminal proline residue, the proline-specific exopeptidase is preferably an aminopeptidase, more preferably an aminopeptidase liberating a single amino acid from the peptide chain (EC 3.4.11 ). Furthermore, it is preferred that the aminopeptidase has a preference for proline (EC 3.4.1 1.5, EC 3.4.14.2 or EC 3.4.14.5). Most preferably an aminopeptidase having a high preference for proline is used (EC 3.4.11.5). Examples of suitable aminopeptidases are described in WO98/51804. The following combinations of enzymes are highly preferred: (a) a proline-specific endoprotease yielding a peptide chain with a carboxy-terminal proline residue and a proline-specific carboxypeptidase; (b) a proline-specific endoprotease yielding a peptide chain with an amino-terminal proline residue and a proline-specific aminopeptidase; (c) a proline-specific endoprotease yielding a mixture of peptide chains containing amino-terminal and carboxy-terminal proline residue and a proline-specific aminopeptidase and a proline-specific carboxypeptidase; (d) a mixture of proline-specific endoprotease yielding a mixture of peptide chains containing amino-terminal and carboxy-terminal proline residue and a proline- specific aminopeptidase and a proline-specific carboxypeptidase.

It has surprisingly been found that the use of such compositions not only improve the flavour of baked cereal products, but also that it enables the flavour to be stabilized for a longer period in time, retaining the desired fresh bread flavour during shelf life. In a third aspect, the invention provides a composition for the improvement of the flavour of a baked cereal product characterized in that the composition comprises a proline- specific endoprotease, described hereinbefore, in combination with an exopeptidase, also described hereinbefore. The composition according to the invention may further comprise additives with bread and/or dough improving properties. The latter comprise chemical agents such as ascorbic acid, emulsifiers etceteras as well as enzymes such as other endoproteases or exopeptidases, amylases, hemicellulases, cellulases, oxidases, lipases, or in general starch degrading enzymes, non-starch polysaccharide degrading enzymes, lipid degrading enzymes, oxidising, reducing and cross-linking enzymes. In a fourth aspect, the invention provides a process for preparing a dough comprising the addition of a composition for the improvement of the flavour of a baked cereal product characterized in that the composition comprises a proline-specific endoprotease described hereinbefore in combination with an exopeptidase, also described hereinbefore. Dough is usually made from well-known ingredients such as cereal, preferably wheat, flour, water, a leavening agent, preferably baker's yeast and optionally other ingredients such as salt, sugar etceteras. The process for making a dough suitable for the production of baked products thereof, such as bread, biscuits etceteras, are well known in the art. Both the proline-specific endoprotease and the exoprotease are added to the dough in effective amounts. This amount is dependent on the type of process, dough temperature and length of proofing of the dough as well as the desired effect, but can in general easily be determined by the skilled person. In a preferred embodiment of the method according to the invention at least 1 unit, more preferably, at least 10 units and even more preferably 100 units of proline-specific endoprotease per kg of flour are added - for the definition of the unit see method section below. In a preferred embodiment of the method according to the invention at least 0.1 unit, preferably at least 1 unit, more preferably at least 10 units and even more preferably 100 units of exopeptidase per kilogram of flour is added to the dough - for the definition of the unit see method section below. In a fifth aspect, the invention provides dough comprising the composition according to the invention. This dough can be stored for a while, for example in a frozen form, or can immediately be used to prepare a baked cereal product by baking the dough The baking of the dough can be done at any generally used temperature for preparation of the desired baked cereal product. The baked cereal product obtained by the process of baking the dough according to the invention is another aspect of the invention, since it has an improved flavour compared with the baked cereal product obtained by the process of baking dough not comprising the composition according to the invention. The baked cereal product can encompass all types of baked goods in which flavour is essential for appreciation, for example such as bread, cookies, cake. Preferably the baked cereal product is bread, since the process according to the invention yields a significant increase in baked crust flavour. Especially bread baked in closed tins and/or produced in a very short process like the Chorleywood bread process yields a significant increase in baked bread flavour. However, the application is not limited to these types of bread, which are only given as examples.

Hereafter the invention is described in the following non-limiting examples.

Materials Proline-specific endoprotease

For the experiments described in the examples below, the proline-specific endoprotease was obtained from a selected Aspergillus niger strain G306 (deposited with the Centraal Bureau voor Schimmelcultures (CBS109712) on 10 September 2001). This strain contains a gene encoding the proline-specific endoprotease described in WO 02/45524. The proline-specific endoprotease is present in an ultrafiltrate obtained after ultrafiltration of a fermentation broth obtained after fermentation of the Aspergillus niger strain. The proline-specific activity of the obtained proline-specific endoprotease concentrate was 8.6 U/ml, determined as described under Methods. The protein concentration was estimated to be 85 mg/ml, based on the specific activity of a sample of proline-specific endoprotease with a purity estimated to be higher than 90%.

Isolation of a proline-specific carboxypeptidase from Xanthomonas species Isolation of a carboxypeptidase from Xanthomonas as described in US 5,693,503 turned out to be not successful in our hands and therefore, a completely new purification protocol had to be developed. Therefore, a cloning procedure was followed using gene banks obtained from microorganisms that are capable of producing an enzyme that can release proline from the synthetic substrate Z-Pro-Pro-OH. In order to induce the activity in the wild-type microorganism strains, they were first grown in a defined rich mineral medium (Yeast Carbon Base, pH=7) with casein as unselective N-source. After a certain period the strains that showed growth were harvested.

Cells were permeabilized and subjected to an activity assay with Z-Pro-Pro-OH as a substrate. The enzyme isolated from Xanthomonas campestris pv campestris showed activity under acid conditions. Also an enzyme isolated from Xanthomonas citrii was found to be active on carboxy terminal proline residues. Methods Spectrophotometric method for determining enzyme activities

To measure the activity of the proline-specific endoprotease, a substrate solution of 2 mM of N-carbobenzoxy-glycine-proline-p-nitro anilide (Z-Gly-Pro-pNA; M.W. 426.43; Bachem, Switserland) made in a 0.1 M citric acid / 0.2 M disodium phosphate buffer pH 5.0 containing 40 % dioxan is used. To 1 mL of buffer pH 5.0, 250 μl of the substrate solution is added followed by 100 μl of the enzyme solution (larger or smaller volume amounts of enzyme solution should be compensated for by buffer solution). The reaction mixture is incubated at 37°C and the release of pNA is followed by measuring the absorbance increase at 410 nm. One unit is defined as the quantity of enzyme that liberates 1 μmole pNA from Z-Gly-Pro-pNA per minute under the described reaction conditions while using a molar extinction coefficient (E) of 8,800 M^"1. cm^"1. The activity of the proline-specific carboxypeptidase from Xanthomonas species was measured by determining the quantity of proline residues released from the synthetic peptide Z-Pro-Pro-OH (Bachem, Switserland) using an amino acid analyser. One unit is the quantity of enzyme that liberates 1 μmole of proline from Z-Pro-Pro-OH per hour at pH 7.0 and 37 ^<€. Analysis of crust flavour

The analysis of crust flavour was carried out by a sensory analysis, done by an in- company trained non-professional panel consisting of at least seven persons. The panellists were requested to smell the crust of three pieces of bread and, to order the pieces in strength of crust flavour (1 = strongest, 5 = least strong). They were also requested to indicate their preference (1 = most preferred, 5 = least preferred). Analysis of crumb firmness

To analyse whether too much gluten was degraded because of protease action, the crumb firmness of the baked breads was measured using a Stevens Texture Analyser according to methods known in the art. Two slices of 2.0 cm thickness from the centre of each loaf were analysed using a probe of 1.5-inch diameter, a compression depth of 5 mm

(25%) and a rate of compression of 0.5 mm/sec.

Analysis of crumb resilience The crumb resilience of all slices was compared to each other by pushing a finger into the crumb of a bread slice of 2.0 cm thickness from the centre and looking at the elasticity of the crumb. This analysis was performed on the two slices as analysed for crumb firmness.

EXAMPLES

Example 1 Effects of the use of a proline-specific endoprotease on the flavour and structure of a Dutch tin bread

For bread making a dough was prepared from 3500 g of wheat flour (80% Kolibri™ and 20 % Ibis™), 1960 ml water (56 %), 77 g compressed yeast (2.2 %), 70 g salt (2 %), 140 mg ascorbic acid (40 ppm), 87.5 mg Bakezyme™ P₅₀o (10 ppm), 40 mg Bakezyme™ HSPeooo (20 ppm) and various quantities of enzymes indicated in Table 1.

The ingredients were mixed into a dough using a Kemper^® spiral mixer for 2 minutes at speed 1 followed by 6 minutes mixing at speed. Dough pieces of 875 g were rounded, proofed for 35 minutes at 34°C and 85 % RH, punched, moulded, panned, proofed for 75 minutes at 38°C and 87 % RH and baked for 20 minutes at 220 °C. Loaf volume was determined by rape seed displacement method. Crumb firmness and resilience was determined as well was the flavour determined as described at the method section. During the total test all bread samples are tested three times. As a reference for proteolytic breakdown of gluten network the bacterial endoprotease Bakezyme B-500 was applied. From these results in Table 1 it is clear that addition of the proline-specific endoprotease at various levels increased crust flavour, especially after 24 hrs of storage. After 68 hours of storage sensory differences are much smaller. Addition of 46 U/kg flour resulted in a stronger and preferred crust flavour albeit also a deviating, not specifically roasty, flavour note is present. Addition of the proline-specific endoprotease apparently did not affect the gluten network and gas-retaining capacity of the dough because the loaf volumes and firmness values were not affected. Crumb softness is improved when proline- specific endoprotease is added. The use of a bacterial non-specific endoprotease (Bakezyme B-500 (ppm) resulted in some decrease of the loaf volume, a strong increase in crumb firmness and a negative effect on crust flavour.

Table 1: Effect of the use of a proline-specific endoprotease on bread flavour and structure

(a) non-bread crust flavour (b) in particular off-flavours (stale bread) (c) fresh bread crumb flavour but also containing some deviating, not specifically roasty, flavour notes Example 2

Effects of the use of a proline-specific endoprotease combined with a proline-specific carboxypeptidase on the flavour and structure of a Dutch tin bread

This example demonstrates that the crust flavour can be further improved and stabilised by adding to the dough a combination of a proline-specific endoprotease and a proline-specific carboxypeptidase from Xanthomonas campestris, which significantly improves and stabilises crust flavour when used in combination with the proline-specific endoprotease that was also used for examples 1. Tin bread is produced and analysed as described in the foregoing example. Tests were performed at different levels of enzymes as indicated in Table 2. The results of the tests are shown in Table 2. Table 2: Effect of additional proline-specific carboxypeptidase on bread flavour and structure

(a) fresh bread crust flavor, but roasty component is specifically intensi led. (b) especially off-flavors (stale bread) present From these results it is clear that combination of the proline-specific endoprotease and the proline-specific carboxypeptidase results in an increase of fresh bread crust flavour and stabilisation of this flavour over a long shelf-life period, whilst maintaining the loaf volume and dough consistency of the untreated reference loaves.

Claims

I . Use of a composition for the improvement of the flavour of a baked cereal product characterized in that the composition comprises a proline-specific endoprotease.

2. Use according to claim 1 wherein the composition comprises a proline-specific endoprotease, which is derived from an Aspergillus species, preferably Aspergillus niger.

3. Use according to anyone of claims 1 or 2 wherein the composition further comprises an exopeptidase.

4. Use according to claim 3, wherein the composition comprises an exopeptidase which has a preference for proline.

5. Use according to any one of claims 3-4, wherein the proline-specific endoprotease yields a carboxy-terminal proline and wherein the exopeptidase is a carboxypeptidase.

6. Use according to any one of claims 4-5, wherein the exopeptidase is derived from Xanthomonas or Escherichia.

7. A composition as defined in anyone of claims 3-6.

8. Process for preparing a dough comprising addition of the composition according to claim 7 to the recipe. ~

9. Dough comprising a composition according to claim 7.

10. Process for preparing a baked cereal product comprising baking a dough according to claim 9.

I I . Baked cereal product obtainable by the process of claim 10 or by the use of anyone of claims 1 -2.